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SAN FRANCISCO--(BUSINESS WIRE)--Notice has been issued to Fishers and Fish processing businesses confirming that their claims are now part of a class action that has been certified by the United States District Court. Information has been mailed to each identified class members and additional information is available on-line, through various trade associations, and various publications.
Background on the May 2015 Refugio State Beach Spill
The Class Action arises from the May 15, 2015 rupture of a corroded underground pipeline owned by Texas-based Plains All American near Refugio Beach. As a result of the spill, the Department of Fish and Wildlife imposed a ban on fishing in a 138-square-mile zone from Gaviota State Beach to Isla Vista. Plains All American has acknowledged that it is the "Responsible Party" as required by federal law. The company and one of its employees also face criminal charges.
The civil litigation, Andrews et al. v. Plains All American Pipeline, L.P. et al. was originally filed shortly after the spill as a series of separate actions that have since been consolidated. The proceedings are pending in the United States District Court for the Central District of California, before Judge Philip S. Gutierrez.
Fisher and Fish Industry Subclass is Certified
On February 28, 2017, Judge Gutierrez issued an Order certifying the Fisheries and Fish Industry Subclass in the litigation, which allows those claims to proceed collectively. This Subclass contains "fishers and those that purchased fish from the commercial fisherman who worked in designated areas of the Santa Barbara coastline" affected by the closure of fishing areas in the Pacific Ocean and attendant damage to those fisheries. The Court also appointed four law firms as Lead Counsel for the Fisher Subclass. Lead Counsel recently submitted a Plan of Notice, to provide information about the litigation to all known members of the certified class.
Those who wish to participate in the class action do not need to do anything more at this time. The notice provides additional information, including instructions for those who do not want to participate, and explains how the class action litigation coordinates with claims made directly to Plains under the federal Oil Pollution Act.
Additional Subclasses are Pending
Class Counsel seeks to certify two additional subclasses on behalf of oil spill victims: an Oil Industry Subclass for workers and businesses who were employed or contracted to work on the offshore drilling platforms or on-shore processing facilities shut down as a result of the oil spill; and a Real Property Subclass that includes beachfront properties, properties with a private easement to the beach, and properties within one-half mile of beach affected by the oil spill.
Notice Mailed to Fishers and Fish Industry
Written notice has been mailed to each identified class member, and should arrive within seven days. The mailed notice will be followed by printed advertisements and postings in four languages. Information is also available online in English, Spanish, Chinese and Vietnamese.
For additional information, class members are encouraged to visit the class action website www.plainsoilspill.com, or call the toll-free number 1-888-684-6801.
Statement from Class Counsel
"The purpose of this lawsuit is to compensate local fisherman, businesses, and other victims, and to hold Plains accountable for all the harm it has wrought on the Santa Barbara community," said Robert Nelson, a partner in Lieff Cabraser's San Francisco office and one of four Lead Counsel appointed by Judge Gutierrez. "Plaintiffs are pleased with the Court's certification of the Fisher and Fish Industry Subclass, and look forward to expanding the class to include other victims of the spill. We are eager to prove their claims in court."
About Class Counsel
Lieff Cabraser Heimann & Bernstein, LLP is a seventy-plus attorney law firm with offices in San Francisco, New York, Nashville, and Seattle. It is among the largest law firms in the United States that only represent plaintiffs. Since its founding in 1972, Lieff Cabraser has litigated and resolved hundreds of class action lawsuits and thousands of individual cases. Lieff Cabraser successfully prosecuted cases against many of the world's most powerful corporations, obtaining compensation for families, businesses, and property owners harmed by toxic environmental exposures.
Keller Rohrback L.L.P. is a national law firm headquartered in Seattle, with offices in Santa Barbara, Phoenix, New York and Missoula/Ronan. The firm's Complex Litigation Group has obtained judgments and settlements, on behalf of clients and class members, in excess of 18 billion dollars, including fishermen and other victims of the Exxon Valdez Oil Spill. Members of the firm currently hold key leadership roles in the landmark Volkswagen "Clean Diesel" Litigation, and Wells Fargo unauthorized bank account litigation, among others.
Cappello & Noël LLP is a nationally-recognized trial firm based in Santa Barbara, California, with an emphasis on lender liability and class actions. As the City Attorney of Santa Barbara, A. Barry Cappello obtained a pioneering settlement against offshore oil companies following the 1969 Santa Barbara Channel Oil Spill. Since its founding in 1977, the firm has obtained jury trial verdicts and settlements in excess of $1 billion for individuals and small businesses against corporations, banks and insurance companies. The firm has successfully litigated and tried class actions challenging discriminatory business practices, environmental contamination, and price-fixing.
Audet & Partners, LLP, is a nationally recognized firm based in San Francisco specializing in complex litigation and class actions. The attorneys at Audet & Partners, LLP, represents individuals, small businesses and institutional shareholders in antitrust, product liability, negligence, consumer, construction defect, securities, environmental, whistle blower, and employment cases. In recognition of their commitment to the legal profession and outstanding abilities to get results for their clients, the firm and its members have served as Court-appointed Plaintiffs' Counsel in dozens of federal and state cases throughout the United States.


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After my first pregnancy, I spent every day trying to get back to my pre-baby body. Does that even exist? I used to walk hours a day, watched every calorie and tried to do everything possible to drop the extra pounds. I lost so much time trying to perfect myself that I forgot that being a mom was my first priority.
After my second pregnancy, I didn't have that luxury anymore. I had two little rugrats now both requiring totally different needs. One of those needs was the necessity to keep them busy. Swimming seemed to be an activity that drained the energy out of any living being and that guaranteed me a good nap time. So off to the Dorval Aquatic centre! They have a really incredible set up for kids. It is a gradual incline into the water and has an indoor splash pad. I hesitated going swimming for months. Why you may ask? Well because of that dreaded moment that I needed to put on a bathing suit.
That day finally came. As I dressed both my children in their suits I marvelled at their beautiful little bodies. They were so incredibly perfect. My body made that. I watched my daughter lovingly look at me and thought what is she going to think about herself if she grows up seeing her mother constantly judging herself? At that moment I was empowered. I owned my body. Every scar, every ripple, every extra little fat cell that wasn't there before; I owned that. I stood confident acknowledging that the woman's body is an incredible thing; something that should be celebrated. My head was held high and I was a new woman. If only the moms from my Facebook Group Perfit Moms could see me now. We'd had discussions in our group about how we struggled with body image and here I was winning that battle.
I have never looked back and I continue to celebrate. I am not the mom who is going to stand on the sidelines no matter what my size is. I will be the one running from across the room ready to cannonball into the water hoping to get all you other beautiful people to join me. So wear the bathing suit, the tight jeans or the dress you've been looking at for months. You deserve to celebrate.

subset: commoncrawl


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[Image courtesy of all-flags-world]
As legislative sessions wind down across the nation, state lawmakers are putting the final touches on election law changes before they adjourn. Earlier this week, I wrote about a Minnesota omnibus that passed the Legislature and headed to the governor. Further south and west in Topeka, Kansas lawmakers passed their own omnibus and some other session-ending legislation – but without the overwhelming bipartisan majorities seen in St. Paul.
One centerpiece of the omnibus is a switch of local nonpartisan elections to coincide with statewide elections. Supporters say it will improve turnout, but opponents (including many local officials) disagree, saying it is more about affecting outcomes. The Wichita Eagle has more:
A bill to shift local elections from the spring to the fall has passed the Kansas Legislature.
The House passed HB 2104 by a vote of 64-58 Thursday after an hour of debate. The minimum for a constitutional majority is 63 votes. The bill has already passed the Senate and now heads to the governor's desk.
The bill is a combination of several changes to election law. It moves city and school board elections to the fall of odd-numbered years, but keeps them non-partisan.
The change is projected to double turnout in local elections. Rep. John Whitmer, R-Wichita, noted that turnout in this spring's election in Wichita hovered around 16 percent despite a mayoral race and a ballot measure about marijuana.
The change has been largely opposed by local elected officials and school boards across the state.
"When did we stop listening to local governments? When did we start deciding that we know best?" asked Rep. Ed Trimmer, D-Winfield.
Lynn Rogers, a member of the Wichita school board, contended in Wichita that the date change was "not being done to increase voter participation. It's being done for control. Plain and simple."
"If they were really concerned about voter turnout, they would change August primaries," Rogers said.
During the House debate, Republicans contended the bill would make local officials more accountable by boosting turnout. Rep. Steve Huebert, R-Valley Center, called low turnout "the ultimate voter suppression tool."
Mirroring action in other states, Kansas would also eliminate the state's largely unused presidential primary to save money:
The bill also would permanently eliminate the presidential primary. That would help enshrine the caucus system, which requires candidates to pay their political party $10,000 in order to participate.
Republicans say primaries, which come at a cost of $3 million to the state, are too expensive.
Kansas has not held a primary since 1992. Rep. Tom Sawyer, D-Wichita, called the elimination of the presidential primary ill-advised, contending that primaries engage more voters. He said that 2016, a year in which both parties have open races for the presidency, would be an ideal time for Kansas to hold a primary.
Legislators also moved to clean up language about candidate withdrawals that contributed to controversy and litigation in the state's 2014 U.S. Senate race:
The bill also addresses a controversy from this past year's election by changing the requirements for a candidate to withdraw from a race after winning a primary.
Democrat Chad Taylor pulled out of the U.S. Senate race in September, a move Republicans saw as meant to boost an independent candidate against Republican incumbent U.S. Sen. Pat Roberts. This sparked a legal battle, with Secretary of State Kris Kobach trying unsuccessfully to keep Taylor on the ballot.
Under the bill, candidates' names could be removed only if they die, if they move out of the state or if they or a member of their immediate family suffer from a medical hardship.
Kobach, who recommended the change, initially wanted death to be the only way off the ballot.
Not part of the omnibus but also enacted yesterday was a bill (SB35) giving the Secretary of State prosecutorial powers in election fraud cases and increasing the penalties upon conviction. Those provisions were extremely controversial and were almost removed in the House during final debate, per the Topeka Capital-Journal:
Debate on the House floor centered on a series of amendments that would have stripped out provisions of the bill opponents found objectionable. The bill's leading proponent, Rep. John Rubin, R-Shawnee, successfully urged lawmakers to reject the amendments.
Rubin argued that amending the bill would force a conference committee with the Senate. This late in the session, a conference committee would likely kill the bill for the year as lawmakers look to adjourn as soon as the state's budget issues are solved.
County attorneys for whatever reason are not interested in prosecuting election crimes, Rubin said. Election fraud is a serious crime, he said, striking at the heart of democracy.
"This bill remains critical to me if we want to stand for free and fair elections," Rubin said.
The closest lawmakers came to amending the bill came in a change offered by Rep. Russell Jennings, R-Lakin, that would have stripped away prosecutorial power for the secretary of state from the bill, which also boosts penalties for election crimes. The amendment failed in a 60-61 vote.
Jennings said having three entities – county attorneys, the attorney general and the secretary of state — going after election crimes could lead to confusion.
"It creates a scenario where you could have a race to the courthouse. And whoever files first gets to prosecute. And there's only [one] of the three in this conversation that really wants to get to prosecute and I really want him to file papers and keep good records, not prosecute," Jennings said.
The measures now head to Governor Sam Brownback, who is likely to sign them both.
It's interesting to note how Kansas and Minnesota have taken such different approaches to election legislation; of course, one major difference is that Kansas Republicans control the legislature and the governorship so there is no need for bipartisan compromise. That said, the fact that Minnesota's governor has said he won't sign any legislation that doesn't enjoy bipartisan support is also undoubtedly a factor.
Nevertheless, as session ends in the two states it's fair to say that the the often difficult relationship that often exists between partisans in a state legislature is probably more difficult these days in Topeka than in St. Paul.
Stay tuned …


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I asked the rain
When I stood at the orphanage wall:
"Why do you pour on me furiously?"
The rain replied: "Don't cry so often!"
And cloudbursts pelt down on the yard.
I asked the sun
When they took me to another orphanage:
"Why don't you shine for me?"
The sun replied: "Keep smiling!"
And disappeared behind the clouds by the sea.
I asked the clouds
When my mother rarely was allowed to look after me:
"Why are you so gloomy above me?"
The clouds replied: "Calm down constantly!"
And gathered turbulently in the country.
I asked the snow
When they took me to another family:
"Why do you treat me so cold-heartedly?"
The snow replied: "Nevertheless, warm your heart up!"
And clashingly a frosty night came over the village.
I asked the wind
When again they took me to another family:
"Why do you blow me back and forth?"
The wind replied: "Everything comes and goes!"
And rushed to the shores of Caledonia.


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When your child has good oral health, it sets them up for good health overall, and helps them with their wellness as they grow into adults. Gum disease is linked to many illnesses like heart disease, diabetes, respiratory disease, osteoporosis, and rheumatoid arthritis. Our mouths are an entryway for infection, and inflammation can let bacteria enter the bloodstream.
Oral health affects your child's speech, how they eat, and their ability to smile and show emotions. It also influences a kid's self-esteem, academics and school attendance. Starting them off with healthy habits to care for their mouths is essential in preventing oral and other chronic illnesses in the future.
Protect Tiny Teeth As Soon As Possible
Your baby's teeth are hiding just beneath their gums as soon as they are born. Every baby is different, but usually the first teeth to appear are in the top and bottom front of their mouth. They usually start to come in anytime between 4 months and 12 months of age. But the arrival of teeth comes with the potential for tooth decay. When you see a tiny tooth peeking out of your baby's gums, that means it's time to begin caring for it.
Here's what you can do to protect tiny teeth before and after they've come in:
- Start cleaning baby's mouth at a few days old. Wipe the gums with a soft, clean, moist gauze pad or washcloth after each feeding.
- When baby is ready for solids, choose foods with no added sugar.
- Brush baby's teeth as soon as they appear, twice per day (morning and night) using a soft-bristled brush with fluoride toothpaste in the size of a grain of rice.
- To find the best toothpaste for your child, use the ADA Seal of Acceptance.
Besides helping your baby chew, speak and smile, baby teeth also hold space in their jaw for the permanent teeth that are growing under their gums. When your baby loses a tooth too early, a permanent one can drift into the empty space making it difficult for others to find room when they come in. This is one of the reasons that adult teeth can be crooked or crowded.
One of the main causes of tooth decay in babies and toddlers is from exposing their teeth to drinks that contain sugar. This is especially the case if they sleep with a bottle or it's used as a pacifier. Also, remember that bacteria from your mouth can introduce germs to your child. For instance, if your baby's pacifier falls on the floor, don't pick it up and put it in your mouth to clean it; the bacteria that causes cavities can pass through your saliva to theirs. The same is true for food. Try not to offer them a bite of your food or serve them from your utensils.
Your little one should see the dentist by their first birthday. At this visit, the dentist will do the following:
- Check your baby's teeth;
- Show you the best way to clean your little one's teeth;
- Talk to you about other ways to keep your child's mouth healthy like healthy diet, fluoride, and dental sealants.
If you're not sure about a dentist for your baby, ask your pediatrician to check their teeth and for a dental referral.
Growing Into Good Oral Health Habits
Tooth decay, or cavities, is still one of the most common chronic diseases for kids from age 6 to 19. About 1 in 5 (20%) children aged 5 to 11 years have at least one untreated decayed tooth. And 1 in 7 (13%) adolescents (aged 12-19) have at least one untreated decayed tooth.
Most children have a full set of 20 primary teeth by the time they are three years old.
Cleaning your child's teeth shouldn't be difficult. If your child is 3 or younger, use a smear of toothpaste (about the size of a grain of rice) on a child-sized toothbrush. For children 3 or older, use a pea-sized amount of fluoride toothpaste. Be sure they spit out the toothpaste rather than swallowing it. Monitor their brushing until around age 6 or until they've mastered the skill.
Other ways you can help ensure your child has the healthiest mouth possible:
- Feed them healthy foods like fruits and vegetables and avoid sugary snacks like cookies and gummy candies. Even pretzels and crackers are particularly bad for baby's teeth.
- Have them drink water, preferably tap water that contains fluoride.
- Clean between their teeth before or after brushing. Kid-friendly plastic flossing tools make it easier until your child learns how or is old enough to floss themselves.
- Talk to their dentist or school about dental sealant options. Sealants provide an extra barrier between cavity-causing bacteria and teeth, reducing the risk of tooth decay by about 80% in molars.
The Truth About Fluoride
You may have heard that fluoride is dangerous, but there is no scientific evidence to prove this. In fact, drinking water with fluoride has been shown to reduce cavities by 25%.
Fluoride is a mineral that occurs naturally in all water sources, including oceans, rivers and lakes. It is safe and effective at preventing tooth decay and is added to some community tap water, toothpastes and mouth rinses. Fluoride strengthens tooth enamel, helping it resist the acid that can cause tooth decay. It also reduces the ability of plaque bacteria to produce acid.
Check with your local water utility agency or find out here if your community's water is fluoridated. Be aware that bottled water may not contain fluoride; therefore, children who drink it or unfluoridated tap water regularly may be missing the benefits of fluoride. Ask your pediatrician or dentist if your child should get a prescription for fluoride drops or chewable tablets.


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No matter how many safeguards are put into place or how clear protocols are disseminated at a medical facility, hospital errors will always occur if the employees implementing those standards are negligent or abusive. In other words, medical malpractice can only be prevented if nurses, doctors, and all other medical professionals follow the required actions at all times.
Unfortunately, far too often individuals in these crucial positions make reckless, or even knowing, mistakes that put dozens of patients at risk. The Washington Post reported on just such a story.
A nurse at a hospital in Minnesota was recently suspending following an investigation into her theft of pain medication. The nurse is suspecting of taking the medication directly from patient IV bags. Apparently the nurse used a syringe to pull out pain medication from the bags. She then replaced the stolen drugs with saline or air to fool observers into thinking the level had not changed.
What's worse is that it is thought she may have introduced an unusual bacterial infection into those patients from whom she was stealing. The bacterium involved is not usually found in people, and so doctors aren't clear what the ultimate consequence will be.
At least 23 patients have been notified of the problem. As of yet no serious problems have resulted from the possible infection-however there remains a clear risk that more deadly complications could develop.
The U.S. Food and Drug Enforcement Administration and Food and Drug Administration have entered the investigation.
Our Chicago medical malpractice lawyers remain shocked by all egregious examples of criminal conduct by healthcare professionals. It is examples like this that should remind all medical administrators of the vital importance of ensuring their employees remain of the highest quality possible-above any thoughts of similar, criminal conduct. As a patient (or family member of a patient), it is important to hold the facility accountable for the egregious actions of its employees. Contact an Illinois medical malpractice attorney to learn more about how the legal system protects victims in these cases.
See Our Related Blog Posts:
State Legislation Seeks to Improve Safety of Illinois Medical Patients


text: RADIATIVE IGNITION OF SOLID PROPELLANTS: A PRACTICAL APPROACH 285-304 10.1615/IntJEnergeticMaterialsChemProp.v9.i4.10 Franck Cauty ONERA Yves Fabignon ONERA−The French Aerospace Lab, Palaiseau, France Charles Erades ONERA−The French Aerospace Lab, Palaiseau, France composite propellant ignition model radiative ignition IR pyrometer ignition delay We were interested in studying solid propellant time-to-ignition determination from two perspectives. First, we experimentally determined the sensitivity of composite propellants to ignition by using CO2 laser radiation instead of the classical convective heat generator used at Onera for many decades. Second, we validated the propellant degradation data using a one-dimensional (1D) simulation model based on a classical relationship set. The setup and the associated measurement systems are described in detail in this paper. High-speed video images were obtained, and surface luminance temperature evolutions from optical-fiber pyrometers were determined. The results showed the inert heating of the sample, then the start of binder degradation, and upon ignition of a first ammonium perchlorate (AP) grain ("first light"), rapid rise of temperature to the stationary combustion level. We comment on the different ignition delay times corresponding to criteria and the physical process of the AP propellant grain ignition. The first AP grain large enough to propagate heat to other grains around it, and close enough to the surface, is the starting element of the ignition process. These experimental results were then compared to a 1D numerical simulation model. The solid propellant thermal and reaction parameters were determined from the literature values of the ingredients [AP, hydroxyl terminated polybutadiene (HTPB), and aluminum (Al)] and their mixture ratio. The model gives the surface temperature evolution and the burning rate variation from 0 to the stationary value. The experimental and numerical results showed that the pre-exponential factor of the Arrhenius law governs the delay time and the ignition surface temperature level.


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How to stop sharing Calendar in Outlook.
1. Open Outlook Desktop.
2. Click on the Calendar icon.
3. Under My Calendar, right-click on your Calendar, then choose properties…
4. Select the tab Permissions.
5. Select the name of the person you want to stop sharing a calendar with, then click Remove.
When you finish, click OK.


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Want to play on the Red Dog Music Acoustic stage?
Want to play in the heart of Edinburgh's Old Town on Saturday 16th September? We've teamed up with Greater Grassmarket and Flow State Music and have been handed the keys to the stage for one afternoon only. We're giving you the chance to play a 45 minute set and showcase your music to the people!
If you're a singer-songwriter, laptop-based trip-hop performer or a full band who can strip things back a bit, we're looking for you! To keep things friendly in the neighbourhood, no full kits please – but we'll supply a cajon! – and keep the lyrics family friendly!
To apply, email email@example.com, tell us a bit about yourselves and send us a link to some of your tunes – links to videos of you playing live are ideal.
The gear will be waiting for you on the stage, so just bring yourselves and your instruments!


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Road To Freedom Pt. 1: EASY RIDER
A few weeks ago, I watched a Brian Wilson biopic called "Love & Mercy" for a review here at Furious Cinema. I remembered that the drummer, Dennis Wilson, also appeared in a film called "Two-Lane Blacktop", which I haven't seen in 7 or 8 years. The only thing I recall about that film is that it's quite similar to Easy Rider since it's quite artsy and needs to be interpretated by the audience. Therefore, this week I'll review not only Two-Lane Blacktop, but also Easy Rider and Vanishing Point (for our "Road To Freedom" series) because what these three movies have in common are motorized engines, innovative storytelling and symbolism.
I decided to kickstart it with Easy Rider since it's one of my favorite movies. Plus, this is one of those movies which I wanted to write an analysis for to see the meaning of it. While many people tend to remember only Steppenwolf's rockin' Born To Be Wild title song and the iconic "biker" image, this is not one of those "Wooo-Hoo! We're gonna ride bikes and have a good time, maaan!" kind of movies. In fact, I categorize Easy Rider as a combination of "road movie" and "arthouse film" since it doesn't have a standard Hollywood storyline, it utilizes innovative editing techniques and is filled with lots of symbolic messages.
The premise here is quite simple. Two hippies, Wyatt aka Captain America (Peter Fonda) and Billy (Dennis Hopper) have just finished selling cocaine to a rich buyer (Phil Spector) and receive lots of money. Thus, they decide to celebrate the "American Dream" by traveling to New Orleans to attend the Mardi Gras festival on their motorcycles. That's it. That's the story. The rest of the film is their adventures making new friends and facing various obstacles. Now, before I start analyzing each scene, there's one thing I have to remind you about. The tagline on the poster and in the trailer is "A man went looking for America. And couldn't find it anywhere…". That is the main theme of what poor Wyatt and Billy have to face.
So after receiving the money and hiding it in his bike's gas tank, Wyatt decides to take off his watch and throw it away. Why? I think this is because he wants to be free from worrying about time and then he go be anywhere, without thinking about whether it is morning or afternoon. After the epic "Born To Be Wild" montage (and being turned away at a cheap motel), Wyatt and Billy arrive at a small farmhouse to repair their bikes and have a meal with the ranch owners' family. What we have here is an archetype of the nuclear family. We have the white father, Mexican mother, and several kids praying to Jesus before eating. This is the antithesis of what Wyatt and Billy are since this family is The Waltons version of "freedom", in which everyone sticks to religion and family values as well as having the nice house and small business in pursuit of All-American happiness. But Wyatt and Billy basically represent the counterculture movement that rejects those 50's values through their clothing, music and drug-fueled lifestyle. Therefore, they can't stay there long. However, Wyatt is nice enough to respect people who have different mindset from them (which is something that's quite rare nowadays, unfortunately) and says…
"No, I mean it. You've got a nice place. It's not every man that can live off the land, you know? Can do your own thing in your own time. You should be proud."
Then, they move on and pick up a hitchhiker (Luke Askew). We later learn that he's trying to run away from his big hometown city and wants to be someone else (he claims that he wants to be Porky Pig). The next day, our heroes take him to his destiny: a small hippie commune. We might infer that he's an example of what American teenagers actually did in the 60's: left home and lived together in a small place that was filled with sex, drugs, astrology, and music. In fact, this commune has all of them! This is what the hitchhiker says about this place…
"These people got here late in the summer. Too late to plant. But the weather was beautiful…and it was easy living. And then came that winter. There were 40 or 50 living in a one-room place. Nothing to eat, out by the side of the road looking for dead horses. Anything they could get ahold of. There's 18 or 20 left. And they're city kids. Look at them. But they're getting this crop in. They'll stay here till it's harvested. That's the whole thing."
However, shortly before that, there are characters that try to analyze this line from their book…
"Starting brings misfortune. Perseverance brings danger. Not every demand for change in the existing order should be heeded. On the other hand, repeated and well-founded complaints…should not fail to a hearing."
I think this might be a slight jab at the hippie movement at the time since it predicted the downfall of it. The revolution of the late 60's was over because, later on it was less about a big change for a better future and more about just getting high and having sex. It became all about whining and not about doing something.
While Wyatt seems to enjoy the "freedom" of the commune, Billy can't stand the goofy rituals of the place and tells him it's time to move on. This is one of the scenes that shows us the difference between each character in the beginning: Wyatt is the optimistic laid-back guy, while Billy is pessimistic and hostile. As the story progresses, their thoughts are slowly swapped.
Later on the road they get arrested since they join a local small town parade on their bikes without permission. They spend some time in jail and befriend a cellmate named George Hanson (played by the great Jack Nicholson). George is an ACLU lawyer who repeatedly gets tossed in jail for drunken behavior. His view of freedom means having sex at a whorehouse in New Orleans and having equality of life like what aliens are doing. Yep. He's a conspiracy theorist who believes in UFOs as well…
"They've been comin' here ever since 1946, when the scientists first started bouncin' radar beams off of the moon. And they have been livin' and workin' among us, in vast quantities, ever since. The government knows all about 'em. […] Well, they are people just like us. From within our own solar system. Except that their society is more highly evolved. I mean, they don't have no wars, they got no monetary system, they don't have any leaders; because, I mean, each man is a leader. I mean, each man – because of their technology, they are able to feed, clothe, house, and transport themselves equally and with no effort. […] Why don't they [the aliens] reveal themselves to us – is because if they did, it would cause a general panic. Now, I mean, we still have leaders upon whom we rely for the release of this information. These leaders – have decided to repress this information because of the tremendous shock that it would cause to our antiquated systems. Now, the result of this has been that the Venutians have contacted people at all walks of life, all walks of life – Yes! It would be a devastin' blow to our antiquated systems. So, now Venutians are mating with people in all walks of life in an advisory capacity. For once, man will have a god-like control over his own destiny. He will have a chance to transcend and to evolve with some equality for all."
It sounds goofy as hell, especially in the age of internet trolls and the post-truthers era, but back then, this was an interesting movement that brought us Bigfoot and fun stuff like that. Nonetheless, at least George has something to believe in. Something about advancing equality for all. Something against the government. However, the next day they go to a diner and, while some young giggly chicks are excited about their appearance, some rednecks make fun of them. As I said, the counterculture of 60's was totally against the traditional values and seen as a threat. That's why the rednecks hate them and they have this following conversation later that night…
"George Hanson: You know, this used to be a helluva good country. I can't understand what's gone wrong with it.
Billy: Man, everybody got chicken, that's what happened. Hey, we can't even get into like, a second-rate hotel, I mean, a second-rate motel, you dig? They think we're gonna cut their throat or somethin'. They're scared, man.
George Hanson: They're not scared of you. They're scared of what you represent to 'em.
Billy: Hey, man. All we represent to them, man, is somebody who needs a haircut.
George Hanson: Oh, no. What you represent to them is freedom.
Billy: What the hell is wrong with freedom? That's what it's all about.
George Hanson: Oh, yeah, that's right. That's what's it's all about, all right. But talkin' about it and bein' it, that's two different thangs. I mean, it's real hard to be free when you are bought and sold in the marketplace. Of course, don't ever tell anybody that they're not free, 'cause then they're gonna get real busy killin' and maimin' to prove to you that they are. Oh, yeah, they're gonna talk to you, and talk to you, and talk to you about individual freedom. But they see a free individual, it's gonna scare 'em.
Billy: Well, it don't make 'em runnin' scared.
George Hanson: No, it makes 'em dangerous. Buhhhh! Neh! Neh! Neh! Neh! Neh! Neh! Swamp!"
But in the end, what does the freedom mean? Freedom to express something? Freedom to say that we disagree about something against our values? Do those rednecks have a freedom to hate our heroes? Anyway, later the rednecks attack our heroes and kill poor George as he sleeps.
Finally, our heroes make it to New Orleans and go to the whorehouse that George recommends to them. They hang out with two hookers (Karen Black and Toni Basil), and hit the streets to see the big Mardi Gras festival, do some drugs and have sex. But before that, Wyatt sees this quote on the wall…
"Death only closes a man's reputation and determines it as good or bad."
Along with flashes that cut to the tragic ending, this foreshadows what's going to happen to our hero. Then, if that's not enough, they later have an LSD trip in a cemetary while we hear someone praying about life and death! After the legendary trippy montage, we hear Wyatt say the famous "We blew it" line (while Billy still thinks that they should use money to start a new life…see how their minds get swapped now?). What does that line mean? Well, they finally arrived in New Orleans, but still have no idea what freedom and the pursuit of happiness is. Several concepts of freedom come and go, but none of them satisfy the main characters. Why? Is it because they don't have a clear concept of happiness and freedom in the first place? Is it because the sex and drug-fueled counterculture got them nothing? Do their lives only reflect the life of all young adults who are chasing something blindly and pretend to believe that it's what they want? There are so many more questions that you can discuss after watching it.
Finally, we see Wyatt and Billy get killed by two rednecks. Again. Some might view it as a tragic ending, but those rednecks are going to abuse the term "freedom" to hate our heroes and kill them anyway. This might be the reason Peter Fonda said that one of the main themes of Easy Rider is…
"Liberty's become a whore, and we're all taking the easy ride."
Because in the end, nobody can say exactly what liberty means! We are going to yell out this word to satisfy ourselves and…that's all! But in the end, no matter how you interpret this film, Easy Rider is a very unique experience. There's no way a film like this will be made again and have the same impact. It's artsy, thought-provoking, and charming. I highly recommend both the film and soundtrack album (I have it on CD and is worth listening to while you are driving).
Tomorrow, we'll take a look at what people call "Universal Pictures' answer to Easy Rider": Two-Lane Blacktop.


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Azali condemns the sale of 'economic passports' but he allows the practice to continue!
With the possibility of early elections in 2019, President Assoumani Azali is engaged in a public relations exercise to denounce the passport sale scheme introduced by his predecessors. The scheme is thought to have cost the state $900 million in [. [...]
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The Journey to TNakakihara.com
I think that growing up in Los Angeles where everyone is a Slash-Something, a student slash actor, a mom slash lifestyle guru, an artist slash barista, I always wanted to know what my Slash-Something was going to be. I also have always been drawn to the concept of blogging – it's better than a diary because you get to share and (if you're lucky) be popular! I grew up really wanting to be a something slash blogger.
I didn't actually end up starting my first blog until I was 20; in three years I have started three separate blogs and let each and every single one die a slow boring death.
July 2020


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Mailing Solutions
Organizations can have compelling reasons for keeping a legacy system, such as:
• The system works satisfactorily, and the owner sees no reason to change it.
• The costs of redesigning or replacing the system are prohibitive because it is large, monolithic, and/or complex.
• Retraining on a new system would be costly in lost time and money, compared to the anticipated appreciable benefits of replacing it (which may be zero).
• The system requires near-constant availability, so it cannot be taken out of service, and the cost of designing a new system with a similar availability level is high. Examples include systems to handle customers' accounts in banks,computer reservations systems, air traffic control, energy distribution (power grids), nuclear power plants, military defense installations, and systems such as the TOPS database.
• The way that the system works is not well understood. Such a situation can occur when the designers of the system have left the organization, and the system has either not been fully documented or documentation has been lost.
• The user expects that the system can easily be replaced when this becomes necessary.
• Newer systems perform undesirable (especially for individual or non-institutional users) secondary functions such as a) tracking and reporting of user activity and/or b) automatic updating that creates "back-door" security vulnerabilities and leaves end users dependent on the good faith and honesty of the vendor providing the updates. This problem is especially acute when these secondary functions of a newer system cannot be disabled.
CloudMail lets you efficiently send e-mails from FileMaker to any size audience - a single recipient, a 30-person department, a 20,000 person newsletter list, or a million person municipal database. In addition to sending, it tracks who receives the message, who views it, and who clicks any links in the message. It handles unsubscribe requests automatically, and if a recipient files a complaint with their ISP, they are automatically excluded from future mailings. Undeliverable e-mail messages are reported as well, so you can keep your mailing list fresh.


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Renee Hector has "come back home" after signing for Watford Ladies to boost their defensive options as the Golden Girls continue their push for Championship football next season.
The former Tottenham Hotspur and Charlton Athletic player has returned to the club where she started her career and made 40 appearances, helping them achieve promotion in 2014.
"It's a really good feeling to sign for Watford," Hector told the club website. "I played here for ten years so it just feels like I went on a bit of a vacation for few years and now I've come back home. I feel great.
"It's a club that's obviously very, very close to my heart. I've worn the badge for a long time. It's a club that means a lot to me and my family.
"Even when I went away and was at other clubs I kept an eye on them and wished them well. It means the world to get the badge back on my chest."
The Golden Girls are currently second in the FA Women's National League Southern Premier Division, six points behind leaders Crawley Wasps but with two games in hand, and Hector knows what's needed to get the job done at the end of a season.
"I feel like I'm a bit of a promotion master," she said. "I've been promoted to different leagues three or four times in my senior career. I definitely feel like it's something I can bring to the team.
"I'm used to be under such intense pressure towards the end of the season where you have to hold your nerve."
Commenting on the signing, head coach Clinton Lancaster said: "Renée has come down and been working really hard. Her hard work and dedication has been fantastic. She's the kind of person and character we want at Watford, we want to continue to work with her and get her back match fit and see her in a Watford shirt before the end of the season.
"She's going to be a fantastic addition for the club and we are looking forward to working with her as a Watford player."


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In this study, we examined the expression and functions of related to testes-specific, vespid, and pathogenesis protein 1 (RTVP-1) in glioma cells. RTVP-1 was expressed in high levels in glioblastomas, whereas its expression in low-grade astrocytomas and normal brains was very low. Transfection of glioma cells with small interfering RNAs targeting RTVP-1 decreased cell proliferation in all the cell lines examined and induced cell apoptosis in some of them. Overexpression of RTVP-1 increased astrocyte and glioma cell proliferation and the anchorage-independent growth of the cells. In addition, overexpression of RTVP-1 rendered glioma cells more resistant to the apoptotic effect of tumor necrosis factor–related apoptosis-inducing ligand and serum deprivation. To delineate the molecular mechanisms involved in the survival effects of RTVP-1, we examined the expression and phosphorylation of various apoptosis-related proteins. We found that overexpression of RTVP-1 decreased the phosphorylation of c-Jun-NH2-kinase and increased the expression of Bcl2 and that the protective effect of RTVP-1 was partially mediated by Bcl2. Finally, we found that RTVP-1 regulated the invasion of glioma cells as was evident by their enhanced migration through Matrigel and by their increased invasion in a spheroid confrontation assay. The increased invasive potential of the RTVP-1 overexpressors was also shown by the increased activity of matrix metalloproteinase 2 in these cells. Our results suggest that the expression of RTVP-1 is correlated with the degree of malignancy of astrocytic tumors and that RTVP-1 is involved in the regulation of the growth, survival, and invasion of glioma cells. Collectively, these findings suggest that RTVP-1 is a potential therapeutic target in gliomas. (Cancer Res 2006; 66(8): 4139-48)
- glioma
- proliferation
- apoptosis
- invasion
Gliomas are the most frequent primary brain tumors, accounting for >50% of all brain tumors ( 1, 2). Glioblastomas, the most malignant form, are characterized by increased proliferation and invasion into the surrounding normal brain tissue ( 3). Current treatment options include surgery, radiation therapy, and chemotherapy. Unfortunately, the prognosis of patients with glioblastomas remains extremely poor, and the median survival of 12 months from the time of diagnosis has not significantly changed during the last years ( 4, 5). Limitations to therapy include the infiltrative nature and enhanced angiogenesis of these tumors ( 6, 7). In addition, glioma cells are resistant to current modalities of irradiation, chemotherapy, and immunotherapy ( 8). Therefore, innovative approaches are essential for the treatment of patients with gliomas, especially because the occurrence of these tumors is increasing.
Related to testes-specific, vespid, and pathogenesis protein 1 (RTVP-1) is a novel gene that was cloned from human glioblastoma cell lines by two groups and was identified as glioma pathogenesis–related protein ( 9) or RTVP-1 ( 10). RTVP-1 was reported to be expressed in high levels in gliomas and glioma cell lines, whereas no expression was observed in other cells or tumors of the central nervous system ( 9, 10). In addition, RTVP-1 has also been implicated as a marker of myelomonocytic differentiation in macrophages ( 11) and has been reported to act as a tumor-suppressor gene in prostate cancer ( 12, 13). RTVP-1 contains a transmembrane domain and a putative signal peptide and it has high homology with the human testis-specific protein, TPX1 ( 14), the venom allergen, antigen 5 ( 15), and with a mouse acidic epididymal glycoprotein–like molecule ( 16). In addition, RTVP-1 is structurally related to group 1 of the plant pathogenesis–related (PR-1) proteins, which play major roles in plant defense response to viral, bacterial, and fungal infection ( 17, 18). Because the TPX-1 and the PR-1 proteins are all secreted ( 14, 18), it has been suggested that RTVP-1 is a secreted protein as well.
The functions of RTVP-1 have been mainly studied in prostate cancer cells ( 12, 13). RTVP-1 has been reported to be regulated by p53 and by DNA-damaging agents ( 12). Overexpression of the human RTVP-1, similar to its mouse homologue, induced apoptosis in various prostate cell lines ( 13) and this effect was dependent on the presence of the signal peptide, suggesting a role of a secreted form of RTVP-1 in this effect ( 12).
Although RTVP-1 has been reported to be expressed in gliomas, its expression in astrocytic tumors with different degree of malignancy and its role in glioma cell function have not been reported. In this study, we show that the expression of RTVP-1 is correlated with the malignancy of astrocytic tumors, and that RTVP-1 is involved in the regulation of the proliferation, survival, and invasion of glioma cells.
Materials and Methods
Materials. An affinity-purified monoclonal anti-FLAG antibody, leupeptin, aprotinin, phenylmethylsulfonyl fluoride (PMSF), and sodium vanadate were obtained from Sigma Chemical Co. (St. Louis, MO) and a polyclonal anti–protein kinase Cε antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Human tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) was from Peprotech (Rocky Hill, NJ), and anti-pAKT, AKT, phosphorylated c-Jun-NH2-kinase (pJNK), JNK, poly(ADP)ribose polymerase (PARP), active caspase-3, Bax, and Bcl2 antibodies were obtained from Cell Signaling Technology (Beverly, MA).
Tumors and primary cultures. Tumors were collected from patients operated at Henry Ford Hospital (Detroit, MI). Tumors were classified according to the WHO criteria into the various subtypes of low-grade astrocytomas, anaplastic astrocytomas, and glioblastomas using formalin-fixed paraffin-embedded H&E-stained tissue slices. Fresh tissues were frozen immediately following surgery in liquid nitrogen and stored at −70°C until processing.
Primary cultures were obtained from freshly resected tissues immediately following 1 hour of surgical removal. Samples were first washed in PBS and then minced into small pieces in DMEM with 10% FCS and were further triturated to obtain maximal cell dispersion. Cells were plated in 25 cm2 tissue culture flasks and were grown for 7 to 10 days. Cultures were used up to passage 7.
Sample collection and processing were done according to the regulations of the committee on research involving human subjects of the Organization Institutional Review Board.
Cell lines. The glioma cell lines A172, U87, LN-18, T98G, and LN-229 were obtained from American Type Culture Collection (Manassas, VA), and the U251 and LNZ308 were obtained from Oliver Bogler (Department of Neurosurgery, Henry Ford Hospital). Cells were grown on tissue culture dishes in medium consisting of DMEM containing 10% heat-inactivated FCS, 2 mmol/L glutamine, penicillin (50 units/mL), and streptomycin (0.05 mg/mL). Medium was changed every 3 to 4 days and cultures were split using 0.25% trypsin.
Normal astrocytes were obtained from Cambrex (Walkersville, MD). Cells were grown in an astrocyte-specific medium provided by Cambrex.
RNA extraction and reverse transcription-PCR. Total RNA was extracted from the tissue samples by RNeasy (Qiagen, Santa Clarita, CA) according to instructions of the manufacturer. One microgram of total RNA was transcribed into cDNA using the Reverse Transcriptase System (Promega, Madison, WI) and pd(N)6 random nucleotides. Relative levels of RTVP-1 mRNA were estimated by a semiquantitative PCR compared with the mRNA levels of the ribosomal protein S-12. PCR amplification was done using Taq DNA Polymerase (Takara, Otsu, Shiga, Japan). Amplification step consisted of 95°C for 2 minutes and 26 (for RTVP-1) or 30 (for S-12) cycles of 95°C for 30 seconds, 65°C for 30 seconds, and 72°C for 90 seconds. In a preliminary study, each cDNA was amplified in series of 20 to 40 cycles to obtain data within the linear range of the assay. PCR products were size fractionated by electrophoresis in 2% agarose gels and stained with ethidium bromide. The specificity of the PCR product was verified by DNA sequencing. Bands from reverse transcription-PCR (RT-PCR) using RTVP-1 and S12 primers were scanned and quantified by Scion Image. RTVP-1 products were normalized to S12 products to control for differences in loading and sample integrity.
The following primers were used for semiquantitative RT-PCR. For RTVP-1, forward primer: 5'-TGAGCACTCAGGCAATCACACTCT; reverse primer: 5′-AGTGCTGGGTCCCAAGTCATGTAT (271 bp product) and forward primer: 5′-CACAAGCTGCACCCAAACTTCACT; reverse primer: 5′-ATGGGCCAGCCTGGATATACAACA (430 bp product). For S12, the following primers were used: forward primer, 5′-GGAAGGCATTGCTGCTGG, reverse primer, 5′-CCTCAATGACATCCTTGG (285 bp product). Primers for S-12 and RTVP-1 span exon-intron junctions to avoid amplification of contaminating genomic DNA.
The expression profile of RTVP-1 mRNA in the different human tissues was determined in a Human Rapid-Scan panel (Origene, Rockville, MD). mRNA levels of S12 were determined to control for equal loading.
Real-time RT-PCR. Total RNA was extracted using RNeasy midi kit according to the instructions of the manufacturer (Qiagen). Reverse transcription reaction was carried out using 2 μg total RNA as described for the RT-PCR analysis. A primer optimization step was tested for each set of primers to determine the optimal primer concentrations. Once the optimal primer concentrations were determined, primers, 25 μL of 2× SYBR Green Master Mix (Invitrogen, Carlsbad, CA), and 30 to 100 ng cDNA samples were resuspended in a total volume of 50 μL PCR amplification solution. The following primers were used: RTVP-1 forward, TGCCAGTTTTCACATAATACAC; RTVP-1 reverse, GGATTTCGTCATACCAGTTT and S12 forward, TGCTGGAGGTGTAATGGACG; S12 reverse, CAAGCACACAAAGATGGGCT.
Reactions were run on an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). The cycling conditions composed of 4-minute polymerase activation at 95°C and 40 cycles, 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute. Cycle threshold (Ct) values were obtained from the ABI 7000 software. S12 or β-actin levels were also determined for each RNA sample as controls. Fold change of relative mRNA expression was determined using the 2−ΔCt method ( 19).
Northern blot analysis. Northern blotting was done on 10 μg total RNA by using 1% formaldehyde-agarose gel and transfer onto nylon membranes (Schleicher & Schuell, Dassel, Germany). DNA probes for the RTVP-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthesized by PCR and 32P-labeled by random priming (Biological Industries, Beit Haemek, Israel). Hybridization was done at 42°C in 5× SSC-50% formamide-5× Denhardt's solution-2% SDS-10% dextran sulfate-100 μg denatured sheared salmon sperm DNA/mL. GAPDH probe was used to standardize loading.
The Multiple Human Tissue Northern blots were obtained from BD Bioscience (Mountain View, CA) and were similarly hybridized with the RTVP-1 and GAPDH probes.
Construction of RTVP-1 plasmids. The full-length RTVP-1 was amplified by RT-PCR on template of RNA obtained from U87 cells using specific primers (5′-TAATAActcgagACCATGCGTGTCACACTTGCTAC-3′ and 5′-TAATAAacgcgtGTCCAAAAGAACTAAATTAG-3′). Xho and MluI sites, indicated by lowercase letters, were incorporated to facilitate cloning. The sequence of the PCR product was found to be identical to the published sequence of RTVP-1 (GI 15217074). The PCR product was cut with Xho, MluI and cloned into pCMV-tag 2B (Stratagene, La Jolla, CA), which was previously constructed to contain the MluI site between the XhoI and the ApaI sites ( 20). RTVP-1 was similarly cloned into the MTH vector ( 21). The integrity of the inserts was verified by DNA sequencing.
Cell transfection. Cells were transfected either with the control vector or with the RTVP-1 expression vector by electroporation using the Nucleofector device, protocol number A29 (Amaxa Biosystems, Gaithersburg, MD). Transfection efficiency using nucleofection was between 70% and 90%. Stable clones were isolated using selection medium containing 750 μg/mL geneticin (Life Technologies, Gaithersburg, MD). All the results were confirmed on one pool and an additional individual clone.
Small interfering RNA transfection. Small interfering RNA (siRNA) duplexes were synthesized and purified by Dharmacon (Lafayette, CO). The siRNA sequence for targeting RTVP-1 mRNA was 5′-AAGACTGCGTTCGAATCCATA-3′ (siRNA1). In addition, we used a pool of four RTVP-1 siRNA duplexes that was also obtained from Dharmacon. A pool of Bcl2 duplexes was obtained from Dharmacon and a scrambled sequence was used as a negative control. Transfection of siRNAs was done using OligofectAMINE (Invitrogen) according to the instructions of the manufacturer. Experiments were done 72 hours after transfection.
Adenovirus preparation and infection. The AdEasy system was kindly provided by Dr. Vogelstein (The Johns Hopkins University School of Medicine, Baltimore, MD; ref. 22). Adenoviruses expressing RTVP-1 were prepared as previously described ( 23). Briefly, RTVP-1 was first cloned into the pShuttle-CMV. The linearized plasmid was transformed into Escherichia coli BJ5183-AD-1 competent cells (Stratagene) carrying the pAdEasy-1 plasmid that encodes the adenovirus-5 backbone. Recombination was confirmed by restriction and PCR analyses. The linearized recombinant plasmid was transfected into HEK293 cells, viruses were collected after 7 to 10 days and were further amplified. Cells were incubated with 5 multiplicity of infection of the recombinant adenovirus vectors for 1 hour. The medium was then replaced with fresh medium and the cells were used 24 to 48 hours post infection.
Generation of anti-RTVP-1 antibody. Anti-RTVP-1 antibody was generated by 21st Century Biochemicals (Marlboro, MA) by immunizing rabbits with synthetic peptides VRIHNKFRSEVKPTAC and CVQLKYPNLVLLD corresponding to amino acids 38 to 53 and 255 to 266 of human RTVP-1, respectively. The anti-RTVP-1 serum was affinity purified and characterized by ELISA and Western blot analysis.
Quantitation of cell growth. Cells (2 × 104) were seeded in triplicate in tissue culture dishes and were harvested at 24-hour intervals using trypsin. Cells were washed in PBS and were then counted using a phase microscope. Cell numbers were then calculated per milliliter of the original dilution. Each assay was done in quadruplets.
Soft agar assays. Cells were harvested, washed, and plated in a soft agar (0.4%) overlaying a 0.9% base agar. The agar plates were incubated at 37°C for 10 days and colonies (containing >50 cells) were viewed by the microscope and counted. Each assay was done in triplicate.
Preparation of cell homogenates and immunoblot analysis. Cell pellets (106 cells/mL) were resuspended in 100 μL lysis buffer [25 mmol/L Tris-HCl (pH 7.4), 50 mmol/L NaCl, 0.5% Na deoxycholate, 2% NP40, 0.2% SDS, 1 mmol/L PMSF, 50 μg/mL aprotinin, 50 μmol/L leupeptin, and 0.5 mmol/L Na3VO4] on ice for 15 minutes. Sample buffer (2×) was added and the samples were boiled for 5 minutes.
Lysates (30 μg protein) were resolved by SDS-PAGE and were transferred to nitrocellulose membranes. The membranes were blocked with 5% dry milk in PBS and subsequently stained with the primary antibody. Specific reactive bands were detected using a goat anti-rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase (Bio-Rad, Hercules, CA) and the immunoreactive bands were visualized by the ECL Western blotting detection kit (Amersham, Arlington Heights, IL).
Measurement of cell apoptosis. Cell apoptosis was measured by flow cytometry after propidium iodide staining as previously described ( 23). Briefly, cells were treated according to the specific experiment. The cells were scraped and centrifuged with the supernatant medium at 3,500 rpm for 5 minutes. Cells were resuspended in PBS and fixed in 70% ethanol for 1 hour on ice. Fixed cells were washed with PBS and stained with propidium iodide (5 μg/mL) solution containing RNase (50 μmol/L). Cells were then analyzed on a Becton-Dickinson FACSCalibur.
Cell apoptosis was also measured using antihistone ELISA (Cell Death Detection kit). For these experiments, extracts of cells containing histone-associated DNA fragments were incubated in 96-well plates coated with antihistone antibodies for 2 hours. Plates were than washed and incubated with anti-DNA antibodies conjugated to peroxidase for an additional 2 hours. Substrate solution was added and absorbance was measured at a wavelength of 405 nm. In addition, cell apoptosis was evaluated by Western blot analysis of PARP and caspase-3 cleavage using anti-PARP antibody and anti–active caspase-3 antibody.
Invasion assay. Boyden chamber chemoinvasion assays were done as previously described by Reich et al. ( 24). Matrigel (reconstituted basement membrane; 25 μg) was dried on a polycarbonated filter (polyvinyl pyrrolidone-free; Nucleopore; Whatmann, Madistone, United Kingdom). Cells were harvested by brief exposure to 1 mmol/L EDTA, washed with DMEM containing 0.1% bovine serum albumin, and added to the Boyden chamber (200,000 cells). Cells were incubated for 6 hours at 37°C in humidified atmosphere of 95% air and 5% CO2. Cells that transversed the Matrigel layer and attached to the filter were stained with Diff Quik kit (Dade Diagnostics, Aguada, Puerto Rico) and counted in five randomized fields. Results are expressed as the mean ± SE of three independent experiments.
Analysis of matrix metalloproteinase isoforms (zymography). Cells (200,000) were incubated for 24 hours in serum-free DMEM, and their supernatant was analyzed for matrix metalloproteinase (MMP) activity on a gelatin impregnated (1 mg/mL; Difco, Detroit, MI), SDS-PAGE 8% gel, as described previously by Reich et al. ( 24, 25). Culture medium samples were separated on substrate-impregnated gels under nonreducing conditions, followed by 30 minutes of incubation in 2.5% Triton X-100 (BDH, Poole, United Kingdom). Gels were then incubated for 16 hours at 37°C in 50 mmol/L Tris, 0.2 mol/L NaCl, 5 mmol/L CaCl2, and 0.02% (w/w) Brij 35 (pH 7.6). At the end of the incubation period, gels were stained with 0.5% Coomassie G250 (Bio-Rad) in methanol/acetic acid/H2O (30:10:60).
Spheroid confrontation model. The spheroid confrontation assay was done as previously described ( 26). Briefly, fetal rat brains were collected from Sprague-Dawley rats at embryonic day 18. Single-cell suspensions were made by digestion of whole brains in 0.1% tyrpsin/HBSS. Cell suspensions were cultured on agar-coated plates for 3 weeks to allow for the development of brain aggregates. Cells of interest are also cultured on agar coated plates for 2 weeks to allow for the development of spheroids. At the beginning of culture and once per week thereafter, either Cell Tracker Red or Cell Tracker Green (Invitrogen) was added to separate wells of aggregates and spheroids to allow for the visualization of confrontations. One aggregate and one spheroid were then transferred to a single agar-coated well of a 48-well culture plate and confronted mechanically. Confrontations were visualized at 24, 48, and 72 hours using an Olympus fluorescent microscope. Green and red fluorescence was visualized separately and merged to visualize invasion.
Statistical analysis. The results are presented as the mean values ± SE. Data were analyzed using ANOVA and a Student's t test. For real-time PCR data, Student's t test (with correction for data sets with unequal variances) was done using Prism 4 (GraphPad Software, Inc., San Diego, CA).
Expression of RTVP-1 in various human tissues. We first examined the expression of RTVP-1 in various human tissues by RT-PCR and Northern blot analysis. Using RT-PCR, we found that the expression of RTVP-1 was very low in normal brain and undetectable in fetal brain. Similarly, RTVP-1 expression was not detected in the colon, pancreas, skin, lymphocytes, and fetal liver, whereas higher levels were detected in the heart, spleen, muscle, lung, bone marrow, placenta, adrenal gland, and prostate ( Fig. 1A ).
Using Northern blot analysis, we observed three main transcripts that were detected by the RTVP-1 DNA probe in the heart, placenta, liver, and skeletal muscle ( Fig. 1B). This observation is similar to the results reported by Rich et al. ( 10). In line with the RT-PCR results, the expression of RTVP-1 in the brain was barely detected by the Northern blot analysis. In contrast, the expression of RTVP-1 in the lung was detected by RT-PCR but only weakly by Northern blot analysis. Similarly, five additional samples of lung tissues expressed RTVP-1 mRNA as detected by RT-PCR (data not shown), suggesting that the discrepancy in the results of the RT-PCR and the Northern blot analysis may represent differences in assay sensitivity or variability in RTVP-1 expression in different individuals.
Expression of RTVP-1 in astrocytic tumors with different degrees of malignancy. RTVP-1 has been previously reported to be expressed in glioblastomas ( 9, 10); however, its expression in other types of astrocytic tumors has not been reported. To characterize the expression of RTVP-1 in different types of astrocytic tumors, we used tumor samples from low-grade astrocytomas (grade 2), anaplastic astrocytomas (grade 3), and glioblastomas (grade 4). The expression of RTVP-1 in these tumors was compared with that of normal brains using semiquantitative RT-PCR. As presented in Fig. 2A , the expression of RTVP-1 in normal brains was barely detected. Similarly, most low-grade astrocytomas expressed very low levels of RTVP-1. In contrast, higher levels of RTVP-1 were observed in anaplastic astrocytomas and glioblastomas ( Fig. 2A). Quantitation of RTVP-1 expression compared with that of S12 clearly shows that the expression of RTVP-1 in significantly higher in glioblastomas and anaplastic astrocytomas compared with low-grade astrocytomas and normal brains ( Fig. 2B).
Similar results were obtained using real-time RT-PCR ( Fig. 2C). Thus, the relative RTVP-1 mRNA expression to S12 in brains and low-grade astrocytomas was very low (1.30 ± 0.22 and 2.2 ± 0.34, respectively), whereas glioblastomas expressed significantly higher levels of RTVP-1 (6.9 ± 0.92).
The expression of RTVP-1 was also examined in glioma cell lines and primary glioma cultures. As presented in Fig. 2D, RTVP-1 was expressed in all the glioma cells examined, whereas it was barely detected in human fetal astrocytes.
Using Northern blot analysis, we observed a strong expression of the 4.2, 3.2, and 1.1 kb transcripts in the U87 and A172 glioma cell lines and a weaker expression of the 1.6 kb transcript in these cells ( Fig. 2E).
Previous studies reported the expression of four distinct mRNAs of 4.2, 3.2, 1.6, and 1.0 kb by using an RTVP-1 DNA probe. To further explore this point, we examined the effect of the RTVP-1 siRNA on the expression of the different mRNAs. We found that the RTVP-1 siRNA, which targets the first exon of RTVP-1, significantly reduced the expression of the 1.6 and 1.0 kb mRNAs, whereas it did not significantly affect the expression of the 4.2 and 3.2 transcripts (data not shown).
The expression of the RTVP-1 protein was examined using an anti-RTVP-1 antibody and Western blot analysis. The anti-RTVP-1 antibody recognized a band around 28 kDa and this band was blocked by the addition of the immunizing peptides. Similar to the mRNA results, RTVP-1 was highly expressed in glioblastoma, whereas low expression was observed in normal brains and low-grade astrocytomas ( Fig. 2F). Thus, the relative RTVP-1 protein expression compared with actin was 4.8 ± 0.7 in glioblastomas, 0.56 ± 0.07 in low-grade astrocytomas, and 0.11 ± 0.2 in normal brains.
Silencing of RTVP-1 decreases cell proliferation and induces cell apoptosis. To examine the role of RTVP-1 in glioma cell function, we first used siRNAs directed against the RTVP-1 mRNA. Transfection of the glioma cell lines, U87 and A172, with a siRNA duplex for 3 days resulted in around 90% and 70% decrease in the expression of the RTVP-1 mRNA, respectively, whereas no decrease in the expression of RTVP-1 was observed with a scrambled control siRNA ( Fig. 3A ). The RTVP-1 siRNA also decreased the expression of the RTVP-1 protein. Transfection of U87 and A172 cells with RTVP-1 siRNA significantly decreased the expression of RTVP-1 after 72 hours as detected by anti-RTVP-1 antibody, whereas a control siRNA did not have a significant effect ( Fig. 3B).
The RTVP-1 siRNA–transfected U87 cells exhibited a significant decrease in cell proliferation ( Fig. 3C) and in cell growth in soft agar ( Fig. 3D). In addition, ∼30% of the U87 cells were apoptotic as determined by propidium iodide staining and fluorescence-activated cell sorting (FACS) analysis ( Fig. 3E). The A172 cells, in which RTVP-1 expression was less reduced by the siRNAs, exhibited a significant decrease in cell proliferation ( Fig. 3C) and cell growth in soft agar ( Fig. 3D), but a smaller degree of cell apoptosis as determined by propidium iodide staining ( Fig. 3E). Similar results were observed with another set of RTVP-1 duplex (data not shown).
To further examine the effect of RTVP-1 silencing, we used established primary glioma cultures. Transfection of the primary cultures with RTVP-1 siRNAs reduced the expression of RTVP-1 mRNA ( Fig. 3A) or protein levels ( Fig. 3B) to a different degree. Similar to the results obtained with the glioma cell lines, cells in which RTVP-1 was significantly depleted (HF1308) exhibited both a decrease in cell number ( Fig. 3C) and an increase in cell apoptosis ( Fig. 3E). In contrast, cells in which RTVP-1 expression was only partially reduced (HF1254) showed a similar decrease in cell number but only a small increase in cell apoptosis ( Fig. 3C and E).
Similar results of cell apoptosis were also observed using additional apoptosis assays, including anti–active caspase-3 antibody ( Fig. 3F) and antihistone ELISA ( Fig. 3G).
Overexpression of RTVP-1 increases glioma cell growth. Recent studies reported that overexpression of RTVP-1 in prostate cancer cell lines induced cell apoptosis in these cells ( 12, 13). To examine the effect of RTVP-1 on glioma cells, we used glioma cells stably expressing RTVP-1 ( Fig. 4A ). For these experiments, we used two clones of control vector cells, CV1 and CV2, and the RTVP-1-overexpressing cells, RTVP1-1 and RTVP1-2. Overexpression of RTVP-1 is presented in Fig. 4A using anti-FLAG antibody. We found that U87 cells stably expressing RTVP-1 (RTVP1-1 and RTVP1-2 cells) exhibited higher degree of cell proliferation compared with control vector (CV1 and CV2) cells ( Fig. 4B). In addition, we found that overexpression of RTVP-1 enhanced the colony-forming ability of the U87 cells on soft agar ( Fig. 4C and D). Thus, RTVP-1-overexpressing cells exhibited an increase in the size and number of colonies formed on the soft agar compared with the control vector cells, suggesting that RTVP-1 increased also the anchorage-independent growth of glioma cells. Similar results were obtained with two clones of A172 cells overexpressing RTVP-1 (data not shown).
Because RTVP-1 expression is higher in glioma cells compared with astrocytes, we examined the effect of RTVP-1 overexpression on cultured normal human astrocytes. For these experiments, we infected human astrocytes with an adenovirus vector expressing RTVP-1 and examined the proliferation of the cells following 1 to 4 days in culture. As presented in Fig. 4E, astrocytes expressing RTVP-1 exhibited higher degree of cell proliferation after 3 and 4 days in culture compared with astrocytes infected with an adenovirus vector expressing LacZ (control vector).
RTVP-1 regulates glioma cell survival. Because silencing of RTVP-1 induced cell apoptosis in the glioma cell lines, we examined the role of RTVP-1 overexpression in glioma cell survival using the apoptotic stimuli TRAIL and serum starvation. U87 cells overexpressing control vector cells (CV1 and CV2) exhibited ∼30% of apoptotic cells in response to TRAIL (100 ng/mL). In contrast, the RTVP1-1 and RTVP1-2 overexpressors exhibited a significantly lower degree of cell apoptosis ( Fig. 5A ). In addition, we found that cells overexpressing RTVP-1 were more resistant to cell apoptosis induced by serum deprivation compared with control vector cells ( Fig. 5B). Similar results were obtained using anti-PARP antibody, which detected PARP cleavage ( Fig. 5C).
Similarly, RTVP-1 protected A172 from TRAIL-induced apoptosis. Thus, TRAIL induced apoptosis (24%) in the A172 cells after 5 hours of treatment, whereas A172 cells infected with adenovirus vector expressing RTVP-1 showed a significant lower level of cell apoptosis (9%; Fig. 5D).
To delineate the mechanisms involved in the survival effect of RTVP-1, we examined the expression and phosphorylation of the apoptosis-related proteins Bcl2, Bax, AKT, and JNK. We found that U87 cells overexpressing RTVP-1 (RTVP1-1 and RTVP1-2) expressed higher levels of Bcl2, whereas no significant differences were obtained in the expression of Bax. In addition, cells overexpressing RTVP-1 exhibited lower levels of pJNK, whereas no significant changes were observed in the phosphorylation or the expression of AKT between the two groups of cells ( Fig. 5E).
To examine the role of Bcl2 in the protective effect of RTVP-1, we transfected the RTVP-1-overexpressing U87 cells (RTVP1-1) with Bcl2 siRNA and examined the response of the cells to the apoptotic effect of TRAIL. The expression of Bcl2 was significantly decreased in RTVP1-1 cells ( Fig. 5F) and the CV1 cells (data not shown) transfected with the Bcl2 siRNA for 2 days. We found that silencing of Bcl2 slightly increased the response of the CV1 cells to the apoptosis induced by TRAIL, whereas it significantly increased the apoptosis of the RTVP-1-overexpressing cells from 12.4% to 22.5%. Thus, the increase in Bcl2 observed in the RTVP-1-overexpressing cells plays a role in the protective effect of RTVP-1 ( Fig. 5G).
RTVP-1 regulates the invasion and MMP2 activity in glioma cells. Glioma cells are characterized by increased invasion and migration ( 27, 28). To examine the role of RTVP-1 in glioma cell invasion, we examined the ability of the cells to migrate through a Matrigel layer using Boyden chamber. Overexpression of RTVP-1 significantly increased the invasion of U87 cells by ∼3-fold compared with control vector cells ( Fig. 6A ). Similar results were observed in A172 cells overexpressing RTVP-1 and these cells exhibited a 4-fold increase in cell invasion compared with control vector cells (data not shown).
To further examine the effect of RTVP-1 on glioma cell invasion, we used the spheroid confrontation assay that reflects more the in vivo conditions ( 25). In this assay, astrocytic and glioma spheroids were coplated and the degree of invasion of the glioma cells into the astrocytic spheroids was than determined. As presented in Fig. 6B, U87 overexpressing RTVP-1 started to invade the astrocytic spheroids within 24 hours of their confrontation, whereas the control vector cells only established a minimal contact with the astrocytic spheroids at that time. After 48 hours, the majority of the astrocytic spheroids were completely invaded by the RTVP-1 overexpressors, whereas only a partial invasion of the astrocytic spheroids was observed by the control vector cells ( Fig. 6B). Similar results were obtained in additional three experiments (data not shown). Thus, overexpression of RTVP-1 increases the invasive response of glioma cells.
MMPs play important roles in the digestion of extracellular matrix and in the invasion of tumor cells ( 28, 29). Using a zymography assay, we examined the activation of MMP2 in conditioned medium of control vector and RTVP-1-overexpressing cells. In accordance with our results of the invasion assays, we found that RTVP-1 increased the activity of MMP2 in the U87 cells ( Fig. 6C). U87 did not express MMP9 and overexpression of RTVP-1 did not induce the expression or activation of this MMP ( Fig. 6C).
In this study, we examined the expression of RTVP-1 in various astrocytic tumors and the role of RTVP-1 in glioma cell function. RTVP-1 has been originally cloned from human glioma cell lines ( 9, 10); however, its expression in human tissues and astrocytic tumors has not been fully characterized and its functions in glioma cells have not yet been reported.
Using RT-PCR and Northern blot analysis, we found that RTVP-1 was expressed in various human tissues, such as prostate, heart, spleen, and bone marrow, whereas its expression in other tissues, such as brain, colon, pancreas, and skin was very low or undetectable. These findings further extend the results reported by Rich et al. ( 10) that RTVP-1 is expressed in a large number of tissues and support their results that the expression of RTVP-1 in normal brain was absent or barely detected. The role of RTVP-1 in the different normal tissues in which it is expressed is currently not understood. Based on its homology to members of the PR-1 proteins ( 9, 17), RTVP-1 may be involved in immune responses to various pathogens.
Although RTVP-1 has been reported to be highly expressed in gliomas, its relative expression in astrocytic tumors with different degree of malignancy was not examined. Using semiquantitative RT-PCR, we found that the expression of RTVP-1 was the highest in glioblastomas (grade 4), whereas most low-grade astrocytomas (grade 2) expressed very low levels of RTVP-1. Similar results were obtained using Western blot analysis and anti-RTVP-1 antibody. Thus, our results show that RTVP-1 is highly expressed in gliomas, similar to previous reports ( 9, 10), and indicate that the expression of RTVP-1 is correlated with the degree of malignancy of the astrocytic tumors.
In agreement with previous reports ( 9, 10), three predominant mRNAs of 4.2, 3.2, and 1.0 kb and a weaker band of 1.6 kb were detected by using an RTVP-1 DNA probe. Suppression of RTVP-1 protein expression was evident by using siRNA, which targets the first exon of RTVP-1. This was coupled with a selective knockdown of the expression of the 1.6 and 1.0 kb mRNAs. Our results, along with database from Genbank, imply for two RTVP-1-encoding transcripts of 1.6 and 1.0 kb; the first contains a longer 3′ untranslated region. These transcripts are likely to be functionally equivalent although they could be differentially regulated and transcribed. The longer transcripts of 3.2 and 4.2 kb are likely to have partial overlap with RTVP-1 sequences, excluding exon 1. Therefore, they remained stable in the presence of siRNA, which targets the first exon of RTVP-1. These transcripts probably represent different genes, such as the 3,359 bases HIV-1 rev binding protein 2 mRNA, which contains sequences that are complementary to RTVP-1 exons 5 to 6 and is being transcribed on the opposite strand.
To delineate the function of RTVP-1 in glioma cells, we first used siRNAs targeting the RTVP-1 mRNA. Silencing of RTVP-1 expression decreased cell proliferation and anchorage-independent growth in the cell lines examined. In addition, the silencing of RTVP-1 induced cell apoptosis in some of the cell lines. Thus, cell lines and primary cultures in which RTVP-1 expression was almost completely reduced exhibited a large degree of cell apoptosis, whereas a small degree of cell apoptosis was observed in cells in which RTVP-1 expression was moderately silenced. Our results point to an important role of RTVP-1 in the control of glioma cell growth and survival and suggest that a decrease in the expression of RTVP-1 affects the proliferation of glioma cells and that even low levels of RTVP-1 are sufficient to maintain the survival of these cells. Alternatively, the differential apoptotic response of the glioma cells to RTVP-1 silencing may be due to differences in the expression of other apoptosis-related proteins or signaling pathways, such as p53 or Bcl2.
Our conclusions regarding the roles of RTVP-1 in the regulation of glioma cell proliferation were further supported by the results obtained with glioma cell overexpressing RTVP-1. Thus, overexpression of RTVP-1 induced an increase in glioma cell proliferation. Similarly, RTVP-1-overexpressing cells displayed an increased anchorage-independent growth in soft agar. Furthermore, we found that overexpression of RTVP-1 increased the proliferation of cultured normal astrocytes. Collectively, these results suggest that RTVP-1 plays an important role in the growth of astrocytes and glioma cells.
We also found that cells overexpressing RTVP-1 by either adenovirus vectors or by stable expression exhibited an increased resistance to the apoptosis induced by serum deprivation and by TRAIL. Thus, these data further support the results of the siRNA studies that RTVP-1 regulates the survival of glioma cells. In an attempt to explore the mechanisms involved in the survival function of RTVP-1, we examined the expression and phosphorylation of various apoptosis-related proteins. We found that overexpression of RTVP-1 induced an increase in the expression of Bcl2, whereas it did not significantly alter the expression of Bax. The increase in Bcl2 partially mediated the protective effect of RTVP-1, because silencing of Bcl2 decreased the protective effect of RTVP-1 against TRAIL-induced apoptosis. The expression of Bcl2 has been associated with cell survival in various cellular systems ( 30), including glioma cells ( 31, 32). Indeed, overexpression of Bcl2 protected glioma cells from diverse apoptotic stimuli, such as FAS ligation, chemotherapeutic drugs, and radiation ( 33).
Overexpression of RTVP-1 also reduced the phosphorylation of JNK, whereas it did not affect the phosphorylation of AKT. JNK has been implicated in the regulation of cell apoptosis in response to various stimuli ( 34, 35). Therefore, the decrease in the phosphorylated form of JNK together with the increased expression of Bcl2 may render the overexpressing cells resistant to the apoptosis induced by serum-deprivation and TRAIL.
Our results regarding the role of RTVP-1 in glioma cell survival are different from those reported by Ren et al. ( 12, 13) for prostate cancer cells. In their studies, Ren et al. ( 12, 13) reported that overexpression of RTVP-1 using adenovirus vectors induced cell apoptosis in prostate cancer cells and concluded that RTVP-1 acted as a tumor suppressor gene in these cells. The reason for the different results obtained in both studies can be probably attributed to the different cellular systems that have been used. Interestingly, the expression of RTVP-1 is higher in the normal prostate compared with prostate tumors, opposite to what is found in normal brain/astrocytes and gliomas, where RTVP-1 expression is barely detected in the normal brain but is highly expressed in the malignant astrocytic tumors. Thus, RTVP-1 may act as a tumor suppressor gene in prostate cells and as a tumor promoter in gliomas and exerts opposite effects on cell apoptosis in these two cellular systems. Alternatively, the opposite effects of RTVP-1 in the two cellular systems may be due to the different processing of RTVP-1 in these cells. Based on their deletion studies, Ren et al. ( 12) suggested that the apoptotic effect of RTVP-1 is mediated by a secreted form of the protein. Thus, differences in the levels of cellular and extracellular forms of RTVP-1 in gliomas and prostate cancer cells may influence the apoptotic potential of RTVP-1. Additional studies using specific anti-RTVP-1 antibodies are essential to explore these possibilities.
In addition to its role in cell growth and survival, RTVP-1 also regulated the invasion of glioma cells in vitro. Thus, RTVP-1-overexpressing cells migrated more through Matrigel compared with control vector cells. In addition, we showed that RTVP-1 overexpressors exhibited stronger invasive properties into astrocytic spheroids compared with control vector cells. One of the mechanisms that contribute to the increased invasion of glioma cells is the expression and activation of MMPs ( 36, 37). MMPs are a family of enzymes involved in the degradation of extracellular matrix and therefore play a role in tumor invasion and metastasis ( 38, 39). We found that in control U87 cells, the active forms of MMP2 and MMP9 were undetected. In contrast, the activity of MMP2 was significantly increased in cells overexpressing RTVP-1, further suggesting that RTVP-1 increases the invasive potential of glioma cells. Our results indicate that although U87 express endogenous RTVP-1, its overexpression further enhances the growth, survival, and invasion of these cells.
The activation of MMP2 is regulated by the interaction between MMP2, MT1-MMP, and the tissue inhibitor of metalloproteinase-2 ( 40, 41). The mechanisms by which RTVP-1 induces the activation of MMP2 are currently under investigation. However, it is noteworthy that overexpression of Bcl2 has been recently shown to increase the activity of MMP2 via a furin-dependent pathway and to regulate the motility and invasiveness of glioma cells ( 42, 43). Thus, the increased expression of Bcl2 in the RTVP-1-overexpressing cells may also play a role in the increased MMP2 activity and invasion of the glioma cells in addition to its antiapoptotic effect.
In summary, we found that RTVP-1 is highly expressed in glioblastomas compared with low-grade astrocytomas and normal brains and that it plays a role in the regulation of the growth, survival, and invasion of glioma cells. Gliomas exhibit poor prognosis mainly due to their invasive potential and resistance to current therapeutic modalities. Thus, defining novel molecular targets is essential for the development of more effective therapeutic strategies. The results of this study suggest that RTVP-1 may serve as a diagnostic marker and a novel therapeutic target in the treatment of gliomas.
Grant support: James S. McDonnell Foundation 21st Century Scientist Award/Brain Cancer Research (C. Brodie), National Cancer Institute grant CA-R21-96965, and William and Karen Davidson Fund, Hermelin Brain Tumor Center.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Sandra Rempel for critical review of the manuscript.
Note: T. Rosenzweig and A. Ziv-Av contributed equally to this work.
- Received August 11, 2005.
- Revision received January 9, 2006.
- Accepted February 10, 2006.
- ©2006 American Association for Cancer Research.