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NPR Newscast
4min 45sec
October 1, 2004 Consumers who signed up for the "do not call" list have been largely free of commercial telemarketing calls since the federal law went into effect a year ago. Telemarketing firms have experienced business losses, but some have managed to adapt to the law. NPR's Larry Abramson reports.
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October 1, 2004 ABC television airs a new primetime soap, Desperate Housewives, this Sunday. The show's premiere comes as the network struggles to introduce new dramas to its primetime schedule. Last year's new dramas were all cancelled by the end of the season. Variety TV critic Brian Lowry has a review.
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October 1, 2004 Before the foreign policy debate between President Bush and Sen. John Kerry was over, the media began interpreting the event. From real-time blogs to mainstream media and morning radio shows, analysts say that poll results had some influence on the story line. NPR's Robert Smith reports.
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October 1, 2004 NPR's Noah Adams talks with Jonathan Freedland of London's The Guardian newspaper about the censure of a John Humphrys, a BBC journalist denounced for interrupting his interview guests too many times.
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September 30, 2004 Award-winning journalist Guy Garcia discusses his new book The New Mainstream: How the Multicultural Consumer Is Transforming American Business. He writes that this "new mainstream" is putting a multicultural spin on everything from business and politics to entertainment and technology.
September 29, 2004 Slate contributor Seth Stevenson reviews the latest ad campaign for Old Spice, the old fragrance giant that's hoping to appeal to a younger crowd.
<iframe src="http://www.npr.org/player/embed/4052773/4052774" width="100%" height="290" frameborder="0" scrolling="no">
September 28, 2004 Media watchers say recent book and film trends suggest a "perfect storm" of politically motivated popular culture, which has been building for years. Books from the left and right top best-seller lists, while films like Fahrenheit 9/11 draw box-office crowds. Hear NPR's Lynn Neary.
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September 27, 2004 NPR's Michele Norris talks with Steve Dworman, publisher of the Infomercial Marketing Report, about the 20th anniversary of the infomercial being celebrated in Las Vegas at the Electronic Retailing Assn. conference. Dworman says major TV networks are starting to incorporate infomercial programming into their traditional lineups. "Oprah did it with the car giveaway," he says. "That was essentially an hour-long infomercial."
<iframe src="http://www.npr.org/player/embed/4049567/4049568" width="100%" height="290" frameborder="0" scrolling="no">
September 27, 2004 NPR's Mike Pesca reports on how one woman in New Jersey sent a political donation through the Internet to a group in Texas, and how that group made an advertisement aimed at influencing voters in Oregon.
<iframe src="http://www.npr.org/player/embed/4048948/4048949" width="100%" height="290" frameborder="0" scrolling="no">
September 26, 2004 NPR's Catrin Einhorn reports on how Chicago-based Jam Productions is struggling to compete against media behemoth Clear Channel Communications, which dominates the U.S. concert promotion business.
<iframe src="http://www.npr.org/player/embed/3936943/3936944" width="100%" height="290" frameborder="0" scrolling="no">
September 25, 2004 The Israeli-Palestinian All for Peace radio currently broadcasts material in Hebrew, Arabic and English, attempting to bridge gaps between cultures and people. The station's managers hope to start FM broadcasts soon. NPR's Linda Gradstein reports.
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September 24, 2004 Who will replace Disney CEO Michael Eisner, and what will it mean for investors? It's the Motley Fool take -- our take on business news that popped up on the Fool radar. This week, we're joined by Motley Fool writer Rick Munarriz.
September 23, 2004 Commentator John McWhorter just started reading New Yorker magazine. He was surprised to find that most of the cartoons depict white New Yorkers while modern day Manhattan is a place full of people of color.
<iframe src="http://www.npr.org/player/embed/3933579/3933580" width="100%" height="290" frameborder="0" scrolling="no">
September 23, 2004 NPR's Noah Adams talks to Tess Vigeland of Marketplace about the advertising industry's opposition to new proposed regulations, including a tax on advertisements.
<iframe src="http://www.npr.org/player/embed/3932743/3932744" width="100%" height="290" frameborder="0" scrolling="no">
September 22, 2004 NPR's Michele Norris talks with Ken Tucker, editor of Entertainment Weekly, about the fall TV lineup. It's populated with some dramas meant to compete for cable viewers -- an airliner crashes and survivors must share an island with a monster in Lost. For reality TV buffs, ABC debuts Wife Swap, in which wives switch families, and socio-economic classes.
<iframe src="http://www.npr.org/player/embed/3931311/3931312" width="100%" height="290" frameborder="0" scrolling="no">
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Jovanna & Thomas
Our Registry
Jovanna Quinones and Thomas Doak's Honeymoon Registry
Couples Swept Away
Thank You for visiting our honeymoon bridal registry and wedding website. We decided to use this honeymoon bridal registry to allow us to pre plan all the wonderful things we want to do on our dream honeymoon. If you have any questions about how our honeymoon bridal registry works, please contact a honeymoon registry specialist at 1-877-699-5884 and they can help with our wedding registry list. Again, Thank You so much for using our honeymoon bridal registry and wedding website. Most of all, thank you for being part of our wedding and helping us have the romantic honeymoon and wedding we've always imagined.


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Select Local Merchants
Champion Cabinets CorpRead MoreGot a problem in the house you don't know how to fix? Supplement your knowledge with a call to Champion Cabinets Corp's handyman service in Walnut. For remodeling projects big and small, the amazing carpenters at Champion Cabinets Corp will help work with you to bring your vision to life. When things need fixing, the friendly handymen at Champion Cabinets Corp are there to help.19950 East Walnut Drive NorthWalnut, CA US
Choice Granite CompanyRead MoreFor the lingering problems in your home, look no further than Choice Granite Company's handyman service in Diamond Bar. Does your lawn need a little tender loving care? If so, you'd be wise to call upon Choice Granite Company's great team to get the job done. Replace any dingy looking floor with the quality construction services by Choice Granite Company. Don't ditch your four-legged pal outside — dogs are more than welcome at Choice Granite Company. When things need fixing, the friendly handymen at Choice Granite Company are there to help.23435 Golden Springs DriveDiamond Bar, CA US
Apco Appliance and AirRead MoreShop a range of large appliances and replace your out-of-date home appliances. Second to none in the washer-dryer world, the unbelievable combos at this store will make doing laundry lovable. Do you need an upgrade to your suite of small appliances? Step your game up at Apco Appliance and Air. Say goodbye to dirt and dust with a new and easy-to-maneuver vacuum cleaner. Parking is accessible and not far from the store.23441 Golden Springs DriveDiamond Bar, CA US
Vacation Pools and SpasRead MoreWork on your speed, form and endurance with a brand new bike from Oak Hills' Vacation Pools and Spas. Get the most out of the great outdoors with quality supplies and equipment from Vacation Pools and Spas. Vacation Pools and Spas is located near endless parking possibilities, allowing drivers to park with ease. So if you're looking to roll out with a brand new set of wheels, check out the awesome selection of bikes and gear at Vacation Pools and Spas today.13760 Wells Fargo StOak Hills, CA US
Tropicana Islands and GrillsRead MoreWith the many parking choices near Tropicana Islands and Grills, finding parking within walking distance is a breeze.249 W Allen AveSan Dimas, CA US
Larry's Lawnmower ShopRead MoreCombining great performance with technology, these large appliances are sure to impress. Give yourself more room in your home and treat yourself to some more compact appliances from Larry's Lawnmower Shop. Clean up messes right when they happen with this reliable and speedy vacuum cleaner. Customers don't need to worry about finding parking, as there are many spots available in the area.244 W Holt AvePomona, CA US


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here welcome to: forums > random > say anything > guess how old the preson above you is
"Vertigo" wrote:maybe.. 13?
"Storm1232" wrote:12?
"Storm1232" wrote:12?
Rylan: *SASS-OVERLOAD*
Violet: Woah.....hawt. o.o
Authors: AHAHAHAHAHA
Riolet and Jaziel are OTP.
Attack on Titan. Bruh.
"crazehgurl12" wrote:
"Storm1232" wrote:12?
I do not like bad grammar, and there is a high chance of me correcting you.
Yes Stormie-chan is back once again.
Kon'nichiwa, futatabi min'na!
(Hello Agin Everyone!)
"Storm1232" wrote:
"crazehgurl12" wrote:
"Storm1232" wrote:12?
Uhm,..Im bad at this game...12?
Rylan: *SASS-OVERLOAD*
Violet: Woah.....hawt. o.o
Authors: AHAHAHAHAHA
Riolet and Jaziel are OTP.
Attack on Titan. Bruh.
Welcome to Minions Lab, the ultimate brain teasing game! In this fun online game...
Uncle George has left you his farm, but unfortunately it's in pretty bad shape. ...
Explore the world or hang out with friends in your own condo. Get a unique look,...
Shoot colorful bubbles with magical experiments. Follow classes at the Academy t...
Welcome to the world of Stella's Fashion Show! - Meet Stella and friends! - D...
Connect cookies to serve your customers! Slide to connect and collect the right ...
Play My Candy Box and tease your brain with this funny and highly entertaining p...
The Hunger Games, a movie based on the book series by Suzanne Collins has been i...


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A goal for Bills starters against Bucs: score a TD - 0 Comments
ORCHARD PARK, N.Y. — Bills running back C.J. Spiller attempted to inject some levity into EJ Manuel's post-practice news conference by asking the quarterback a question.
A smiling Spiller wondered earlier this week what the offense's focus would be for Saturday's exhibition game when the Bills (1-2) host the Tampa Bay Buccaneers (0-2).
Manuel didn't miss a beat.
"It's an extremely important game for us, especially down in the red zone," he said. "We haven't scored a touchdown there yet."
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Nashville Predators defenseman Seth Jones, the team's first round draft pick, during Predators Development Camp at Centennial Sportsplex on Tuesday, July 9, 2013.
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Overall Experience:
My overall experience at my school has been good. I was expecting more school spirit and school actives and life on campus, but thats not exactly what school is for so thats okay. The school turned out to be to expensive as well. I enjoyed when Bill Clinton visited our campus. Our school is known well in Arkansas because we are looked at as the Razorbacks backup. If I could do it all over again, I would more than likely go to my dream school in Florida since I'm already going to be in debt regardless.
Overall Experience:
Arkansas State University is awesome. A lot of friends a lot of fun. Easy to understand professors. Really good choice!
Overall Experience:
The professors really want to see every student succeed!
Overall Experience:
I love my degree program and professors. It's a great school near home.
Overall Experience:
I would chose my school over again, because I would not want to change where my life has ended up today.
Overall Experience:
Everything on campus is great! The teachers, classes, the other students, activities, and everything else on campus. the complaint that I have is the financial aid offices. We had a problem one semester and they had put one of my scholarship checks in the wrong account, but the is the only bad thing about my school.
Overall Experience:
I love the overall university! The Nursing Program really has just made me at home and I would choose this school for that program any day!
Campus Housing:
Living on campus is okay. The dorms are nice but the housing process can be a headache.
Off-Campus Housing:
This will be my first year to rent an apartment. But so far the apartment and neighborhood are nice. I will be about 10 min from college campus.
Party Scene:
I don't really attend the parties. I have attended a few and they were okay.
Off-Campus Dining:
There are different restaurants from hamburger to steak and seafood. I love all the options that are available off campus.
They are very strict when spring break or Christmas break comes around. If you don't pay to stay during break and the college student gets caught in the dorm, the student will have to pay a hefty fine.
The process has always been easy for me.
Campus Food:
I go to the caf and other restaurants on and off campus.
Campus Housing:
My freshmen year was tough because my dorm is the oldest, it smells, the shower always dripped water, the air unit was not consistent in keeping the room cool or warm when needed.
Off-Campus Housing:
It's better than on campus. Don't have to worry about as much. More privacy
Off-Campus Dining:
Lots of chains so there's not a lot of different things to try for out of town people
Party Scene:
The parties are fun. But campus police like to ruin the whole campus sometimes.
Sometimes it's really unnecessary stuff that we get I trouble for. But other stuff I see why it is enforced.
It really is a hassle. It seems like it takes forever just for that. Makes you stress out a lot.
Campus Food:
Food court is awesome. I mean same stuff but it's got a pretty good variety for a college. The caf is really just not that great. But if it's the only thing you have to eat on then it's good
Campus Housing:
It's alright. It's just usual college living. The upperclassman living areas are much better looking on the outside. But the inside isn't as great as the outside looks. Cleanliness Is ok. Could be better.
Health & Safety:
The security for the campus is wonderful.
The students along with the fans are extremely involved with the athletics, and the team is good as well. They are currently doing some updates to the football field that will add a great look to their Stadium for home games as well as other school events.
Off-Campus Housing:
I love living off campus even if it takes twenty minutes to get to school. The area is stunning, and the neighbors are friendly. Everything you need is within a ten minute distance except the school, but it is worth it.
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ResidentUtility™ is a robust billing and utility management solution built to increase your asset value without increasing your workload. Its paperless convergent billing and collection system accelerates property cash flow, cuts expenses, pleases residents, and generates new ancillary revenue. In short, ResidentUtility will grow your NOI and asset value.
Reduce the amount of working capital needed to manage billing and utilities while reducing rent delinquencies by an average of 20%.
Boost your properties value by increasing collection rates and ancillary income.
A single bill contains all utility and lease charges the property wants to collect. Pet fees, cable, trash removal, rent, utilities can all be included and paid online.
An intuitive vacant cost management interface minimizes the amount of energy consumed by more than 50 percent in vacant units.
Real-time reports let you check key performance metrics for your whole portfolio or a specific property.
See old balances cleared up within the first three months when switching to convergent billing from a bill and collect services. Collections then stay at 95%.
From the experiences we've had using other Entrata applications, we saw ResidentUtility as an opportunity to add another valuable business tool to our mix. ResidentUtility will help us manage our vacant cost recovery and also grant our residents increased transparency in the billing process.
Precise data entry and quick access to all of your utility bills
Your property's invoices are mailed directly to our utility experts who then carefully enter readings into the system. The data integrates with your accounting software and is digitally stored for future access.
Before the final bill is sent to your property, it is audited twice to ensure proper data entry.
All bills are processed and available for payment within 24 hours of receiving them, guaranteeing accurate and up-to-date accounts payable and helps eliminate late fees.
Our clients trust us with over $9 billion in transactions annually. Our billing system enforces all relevant laws and regulations according to your property's zip code, so you never have to worry about billing errors.
With PCI, MITS, and SAS70 Certifications you can trust us to process your payments securely and accurately.
Our team of lawyers compile and maintain a regulatory and legal database for all US jurisdictions.


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$350 hdfjfhfjfhf Healthy Cute shih tzu puppies for saleShih-Tzu · Pike Road, ALhdjfgfjfffbfhf Healthy Cute shih tzu puppies for sale. If interested contact or text.. (two 0 nine five 00 one six six one)Salah P. · 7 hours ago
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$280 Black pug puppies male and female already home trained for adoptionPug · Chicago, IL… . They are in a perfect state of health and will come with all documen...May J. · 2 hours ago
$300 Attractive and home trained yorkshiere terrier puppies male and femalePug · Boardman, OH… . Puppies have all health papers and shots. familiar with other pet...Moumis I. · 3 hours ago
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Edit hours
Publix is the largest and fastest growing employee-owned supermarket chain in the US. It's a great place to work and shop. For any Publix Pharmacy inquiries please call (407) 897-7373.
The service is great and everybody is really nice!
This is a GREAT Publix as long as you don't go around 5pm. It's super packed!
Clean and well organized.
Great beer selection and soon they will be getting a liquor store!!
Adding more craft beers to the mix.
You can make your own six pack with an assortment(50 or so) of craft beers
Moving into the Park and tested the customer service; friendly and helpful as ever.
You don't have to be rich or a snob to shop here. It's just like every publix.
All the hot MILFS shop here.
You should try Publix sushi, it is so good!
All the rich Baldwin snobs shop here.
This Publix ALWAYS has a great Carvel Ice Cream Cake selection. Mmmm, eases the pain!
Jordan in customer service is rude and snippy. Avoid her.
Baldwin park rocks and so does the Publix!
Just got done with the week's grocery shopping? Bring back your plastic grocery bags to Publix next time for recycling!! #bagslife
Avoid the brown rice sushi, you are a jerk if you eat that.
The deli crew ROXXOR Read more
Tell Seth not to forget Milk, Bread, coffee cake jr's, and sweet tea.
You should eat while you're here. It doesn't really matter what sub you get here. You're gunna be happy
Publix now has a Greenwise Bulk section.
Public accepts expired coupons - how cool is that?


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Some are touting New York's yogurt empire as the equivalent to California's Silicon Valley. Editor Susan Crowell says let's hope it keeps growing, one spoonful at a time.
Anti-GMO sentiment is based on faulty science, emotion, a mistrust of big corporations, and even conspiracy theories. Editor Susan Crowell serves up her thoughts.
Any time we can increase the consumption of fruits and vegetables, we win — both from a health and an agricultural production standpoint.
Today, we want it all: a healthy environment, low taxes, cheap food, profitable agriculture community and a solid rural economy. But if we looked long-term, what is it that would be "worth as much to agriculture 25 years from now" as it is today?
By: TOM DOWNING The activities at steam and engine shows take place primarily outside and so are greatly subject to weather conditions. Some might argue this fact is a good argument for having a four-day show or longer, as most rainy spells don't commonly last that long, so the chances of having a decently profitable […]
No two farmers manage their farms the same way — one organic grower could have higher risk of bacterial contamination than another, and the same could be said of a nonorganic grower.
Editor Susan Crowell is going to quit her day job and become a professional dairy showman. All someone needs to do is to: a) train her; and b) keep her in M&Ms.
What can we learn for our own farm's success from the missteps of Hostess, Kodak and J.C. Penney? Editor Susan Crowell weighs in.
Editor Susan Crowell says we need new uses of agricultural commodities, to keep that new blood and passion to continue to build our rural communities.
Short on farm labor? Just create a "working mixer" for singles, says Editor Susan Crowell. (Hey, it worked for a farmer in Idaho!)


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Moon CalledPenguin Publishing Group 2006; US$ 7.99
Mercy Thompson's life is not exactly normal. Her next-door neighbor is a werewolf. Her former boss is a gremlin. And she's fixing a VW bus for a vampire. But then, Mercy isn't exactly normal herself. more...
Blood BoundPenguin Publishing Group 2007; US$ 7.99
The second novel in the #1 New York Times bestselling series. A demon-riding vampire has gone on a killing spree unlike any the Tri- Cities has ever seen-and the undead and werewolves sent to stop him haven't returned. A coyote is no match for a demon, but shapeshifting mechanic Mercy Thompson is immune to many vampiric powers-and those are... more...
Iron KissedPenguin Publishing Group 2008; US$ 7.99
When her former boss and mentor is arrested for murder and left to rot behind bars by his own kind, it's up to shapeshifting car mechanic Mercy Thompson to clear his name, whether he wants her to or not. And she'll have to choose between the two werewolves in her life-whether she wants to or not. more...
Cry WolfPenguin Publishing Group 2008; US$ 7.99
Now Briggs begins an extraordinary new series set in Mercy Thompson?s world?but with rules of its own. INTRODUCING THE ALPHA AND OMEGA NOVELS... Anna never knew werewolves existed until the night she survived a violent attack?and became one herself. After three years at the bottom of the pack, she?d learned to keep her head down and never, ever... more...
Alpha & OmegaPenguin Publishing Group 2008; US$ 2.99
More information to be announced soon on this forthcoming title from Penguin USA. more...
Bone CrossedPenguin Publishing Group 2009; US$ 7.99
View our feature on Patricia Briggs?s Bone Crossed .Shapeshifter Mercy Thompson hit the #1 spot on the bestseller list with Iron Kissed ... And she's planning a return trip. Marsilia, the local vampire queen, has learned that Mercy crossed her by slaying a member of her clan. Now, she's out for blood. But since Mercy is protected from... more...
From Potter's FieldScribner 1995; US$ 6.99
#1 bestselling author Patricia Cornwell returns to the chilling world of gutsy medical examiner Kay Scarpetta in this suspense classic. An unidentified nude female sits propped against a fountain in Central Park. There are no signs of struggle. When Dr. Kay Scarpetta and her colleagues Benton Wesley and Pete Marino arrive on the scene, they instantly... more...
Book Of The DeadLittle, Brown Book Group 2008; US$ 13.30
The 'book of the dead' is the morgue log, the ledger in which all cases are entered by hand. For Kay Scarpetta, however, it is about to have a new meaning. Fresh from her bruising battle with a psychopath in Florida, Scarpetta decides it's time for a change of pace. Moving to the historic city of Charleston, South Carolina, she opens a unique private... more...
Port MortuaryLittle, Brown Book Group 2010; US$ 13.30
Kay Scarpetta has been training at the Dover Port Mortuary, mastering the art of 'virtual autopsy' - a groundbreaking procedure that could soon revolutionise forensic science. And it is not too long before these new skills urgently need to be put into practice. A young man drops dead, apparently from a heart condition, eerily close to Scarpetta's... more...
Hornet's NestLittle, Brown Book Group 2008; Not Available
Deputy Chief Virginia West likes and respects her boss, Hammer, but with an increasing number of visiting businessmen being murdered in her city by a maniac with a penchant for painting his victims bright orange, she finds it hard to accept Hammer's edict that a rookie reporter should ride on patrol with her to better relations with their citizens.... more...


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P.E.Review:Transission & Distribution (CD-ROM)E R Collins
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Get the added professional edge by becoming licensed as a Professional Engineer (P.E.)! PE. licensure opens the doors to obtaining career opportunities and promotions, engineering consulting, and gaining respect of your peers. The IEEE can help you prepare for the exam to obtain this important licensing with these first two modules of a series. These videos will assist a practicing electrical engineer to prepare for the Power Engineering portion of the Professional Engineering Licensing Examination. Presenter Randy Collins provides detailed solutions to sample examination questions. The first module concentrates on Transmission & Distribution section. The second takes a close look at the Machinery and Applications section. Since engineering licensure specifications vary from state to state, please refer to your state's licensure board for requirements and regulations. State licensing board use examinations prepared by the National Council of Examiners for Engineering and Surveying (NCEES).
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Company Overview of Center For Health Value Innovation
Executive Profile
Robert E. Campbell Ph.D.
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Dr. Robert E. Campbell, Ph.D., is a Strategic Advisor and Director of Center For Health Value Innovation. He served as the Vice President of Finance at Johnson & Johnson. He served at Johnson & Johnson for about 40 years. He is a Director at Mesoblast, Inc. Dr. Campbell is a Member of Advisory Board of Plebys International, LLC. He served as the Chairman of Board of the Robert Wood Johnson Foundation since July 1999 and Chairman of Board of The Cancer Institute of New ...
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Do these earphones have total control over the iPhone 5/5th gen iPod (ability to change track from the remote)
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Will the remote just control the iPhone,s iPod function?
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Let's Discuss the Insane 'Daughter Shot-Put' Scene From No Escape
Even Owen Wilson thought it was crazy.By Kyle Buchanan and Jennifer Vineyard
Even Owen Wilson thought it was crazy.By Kyle Buchanan and Jennifer Vineyard
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Long in the archives, Marc Maron reposted it last night.By Jesse David Fox
Featuring the least likely Marvel character possible.By Jesse David Fox
Checking in a year after he came out on Marc Maron's "WTF" podcast.By Jesse David Fox
First, there was Louis C.K. Then, Robin Williams. Now ... who? Wait, seriously?By Eliot Glazer
He feels their pain from his garage.By Jada Yuan
The first 100 episodes in a nice little package.By Zach Dionne
Terrific.By Margaret Lyons
Available for purchase in NYC, Los Angeles, and San Francisco.By Sarah Bennett
Makes about as much sense as 50 Cent shilling for Vitaminwater, right?By Sarah Bennett
Because The Antiques Roadshow is just too edgy.By Sarah Bennett
Well, her voice anyway. But still!By Margaret Lyons
Bill Cosby is bloated or something. We need to help Grandpa!By Eliot Glazer
For the song of that name.By Amanda Dobbins
Watch his new fake talk show, "Night Sweats."By Amanda Dobbins
Don't ask why.By Amanda Dobbins
Is it for a Funny or Die video? Or is he going Carrey?By Amanda Dobbins
New music from a real WTF supergroup.By Amos Barshad
It's about him.By Margaret Lyons


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3 hour guided bus tour of James Bond film locations including Mi6 and sites from SPECTRE, Skyfall, Goldeneye, The World is Not Enough, Die Another Day and more.
Walking tour of James Bond film locations in London including Skyfall, Die Another Day, SPECTRE as well as real life spy sites.
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Great views and great staff,Antonio(security) was a blessing.You want something or need help with anything go find Antonio he will get it done. He appears out of nowhere,made our stay there so much more comfortable he makes building #5 complete. Every tues hit the market behind the casino, go early. shopping mall up the street has everything.Restaurants are plentiful,and walking... more
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Page 1
Caspase-3-Dependent Proteolytic Cleavage of Protein Kinase C? Is
Essential for Oxidative Stress-Mediated Dopaminergic Cell Death
after Exposure to Methylcyclopentadienyl Manganese Tricarbonyl
Vellareddy Anantharam, Masashi Kitazawa, Jarrad Wagner, Siddharth Kaul, and Anumantha G. Kanthasamy
Parkinson Disorders Research Program, Department of Biomedical Sciences, Iowa Sate University, Ames, Iowa 50011
In the present study, we characterized oxidative stress-dependent
cellular events in dopaminergic cells after exposure to an organic
form of manganese compound, methylcyclopentadienyl manga-
nese tricarbonyl (MMT). In pheochromocytoma cells, MMT expo-
sure resulted in rapid increase in generation of reactive oxygen
species (ROS) within 5–15 min, followed by release of mitochon-
drial cytochrome C into cytoplasm and subsequent activation of
caspase-3 (15- to 25-fold), but not caspase-8, in a time- and
dose-dependent manner. Interestingly, we also found that MMT
exposure induces a time- and dose-dependent proteolytic cleav-
age of native protein kinase C? (PKC?, 72–74 kDa) to yield 41 kDa
catalytically active and 38 kDa regulatory fragments. Pretreatment
with caspase inhibitors (Z-DEVD-FMK or Z-VAD-FMK) blocked
MMT-induced proteolytic cleavage of PKC?, indicating that cleav-
age is mediated by caspase-3. Furthermore, inhibition of PKC?
activity with a specific inhibitor, rottlerin, significantly inhibited
caspase-3 activation in a dose-dependent manner along with a
reduction in PKC? cleavage products, indicating a possible posi-
tive feedback activation of caspase-3 activity by PKC?. The pres-
ence of such a positive feedback loop was also confirmed by
delivering the catalytically active PKC? fragment. Attenuation of
ROS generation, caspase-3 activation, and PKC? activity before
MMT treatment almost completely suppressed DNA fragmenta-
PKC?K376R(dominant-negative mutant) prevented MMT-induced
apoptosis in immortalized mesencephalic dopaminergic cells. For
the first time, these data demonstrate that caspase-3-dependent
proteolytic activation of PKC? plays a key role in oxidative stress-
mediated apoptosis in dopaminergic cells after exposure to an
environmental neurotoxic agent.
Key words: apoptosis; oxidative stress; Parkinson's disease;
environmental factors; manganese; dopaminergic degeneration
Parkinson's disease (PD) is an idiopathic neurodegenerative dis-
order characterized by profound loss of dopaminergic neurons in
the nigrostriatal tract. Although debated, most studies have con-
cluded that aging, environmental neurotoxicant exposures, and
genetic alterations are potential risk factors in the development of
PD (Oertel and Kupsch, 1993; Langsten and Hill, 1998; Aschner,
2000; Simon et al., 2000). Recently, a study conducted on
thousands of twins concluded that genetic factors do not play a role
in the pathogenesis of geriatric onset of PD, which further supports
the view that environmental factors are dominant risk factors
in the etiology of PD (Tanner et al., 1999). Results of several
epidemiological studies conducted in rural areas have also sug-
gested that certain pesticides and other environmental factors,
including transition metals such as manganese, have a positive
association with increased incidences of PD (Seidler et al., 1996;
Liou et al., 1997; Gorell et al., 1999). Occupational exposure to
manganese during mining was shown to cause a Parkinson's-like
syndrome known as Manganism (Mena et al., 1967; Barbeau, 1984;
Donaldson, 1987; Gorell et al., 1999). Furthermore, exposure to
manganese-containing compounds such as manganese ethylene-
bis-dithiocarbamate (a fungicide) and Bazooka (a cocaine-based
drug) among farm workers and abusers, respectively, has been
shown to result in adverse neurological defects (Roels et al., 1987;
Ferraz et al., 1988; Wang et al., 1989; Thiruchelvam et al., 2000).
Methylcyclopentadienyl manganese tricarbonyl (MMT) has
been used in Canada as an anti-knock gasoline agent and has
been recently legalized for use in the United States as a replace-
ment for tetraethyl lead [(CH3CH2)4Pb] in gasoline (Lynam et al.,
1999; Zayed et al., 1999). Because MMT is a manganese-
containing compound, its use has raised great a concern regarding
increased exposure to the public and its possible adverse health
effects (Frumkin and Solomon, 1997; Davis, 1998; Lynam et al.,
1999; Zayed et al., 1999). Exposure to MMT produces a pro-
longed and more pronounced accumulation of manganese in rat
brain as compared with manganese derived from an inorganic
source, for example, MnCl2(Zheng et al., 2000). Administration
of MMT produces seizures in mice (Fishman et al., 1987) and
also results in depletion of dopamine in the mouse striatum
(Gianutsos and Murray, 1982). Furthermore, MMT administra-
tion has been shown to be an effective inhibitor of complex I in
mitochondrial electron transport chain (Autissier et al., 1977), an
action similar to the pyridinium metabolite of 1-methyl-4-phenyl-
1,2,3,6-tetrahydropyridine (MPTP), a Parkinsonian toxin. Re-
cently, we demonstrated that MMT exposure induces reactive
oxygen species (ROS) generation, dopamine depletion, and cell
death in dopamine-producing rat pheochromocytoma (PC12)
Received Oct. 26, 2001; revised Dec. 7, 2001; accepted Dec. 12, 2001.
This work was supported by the National Institute of Environmental Health
Sciences Grant RO1-ES10586. We acknowledge Dr. Palur Gunasekar (Operational
Toxicology, Air Force Research Laboratories, Dayton, OH) for his initial assistance
in some experiments. We thank Dr. Michael L. Kirby, Dr. Arthi Kanthasamy, and
Mr. Siddharth Ranade in the preparation of this manuscript and Dr. Donghui Cheng
for help with flow cytometry.
Correspondence should be addressed to Dr. A. G. Kanthasamy, Parkinson
Disorders Research Laboratory, Department of Biomedical Sciences, 2062 Vet-
erinary Medicine Building, Iowa Sate University, Ames, IA 50011. E-mail:
J. Wagner's present address: Department of Chemistry, California State Univer-
sity, Fresno, CA.
Copyright © 2002 Society for Neuroscience 0270-6474/02/221738-14$15.00/0
The Journal of Neuroscience, March 1, 2002, 22(5):1738–1751
Page 2
cells, which can be protected by pretreatment with antioxidants
(Wagner et al., 2000). To further understand the cellular mech-
anism of MMT-mediated apoptosis, we investigated whether
oxidative stress induced by MMT can activate a series of cellular
factors associated with apoptotic pathways, which could subse-
quently lead to programmed cell death in dopaminergic cells.
Herein, we report that MMT exposure activates a novel apoptotic
pathway in dopaminergic cells through caspase-3-dependent pro-
teolytic cleavage of PKC?.
Reagents. MMT was obtained from Sigma-Aldrich (St. Louis, MO);
rottlerin was purchased from Calbiochem (San Diego, CA); acetyl-Asp-
Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), acetyl-Iso-Glu-Thr-Asp-7-
amino-4-methylcoumarin (Ac-IETD-AMC), acetyl-Leu-Glu-His-Asp-7-
amino-4-methylcoumarin (Ac-LEHD-AMC), and Z-Asp-Glu-Val-Asp-
fluoromethyl ketone (Z-DEVD-FMK) were obtained from Alexis
Biochemicals (San Diego, CA); Z-Val-Ala-Asp-fluoromethyl ketone (Z-
VAD-FMK) was obtained from Enzyme Systems (Livermore, CA).
was obtained from Bachem (King of Prussia, PA); fluorescein isothiocya-
nate conjugated to VAD-FMK (FITC-VAD-FMK) was purchased from
Promega (Madison, WI); antibodies to PKC?, PKC?, PKC?I, and PKC?II
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), cyto-
chrome C (mouse monoclonal) from PharMingen (San Diego, CA), green
fluorescent protein (GFP) (mouse monoclonal) from Clontech (Palo Alto,
CA), and ?-actin (mouse monoclonal) from Sigma (St. Louis, MO). ECL
chemiluminescence kit was purchased from Amersham Pharmacia Biotech
(Piscataway, NJ). PC12 cells were purchased from American Type Culture
Collection (ATCC) (Rockville, MD), and immortalized rat mesencephalic
dopaminergic neuronal cell line (1RB3AN27) was a kind gift of Dr. Kedar
N. Prasad (University of Colorado Health Sciences Center, Denver, CO).
Hydroethidine and Hoechst 33342 were purchased from Molecular Probes
(Eugene, OR). Cell Death Detection ELISA Plus assay kit was purchased
from Roche Molecular Biochemicals (Indianapolis, IN). PKC? catalytic
fragment, acridine orange, histone H1, ?-glycerophosphate, superoxide
dismutase (SOD), ATP, Protein-A-Sepharose, phosphatidylserine, and dio-
leoylglycerol were purchased from Sigma. Mn(III)tetrakis(4-Benzoic acid-
)porphyrin chloride (MnTBAP) was purchased from Oxis Health Products
(Portland, OR). [?-32P]ATP was purchased from NEN (Boston, MA).
Bradford protein assay kit was purchased from Bio-Rad (Hercules, CA).
Lipofectamine Plus reagent, Roswell Park Memorial Institute (RPMI)-
1640 medium, horse serum, fetal bovine serum, L-glutamine, penicillin,
streptomycin, and PCEP4 plasmid were purchased from Invitrogen (Gaith-
ersburg, MD). BioPORTER, protein delivery reagent was purchased from
Gene Therapy Systems (San Diego, CA), and plasmids PKC?K376-GFP
fusion protein and pEGFP-N1 were kind gifts of Dr. Stuart Yuspa (Na-
tional Cancer Institute, Bethesda, MD).
Cell culture. PC12 (ATCC CRL1721) cells were grown in RPMI
medium supplemented with 10% horse serum, 5% fetal bovine serum,
1% L-glutamine, penicillin (100 U/ml), and streptomycin (100 U/ml) and
maintained at 37°C in a humidified atmosphere of 5% CO2. Immortal-
ized rat mesencephalic cells (1RB3AN27) were grown in RPMI medium
supplemented with 10% fetal bovine serum, 1% L-glutamine, penicillin
(100 U/ml), and streptomycin (100 U/ml), maintained at 37°C in a
humidified atmosphere of 5% CO2(Prasad et al., 1998).
Stable transfection. Plasmid pPKC?K376R-GFP encodes protein kinase
C?-GFP fusion protein, the number K376R refers to the mutation of
lysine residue at position 376 to arginine in the catalytic site of PKC?
rendering it inactive (Li et al., 1999). Plasmid pEGFP-NI encodes the
green fluorescent protein alone and used as vector control. pEGFP-N1
and pPKC?K376R were transfected into 1RB3AN27cells using Lipo-
fectamine Plus reagent according to the procedure recommended by the
manufacturer. In brief, 8 ?g of DNA, 24 ?l of lipid, and 24 ?l of Plus
reagent were used to transfect 1RB3AN27cells in 100 mm tissue culture
dishes at 50% confluency in 4 ml of culture medium without serum. Fresh
medium containing serum was added 3 hr later. For stable cell lines, the
1RB3AN27cells were selected in 400 ?g/ml hygromycin, 48 hr after
cotransfection with PCEP4 plasmid, which confers hygromycin resis-
tance. Colonies were isolated with trypsin and glass cloning cylinders,
and they were then replated and grown to confluence in T75 flasks.
Subsequently, the stable cell lines were maintained in 200 ?g/ml
Treatment paradigm. After 2–4 d in culture, PC12 cells and 1RB3AN27
were harvested and resuspended in serum-free growth medium at a cell
density of 1–3 ? 106/ml. Cell suspensions were treated with varying
concentrations of MMT (30–500 ?M) over a period of 0.5–5 hr at 37°C.
In inhibitor studies SOD (ROS inhibitor, 100 U/ml), MnTBAP (ROS
inhibitor, 10 ?M), rottlerin (PKC? inhibitor, 5–20 ?M), Ac-DEVD-CHO
(caspase-3-specific inhibitor, 100–300 ?M), Z-DEVD-FMK (caspase-3-
specific inhibitor, 10–50 ?M), or Z-VAD-FMK (a broad spectrum
caspase inhibitor, 30–100 ?M) were added 30–90 min before the addition
of MMT. The reaction samples were removed at 0.25, 0.5, 1, 2, 3, and 5
hr, then spun at 200 ? g, and after 5 min, the cell pellets were used for
assessing cytochrome C release, caspase-3, caspase-8, and caspase-9
enzymatic activities, extent of PKC? cleavage, and DNA fragmentation.
Dimethylsulfoxide (DMSO) (0.5–1%) was used as a vehicle in control
Lactate dehydrogenase assay. Lactate dehydrogenase (LDH) activity in
the cell-free extracellular supernatant was quantified as an index of cell
death (Vassault, 1983). We modified the original method to a 96-well
format (Kitazawa et al., 2001). Briefly, PC12 cells were plated in 96-well
plate, and after treatment 10 ?l of the extracellular supernatant was
added to 200 ?l of 0.08 M Tris buffer, pH 7.2, containing 0.2 M NaCl, 0.2
mM NADH, and 1.6 mM sodium pyruvate. LDH activity was measured
continuously by monitoring the decrease in the rate of absorbance at 339
nm using a microplate reader (Molecular Devices, Sunnyvale, CA), and
the temperature was maintained at 37°C during reading. Changes in
absorbance per minute (?A/?T) were used to calculate LDH activity
(U/I), using the following equation: U/I ? (?A/?T) ? 9682 ? 0.66, where
9682 was a coefficient factor, and 0.66 was a correction factor at 37°C.
Detection of reactive oxygen species and lipid peroxidation by flow cytom-
etry. Flow cytometry analysis was performed on a Becton Dickinson (San
Francisco, CA) FACScan flow cytometer. Hydroethidine, a sodium
borohydride-reduced derivative of ethidium bromide, is used to detect
ROS produced specifically inside the cell (Narayanan et al., 1997). When
hydroethidine is loaded in the cells, it binds to cellular macromolecules.
Once O2
and increases red fluorescence (620 nm). A 15 mW air-cooled argon–ion
laser was used as an excitation source for hydroethidine at 488 nm, and
the optical filter was 585/42 nm bandpass. Cells were detected and
distinguished from the background by forward-angle light scattering
and orthogonal light scattering characteristics. All the flow cytometric
data were analyzed by Cellquest data analysis software to determine the
significant increase or decrease of fluorescence intensity.
PC12 cells and engineered 1RB3AN27cells expressing kinase inactive
PKC? protein were resuspended with HBSS with 2 mM calcium at a
density of 0.5 ? 106cells/ml. Cells were then incubated with 10 ?M
hydroethidine for 15 min at 37°C in the dark to allow dye loading into the
cells. After incubation with dye, excess dye was removed, and the cells
were resuspended with HBSS. After addition of MMT (30–500 ?M) ROS
generation was measured at 0, 5, 15, 30, and 45 min after the exposure.
In inhibitor studies, cells were incubated with SOD (100 U/ml) and
MnTBAP (10 ?M) 10–30 min before MMT exposure.
Quantification of cytochrome C release. Cytochrome C release was
quantified using a recently developed ELISA kit developed by MBL
(Watertown, MA). This is a fast, highly sensitive and reliable assay for
the detection of early changes in cytochrome C levels. Briefly, after 2–4
d in culture, PC12 cells were harvested and resuspended in serum-free
growth medium at a cell density of 5 ? 106/ml. Cell suspensions were
exposed to 200 and 500 ?M MMT for 15–30 min at 37°C. After treatment
the cells were spun at 200 ? g, and after 5 min, washed once with 1?
ice-cold PBS and resuspended in 1 ml of ice-cold homogenization buffer
(10 mM Tris HCl, pH 7.5, 0.3 M sucrose, 1 mM phenylmethylsulfonyl
fluoride, 25 ?g/ml aprotinin, and 10 ?g/ml leupeptin) and homogenized
on ice. Cells were then centrifuged for 10,000 ? g for 60 min at 4°C. The
resulting supernatants were collected as cytoplasmic fraction and used
for cytochrome C release measurements. The MBL ELISA kit measures
cytochrome C by one-step sandwich ELISA. The assay uses affinity-
purified two polyclonal antibodies against cytochrome C. The cytoplas-
cytochrome C polyclonal antibody in the 96-well microtiter for 60 min at
room temperature (RT). After washing with buffer (provided with the
kit), the peroxidase substrate is mixed with the chromogen and allowed
to incubate for an additional 15 min. An acid solution provided with the
kit is then added to each well to terminate the enzyme reaction and to
stabilize the developed color. The optical density of each well is then
measured at 450 nm using a microplate reader. The concentration of
?is generated, it converts hydroethidine to ethidium bromide
peroxidaseconjugated anti-
Anantharam et al. • MMT Cleaves Protein Kinase C?
J. Neurosci., March 1, 2002, 22(5):1738–1751 1739
Page 3
cytochrome C is calibrated from a standard curve based on reference
Confocal analysis of in situ caspase activity. For this study, we used
CaspACE kit (Promega) to label PC12 cells. The kit uses FITC-VAD-
FMK, an FITC conjugate of the cell-permeable caspase inhibitor
Z-VAD-FMK, which binds to activated caspase and serves as an in situ
marker for apoptosis. The experiment was performed as per the manu-
facturer's protocol with slight modifications. Briefly, PC12 cells were
grown on laminin (5 ?g/ml)-coated slides for 2–3 d in a 37°C, 5% CO2
incubator. Cells were then exposed to 200 ?M MMT for 1 hr in the dark.
After exposure, the cells were treated with 10 ?M FITC-VAD-FMK for
20 min at 37°C. Cells were then rinsed with 1? PBS and fixed in 10%
buffered formalin for 30 min at RT in the dark. After fixing, the cells
were washed three times with PBS to remove formalin and then mounted
with medium and coverslips, and observed under a Leica TCS-NT
confocal microscope (Leica Microsystems Inc., Exton, PA).
Enzymatic assay for caspases. Caspase-3, caspase-8, and caspase-9
activities were performed as previously described by Yoshimura et al.
(1998). Briefly, after treatment cells were spun and the cell pellets were
lysed with Tris buffer, pH 7.4 (50 mM Tris HCl, 1 mM EDTA, and 10 mM
EGTA) containing 10 ?M digitonin for 20 min at 37°C. Lysates were
centrifuged at 900 ? g for 3 min, and the resulting supernatants were
incubated with specific fluorogenic caspase substrates at 37°C for 1 hr.
Ac-DEVD-AMC (50 ?M), Ac-IETD-AMC (50 ?M), and Ac-LEHD-
AMC (50 ?M) were used as substrates for determining caspase-3-,
caspase-8-, and caspase-9-like protease activities, respectively. Levels of
cleaved (active) caspase substrate were monitored at excitation ? 380 nm
and emission ? 460 nm using a fluorescence plate reader (model: Fluo-
roskan-11; Titertek). Caspase activities were expressed as fluorescence
units per milligram of protein per hour. The protein concentrations were
determined using the Bio-Rad protein assay kit.
Isolation of cytoplasmic fractions. After incubation, the PC12 cells were
spun at 200 ? g for 5 min. Cell pellets were then washed once with
ice-cold Ca2?-free PBS saline and resuspended in 2 ml of homogeniza-
tion buffer (20 mM Tris-HCl, pH 8.0, 10 mM EGTA, 2 mM EDTA, 2 mM
dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 25 ?g/ml aprotinin,
and 10 ?g/ml leupeptin). The suspensions were sonicated for 10 sec, and
centrifuged at 100,000 ? g for 1 hr at 4°C. The supernatants were
collected as cytosolic fractions. Protein concentration of each sample was
determined, and the SDS-gel electrophoresis was performed as described
Western blotting. Cytoplasmic fractions containing equal amounts of
protein were loaded in each lane and separated on a 10–12% SDS-
polyacrylamide gel. Proteins were then transferred to nitrocellulose
membrane by electroblotting for 75–90 min at 100 V. Nonspecific binding
sites were blocked by treating the nitrocellulose membranes with 5%
nonfat dry milk powder for 2 hr before treatment with primary antibod-
ies. The nitrocellulose membranes containing the proteins were incu-
bated with primary antibodies for 1 hr at RT with antibody directed
against PKC? (1:2000 dilution), PKC? (1:2000 dilution), cytochrome C
(1:2000 dilution), or GFP (1:1000 dilution). The primary antibody treat-
ments were followed by treatment with secondary HRP-conjugated anti-
rabbit or anti-mouse IgG (1:2000 dilution) for 1 hr at RT. Secondary
antibody-bound proteins were detected using an ECL chemilumines-
cence kit (Amersham). To confirm equal protein loading, blots were
reprobed with a ?-actin antibody (1:5000 dilution). Gel photographs were
taken with a gel imaging system and quantification of bands was per-
formed using the imaging software from Scion Corp. (Frederick, MD).
Immunoprecipitation kinase assays. PKC? enzymatic activity was as-
sayed using an immunoprecipitation kinase assay as described by Rey-
land et al. (1999) and Vancurova et al. (2001). Briefly, after treatment
with MMT, PC12 cells were washed once with PBS and resuspended in
1 ml of PKC lysis buffer (25 mM HEPES, pH 7.5, 20 mM
?-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1% Triton X-100,
0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 10 mM NaF,
and 4 ?g/ml each aprotonin and leupeptin). In inhibition experiments,
cells were pretreated with 10 ?M rottlerin before the addition of 200 ?M
MMT. The cell lysates were allowed to sit on ice for 30 min and
centrifuged at 13,000 ? g for 5 min, and the supernatants were collected
as cytosolic fraction. Protein concentration was determined using a
Bradford assay. Cytosolic protein (0.25–0.5 mg) was immunoprecipitated
overnight at 4°C using 2 ?g of anti-PKC?, anti-PKC?, anti-PKC?I, or
anti-PKC?II antibodies. The immunoprecipitates were then incubated
with Protein-A Sepharose (Sigma) for 1 hr at 4°C. The protein A bound
antigen–antibody complexes were then washed three times with PKC
lysis buffer, three times with 2? kinase buffer (40 mM Tris, pH 7.4, 20 mM
MgCl2,20 ?M ATP, and 2.5 mM CaCl2), and resuspended in 20 ?l of 2?
kinase buffer. Reaction was started by adding 20 ?l of reaction buffer
containing 0.4 mg Histone H1, 50 ?g/ml phosphatidylserine, 4.1 ?M
dioleoylglycerol, and 5 ?Ci of [?-32P] ATP (3000 Ci/mM) to the immu-
noprecipitated samples and incubated for 10 min at 30°C. SDS gel-
loading buffer (2?) was added to terminate the reaction, the samples
were boiled for 5 min, and the products were separated on a 12.5%
SDS-PAGE gel. For in vitro inhibition of PKC? kinase activity, 5–20 ?M
rottlerin was added to 200 ?M MMT-treated immunoprecipitated sample
15 min before the addition of 2? reaction buffer containing 5 ?Ci of
[?-32P] ATP (3000 Ci/mmol). The H1 phosphorylated bands were de-
tected using a Personal Molecular Imager (FX model; Bio-Rad), and
quantification was done using Quantity One 4.2.0 software.
Intracellular delivery of PKC? catalytic fragment. Intracellular delivery
of PKC? fragment was performed using a recently developed lipid-
mediated delivery system (BioPORTER; Gene Therapy Systems, San
Diego, CA). This is a fast and reliable procedure that delivers proteins in
a functionally active form into the cytoplasm of cells (Zelphati et al.,
2001). The protein delivery system is composed of a new trifluoroacety-
lated lipopolyamine (TFA-DODAPL) and dioleoyl phosphatidylethano-
lamine. This cationic formulation has recently been used for delivery of
various bioactive molecules, including antibodies, enzymes (caspase-3,
caspase-8, ?-galactosidase, and granzyme B), cytochrome C, dextran
sulfates, phycobiliproteins, and albumins into the cytoplasm of numerous
adherent and suspension cells (Zelphati et al., 2001). Active PKC?
catalytic fragments were delivered into cells using the protein delivery
reagent by following the manufacturer's protocol. PC12 cells (?1–2 ?
105cells/ml) were subcultured in 24-well tissue culture plate for 24 hr.
PKC? catalytic fragment (5 ng) was mixed with 3 ?l of protein delivery
reagent and 300 ?l of serum-free DMEM media and added to each well.
The cells were incubated at 37°C for 4 hr. Cells were then lysed, and
caspase-3 activity measured as described above. Heat-inactivated PKC?
catalytic fragment was used as negative control and inactivation was
performed by incubating the active PKC? fragment at 95°C for 15 min.
The delivery efficiency was ?70% in PC12 cells as determined using a
FITC-tagged antibody control (supplied with the assay kit). Also, the
protein delivery system produced no significant cytotoxic response as
measured by Trypan blue dye exclusion method.
In situ assessment of apoptosis. To assess nuclear morphology and DNA
damage, we stained the cells with fluorescent DNA-binding dyes acridine
orange and Hoechst 33342. Acridine orange, a useful probe for detecting
apoptotic cells, exhibits metachromatic fluorescence that is sensitive to
DNA conformation. Apoptotic cells stained with acridine orange show
reduced green and enhanced red fluorescence in comparison with nor-
mal cells (Pulliam et al., 1998). Briefly, PC12 cells were grown on laminin
(5 ?g/ml)-coated slides for 2–3 d in a 37°C, 5% CO2incubator. Cells
were washed twice with PBS and after 1 hr treatment with 200 ?M MMT,
the cells were incubated with 10 ?M acridine orange for 15 min at RT in
the dark. The cells were again washed with PBS, mounted with cover-
slips, and observed under a Nikon DiaPhot microscope, and pictures
were captured with a SPOT digital camera (Diagnostic Instruments,
Sterling Heights, MI).
Morphological changes associated with apoptosis were also assessed by
staining with Hoechst 33342. Cells stained with Hoechst 33342 dye
fluoresce bright blue after binding to DNA in the nucleus. The nucleus
of apoptotic cells exhibit strong blue staining and staining pattern is
heterogeneous and occurs in patches, indicative of chromatin condensa-
tion, whereas the nucleus of nonapoptotic cells exhibit more diffused,
weak and homogenous staining (Shimizu et al., 1996; Du et al., 1997).
Briefly, PC12 cells were plated on collagen (6 ?g/cm2)-coated cover
slides and treated with 200 ?M MMT. After 1 hr of exposure, the cells
were fixed with 10% buffered formaldehyde for 30 min at room temper-
ature and stained with Hoechst 33342 (10 ?g/ml) for 3 min in dark. The
cells were again washed three times with PBS, mounted with coverslips,
and observed under a Nikon DiaPhot microscope under UV illumina-
tion, and pictures were captured with a SPOT digital camera (Diagnostic
Quantification assay for DNA fragmentation. DNA fragmentation assay
was performed using a recently developed Cell Death Detection ELISA
Plus assay kit. This is a fast, highly sensitive and reliable assay for the
detection of early changes in apoptotic cell death and measures the
appearance and amount of histone-associated low molecular weight
DNA in the cytoplasm of cells. This assay has been recently used in
quantitation of apoptosis because of its reliability and high sensitivity
1740 J. Neurosci., March 1, 2002, 22(5):1738–1751Anantharam et al. • MMT Cleaves Protein Kinase C?
Page 4
(Reyland et al., 1999). Briefly, PC12 and engineered 1RB3AN27cells
were exposed to 200–500 ?M MMT for 1–3 hr. In inhibitor studies, SOD
(100 U/ml), MnTBAP (10 ?M), rottlerin (10 ?M), Z-DEVD-FMK (50
?M), or Z-VAD-FMK (100 ?M) were treated for 30 min at 37°C before
the addition of MMT. After MMT treatment, cells were spun down at
200 ? g for 5 min and washed once with 1? PBS. Cells were then
incubated with a lysis buffer (supplied with the kit) at RT. After 30 min,
samples were centrifuged, and 20 ?l aliquots of the supernatant were
then dispensed into streptavidin-coated 96-well microtiter plates fol-
lowed by addition of 80 ?l of antibody cocktail and incubated for 2 hr at
RT with mild shaking. The antibody cocktail consisted of a mixture of
anti-histone biotin and anti-DNA-HRP directed against various histones
and antibodies to both single-stranded DNA and dsDNA, which are
major constituents of the nucleosomes. After incubation, unbound com-
ponents were removed by washing with the incubation buffer supplied
with the kit. Quantitative determination of the amount of nucleosomes
retained by anti-DNA-HRP in the immunocomplex was determined
spectrophotometrically with 2,2?-azino-di-(3-ethylbenzthiazoline sulfo-
nate (6)) diammonium salt (ABTS) as an HRP substrate (supplied with
the kit). Measurements were made at 405 nm against an ABTS solution
as a blank (reference wavelength ?490 nm) using a Molecular Devices
Spectramax Microplate Reader.
Data analysis. Data analysis was performed using Prism 3.0 software
(GraphPad Software, San Diego, CA). Data from caspase enzymatic
activities and DNA fragmentation assays were first analyzed using one-
way ANOVA. Neuman–Keuls or Dunnett's post-tests were then per-
formed to compare control with MMT-treated groups, and differences
with p ? 0.05 were considered significant. For individual comparisons,
student t test or Welch's corrected t test where appropriate was used.
MMT-induced cytotoxicity
PC12 cells were exposed to 200 and 500 ?M MMT for varying
amounts of time. The amount of LDH released into the extra-
cellular media was measured as an index of cytotoxicity (Vassault,
1983; Kanthasamy et al., 1995). Extracellular LDH activity
showed a dose- and time-dependent increase with MMT treat-
ment ranging from 2- to 20-fold over the control group (Fig. 1).
For example, exposure to 200 ?M MMT resulted in 3-, 10-, and
17-fold increase in LDH release over untreated cells at 1, 3, and
5 hr, respectively. To determine the percent cell death, total LDH
content of untreated cells (?2000 U/I) was normalized to 100%.
PC12 cells exposed to 200 ?M MMT for 3 hr produced ?50% cell
death. Similarly, exposure to 500 ?M MMT resulted in a signifi-
cant ( p ? 0.05) toxicity in MMT-treated groups as compared
with vehicle-treated cells at all time points. There were no sig-
nificant differences in LDH release between vehicle-treated and
untreated PC12 cells during the 5 hr exposure. These data suggest
that MMT induces a dose- and time-dependent cell death in
dopamine-producing cells.
Generation of ROS after MMT treatment
Flow cytometric analysis using the ROS-sensitive fluorescence
probe hydroethidine revealed that MMT treatment induces ROS
generation. Figure 2A depicts a representative flow cytometric
histogram of 200 ?M MMT-treated PC12 cells exhibiting time-
dependent increases in red fluorescence. MMT treatment in-
creased ROS production in a dose- and time-dependent manner
(Fig. 2B). For example, a 15 min exposure to 30, 100, and 200 ?M
MMT resulted in a 10, 41, and 76% increase in ROS production,
respectively. Exposure to 200 ?M MMT resulted in 40, 76, and
80% increase in ROS production over vehicle treatment at 5, 15,
and 30 min, respectively. The time course study also revealed that
500 ?M MMT treatment induced a rapid and dramatic increase in
ROS generation (?200% of control) within 5 min and then the
response rapidly declined over time (data not shown). Pretreat-
ment with SOD (100 U/ml) or MnTBAP (SOD mimetic, 10 ?M)
significantly ( p ? 0.05) reduced MMT-induced ROS production,
indicating that MMT predominantly generates superoxide spe-
cies (Fig. 2C).
Accumulation of cytochrome C in the cytosol after
MMT treatment
ROS production in the cells is known to activate many cellular
factors including cytochrome C, which subsequently triggers ap-
optotic cell death (Tan et al., 1998; Cassarino et al., 1999).
Release of cytochrome C from the mitochondria into the cyto-
plasm is an early event that occurs during programmed cell death
(Muller-Hocker, 1992; Crompton, 1999), and therefore we deter-
mined whether MMT induces release of cytochrome C in PC12
cells. Figure 3A shows a time-dependent increase of cytochrome
C in the cytoplasmic fractions of PC12 cells treated with MMT.
No detectable levels of cytochrome C were detected in the cytosol
of vehicle (DMSO)-treated cells up to 3 hr, whereas a profound
release of cytochrome C was observed as early as 1 hr in MMT-
treated cells. Nitrocellulose membranes were reprobed with
?-actin antibody, and the density of 43 kDa ?-actin bands was
identical in all lanes confirming equal protein loading. To further
accurately quantify how soon cytochrome C is released, we used
a highly sensitive cytochrome C ELISA sandwich assay. MMT
exposure resulted in a dose-dependent increase in cytosolic cy-
tochrome C as early as 15 min (Fig. 3B). A 15 min exposure to 200
and 500 ?M MMT resulted in an increase in cytosolic cytochrome
C by 40 and 200% over the vehicle-treated group, respectively,
and a 30 min exposure resulted in an increase in cytosolic cyto-
chrome C by 70 and 170%, respectively. The reason for the
decrease in amount of cytochrome C released at 30 min after 500
?M MMT exposure might be attributed to loss of cellular integ-
rity caused by the observed necrotic cell death (Fig. 1).
Activation of caspase-3 and caspase-9 but not
caspase-8 after MMT treatment
Because the release of cytochrome C is known to activate a group
of cysteine proteases, namely caspases (Cohen, 1997; Earnshaw et
al., 1999; Schultz and Andreasen, 1999; Jellinger, 2000), we ex-
amined whether caspase-8, caspase-9, and caspase-3 are activated
during MMT exposure. PC12 cells exposed to MMT showed a
Figure 1.
200 and 500 ?M of MMT for 0.5–5 hr at 37°C. After the exposure, cell-free
extracellular supernatants were collected, and LDH activity was mea-
sured by spectrophotometer. Values represent mean ? SEM for three to
five separate experiments in triplicate. Significance was determined by
ANOVA followed by Dunnett's post-test between the vehicle-treated
group and each treatment group (*p ? 0.05).
MMT exposure induces cell death. PC12 cells were exposed to
Anantharam et al. • MMT Cleaves Protein Kinase C?
J. Neurosci., March 1, 2002, 22(5):1738–1751 1741
Page 5
significant increase in caspase-9 activity, however, no significant
increase in caspase-8 enzyme activity was observed (data not
shown). A 30 min exposure to 200 and 500 ?M MMT produced a
threefold and twofold increase in caspase-9 activity, respectively.
The lack of dose–response in caspase-9 enzymatic activity at 500
fluorescent intensity was measured at 0, 5, 15, and 30 min. Data represent
the mean ? SEM of two to five separate experiments in triplicate.
Asterisks (*p ? 0.5 and **p ? 0.01) indicate significant differences
compared with the time-matched vehicle-treated cells. C, Effect of SOD
and MnTBAP on ROS production. Cells were pretreated with ROS
inhibitors, SOD (100 U/ml) and MnTBAP (10 ?M), and then exposed to
100 or 200 ?M MMT for 15 min. The value of each treatment group is the
mean ? SEM from two to three separate experiments performed in
triplicate. Asterisks (*p ? 0.05) indicate significant differences compared
with MMT-treated cells.
Figure 2.
suspended in HBSS supplemented with 2 mM Ca2?at a density of
0.5–0.75 ? 106cells/ml. A concentration of 10 ?M hydroethidine was
added to the cells and incubated for 15 min at 37°C in the dark. A,
Time-dependent change in hydroethidine fluorescent intensity in PC12
cells treated with MMT. A concentration of 200 ?M MMT was added, and
fluorescent intensity was measured at 0, 15, and 30 min by flow cytometry
as described in Materials and Methods. The data are a representative flow
cytometric histogram of MMT-treated PC12 cells exhibiting a time-
dependent increase in red fluorescence. B, Dose- and time-dependent
increase in ROS production. Various doses of MMT were added, and
MMT treatment generates ROS in PC12 cells. PC12 cells were
Figure 3.
chrome C in MMT-treated PC12 cells. A, Western blot. B, Cytochrome C
ELISA assay. A, Subconfluent cultures of undifferentiated PC12 cells were
harvested at 1 and 3 hr after treatment with 200 or 500 ?M MMT. The
cytosolic fractions were obtained as described in Materials and Methods.
Cytosolic fractions were separated by 12% SDS-PAGE, transferred to a
nitrocellulose membrane, and cytochrome C (Cyt C) was detected using
polyclonal antibody raised against cytochrome C. For ?-actin measure-
ments, the membrane used for cytochrome C was reprobed with ?-actin
antibody to confirm equal protein loading in each lane. The immunoblots
were visualized using ECL detection agents from Amersham. B, Subcon-
fluent cultures of undifferentiated PC12 cells were harvested at 15 and 30
min after treatment with 200 or 500 ?M MMT. The cytosolic fractions
were obtained as described in Materials and Methods. The value of each
treatment group is the mean ? SEM from two separate experiments in
triplicate. Asterisks (*p ? 0.05) indicate significant differences compared
with vehicle-treated cells.
Dose- and time-dependent accumulation of cytosolic cyto-
1742 J. Neurosci., March 1, 2002, 22(5):1738–1751 Anantharam et al. • MMT Cleaves Protein Kinase C?


text:
A special issue on sepsis has been released by the publisher Landes Bioscience (Austin, TX USA). The articles contained in this special issue of the journal Virulence have been authored by world-class investigators and provide new insights into both the pathogen-related factors and the host defense mechanisms that lead to septic shock and contribute to its resolution or fatal outcome.
Sepsis, or septic shock, is an excessive inflammatory response in answer to a serious infection, most commonly by bacteria, but also fungi, viruses, and parasites. Different microbial components, such as bacterial lipopolysaccharide (LPS), endotoxins, or exotoxins can cause the typical septic inflammatory cascade. The condition can continue even after the infection that caused it is gone. It is a significant cause of morbidity and mortality worldwide. Characteristics of sepsis are massive release of proinflammatory cytokines resulting in tissue damage, an uncontrolled reduction in blood-pressure, multiple organ failure, and death. Sepsis can originate anywhere microbes can gain entry to the body, common sites including the genitourinary tract, the liver and its bile ducts, the gastrointestinal tract, and the lungs. The condition is often life-threatening, especially in people with a weakened immune system or other medical issues.
Dr. Steven Opal from Alpert Medical School of Brown University (Pawtucket, RI USA) has worked with the editorial team of Virulence to organize this special issue on sepsis. Over 20 papers in this issue discuss many different aspects of sepsis and septic shock, such as epidemiology of severe sepsis (Sachin Yende), gender differences in sepsis (Irshad Chaudry), systemic inflammatory response syndrome (Robert Balk), the PIRO model (John Marshall), host innate immune responses to sepsis (Willem Joost Wieringa), the changing immune system in sepsis (Jonathan Boomer), pathogenesis of bacteremia (Eirini Christaki), role of mitocho
|Contact: Andrew Thompson
Landes Bioscience