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Thai Restaurant {branch} | Doytao Thai Restaurant | {branch} Menu | Order Online
Menu Selection
- Only at Doytao
- Food From The Sea
- Entree
- Soup
- Salmon Specials
- Thai Salad
- Classic Curries
- Stir-Fried
- Rice And Noodles
- Duck Specials
- Lunch Specials
- Thai Desserts
Sizzling Set with B.B.Q Pork or Lamb
Only at Doytao
Soft Shell Crab Salad
Shredded carrots, red onion, tomatoes, celery tossed together in sweet and sour dressing, this salad makes an excellent accompaniment to the crispy crab with moist insides.
Marinated Lamb Cutlet
Succulent cutlets of lamb marinated in Doytao's special sauce, this dish is often requested by our customers. Grilled yet tender and juicy, it is served with a side of mixed salad and sweet & sour dipping sauce.
Nutri Fried Rice$14.90
Get healthy! Try our well known fried rice with egg; Balanced mixed Cereal, onions, carrots, corn kernels and peas.
Sizzling Seafood
Aromatic Soft Shell Crab
A Refreshing and intensely aromatic dish consisting of chopped Celery, Shallot, sliced Onion, capsicum stir-fried with egg in curry powder then drizzled on top of deep fried Soft Shell Crab. Spices from the mild curry powder
Tom Yum Fried Rice
One of Thailand's most famous flavours is now in rice form, only at Doytao, this unique dish offers the same Tom Yum flavour of hot and sour with fragrant herbs, seafood cooked to perfection with rice.
Sizzling Seafood
Doytao Thai's Sizzling king prawns, scallops, fish fillet and squid stir-fried with only the best vegetables served on a hot plate.
Volcanic Chicken
Whole boneless chicken marinated in a special home made sauce covered with honey and flamed with brandy.
Doytao Seafood Special
Exotic Fish
Steamed whole snapper fish, topped with minced chicken and tasty sauce with ginger and vegetables.
Basil Fish Fillet
Crispy fish fillet stir-fried with chili and chili jam, bamboo shoots and mixed vegetables in hot and sweet basil sauce.
Doytao Seafood Platter
Food From The Sea
Basil Fish (whole snapper)
Whole snapper fried until golden then covered with a Thai basil sauce that really compliments the flavor of the fish and let's its delicate tenderness shine through.
Doytao Seafood Platter
Grilled Royal king prawns, calamari, scallops and green mussel half shells served with three authentic 'one in town' sauce and a side of only the healthy vegetables.
Royal King Prawns (4 large prawns)$25.90
Grilled prawns in a Thai style marinade of Thai herbs, garlic, coriander roots and butter served with special sauce and a side of only the healthiest vegetables.
Choo Chee Fish
Deep fried whole snapper, saturated in Choo Chee curry, an authentic Thai red curry cooked with coconut milk and sprinkled with lime leaves.
Steamed Barramundi (whole)
Prawns And Mussels$25.90
Very aromatic prawns and mussels marinated and steamed with lemon grass and basil.
Salt and Pepper Fish
Crispy and mouth watering, this whole Barramundi fish is deep fried until perfectly golden brown. Cooked with garlic, shallots, salt and pepper, sprinkled with or without mild chili.
Phuket Grilled Fish (whole)
Grilled Barramundi marinated in Thai herbs and served with an authentic sauce and a side of only the healthiest vegetables.
Steamed Barramundi (whole)
A very popular Thai style fish that is steamed with ginger and celery then poured over with our tasty sour soup.
Exotic Fish
Steamed whole snapper fish, topped with minced chicken and tasty sauce with ginger and vegetables.
Salt and Pepper (Squid)
Sweet and Sour Fish
Whole snapper fried until golden brown and topped with delicious sweet and sour sauce.
Hot Pattaya
A mild-spiced dish, stir-fried succulent green mussels in fresh chili and Thai chili jam sauce, covered with basil leaves and topped with Thai vegetables.
Salt and pepper (Prawns)
A well-known favorite, lightly battered in salt, pepper, capsicum, onion and shallot with or without mild chili.
Salt and Pepper (Squid)
A well-known Thai favorite, lightly battered in salt, pepper, capsicum, onion and shallot with or without mild chili.
Salt and Pepper (Seafood)
A well-known favorite, lightly battered in salt, pepper, capsicum, onion and shallot with or without mild chili.
Prawn Cakes (6pcs)
Curry Puffs (4pcs)
Onions, potatoes and carrots with curry powder, wrapped in pastry and deep-fried.
Prawn Cakes (6pcs)
Prawn mince with Thai herbs served with sweet chili sauce, topped with crushed peanuts.
Chicken Wings with Minced Stuffing (2pcs)
Golden Chicken Wings (6pcs)
Chicken wings marinated in Thai herbs topped with special home-made sauce, sprinkled with sesame seeds and shallots.
Chicken Satay (4pcs)
Tender brochettes of grilled chicken marinated in 12 different spices, served with peanut sauce.
Spring Rolls (4pcs)
Vermicelli, carrots, bamboo shoots and and mushroom, wrapped then deep fried, served with sweet chili sauce.
Spring Rolls Meat (4pcs)
Chicken & prawn mince, wrapped then deep fried, served with sweet chili sauce.
Sour Prawn Soup (Tom Yum Goong)
Coconut Chicken Soup (Tom Kha Gai)
Chicken in coconut soup with galangal, lemongrass and mushroom.
Sour Prawn Soup (Tom Yum Goong)
Famous Thai spicy soup cooked with king prawns, mushrooms, coriander and lemongrass.
Sour Seafood Soup (Tom Po Taek)
Seafood soup, king prawns, scallops, squid and fish, flavored with Thai spicy soup and herbs.
BBQ Chicken (Boneless!) half/whole
BBQ Pork$9.90
Marinated with Thai herbs and served with a small side salad.
BBQ Lamb
Marinated with Thai herbs and served with a small side salad.
Charcoal Wings
Charcoal Wings
Marinated Chicken Wings cooked on a charcoal grill allowing all flavors to penetrate into the meat. Served with grilled sticky rice and a side of salad with mayonnaise, this dish is the perfect appetizer.
Salmon Salad
Salmon Specials
Crispy deep fried salmon cutlet with prawns covered in mouth watering, authentic home-made sauce, with ginger, mushrooms and healthy vegetables.
Salmon Salad
A large salmon cutlet grilled with lemon grass, sided with a very famous Thai "Som Tum" salad with sliced carrots, cherry tomato, beans, mild chilli, topped with cashew nuts.
Salmon Soup
Hot and sour soup with salmon, fresh Thai herbs, mushrooms, lemongrass, coriander and cherry tomatoes.
Salmon in Coconut Soup
Famous Thai coconut soup with salmon, fresh Thai herbs, mushrooms, lemongrass, coriander and cherry tomatoes.
Salmon in Red or Green Curry
Yummy Beef (Yum Nua)
Thai Salad
Thai Salad dressing is a tasty sweet and sour juice made with onion, coriander, roasted chilli, mint leaves and shallots and chilli jam.
Succulent Lamb Salad
Grilled marinated lamb fillet, tossed with mint, shallots, sliced celery and Thai salad dressing.
Chicken or Beef Salad (Larb)
Minced beef/chicken/pork/lamb cooked in Thai Salad dressing (with toasted ground rice, without chili jam).
Yummy Beef (Yum Nua)
A mild salad with grilled beef finely sliced in Thai Salad dressing together with cucumber and tomatoes.
Waterfall Salad (Nam Tok)
A mild salad with thinly sliced grilled beef, mixed in Thai Salad with slices of tomato and herbs.
Seafood Thai Salad
Squid Salad (Yum Pla Muek)
Grilled squid mixed with 'Thai Salad' dressing.
Special Prawn Salad (Plah Koong)
Grilled prawns mixed in 'Thai Salad' dressing.
Vermicelli Chicken Salad
Minced chicken, prawn cooked with Thai Salad dressing and vermicelli noodles.
Choo Chee Prawns
Classic Curries
- Beef/Chicken/Pork/Lamb
- Seafood/King Prawns
- Vegetables with/without tofu
Choo Chee Prawns
Fresh prawns cooked with Thai curry paste, coconut cream, vegetables and topped with coriander.
Masman (Beef Only)
A rich aromatic Thai beef curry cooked with coconut milk, potatoes and onion.
Meat or tofu with Thai curry paste in coconut milk, lime leaves and vegetables topped with crushed peanuts.
Red Curry
Meat or tofu in Thai red curry paste, coconut milk, peas, bamboo shoots, mixed vegetables and fresh basil.
Thai Curry with Roti
Green Curry
Fine slices of meat or tofu with Thai green curry paste, coconut milk, peas, basil, bamboo shoots and mixed vegetables.
Yellow Curry (Chicken Only)
Original dish from the south of Thailand. Chicken with yellow curry in coconut milk, potatoes and onion served with cucumber sauce.
Jungle Curry
Hot Thai country style curry with meat or tofu and vegetables without coconut milk.
Stir-Fry with Oyster Sauce
- Beef/Chicken/Pork/Lamb
- Seafood/King Prawns/Squid
- Vegetables with/without tofu
Stir-Fry with Oyster Sauce
A tasty Thai stir-fried with mushrooms, capsicum, shallots and mixed vegetables with oyster sauce.
Hot Basil Stir-Fry (Pad Krapao)
Stir-fried cooked with mixed vegetables, basil leaves, fresh chili, chili jam and bamboo shoots.
Stir-Fry with Chili
Stir-fried with mixed vegetables, onions, shallots, fresh chili and chili jam.
Stir-Fry with Cashew Nuts (Pad Med Mamuang)
Sweet and Sour Stir-Fry (Pad Preo Wan)
Stir-fried tomato, cucumber, pineapple, capsicum and mixed vegetables with Thai style sweet and sour sauce.
Stir-Fry with Cashew Nuts (Pad Med Mamuang)
Stir-fried with vegetables, cashew nuts with a special home-made sauce.
Plik Khing Stir-Fry (N/A for Vegetarians)
Delicious home-made curry paste stir-fried with mixed vegetables and lime leaves.
Ginger Stir-Fry
A tasty Thai stir-fried with sauteed ginger, onion and mixed vegetables.
Doytao Stir-Fry
Stir-fried with mixed vegetables, chili jam.
Doytao Pan-Fry
Pad Thai
Rice And Noodles
Chicken/beef/pork/lamb or with mixed vegetables, Coconut curry soup with rice vermicelli noodles, bean sprouts on a Doytao Thai original recipe. A popular noodle soup dish.
Prawn/Seafood Laksa$18.90
Coconut curry soup with egg noodles, bean sprouts on an original recipe.
Pad Thai
Popular Thai rice noodles with chicken/beef/pork/lamb with one king prawn, egg or with mixed vegetables with no egg, with/without tofu, roasted peanuts and bean sprouts.
Pad Thai Prawn/Seafood
Popular Thai rice noodles with egg, roasted peanuts and bean sprouts.
Pad See Eew
Pad See Eew$12.90
Beef/Chicken/Pork/Lamb or with mixed vegetables with no egg, with/without tofu pan fried flat rice noodles with egg, sprouts, chinese broccoli and sweet sauce.
Pad See Eew Prawn/Seafood$18.90
Pan fried flat rice noodles with egg, sprouts, chinese broccoli and sweet sauce.
Pad Kee Mao - Spicy Noodles!$12.90
Beef/Chicken/Pork/Lamb or with mixed vegetables with no egg, with/without tofu pan fried noodles with homemade chili basil sauce, egg, chili jam and seasonal vegetables.
Pad Kee Mao Prawn/Seafood$18.90
Pan fried noodles with homemade chili basil sauce, egg, chili jam and seasonal vegetables.
King St Fried Rice$12.90
Fried rice with egg, ham, peas, pineapple, capsicum, sultanas and cashew nuts.
Roti bread (2pcs)
Beef/Chicken/Pork/Lamb/Vegetarian Fried Rice$12.90
Thai fried rice with onion, tomato, peas and egg or with mixed vegetables with no egg, with/without tofu
Prawn / Seafood Fried Rice$18.90
Thai fried rice with King prawns or seafood, onion, tomato, peas, eggs and topped with cashew nuts.
Steamed Jasmine Rice (Per Person)
Red Duck Curry or Green Duck Curry
Duck Specials
Red Duck Curry or Green Duck Curry
Delicious traditional Thai dish consisting of roast duck cooked in red or green curry paste with coconut milk, vegetables, tomato, pineapple and lychee.
Roasted Duck Salad in Doytao's Spicy Sauce
A mild salad with chopped roasted duck in Thai salad dressing together with slices of onion, tomatoes and Thai herbs.
Basil Chicken with Fried Egg
Lunch Specials
Basil Chicken with Fried Egg$12.90
An all time favorite dish. Savory, spicy, and redolent of garlic and basil, but what really sets this dish off is the fried egg on top. Sunny-side-up and yolk still runny, the egg adds just a touch of velvety richness to this humble meal.
Stir-Fried or Curry on Rice$8.90
* Lunch delivery services only avalible from Newtown & Waterloo Branch ( Minimum for delivery $20 )
Mango and Sticky Rice (Seasonal)
Thai Desserts
Coconut Caviar Pudding
A refreshing and deliciously unique dessert of Tapioca pearl with taro in coconut milk, served with ice-cream to give this textured pudding extra richness.
Roti Wrap
Crunchy crispy roti, topped with sweetened condensed milk and sugar


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If you know anything about Vegas at all, you know that we love to have a good time. As a matter of fact, we are pros at that. And among out favorite ways to get our good time rocking is by laughing our asses off with some of the funniest people in the world. That's right. Right here in Sin City, we have dozens of resident headliners as well as touring acts that swing in for a performance here and there. But no matter the comedian, it's a sure bet that each time we head out to a comedy show, we are going to have a damn good time.
So, to make sure that you have as much fun as we do while you're here in the Entertainment Capital of the world, we have listed (in no particular order) our top five favorite comedy shows that will start your night off with some major laughs… unless you're like the grumpiest human in the world.
Mac King Comedy Magic Show at Harrah's
Combining the whimsical worlds of magic and comedy, Mac King has mastered the art of family friendly entertainment complete with goldfish…and guinea pigs…and bears…er… oh my! King will hold you spellbound in his old Vegas-style showroom while you're laughing at his hilarious antics and how-did-he-do-that magic. His flawless sleight-of-hand magic act and a quirky imagination, paired with a Southerner's charm and offbeat showmanship is the perfect combination for a fun time. This show is perfect for kids but will keep older audience members entertained as well with subtle adult jokes that are guaranteed to make you crack a grin.
Carrot Top at Luxor
When Carrot Top comes out on stage there's no telling what crazy contraption he's going to pull out next, but classifying him as just a prop comic is an understatement. His wacky inventions are merely an extension of his outrageous and hilariously funny personality. While some jokes are based on items that he pulls from his prop trunks, others rely on his talent as a stand-up comedian and comical commentary on everything from tourists to NASCAR, celebrities and current events. A comedy show that will leave your abs aching, this is definitely not a show for the whole family… unless you're the type of parent that Carrot Top pokes fun at.
Jeff Civillico: Comedy in Action at The LINQ Hotel & Casino
Jeff Civillico gives a high energy, hilarious show that is fun for the whole family. A champion juggler, professional balancer (like ladders on chin-type balancing) and unicycle rider, this multifaceted, all-ages entertainer is nothing short of well, entertaining. Pulling up audience members to help with a couple of acts as well as utilizing the help of one lucky youngster, Civillico's show is a perfect family outing. Between his razor-sharp wit and his superb eye-hand coordination, his zany humor is the exact recipe for laughter. Oh, and keep an eye out for his signature high kick and wedgie pull.
Laugh Factory at Tropicana
The world-famous Laugh Factory has a home in Vegas at the Tropicana. The comedy club features a rotating roster of established comedians and new rising stars of comedy. The original Laugh Factory opened in 1979 in Hollywood and soon became known for spawning some of the hottest stand-up comedians in the world. Countless comics have performed at the Laugh Factory since its inception including: Tim Allen, Louie Anderson, Roseanne Barr, Milton Berle, David Brenner, Nick Cannon, Drew Carey, George Carlin, Jim Carrey, Dave Chappelle, Dane Cook and many more.
Brad Garrett's Comedy Club at the MGM Grand
There is no shortage of funny in this dynamic stand-up comedy show. With a slew of notable performers rotating almost every night of the week, Brad Garrett's Comedy Club is always a good option. Oh, and did we mention that the club's namesake himself comes to perform on a regular basis as well? You read that correctly. Funnyman Brad Garrett isn't just the name of the joint, but a headlining act on various nights each month.

subset: commoncrawl


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You are here: Home → Forum Home → The Mac Observer Forums → Apple Finance Board → Thread
The value of iPod and iPhones in expanding the MAC base.
Mac Convert….if anyone has ever questions the value of iPod and iPhones to bring PC users into the fold, this should answer it
Subsequent articles will be interesting reading.[ Edited: 10 April 2009 01:22 PM by ldrhawke ]
Never Underestimate the Power of Stupid People in Large Groups or of Hedge Funds that Naked Short Sell aapl


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The Coupon Workshop - Networking Event! (June 30)
the budget author
Wednesday, June 30, 2010 from 6:00 PM to 8:00 PM (EDT)
Event Details
550 Trackside presents The Coupon Workshop summer series!
The Budget Author is thrilled to partner with some amazing, local sponsors to provide you with an outstanding event! A few of the perks? You'll get to tour this incredible facility, and taste amazing food from Doc Green's, Costco, and Truffleluscious. Have kids? No worries. Our friends at College Nannies & Tutors will take care of them for you, for free.
- What is The Budget Author?
The Budget Author is a financial coaching firm that specializes in writing custom budgets for families & individuals. The Budget Author teaches people how to eliminate debt and live on a spending plan that actually works. And guess what, it's way more fun than you think.
- What is The Coupon Workshop?
The Coupon Workshop is the opportunity for you to learn how to reduce your grocery budget by 50-70%, without spending more than 30 minutes per week. Yep, it's that easy. And no, it shouldn't become your part-time job. We're talking simple couponing. No clipping coupons every day, no stress, no chaos. The savings our clients experience on their first grocery trip after attending this workshop are on average 40-50%.
- Is this event just about coupons?
No! We are excited to incorporate business networking into our workshops because we know building relationships is key, and we love to help other businesses grow. Our sponsors (who we're kinda crazy about!) & many other business owners will be present at our summer series events to network with you. So bring your business cards, and be ready to meet a ton of amazing people. (Is networking not your thing? It's okay, you can come just for the food.)
- How do I get in on the free childcare?
Glad you asked! Since we all know it's tough to get a sitter on a week night, our friends at College Nannies & Tutors in Lawrenceville will be providing free childcare for you! All you have to do is select the number of free tickets you need for your children, so we can reserve your spot. College Nannies & Tutors is located right around the corner from our facility, and drop off is between 5:30-6:00 on the night of each event. Click here for directions. (You'll only be a couple blocks away.)
- Do I have to register or can I just show up?
Please (we are begging!) register online in advance so we can make sure we have a seat for you. (And you want to eat, right?) Tickets are $35, and when you purchase 2 tickets (bring a friend or spouse!), your price per ticket drops to $25.
- Problems with registration or additional questions?
Contact us here, or call 770.356.2441.
Can't wait to see you soon!
When & Where
550 Trackside
550 North Clayton Street
Wednesday, June 30, 2010 from 6:00 PM to 8:00 PM (EDT)
Add to my calendar


text: Jan. 24 (Bloomberg) -- New York Mayor Michael Bloomberg speaks at a news conference about the city's plans for events leading to the National Football League's Super Bowl championship game at MetLife Stadium in East Rutherford, New Jersey, on Feb. 2, 2014. NFL Commissioner Roger Goodell, New York Giants co-owners John Mara and Jonathan Tisch, New York Jets owner Woody Johnson, Al Kelly, chief executive officer of the New York/New Jersey Super Bowl Host Committee, Mary Pat Augenthaler, the NFL's vice president of events, and Manhattan Borough President Scott Stringer also speak. The mayor is founder and majority owner of Bloomberg News parent Bloomberg LP. (Source: Bloomberg)


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One of the most interesting footwear collaborations in the last few days, the colette x Timberland 6 Inch Boot enjoys a closer look here. The classic shape of the 6 inch remains un-changed with "colette blue" integrated into the upper via the laces and heel portions. A debossed colette logo can also be seen on the tongue. Releasing exclusively at colette, the boots will be limited to 40 pairs with a retail price of €235 EUR (approximately $334 USD).
Source: Shoes-Up


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Borda Count
Borda Count
• Used for the election of two ethnic-minority members of the National Assembly of Slovenia.
• The Parliament of Nauru.
• Presidential elections in Kiribati.
• Various private organisations and awards, such as the NBA's Most Valuable Player award and the Eurovision Song Contest.
How does Borda Count work?
The Borda Count is a form of preferential voting in single member constituencies where voters rank candidates in order of preference and the rankings are converted into points.
Candidates score one point for being ranked last, two for being next-to-last and so on. The candidate who receives the most points is declared the winner.
The Pros and cons of Borda Count
The case for
The arguments against
The Borda Count shares the advantages of other preferential voting systems such as the Alternative Vote and the Single Transferable Vote, in that all MPs would have the support of a majority of their voters.
It encourages a strategic approach by parties to nominations. An extra candidate increases the cardinal number of points in the system and alters the relativities between other candidates. A minority faction, by standing more candidates, can increase its chances of prevailing over a majority.
It tends to elect broadly acceptable candidates, rather than those supported by the majority.
It encourages tactical voting to an even greater extent than First Past the Post as in Borda Counts your expression of lower preferences can harm your first choice.
Voting Systems
|Proportional Representation
|Mixed Systems
|Majoritarian Systems
|More representative as seats are distributed according to vote share.
|Combines the features of majoritarian-style systems and Proportional Representation.
|Systems that are highly disproportional.
|Single Transferable Vote
|Additional Member System
|First Past the Post
|Party List PR
|Alternative Vote Plus
|Alternative Vote
|Borda Count
|Block Vote
|Limited Vote
|Supplementary Vote
|Two-Round System


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|Property type
|Minimum Stay
|4 nights
Sitting on 8 acres of water front property, Our Home on the Cove is a cottage with 5 bedrooms and room for a family of 4 or 15 to run and play. "The Pines" provides space for volleyball, tag, horseshoes and campfires (yum- s'mores), as well as lounge chairs to watch the water drift by. Swimming in our Cove is a delight and sunbathing on the dock is equally relaxing. The fenced in swing set is a great place to let the little ones play. Don't be surprised to catch a few fish, crabs or even a turtle right off of the pier. Paddling around in the canoe will provide a unique view of our home from the water, and wandering our nature trail will provide an insight into the local wildlife. When all this activity makes you hungry, the kitchen is stocked with enough cookware to feed upward of fifteen people; eat at the kitchen table, or on the screened in porch. After dark, star gazing from the dock is an ideal activity, or make a bonfire! Our Home is truly a place to make memories.
Following are comments provided by our guests in our Guestbook at our property.
Our entire family enjoyed our stay at your beautiful home. You were extremely helpful and accomodating. We loved the firepit, the dock, and the gas grill. The house is in a beautiful, secluded location and is very calm and peaceful. Thanks for a relaxing, fun week! The Johnson Family
Everyone had a great time!! Our crabbing experience was priceless, catching as well as eating. The house was big enough to accommodate our big family. We will certainly recommend.
We all enjoyed our stay at the shore house. The kids had lots to do and spent a lot of time swimming and jumping off the dock. The house was large enough for everyone and was kid friendly. Abby was very kind and easy to work with. She was prompt in returning our deposit as well.
We stayed for 1 week at this property for Christmas 2012. The property and waterfront was beautiful. We saw white tail deer while walking the trails in the woods. We saw a King Fisher just off shore. The kitchen is well equipped with ample equipment for cooking large meals for many guests. The owner was pleasant, accomodating, and very flexible. We will definitely return. Don't hesitate in renting this property. We really enjoyed it.
We had a spectacular vacation at the shore house! The property is beautiful. The house is so homey and was large enough to accommodate all of us. I look forward to our next trip to the shore house!!
Great place to experience "Bay Country" living. Wildlife abounds on this secluded creekside property sheltered by a small island. Just about everything you can imagine and more are provided. The owners are very freindly and helpful. My family looks forward to returning next year.
Our stay at 'The Shore' was fantastic!
We came for the 2004 USNA Commissioning Week and our experience couldn't have been better. As with any vacation rental, the experience begins with the rental agents/owners and ends with the property. The property owners were extremely attentive. Everything they did, from sending area information before our arrival to providing linen service at the midpoint of the stay, was designed to please! The property itself was in excellent condition and left us in need of nothing!
We highly recommend this property and hope to stay there again on a future visit to the Annapolis area.
Our Home is conveniently located 30 minutes away from the Naval Academy, making it a perfect accommodation for families attending Commissioning Week. Historic Ellicott City is a mere half hour away, where one can find plenty of antiquing opportunities, wonderful restaurants (our favorite is Cocoa Lane), and beautiful architecture. In Baltimore, enjoy all the Inner Harbor has to offer, including the Science Center, Baltimore Aquarium and plentiful shopping. Near by is the American Visionary Art Museum and Walters Art Gallery. Last but not least, don't pass up the opportunity to day trip to Washington D.C. to walk the streets of our nations capital, explore the Lincoln Memorial, the Washington Monument, The National Spy Museum and catch a glimpse of the White House.
|Rate Period
|Weekend Night
Year Round
Jul 5 2016 - Jan 1 2018
4 night minimum stay
Personal check. Rates subject to change.
Holiday Rates (Commissioning Week, Forth of July, Memorial Day, etc.) will vary. Please inquire.


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Philippa Ballantine
About the Author
Philippa Ballantine is a writer, podcaster, and librarian. Her podcast of Chasing the Bard won the Sir Julius Vogel Award in 2009, and two of her novels have been short-listed for it. She is currently living in her hometown of Wellington, New Zealand, where her two Siberian cats keep her in line.


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Its crazy how many guys are signing up to the forum before most of big game drawings, or after they have drawn their tag looking for spots. I don't even know why they waste their time signing up, most of the info they are looking for has already been said in other threads. Just an observation.
I am new also but I plan on stickin around giving my two cents every now and again.


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el matadoro supremo!
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el matadoro supremo!
ugh can someone make a shot
These slow starts are killing us.
Not true, they have been playing D all year.
I do not like this rejuvenated Knicks team..
WTG DC x2
Frank Vogel says "Killer instinct, start strong, build a lead and then step on their throats."
It wasn't out of hand then, though. At the moment, we should really come back though.
I am so tired of Roy's inconsistency.
Why DC is still in?
I could see this game getting chippy
Looks like crappy Hibbert showed up tonight. Wonderful.
oh for god sakes just put the whole bench in.
Hibbert sucks the big one right now.
Roy 1 for 8. Lol
I like this lineup.
i am so tired of collison and hibbert. collison can't even dribble anymore and hibbert misses more shots than any 7 footer i've ever seen
Eff this...
Glad the Pacers showed up.
Super Bowl XLI Champions
2000 Eastern Conference Champions
Dear Indiana Pacers,
You all suck.
pacers look like dog****
hibbert looks terrible.
Novak made his shot. That's not looking good
i better go to sleep. this game doesn't look like worth staying up.
Tyler is a great passer
And now we can't even make a trade for Kaman just in case t:


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Send your little one off to sleep in style with this cozy pajama suit from Carter's! Details include a tagless neckline interior, ribbed trim, and grippy feeties. The full zip has a snap at neck. Cotton jersey construction.
Customer with a shipping address in eligible NY Metro areascan choose
at checkout, to get their order delivered on the same business day.
Just place your order by 9AM and choose "Same-Day Delivery".Orders placed after 9AM or on a weekend/holiday will be delivered the next business day.


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Have drinks ready to go using this portable, compact, 2-shelf bar cart complete with a removable top tray.More Details >
Compact in size and always stylish, this black 2-shelf bar cart delivers drinks with modern flair. Boasting pine construction with a black finish and a zebrawood veneer lower shelf, this cart tucks into a corner when not in use. The top is a removable tray that's great for pouring drinks and serving them up to friends and family. Silver hardware and large, stable wheel finish the chic look.


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More Sherlock? Bring On The Cumberbatch!
The bummer with this amazing show is…that seasons are only three episodes long. Granted, each one of the shows is 90 minutes and they are packed with awesomeness. Even as I re-watch each episode to catch the clues that I might have missed. I still can't help to think that, "We need MORE of this show!" More Sherlock! More Cumberbatch.
The dynamic between Benedict Cumberbatch and Martin Freeman is amazing. These are two actors that really work well together. Also, I love that they use Benedict's real parents to play Sherlock's parents and Martins real-life wife to play his wife on the show. It's those "off screen" relationships that have developed more of a harmony on the set, which definitely carries into what happens on screen.
Will we be getting more? Here's what Martin Freeman has to say about it?
If we have to wait for season 4…We will be waiting until 2016.


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The hallmark of Parkinson's disease (PD) is the selective loss of dopamine neurons in the ventral midbrain. Although the cause of neurodegeneration in PD is unknown, a Mendelian inheritance pattern is observed in rare cases, indicating a genetic factor. Furthermore, pathological analyses of PD substantia nigra have correlated cellular oxidative stress and altered proteasomal function with PD. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive Parkinsonism, one of which is a large deletion that is likely to lead to loss of function. Here we show that embryonic stem cells deficient in DJ-1 display increased sensitivity to oxidative stress and proteasomal inhibition. The accumulation of reactive oxygen species in toxin-treated DJ-1-deficient cells initially appears normal, but these cells are unable to cope with the consequent damage that ultimately leads to apoptotic death. Furthermore, we find that dopamine neurons derived from in vitro–differentiated DJ-1-deficient embryonic stem cells display decreased survival and increased sensitivity to oxidative stress. These data are consistent with a protective role for DJ-1, and demonstrate the utility of genetically modified embryonic stem cell–derived neurons as cellular models of neuronal disorders.
Citation: Martinat C, Shendelman S, Jonason A, Leete T, Beal MF, Yang L, et al. (2004) Sensitivity to Oxidative Stress in DJ-1-Deficient Dopamine Neurons: An ES- Derived Cell Model of Primary Parkinsonism. PLoS Biol 2(11): e327. https://doi.org/10.1371/journal.pbio.0020327
Academic Editor: Huda Y. Zoghbi, Baylor College of Medicine
Received: April 28, 2004; Accepted: July 29, 2004; Published: October 5, 2004
Copyright: © 2004 Martinat et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Competing interests: The authors have declared that no conflicts of interest exist.
Abbreviations: 6-OHDA, 6-hydroxydopamine; DAT, dopamine transporter; DHR, dihydrorhodamine-123; DIV, d in vitro; DN, dopamine neuron; E[number], embryonic day [number]; ES, embryonic stem; FACS, fluorescence-activated cell sorter; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly(ADP-ribose)polymerase-1; PI, propidium iodide; PD, Parkinson's disease; RNAi, RNA interference; ROS, reactive oxygen species; SDIA, stromal cell–derived inducing activity; SEM, standard error of the mean; shRNA, short hairpin RNA; TH, tyrosine hydroxylase; WT, wild-type
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by rigidity, slowed movement, gait difficulty, and tremor at rest (Dauer and Przedborski 2003). The pathological hallmark of PD is the relatively selective loss of dopamine neurons (DNs) in the substantia nigra pars compacta in the ventral midbrain. Although the cause of neurodegeneration in PD is unknown, a Mendelian inheritance pattern is observed in approximately 5% of patients, suggesting a genetic factor. Extremely rare cases of PD have been associated with the toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, which is taken up specifically by DNs through the dopamine transporter and is thought to induce cellular oxidative stress. Population-based epidemiological studies have further supported roles for genetic and environmental mechanisms in the etiology of PD (Dauer and Przedborski 2003; Jenner 2003).
The identification of several genes that underlie familial forms of PD has allowed for the molecular dissection of mechanisms of DN survival. Autosomal dominant mutations in α-synuclein lead to a rare familial form of PD (Polymeropoulos et al. 1997), and there is evidence that these mutations generate toxic, abnormal protein aggregates (Goldberg and Lansbury 2000) and cause proteasomal dysfunction (Rideout et al. 2001). A majority of patients with sporadic PD harbor prominent intracytoplasmic inclusions, termed Lewy bodies, enriched for α-synuclein (Spillantini et al. 1998), as well as neurofilament protein (Trojanowski and Lee 1998). Mutations in a second gene, Parkin, lead to autosomal recessive PD (Hattori et al. 2000). Parkin is a ubiquitin ligase that appears to participate in the proteasome-mediated degradation of several substrates (Staropoli et al. 2003).
Homozygous mutations in a third gene, DJ-1, were recently associated with autosomal recessive primary parkinsonism (Bonifati et al. 2003). DJ-1 encodes a ThiJ domain protein of 189 amino acids that is broadly expressed in mammalian tissues (Nagakubo et al. 1997). Interestingly, DJ-1 was independently identified in a screen for human endothelial cell proteins that are modified with respect to isoelectric point in response to sublethal doses of paraquat (Mitsumoto and Nakagawa 2001; Mitsumoto et al. 2001), a toxin that generates reactive oxygen species (ROS) within cells and has been associated with DN toxicity (McCormack et al. 2002). Gene expression of a yeast homolog of DJ-1, YDR533C, is upregulated in response to sorbic acid (de Nobel et al. 2001), an inducer of cellular oxidative stress. These results suggest a causal role for DJ-1 in the cellular oxidative stress response.
Surprisingly, animal models that harbor genetic lesions that mimic inherited forms of human PD, such as homozygous deletions in Parkin (Goldberg et al. 2003; Itier et al. 2003) or overexpression of α-synuclein (Masliah et al. 2000; Giasson et al. 2002; Lee et al. 2002), have failed to recapitulate the loss of dopamine cells. An alternative approach, the genetic modification of midbrain DNs in vitro (Staropoli et al. 2003), is potentially useful but limited by the difficulty and variability in culturing primary postmitotic midbrain neurons. Other studies have focused on immortalized tumor cell lines, such as neuroblastoma cells, but these may not accurately model the survival of postmitotic midbrain neurons.
Here we show that DJ-1-deficient cells display increased sensitivity to oxidative stress. DNs appear to be particularly sensitive to the loss of DJ-1. The initial accumulation of ROS is normal in DJ-1-deficient cells, but subsequent cellular defenses to ROS are impaired, leading to increased apoptosis.
Generation of DJ-1-Deficient ES Cells
To investigate the normal cellular function of DJ-1 and the pathogenic mechanism of the PD mutations, we generated cells deficient in DJ-1. A murine embryonic stem (ES) cell clone, F063A04, that harbors a retroviral integration at the DJ-1 locus was obtained through the German Gene Trap Consortium (http://tikus.gsf.de) (Figure 1A; Floss and Wurst 2002). This integration is predicted to disrupt the normal splicing of DJ-1, leading to the generation of a truncated protein that lacks the carboxy-terminal domain required for dimerization and stability (unpublished data). Of note, a mutation that encodes a similarly truncated protein (at the human DJ-1 exon 7 splice acceptor) has been described in a patient with early-onset PD (Hague et al. 2003).
(A) Schematic map of the murine DJ-1 gene in clone F063A04. The retroviral insertion places the engrailed-2 (En2) intron, the splice acceptor (SA), and the β-galactosidase/neomycin resistance gene fusion (β-geo) between exons 6 and 7.
(B) Southern blot analysis of KpnI-digested genomic DNA from DJ-1 homozygous mutant (–/–), WT (+/+), and heterozygous (+/–)cells, probed with murine DJ-1 cDNA. WT DNA shows a predicted 14-kb band (WT), whereas the mutant allele migrates as a 9-kb band (insertion).
(C) Western blot (WB) of ES cell lysates from WT (+/+), DJ-1 heterozygous (+/–), and mutant homozygous (–/–) clones with antibodies to murine DJ-1 (α-DJ-1) or β-actin (α-β-actin). DJ-1 migrates at 20 kDa, β-actin at 45 kDa.
(D) ES cells were exposed to 0, 5, 10, and 20 μM H2O2 for 15 h and viability was assayed by MTT. Responses of DJ-1 heterozygous cells (diamonds) and DJ-1 knockout clones 9 (open circles), 16 (solid circles), 23 (squares), and 32 (triangles) are shown. ** p ≤ 0.01; *** p ≤ 0.0001.
(E and F) Cell death of DJ-1 heterozygous and DJ-1-deficient cells (clone 32) after exposure to H2O2 (10 μM) was quantified by staining with PI and an antibody to AV with subsequent FACS analysis. AV staining marks cells undergoing apoptosis, whereas PI staining indicates dead cells. * p ≤ 0.05.
(G) DJ-1 heterozygous (+/–) and knockout (clone 32; –/–) cells were assayed at 1, 6, and 24 h after treatment with 10 μM H2O2 by Western blotting for cleaved PARP (89 kDa), which indicates apoptosis. No band is seen for cleaved PARP or β-actin for the DJ-1-deficient cells at 24 h due to cell death. Data represent means ± standard error of the mean (SEM) and were analyzed by ANOVA with Fisher's post-hoc test.
To generate ES cell subclones homozygous for the trapped DJ-1 allele, clone F063A04 was exposed to a high dose of the antibiotic G418, which selects cells that are homozygous for the neomycin resistance gene insertion (Mizushima et al. 2001). Several homozygous mutant ES cell subclones (that had undergone gene conversion at the DJ-1 locus) were identified by Southern blotting (Figure 1B). To confirm that the trapped allele leads to the loss of wild-type (WT) DJ-1 protein, cell lysates from homozygous DJ-1-deficient (also termed "knockout" here) ES cell clones as well as the parental heterozygous clone were analyzed by Western blotting using polyclonal antibodies to the amino-terminal region of DJ-1 (amino acids 64–82) or full-length DJ-1 protein (unpublished data). Neither full-length nor truncated DJ-1 protein products were detected in knockout clones (Figure 1C), consistent with instability of the predicted truncated DJ-1 product. In addition, no full-length DJ-1 RNA was detected in cultures of knockout cells (Figure S1). In contrast, heterozygous and WT ES cells express high levels of DJ-1. Initial phenotypic analysis of knockout subclones indicated that DJ-1 is not essential to the growth rate of ES cells in culture, consistent with the viability of humans homozygous for DJ-1 mutations.
DJ-1 Protects Cells from Oxidative Stress and Proteasomal Inhibition
DJ-1 has been hypothesized to function in the cellular response to oxidative stress. To investigate the role of DJ-1 in the oxidative stress response in vivo, DJ-1-deficient knockout and heterozygous ES cell clones were analyzed for cell viability in the context of increasing concentrations of H2O2. Heterozygous cells were used as controls because the knockout subclones were derived from these. Cell viability was initially determined by MTT assay (which detects reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide [MTT] by metabolic enzymes) in triplicate (Fezoui et al. 2000). Exposure to H2O2 led to significantly greater toxicity in the DJ-1-deficient cells; similar results were obtained with multiple knockout subclones in independent experiments (Figures 1D and 2A). In contrast, in the absence of toxin, heterozygous and knockout cells displayed comparable viability in the MTT assay (Figure S2). Consistent with the MTT assay, fluorescence-activated cell sorting (FACS) analysis of cells stained with annexin V (AV) and propidium iodide (PI) revealed increased death of knockout cells compared to heterozygous cells in the context of H2O2 exposure (Figure 1E). The increase in AV-positive cells implicated an apoptotic mechanism of cell death (Figure 1F). Furthermore, when exposed to H2O2, knockout cells displayed potentiated cleavage of poly(ADP-ribose)polymerase-1 (PARP) in a pattern indicative of an apoptotic death program (Gobeil et al. 2001) (Figure 1G).
(A–C) Cell viability of DJ-1 heterozygous cells (solid bar) and DJ-1-deficient knockout clone 32 cells (open bar) after 15 h exposure to H2O2 (A), lactacystin (B), or tunicamycin (C) as assayed by MTT reduction. * p ≤ 0.05.
(D) DJ-1-deficient knockout cells (clone 32) were transiently transfected with plasmids containing WT human DJ-1 vector (solid bar) and PD-associated L166P mutant DJ-1 vector (gray bar); as a control, knockout cells were also transfected with vector alone (open bar). 48 h after transfection, cells were exposed to 10 μM H2O2 for 15 h and then assayed by MTT reduction. WT human DJ-1 significantly enhanced survival of the knockout cells, whereas the L166P mutant did not. Similar results were obtained at 20 μM H2O2 and with a second DJ-1-deficient clone (unpublished data). Transfection efficiency exceeded 90% in all cases and protein expression level was comparable for human WT and L166P mutant DJ-1 as determined by Western blotting (Figure S1). * p ≤ 0.05.
(E) DJ-1-deficient cells (clone 32; open bar) and control heterozygous cells (solid bar) were assayed for intracellular formation of ROS in response to H2O2 treatment (15 min, 1 or 10 μM) using DHR and FACS analysis.
(F) Protein carbonyl levels were measured by spectrophotometric analysis of DNP-conjugated lysates from DJ-1-deficient (clone 32; solid red line) and control heterozygous cells (dashed blue line). Data are shown as the mean ± SEM and were analyzed by ANOVA with Fisher's post-hoc test.
Additional toxin exposure studies demonstrated that DJ-1-deficient cells were sensitized to the proteasomal inhibitor lactacystin (Figure 2B), as well as to copper (Figure S2), which catalyzes the production of ROS. We did not observe altered sensitivity to several other toxins, including tunicamycin (an inducer of the unfolded protein response in the endoplasmic reticulum; Figure 2C), staurosporine (a general kinase inhibitor that induces apoptosis) (Figure S2), or cycloheximide (an inhibitor of protein translation) (unpublished data).
WT but Not PD-Associated L166P Mutant DJ-1 Protects Cells from Oxidative Stress
To confirm that altered sensitivity to oxidative stress is a consequence of the loss of DJ-1, we performed rescue experiments by overexpressing WT or mutant human DJ-1 in knockout ES cells. Plasmids encoding human Flag-tagged WT DJ-1, Flag-tagged PD-associated L166P mutant DJ-1, or vector alone, were transiently transfected into DJ-1-deficient clones, and these were subsequently assayed for sensitivity to H2O2 using the MTT viability assay. DJ-1-deficient cells transfected with a vector encoding Flag-WT human DJ-1 were effectively rescued in terms of viability in the presence of H2O2 (Figure 2D); Thus, viability in rescued knockout cells mimicked the viability of untransfected heterozygous cells in the context of H2O2 treatment (Figure 2A and 2D). In contrast, transfection of knockout cells with a vector encoding the PD-associated L166P mutant DJ-1 did not significantly increase the viability of H2O2-treated knockout cells (Figure 2D). Baseline cell viability in the absence of toxin exposure was not altered by DJ-1 overexpression, and Western blotting of lysates from transfected cells with an antibody specific to human DJ-1 demonstrated that transfected Flag-WT DJ-1 and Flag-L166P mutant DJ-1 accumulated comparably (Figure S2).
DJ-1 Deficiency Does Not Alter the H2O2-Induced Intracellular ROS Burst
We hypothesized that DJ-1 either alters the initial accumulation of intracellular ROS in response to H2O2 exposure, or that it functions downstream of the ROS burst and protects cells from consequent damage. Therefore, we quantified the accumulation of ROS in response to H2O2 treatment in knockout and heterozygous cells using the ROS-sensitive fluorescent indicator dye dihydrorhodamine-123 (DHR) and FACS analysis. Initial ROS accumulation (at 15 min after stimulation) appeared unaltered in the DJ-1-deficient cells in comparison to control heterozygous cells (Figure 2E). Consistent with this, accumulation of protein carbonyls, an index of oxidative damage to proteins (Sherer et al. 2002), appeared normal initially (at 1 h after toxin exposure; Figure 2F). However, at 6 h after toxin exposure, a point at which knockout cells already display increased apoptosis (as indicated by PARP cleavage; see Figure 1G), protein carbonyl accumulation was robustly increased in the DJ-1-deficient cells. These data suggest that initial ROS accumulation is not altered by DJ-1 deficiency, but that the mutant cells are unable to appropriately cope with the consequent damage. Consistent with this result, no antioxidant or peroxiredoxin activity with purified DJ-1 protein in vitro was detected (S.S. and A.A., personal communication).
DJ-1 Is Required for Survival of ES Cell–Derived DNs
Several methods have been established for the differentiation of ES cells into DNs in vitro (Morizane et al. 2002). To extend our analysis of DJ-1 function to DNs, we differentiated DJ-1-deficient ES cells or control heterozygous cells into DNs in vitro by coculture with stromal cell–derived inducing activity (SDIA; Figure 3A) (Morizane et al. 2002; Barberi et al. 2003). DNs were quantified by immunohistochemistry for tyrosine hydroxylase (TH; a marker for DNs and other catecholaminergic cells), or by analysis of dopamine transporter uptake activity (a quantitative DN marker) (Han et al. 2003). Production of DNs appeared to be significantly reduced in knockout ES cell cultures compared to parental heterozygous cultures at 18 days in vitro (DIV) as determined by both dopamine uptake and TH immunoreactivity (Figures 3B and 3C; 4A–4L). In contrast, general neuronal production did not appear altered in this assay in terms of the postmitotic neuronal marker TuJ1 (a monoclonal antibody specific to neuronal, not glial, class III β-tubulin) (Figures 3E and 4A–4L′); other neuronal subtypes also appeared normal, including GABAergic (Figures 3D and 4A′–4L′) and motor neurons (HB9-positive; Figure S3). To investigate whether the reduction in DNs in DJ-1-deficient cultures is due to defective generation or survival, a time course analysis was performed. At early time points (8 and 12 DIV), dopamine uptake activity was comparable in WT and DJ-1-deficient cultures, whereas subsequently the DJ-1-deficient cultures appeared defective (Figure 3F). Consistent with this, intracellular dopamine accumulation (as quantified using high-performance liquid chromatography) was significantly reduced in DJ-1-deficient cultures (6.4 ± 1.5 ng dopamine/mg protein) relative to control heterozygous cultures (66.0 ± 17.4 ng/mg) at 35 DIV. These data strongly suggest that DJ-1 deficiency leads to loss of DNs, rather than simply to downregulation of cell marker expression.
(A) The SDIA coculture method. DJ-1 knockout or control heterozygous ES cells are cocultured with mouse stromal cells (MS5) in the absence of serum and leukemia inhibitory factor for 18 DIV.
(B) DN production was quantified at 18 DIV by 3H-dopamine uptake assay. DJ-1-deficient ES cell cultures were defective relative to heterozygous control cultures.
(C–D) Neuron production was quantified by immunohistochemical analysis as a percent of TuJ1-positive colonies that express TH (C) or GABA (D). Quantification of TH and GABA immunostaining was performed on all colonies in each of three independent wells. Colonies were scored as positive if any immunostained cells were present. * p ≤ 0.05.
(E) The absolute number of TuJ1-positive colonies was not significantly different between the two genotypes.
(F) Kinetic analysis of DN differentiation in DJ-1-deficient cultures (clone 32, solid square) and heterozygous controls (open circle) as quantified by 3H-dopamine uptake assay. * p ≤ 0.05.
(G) DJ-1-deficient (open bar) and heterozygous control (closed bar) cultures differentiated for 9 DIV and then exposed to 6-OHDA at the indicated dose for 72 h. DNs were quantified by 3H-dopamine uptake assay. Data represent the means ± SEM and were analyzed by ANOVA followed by Fisher's post-hoc test. * p ≤ 0.05.
(A–L) DJ-1 heterozygous (+/–; A–F) and knockout (–/– [clone 32]; G–L) cultures were differentiated by SDIA for 18 DIV and immunostained with antibodies to TH (green) and TuJ1 (red). Images of both (Merge) are also shown.
(A′–L′) Immunostaining of DJ-1 heterozygous (+/–, A′–F′) and deficient (–/–, G′–L′) cultures with antibodies for GABA (green) and TuJ1 (red). Scale bar, 50 μm. Images of both (Merge) are also shown.
We hypothesized that DJ-1-deficient DNs may be sensitized to oxidative stress, akin to DJ-1-deficient undifferentiated ES cells. To test this, DN cultures from DJ-1-deficient or heterozygous control ES cell cultures at 9 DIV were exposed to oxidative stress in the form of 6-hydroxydopamine (6-OHDA), a DN-specific toxin that enters DNs through the dopamine transporter and leads to oxidative stress and apoptotic death (Dauer and Przedborski 2003). DJ-1-deficient DNs displayed an increased sensitivity to oxidative stress in this assay (Figure 3G). Post-hoc analysis of the data indicates that the difference among genotypes is maximal at an intermediate dose of toxin (50 μM); at the highest dose of 6-OHDA employed (100 μM), the difference is lessened (because the heterozygote is increasingly affected as well), indicating that DJ-1-mediated protection is limited. Although we cannot exclude a role for DJ-1 in the late-stage differentiation of DNs, these data suggest that DJ-1 deficiency leads to reduced DN survival and predisposes these cells to endogenous and exogenous toxic insults.
RNAi "Knockdown" of DJ-1 in Midbrain Embryonic DNs Leads to Increased Sensitivity to Oxidative Stress
To confirm the role of DJ-1 in primary midbrain DNs, DJ-1 expression was inhibited by RNA interference (RNAi) in embryonic day 13.5 (E13.5) murine primary midbrain cultures by lentiviral transduction of short hairpin RNAs (shRNAs) (Figure 5) (Rubinson et al. 2003). E13.5 midbrain cultures (Staropoli et al. 2003) were transduced with a lentiviral vector that includes a gene encoding the green fluorescent protein marker eGFP, along with shRNAs homologous to murine DJ-1. DJ1-shRNA virus-infected cells displayed efficient silencing of DJ-1 gene expression to 10%–20% of control vector-infected cultures (as determined by Western blotting [Figure 5Q]). Transduction efficiency, as assessed by visualization of the fluorescent eGFP marker, exceeded 95% in all cases (Figure 5I and unpublished data). After 7 DIV, cultures were exposed to H2O2 for 24 h and then evaluated for DN survival as quantified by immunostaining for TH and dopamine transporter (DAT).
(A–P) Primary midbrain cultures from E13.5 embryos were infected with lentiviral vectors encoding DJ-1 shRNA (or vector alone) under the regulation of the control vector (A–H) or the U6 promoter (I–P). Cells were cultured for 1 wk after infection and then exposed to H2O2 (5 μM; E–H and M–P) for 24 h. Cultures were immunostained for TH (B, F, J, and N) or DAT (C, G, K, or O) and visualized by confocal microscopy. Images containing all stains are included (Merge; D, H, L, and P). Scale bar, 100 μm.
(Q) Cell lysates prepared from midbrain primary cultures infected with DJ-1 shRNA lentivirus (or control vector) were analyzed by Western blotting for murine DJ-1 or β-actin.
(R–T) Quantification of TH, DAT, and GFP signal was performed on ten randomly selected fields in each of three wells for each condition. Red triangles, DJ-1 shRNA treated; black circles, control vector. Data represent the means ± SEM and were analyzed by ANOVA followed by Fisher's post-hoc test. * p ≤ 0.05.
Midbrain cultures transduced with DJ-1 shRNA virus and with control vector displayed similar numbers of TH-positive neurons in the absence of exposure to H2O2 (Figure 5A–D, 5I–L, and 5R–S). In contrast, in the presence of H2O2, DJ-1-deficient cultures displayed significantly reduced DN survival as quantified by immunohistochemistry for TH or DAT (Figure 5E–H, 5M–P, 5R–S). These studies were repeated three times with similar results. The reduction in DAT immunoreactivity appears to be more robust than the reduction in TH-positive cell number in the context of H2O2; this may reflect the differential localization of DAT to DN processes, whereas TH is primarily in the cell body. As we described in a previous manuscript, nondopaminergic cells in the E13.5 primary midbrain cultures are predominantly GABAergic neurons (90%–95%) (Staropoli et al. 2003). Total embryonic midbrain neurons transduced with either DJ-1 shRNA or vector displayed comparable survival in the context of toxin exposure, suggesting that DJ-1 deficiency leads to a relatively specific alteration in DN survival (Figure 5T). These data are consistent with the analyses of ES cell–derived DNs above and indicate that DJ-1 is required for the normal survival of midbrain DNs in the context of toxin exposure.
In this study we present evidence that DJ-1 is an essential component of the oxidative stress response of DNs. DJ-1-deficient cells display an apparently normal initial burst of ROS in response to H2O2, but they are unable to cope with the consequent toxicity, culminating in apoptosis. Additionally, we find that DJ-1 deficiency sensitizes cells to the proteasomal inhibitor lactacystin but not other toxic stimuli such as tunicamycin. Proteasomal inhibition induces the accumulation of short-lived and misfolded cytoplasmic proteins, leading to oxidative stress and apoptosis (Demasi and Davies 2003). ROS and proteasomal inhibition have previously been correlated with PD pathology (Dauer and Przedborski 2003), and it is therefore tempting to hypothesize that DJ-1 mutations lead to PD because of an increased sensitivity to such stressors.
The apparent cell-type specificity of DN impairment in patients with the Parkinsonism-associated DJ-1 mutation is not predicted by the ubiquitous expression of DJ-1 (Nagakubo et al. 1997). In this study, we find that DJ-1 protects both dopaminergic and nondopaminergic cells from oxidative insult. However, DJ-1-deficient DNs appear to be especially sensitive to oxidative insult, suggesting relative cell-type specificity to the consequences of DJ-1 deficiency. Similar results are observed in DJ-1-knockout ES cell–derived DNs (which are devoid of any detectable DJ-1) and in primary DNs with DJ-1 levels reduced by RNAi "knockdown." However, we find that even in the absence of exogenous toxin exposure, the knockout ES cell–derived DNs display reduced survival, whereas survival of the primary embryonic midbrain RNAi knockdown DNs appears to be similar to WT cells. We hypothesize that this discrepancy reflects the activity of residual DJ-1 (approximately 10%–20%) in the RNAi knockdown cultures. Alternatively, the knockout ES cell–derived DNs may be exposed to a greater degree of oxidative stress in vitro than are the knockdown-derived DNs even in the absence of added toxin. The mechanism by which DNs are preferentially targeted for destruction in the absence of DJ-1 is unclear. It has been proposed that DNs are subject to high levels of endogenous oxidative stress that may relate to dopamine metabolism (Jenner and Olanow 1998).
DJ-1 is structurally modified in the context of cellular oxidative stress (Mitsumoto and Nakagawa 2001), suggesting a possible function. Two recent studies (Yokota et al. 2003; Taira et al. 2004) investigated the role of DJ-1 in the oxidative stress response of neuroblastoma tumor cells. Both studies used RNAi to perturb the expression of DJ-1 in neuroblastoma tumor cell lines, and suggested that DJ-1 deficiency sensitizes cells to oxidative stress; these results are consistent with our data. Taira et al. (2004) further reported that overexpression of DJ-1 in neuroblastoma cells leads to a reduction in ROS accumulation and hypothesized that DJ-1 may harbor antioxidant activity in vivo. In contrast, we find that ES cells that are deficient in DJ-1 display a normal initial burst of ROS in the context of H2O2. Consistent with this, we fail to detect DJ-1 antioxidant activity in vitro (Shendelman et al. 2004).
Finally, this study presents a novel, ES cell-based genetic approach to the study of neurodegenerative disorders. Mouse genetic models of disease are often limited by the inherent variability of animal experiments, the limited mouse life span, and the difficulties in manipulating whole animals. For instance, genetic rescue experiments and toxicological dose-response studies are impractical in whole animals. Furthermore, genetic cell models are more readily amenable to molecular dissection of disease mechanisms than are whole animals. Thus, genetically altered, ES cell–derived neurons are likely to be generally useful as cellular models of neurodegenerative disorders. Future studies may also utilize available human ES cells to investigate species differences.
Materials and Methods
Cell culture.
Undifferentiated ES cells were cultured using standard techniques (Abeliovich et al. 2000). SDIA differentiation of ES cell cultures to DNs was performed as described in Kawasaki et al. (2000), except that ES cells were plated at a density of 500 cells/cm2 rather than approximatively 1,000 cells/cm2, and were cocultured with the MS5 mouse stromal cell line (Barberi et al. 2003). For rescue experiments, cells were plated at a cell density of 1.4 × 106 cells/well. Transfections with plasmids encoding human Flag-WT DJ-1, PD-associated L166P mutant DJ-1, or vector alone, were performed using Lipofectamine 2000 (Life Technologies) for 18–36 h according to the manufacturer's instructions (Staropoli et al. 2003). 24 hours post-transfection, cells were split into 96-well plates and treated as described below. Primary cultures and lentiviral transductions were performed as described in Staropoli et al. (2003).
Generation of knockout ES cell clones.
The pT1ATGβgeo gene trap vector, which includes βgeo, a fusion of the genes for β-galactosidase and neomycin resistance, is present between exons 6 and 7 of the murine DJ-1 gene, as determined by cDNA sequencing of trapped transcripts and genomic analysis (Figure 1A). To select for ES cell subclones homozygous for the trapped DJ-1 allele, we treated clone F063A04 with 4 mg/ml G418. Several subclones that were homozygous for the mutant DJ-1 allele were identified by Southern blotting (see Figure 1B), and three were chosen for further experimentation: clones 9, 23, and 32. To confirm that the trapped allele leads to the loss of wild-type (WT) DJ-1 protein, cell lysates from these clones, as well as from the parental heterozygous clone, were analyzed by Western blotting using polyclonal antibodies to the amino-terminal region of DJ-1 or the full-length DJ-1 protein. For Western blotting, cells were resuspended in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.2% Triton X-100, and incubated at 4 °C, rotating for 20 min. Cleared lysate was prepared by centrifuging the lysate at 13,000 rpm for 10 min at 4 °C.
A rabbit polyclonal antibody to DJ-1 was generated against the synthetic polypeptide QNLSESPMVKEILKEQESR, which corresponds to amino acids 64–82 of the mouse DJ-1 protein. Antiserum was produced by the Polyquick polyclonal antibody production service of Zymed Laboratories (South San Francisco, California, United States). The antiserum was affinity-purified on a peptide-coupled Sulfolink column (Pierce Biotechnology, Rockford, Illinois, United States) according to the manufacturer's instructions. Antibody was used at a dilution of 1:200 for immunohistochemistry and Western blotting as described (Staropoli et al. 2003). Immunohistochemistry was performed with a rabbit polyclonal antibody to TH (PelFreez, Rogers, Arizona, United States; dilution 1:1000), the mouse monoclonal antibody to neuronal class III β-tubulin TuJ1 (Covance, Princeton, New Jersey, United States; dilution 1:500), and a rabbit polyclonal antibody to GABA (Sigma, St. Louis, Missouri, United States; dilution 1:1000). Western blotting was performed using a polyclonal antibody to cleaved PARP (Cell Signaling Technology, Beverly, Massachusetts, United States; dilution 1:500), a monoclonal antibody to DJ-1 (Stressgen Biotechnologies, San Diego, California, United States; dilution 1:1000), and a mouse monoclonal antibody to β-actin (Sigma, 1:500).
In vivo assays.
ES cells plated in 96-well format (2.3 × 104 cells/well) were treated for 15 h with H2O2 in ES cell medium deficient in β-mercaptoethanol (Abeliovich et al. 2000). Cell viability (as a percent of untreated control) was determined by MTT assay in triplicate (Fezoui et al. 2000). AV/PI (Molecular Probes, Eugene, Oregon, United States) staining was performed according to the manufacturer's instructions. For DHR staining (Molecular Probes) (Walrand et al. 2003), cells were preincubated for 30 min with DHR (5 μM), washed with PBS, then treated with H2O2 in ES cell medium deficient in β-mercaptoethanol for 15 min at 37 °C. The FACS analysis was performed using a FACSTAR sorter (Becton-Dickinson, Palo Alto, California, United States). Dopamine uptake assays were performed as described (Farrer et al. 1998). Reported values represent specific uptake from which nonspecific uptake, determined in the presence of mazindol, was subtracted. Uptake values were normalized for protein content with the BCA kit (Pierce).
For 6- hydroxydopamine (6-OHDA, Sigma) treatment, the drug was diluted in the differentiation medium (Kawasaki et al. 2000) and medium was changed every day for 72 h.
Primary midbrain embryonic cultures were prepared and transduced with lentiviral vectors as described in Staropoli et al. (2003). The DJ-1 shRNA vector was generated by insertion of annealed oligonucleotides 5′-TGTCACTGTTGCAGGCTTGGTTCAAGAGACCAAGCCTGCAACAGTGACTTTTTTC-3′ and 5′-ACAGTGACAACGTCCGAACCAAGTTCTCTGGTTCGGACGTTGTCACTGAAAAAAGAGCT-3′ into the LentiLox vector (Rubinson et al. 2003). For cellular dopamine quantification, cultures were incubated in standard differentiation medium supplemented with L-DOPA (50 μM) for 1 h to amplify dopamine production, as described in Pothos et al. (1996). Subsequently, cells were washed in PBS and then lysed in 0.2 M perchloric acid. Dopamine levels were quantified by HPLC (Yang et al. 1998) and normalized for protein content as above.
Expression vectors.
The cDNA for human DJ-1 was PCR-amplified from a human liver cDNA library (Clontech, Palo Alto, California, United States). For expression of DJ-1 in ES cell rescue experiments, DJ-1 was cloned into the expression vector pcDNA3.1 (Invitrogen, Carlsbad, California, United States) containing a Flag peptide sequence at the N-terminus using standard cloning techniques. Flag-L166P DJ-1 (pcDNA3) was generated by PCR-mediated mutagenesis.
Protein carbonyl analysis.
For protein carbonyl quantitation (Bian et al. 2003), cells were plated (1.4 × 105 cells per well), grown for 24 h, and then treated with 10 μM H2O2 as indicated. Cells were lysed in 200 μl lysis buffer and cleared lysate was prepared as described above. An aliquot of 40 μl from each time point was added to 2 M HCl (120 μl) with or without 10 μM 2,4-dinitrophenyl-hydrazine and incubated for 1 h at 24 °C with shaking. Proteins were then TCA-precipitated and resuspended in 200 μl of 6 M guanidinium chloride. Absorbance was measured at 360 nm, and DNP-conjugated samples were normalized for protein concentration with the underivitized control samples.
Supporting Information
Figure S1. Quantitative Real-Time PCR for DJ-1 Gene Expression
(A) Real-time PCR analyses of DJ-1 cDNA in WT (+/+), heterozygous (+/–), and knockout (–/–) cultures. Each expression value was normalized to that of β-actin and expressed relative to the respective value of the WT (+/+) control. These gene expression patterns were replicated in at least three independent PCR experiments. Total RNA from ES cells differentiated with the SDIA method for 18 days was isolated using the Absolutely RNA Miniprep kit (Stratagene, La Jolla, California, United States). Synthesis of cDNA was performed using the SuperScript first strand synthesis system for RT-PCR (Invitrogen). Real-time PCR reactions were optimized to determine the linear amplification range. Quantitative real-time RT-PCRs were performed (Stratagene MX3000P) using the QuantiTect SYBR Green PCR Master Mix (Qiagen, Valencia, California, United States) according to the manufacturer's instructions. DJ-1 primer sequences were 5′-CGAAGAAATTCGATGGCTTCCAAAAGAGCTCTGGT-3′ and 5′-CAGACTCGAGCTGCTTCACATACTACTGCTGAGGT-3′; primers used for β-actin were 5′-TTTTGGATGCAAGGTCACAA-3′ and 5′-CTCCACAATGGCTAGTGCAA-3′. For quantitative analyses, PCR product levels were measured in real time during the annealing step, and values were normalized to those of β-actin.
(B) Ethidium bromide staining of the PCR products obtained after 29 cycles for DJ-1 (625 bp) and β-actin (350 bp).
Figure S2. Analysis of DJ-1-Deficient ES Cells
(A and B) Cell viability of DJ-1 heterozygous cells (solid bar) and DJ-1-deficient knockout clone 32 (open bar) after exposure to CuCl2 or staurosporine at the doses indicated.
(C) MTT values of untreated DJ-1-deficient ES cell clones and the control heterozygous cells. Assays were performed exactly as in Figure 2, but in the absence of toxin.
(D) MTT values of untreated DJ-1-deficient ES cells transfected with vector alone or various DJ-1-encoding plasmids. Transfection and expression of WT DJ-1 or mutant forms of DJ-1 does not alter the basal metabolic activity or viability of the cells.
(E) Western blotting of extracts from ES cells transfected with vectors harboring WT human DJ-1 or the L166P mutant.
Figure S3. Immunocytochemistry for HB9 and GABA Neurons in DJ-1-Deficient and Control Heterozygous ES Cells
Both cell cultures were differentiated by SDIA for 18 DIV. Cells were fixed with 4% paraformaldehyde and stained with mouse monoclonal antibodies against HB9 (gift from T. Jessell, dilution 1:50) and rabbit polyclonal antibodies against GABA (Sigma, dilution 1:1000) as in Figure 5. Scale bar, 50 μM.
Accession Numbers
The GenBank (http://www.ncbi.nlm.nih.gov/) accession numbers of the genes discussed in this paper are α-synuclein (NM_000345), Parkin (AB009973), and DJ-1 (AB073864).
We thank O. Hobert and L. Clark for critical reading of the manuscript. SS is funded by the Integrated Graduate Program Training Grant (National Institutes of Health); CM is funded by the American Parkinson's Disease Association; AA receives funding from the Spitzer, Rockefeller Brothers, Taub and M. J. Fox Foundations, National Institute of Neurological Disorder and Stroke (NINDS) and National Institute of Aging (NIA), and is a Culpeper and Beeson scholar.
Author Contributions
CM, SS, and AA conceived and designed the experiments. CM, SS, AJ, TL, and AA performed the experiments. CM, SS, AJ, and AA analyzed the data. CM, SS, AJ, MFB, LY, TF, and AA contributed reagents/materials/analysis tools. CM, SS, and AA wrote the paper.
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SENATOR CARAWAY DIES IN HOSPITAL; Junior Senator From Arkansas Began Work as Farmhand at 7 After Father Was Slain. CAPITOL CAREER ACTIVE Supporter of Wilson and Ardent Democrat, He Led in Fight on Harding Administration. SENATOR CARAWAY DIES IN HOSPITAL Hard Fights in Senate. Was Foe of Daugherty.
LITTLE ROCK, Ark., Nov. 6.-- Fighting Thaddeus H. Caraway, Senator and Democrat of the first rank, died suddenly tonight in a hospital here, bringing to an abrupt end a spectacular political career.
November 07, 1931
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Japan's 'Fiscal Cliff': National Crisis or Political Kabuki?
By Mitsuru Obe and Paul Jackson
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At first glance it looks like Japan's own version of the U.S. fiscal cliff. A looming fiscal crisis lies on the horizon that could hit Japan's faltering economy with spending cuts and threaten its already precarious sovereign debt status.
But is Japan's lurking precipice more political kabuki than fiscal crisis? Analysts suggest the long-term danger of Japan's reticence to tackle the industrialized world's largest debt load is more significant than the short-term implications of the potential funding shortage.
The problem centers on a debt-ceiling stalemate in Japan that is starving the government of funds to finance some 40% of the current fiscal year's budgeted spending. Prime Minister Yoshihiko Noda's administration says it will have to halt some spending unless a bill enabling the issuance of deficit-financing debt is passed by the end of November.
But the imminent funding crisis has had little effect on financial markets to date.
"The lack of market volatility indicates there is a tacit understanding in the market that there won't be disruption to the government funding," Takahira Ogawa, sovereign debt analyst at Standard & Poor's, told JRT. "The government and opposition will find a compromise at some point."
Far from being Tokyo's version of a fiscal cliff, Mr. Ogawa says the situation isn't even like the U.S. debt ceiling crisis in the summer of 2011.
Indeed, it seems nobody wants to push the debate to the point of risking a potential default. As Japan came within a month of its funding deadline, the nation's main parties finally agreed Friday to discuss the deficit-financing bill after months of opposition stonewalling.
Rather than objecting to the draft legislation from a budget-balancing Tea Party-like stance, the opposition has tried to take the bill hostage as it seeks to force an early election. But the bill's use as a political weapon could backfire, since a spending freeze triggered by opposition obstinance is hardly a recipe for election success. The leader of the main opposition Liberal Democratic Party suggested for the first time Monday that the issues of the election timing and bond issuance could be separated, according to local media reports.
Some of the possible consequences of the situation may also have been overstated.
The finance ministry emphasizes it has enough reserves to meet coming interest payments and redemptions. Compare this to a blunt warning by U.S. Treasury Secretary Timothy Geithner in January last year that "failure to raise the (debt ceiling) limit would precipitate a default by the United States."
While the finance ministry has warned it might have to suspend bond offerings starting from a 10-year JGB issuance on Dec. 4, the ministry can always issue short-term bills to overcome temporary difficulties in issuing long-term bonds, experts say.
So the "cliff" seems more an example of Japan's political theater than of upcoming fiscal doom. The nation's political gridlock stems from its divided parliament, as voters repeatedly punish incumbent governments by electing opposition parties to parliament's second house in midterm elections, enabling them to block most draft legislation.
While this political gridlock isn't likely to lead to an immediate downgrade of Japan's sovereign debt, the window of opportunity for tackling the nation's huge debts is narrowing, says Mr. Ogawa.
The outlook on Japan's AA- rating has been held by S&P at "negative" since April 2011. Mr. Ogawa said a final decision will be made after taking into account factors such as whether the current political stalemate is resolved in upper and lower house elections to be held by next summer.
A downgrade would have serious repercussions for Japan, which is already placed by Fitch Ratings in the single-A class. Under Basel international banking standards, another downgrade to single-A level would force foreign banks to change Japan's risk category and cut their JGB holdings, a move that could be followed by foreign pension funds and insurers. The finance ministry doubts, though, that U.S. banks would follow the rules, especially for the debt of an OECD government, adding that Japanese banks, not bound by the standards, are unlikely to take action that would directly hurt their balance sheets — they own $4 trillion of JGBs.
Still, a plunge in JGB prices could force the government to make a capital injection into the banks, due to their large JGB holdings. This would quickly increase the government's debt burden and further undermine its creditworthiness, setting off a vicious cycle of higher debt and higher interest rates, Mr. Ogawa warns.
So fiscal cliff or not, the current market calm doesn't necessarily bode well for Japan's long-term fiscal health even if it's more of a wake-up call than an emergency alarm.
As Mr. Ogawa says, when it comes to nation's growing mountain of sovereign debt, "Japanese politicians have gotten inured to the potential risks."


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Standard & Poor's pledges to reform the credit rating process
Daily news headlines
NEW YORK – Ratings agency Standard & Poor's (S&P) has announced plans to reform its credit ratings process, providing improved strength, transparency, and independence.
Credit rating agencies (CRAs) have been at the centre of increased market and regulatory pressure as their mark-to-market ratings were subject to damaging fluctuations due to the subprime crisis and following credit crunch.
S&P aims to address the challenges facing rating agencies through reform of its governance policies, and the analysis and development of credit rating models. It plans to do this in addition to efforts towards improving education and information to increase transparency, market understanding and, ultimately, investor confidence.
"The ongoing transformation of the financial markets requires us to continue to bring more innovative thinking, greater resources, and improved analytics to the ratings process," says Deven Sharma, president of S&P.
"By further enhancing independence, strengthening the ratings process, and increasing transparency, the actions we are taking will serve the public interest by building greater confidence in credit ratings and supporting the efficient operation of the global credit markets," said Sharma.
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MMA Forum - UFC Forums - UFC Results - MMA Videos (http://www.mmaforum.com/forumindex.php)
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ESPN Coverage Of Fight Night Is Disgraceful
Adverts between rounds again.. But whats worse is the sound! Its sooooo quite that i have to turn it up about 20 notches so i can hear it, and then as soon as the adverts hit i have to make a dash for the volume button so that they neigbours dont get woken by how loud the adverts are!
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Bahaha same problem here man. I swear it happpened on a fight night when Setanta were broadcasting as well. Be fast with the mute!!
Spike is commin through fine in HD here.....:confused05:
Sorry guys....
They will get things sorted out over there sooner or later.
FTR...we have commercials between rounds as well......:thumbsup:
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Sound is fine now. Wasn't a problem, just to remember to turn tv down during adverts.
Yeah CC, I know there are ALOT of breaks during fight nights. Whenever spike goes to a break so do we.
I thought ESPN had listened to the criticism of their coverage of UFC 101. They did a much better job with UFC 102. Tonights Fight Night coverage is terrible! Adverts between rounds -the majority of which seem to be station idents, not even paying advertisers- and over the fighters' entrances. And as for that bloody sound level... WTF? We're clearly back to square one, and I fear for the coverage of UFC 103. This settles it for me; I'll record Saturday's event, then I can fast forward through the adverts when I watch it on Sunday.
If the Nate Diaz fight goes to a decision then ESPN coverage is going to end before the fight...
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text:
October 29, 2007, 12:00 am ET - Amendment SA 3473 proposed by Senator Lott for Senator Sanders.
October 29, 2007, 12:00 am ET - Amendment SA 3473 agreed to in Senate by Unanimous Consent.
Full Text of this Amendment
On page 66, line 10, insert ``, including projects that involve the purchase of environmentally sensitive, fuel-efficient, and cost-effective passenger rail equipment'' before the period.
(As printed in the Congressional Record for the Senate on Oct 29, 2007.)


text:
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See our article in Mimi Vanderhaven!
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