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- Kings' Slava Voynov will face felony assault charge
- Tim Howard earns U.S. Male Soccer Athlete of the Year honor
- Is Robert Griffin III an NFL bust?
- Sharks close trip with uninspired loss 4-1 loss to Sabres
- Chargers notes: Seyi Ajirotutu fined, Ryan Mathews and Manti Te'O to return after bye
- Clippers' concerns revolve around defense and rebounding
- Do the 49ers have an identity crisis?
- Marcus Lattimore injury: 49ers running back has bigger issue
- Klay Thompson signs 4-year, $70 million extension with Warriors
- VIDEO: Steve Ballmer scares his son with enthusiasm after Clippers' win
Lakers' Kent Bazemore has prime opportunity to prove himself
- Updated: March 24, 2014
LOS ANGELES — With the Los Angeles Lakers down two guards heading into Sunday's game against the Orlando Magic somebody had to step up, and that player was Kent Bazemore.
In a hybrid role off the bench that featured him as a scorer and sharing the responsibility of backup point guard duties with Jodie Meeks, the 24-year-old excelled and had arguably his most well-rounded performance as a member of the Lakers as he scored eight points with a career-high eight assists, two rebounds, and one steal in 24 minutes played on Sunday.
"It's probably the first time that I have gotten that assignment and played pretty well with it," Bazemore said. "I tried earlier in the season with Golden State and it didn't work. I tried it a few games ago when I first got here (in Los Angeles) and I was kind of shaky. The guys were just in the right spots all night and I had the easy part by just giving it them to make easy shots."
As for head coach Mike D'Antoni, it was a performance that he was impressed with but also one that he would like to see more of over the remainder of the season.
"I thought Kent Bazemore played one of his better games," D'Antoni said. "He had something like eight assist no turnovers, which is what he needs to do. He has to make plays; he can't just make it for himself. Defensively he's tough.; he gets out there, and gets after it and keeps his energy up. There were some good things, but you just have to repeat it and it a few more times."
Bazemore has been a consistent contributor for the Lakers offensively with 11 double digit outputs in 14 contests with team, but has not had a chance to truly break through. So with a solid performance for the second-year guard on Sunday, it could be the type of game that he needs to get himself going in other aspects of his game outside of points.
It also helps his cause given the fact that he now has the opportunity to do that in consistent minutes off the bench with a couple of his teammates out in Xavier Henry, who will miss an extended period of time with a torn ligament in his left wrist, Jordan Farmar, who will miss at least another week with a right groin strain, and Steve Nash, who is day-to-day with a right hamstring issue.
So with that said, Bazemore knows what lies ahead of him it is now up to him to show that he can strive in that role for the remainder of the season.
Photo Credit: Mark J. Terrill/Associated Press
Bob Garcia IV
Latest posts by Bob Garcia IV (see all)
- 5 things we learned from Lakers' 99-93 loss to Grizzlies - November 27, 2014
- Lakers nearing a one-year deal with forward Earl Clark - November 26, 2014
- Xavier Henry injury: Lakers forward out for season with torn Achilles - November 26, 2014


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Editing of Bulbapedia is currently restricted. Please see this message for more details.
Pokémon Omega Ruby and Alpha Sapphire have arrived!
Check BNN and Bulbanews for up-to-date Pokémon news and discuss it on the forums or in our IRC channel.
Cyndaquil (disambiguation)
From Bulbapedia, the community-driven Pokémon encyclopedia.
Cyndaquil is a species of Pokémon. It may also refer to specific Pokémon of that species:
- A Pokémon which belonged to Ash Ketchum before evolving: Ash's Cyndaquil
- A Pokémon which belonged to Dawn before evolving: Dawn's Cyndaquil
- A Pokémon which belonged to Jimmy before evolving: Jimmy's Cyndaquil
- A Pokémon which belonged to Gold before evolving: Exbo (Explotaro in Chuang Yi's translation)
- A group of cards in the Pokémon TCG: Cyndaquil (TCG)

subset: commoncrawl


text: YVCC Community Relations Image [ YVCC Diversity Series Continues on April 24th By Brian Teegarden April 2, 2013 Yakima Valley Community College's 2012-2013 Diversity Series "Sustainability as Cultural Imperative" continues on Thursday, April 24, 2013 with YVCC Student Video Competition, "Why is Diversity Important to Me?". Diversity Series events are free and open to the public. Read More|Comment Category: Education, Human Interest Tags: community education, education, yvcc Send to a friend! Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!


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Taxi Driver Hits 5-Year-Old In Brooklyn
A 5-year-old child remains in critical condition after being struck by a Ford Escape Taxi traveling Northbound on Hicks Street in Cobble Hill, Brooklyn.
The child was carried to nearby Long Island College Hospital by bystanders.
The driver remained at the scene. So far, he has not been charged with any criminality pending further investigation.Copyright © 2014, Sun Sentinel


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--Meteorite Silver with Obsidian Black leather with contrasting stitching with Obsidian Black carpeting, Rare 6-speed manual gearbox (less than 5% of DB9's were produced... More
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Classiche Certified and Numbers Matching. Mechanically sorted. Excellent paint, chrome and interior. Original colors (Rosso 20200 S Italver / Pelle Nera). Great ownership history. More pics at WWW.WELOVECOOLCARS.COM! Call Ken for... More
Low miles and a great history. This car has been comprehensively serviced and sorted by the best in the business. It is the best... More
I am very pleased to offer this 1988 Ferrari 328 GTB. This particular 328 GTB is an extremely rare color combination of Argento (silver)... More
Corvette Roadster. 283 with 2 4bl carbs, . The chrome is excellent. All instruments and speedo work as normal. Great intrior seats, carpet, and dash.... More
This 1969 Dodge Coronet Super Bee is for sale in our Louisville KY showroom. Occasionally a car will come into the showroom that just... More
Rare 6-speed manual 2006 Aston Martin V8 Vantage. Meteorite Silver over Obsidian Black with silver interior stitching, Bluetooth telephone connectivity, satellite navigation, heated front... More
IM CLASSICS IS PROUD TO PRESENT THIS 1970 CHEVROLET CAMARO Z28. FRESH OFF THE ROTISSERIE THIS BEAUTIFUL Z28 IS FINISHED IN ITS ORIGINAL COLORS... More
This 1970 Volkswagen Pro Sand Rail Buggy, built in 2012, (Stock # P10047) is available in our Pleasanton, CA showroom and any inquiries may... More
1965 Porsche 356C, An unrestored 356C in excellent condition. Production date January 20, 1965. The blue leatherette interior, headliner, dash, door panels, and sun... More
After 13 years of selling classic cars, muscle cars & hot rods, it's becoming hard to get excited when a new vehicle arrives on... More
Vehicle Description 2006 Ford GT FOR SALE!!!! Finished in BLACK with SILVER Stripes. Black interior Options on this GT include: *Lightweight BBS Wheels *Silver Racing Stripes *McIntosh Stereo This is a Like new,... More
Produced in 1974, the Carrera 3.0 RS picked up where the legendary 2.7 RS left off. Rightly a legend in its own right, the... More
Early 911s embody a combination that is rare among collector cars today. They have unparalleled drivability and usability, especially when compared with other cars... More


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How are you? What have you been up to? My ex boyfriend is out of jail. I moved so he can't find me. Other than that, I am doing well. I am happy for the first time in a long time.
sabrina! *hugs you randomly* ...................................
.................................................. .................................................. ..................
Thats good to hear. I'm doing fine and there is nothing new on my end, not at the moment any way.
thanks i try not too but im not always on here all the time and stuff
Yep i guess so but i just had to deal with it so what new with u
yea my grandads the same he may be startin to get clots now tho im really upset too but ur aunt will live longer than them few weeks my grandads been suffering with it the last 3 years but only really hittin him now tbh but we dont belive he will last long now tho....
ok healing now i just have to work on a frew things and be in a good mood so nobody barks at me for being me life sucks right now.
Deth note is alright but i'v only seen the dub i'v recently started to watch hellsing
Devoted Otaku


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Armond Armstead, Mark Harrison remain on NFI lists for now - 0 Comments
Rookies Armond Armstead and Mark Harrison will have to wait their turn, for now.
Armstead, a defensive tackle from the Canadian Football League by way of USC, and Harrison, a Rutgers product, will not begin practicing today. Armstead is on the non-football illness list with an infection, and Harrison is on the non-football injury list after injuring his foot while working out after the combine.


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Space Sci-Fi Features #78Ow by lingy-0Voyager by KaioshenGrand Eclipse by JustV23Supernova by swat3dLMS by isra-acSupernova Explosion by charmedyAncient Discovery by juliedillonSpace Station by Long-PhamCity of Light by Samuel-NordiusUnderwater Cyberpunk City by nkabutoMoon by Gabriel-BS
I got DD YAAAY!Hey what's up everyone?So I was checking DDs of today, then while I was swiping down slowly I saw my work and got so surprised and happy ... ya it's true because I assume you will wonder how would I become happy and surprised while having over 5 DDs!!! Well, because I am an improving artist and I care about my art... so when I make something I'm so happy about I'd be happy if it gets DD but not everytime... I dunno! Anyways, I'm so so so happy and thanks a lot to both KPEKEPand TehAngelsCry for making it happen.And thanks to everyone who have been supporting me and I love you and there will be new stuff coming out soon!!!!
I have a question for macbook users!Hi!Thanks a lot everyone for the birthday wishes and I love you very much Lately I've been a little busy ... well busy enough to make a new work but I will try these days!Anyways, I have a question for macbook users cuz it's about it!Is there a screen contrast adjustment in it?
Your Plans on The Summer?Hey guys!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!Haven't posted a journal to talk to you in a while and i've been so busy and just lately trying opening my computer everyday to quickly check out DDs and then other stuff like my groups ... etc! Usually I become busy from weekdays, but anyways let's leave this aside and talk about plans on the Summer!!!!!!!!!!My plans are: traveling to some place to chill with my brother and more people maybe still don't know! Buying a new freaking computer... cuz this one is like 7 years old!!!!!!!! And other stuff so what about you guys? And please let's talk here and you can talk about other stuff like what do you think of my recent wallpapers and what good movies u saw lately.... etc!
New Space And Sci-Fi ContestThe contest's name:Sunrise.The Theme of The Contest: Basically and simply showing a nice and beautiful sunrise, when the sun starts brightening the world ( whether from space or sky!) The sunrise can be in a space scene (or planetary) or a terra-space!Rules:You must be member of the group first ForgottenGalaxyLimited 2 submissions per person.Submissions must be related to the theme (Sunrise)Submissions must be created anytime and submitted after this blog post and must be submitted before the deadline of the contest.Submissions must be created entirely by you and you must state and link to whatever resources you may have used in your submission.How to participate:Submit your entry at the "Sunrise Contest's Entries" folder
Wish You Guys A Happy 2014 Hi guys! I was trying to post this earlier but I was mostly out+travel to home... so I only had my blackberry Q10 and tried posting a journal from it but couldn't, I don't know why!!!!!!!!!! There wasn't actually there the submit button at all .. ok anyways, I wish you guys a happy 2014 and sorry I'm late maybe and sure no need mentioning even new year's resolutions and I think you know why lol
3 Premium Memberships Go To...Hi! So many good participations so I had to pick 3 because I felt bad!The three premium memberships go to:poeticperfectionistSaito-san97Saskank91And sorry for the rest but thanks a lot for your participations
Giving Away A Premium Membership.So I'm only giving who has none and if you want it leave a comment telling why and then I will give the person who I think should get it.... You guys got 24 hours or I dunno cuz it depends on how much time left for the "give one and get free" offer!
Omg, I just knew RIP Paul WalkerThis is very horrible and sad, the world has lost a great actor and the star of Fast And Furious in a car accident! America is probably crushed from his death and I'm sorry for their lost, he was a very loveable person and I didn't know much about him but I remember on FB he posted his pics with animals and he loved them and that's a very nice thing from a person and he was just very nice and kind and that's very sad he died and even in such a young age!I didn't watch TV or used internet lately to know about his death earlier, but I was sick and still a bit but anyways RIP Paul Walker
Jurassic PokemonShare|ArchiveJurassic PokemonBy techgnoticRealistic Pokemon Season 1 by arvalisarvalis aka RJ Palmer is a long time deviant who has created his own unique niche at DeviantArt.He was first known as the artist who drew incredibly realistic dinosaurs. Somehow his depictions of dinosaurs began morphing into Pokemon characters. He is now renowned as dA's master of realistic Pokemon illustrations. His has been quite a success story through art. His rise has been quite a success story on how to build a following in the community. RJ has wonderful tutorial drawing sessions on his Dev
Pokemon is ForeverShare|ArchiveForeword by techgnoticKay (aka damphyr) is DeviantArt's go-to expert on everything "Pokémon." She also occupies the adventurous position of being one of DeviantArt's main enforcers of standards and practices. We thank her for being a guardian of artistic erotica and especially, for being able to answer every question we get asked about Pokémon and just about anything else for that matter. Supreme Keeper of all pop culture knowledge, Kay must be protected in case of global disaster.by damphyrAn incongruous collection of people lined the sidewalk outside the local video game retailer just before opening on March 6th, 2011. The first in line w
The Hunger Games: Accidental Statement of Protest?Share|ArchiveThe Hunger Games: An Accidental Statement of Protest?By techgnoticCatching fire by Kris-KamikakushiIn Bangkok, Thailand, students are being arrested for raising their hands in the air to flash Katniss' three–finger "freedom" salute.That's right, the Katniss who's the fictional heroine of the popular dystopian sci–fi "Hunger Games" movie series. The world has changed over the last few decades in a very big way, but some are apparently unaware of it or undisturbed by the stunning ramifications.Advanced sur
Star Wars: A New HopeShare|ArchiveStar Wars: A New HopeBy techgnoticThe Empire Bargains by dvsdesignerWho's the most important person now in the Star Wars franchise? JJ Abrams? Think again!It begins in mere weeks from now: The countdown to the first of the next Star Wars sequels, Star Wars VII: The Force Awakens, the first of a new trilogy for a new generation. The first 1 minute trailer, a mostly wordless montage of series' heroes, will debut attached to Hunger Games: Mockingjay Part 1 or on Disney-owned ABC Television. American culture stirs in its sleep. Georg
Burying A Sci-Fi ClicheShare|ArchiveBurying A Sci-Fi ClichéBy techgnoticif I were a writer by In-InkFrom the Dusty Annals of Sci-Fi Cliché HistoryJames Tiptree, Jr. was a writer of science fiction, mostly short stories, whose first work, "Birth of a Salesman," was published in the May 1968 issue of Analog Science Fiction.Analog Science FictionWarm Worlds & OtherwiseDangerous Visions
There Are No Strings On Me!Share|ArchiveThere Are No Strings On Me!By techgnoticO.A.M First Blood by Ku-OnHal9000 - Wallpaper by excursioner12001: A Space Odyssey reduxby jubjubjedi
Collection: Hammer Time!Share|ArchiveHammer Time!There is always a reason. A level of insult. An unprovoked assault. A threat of invasion. A refusal to negotiate in good faith. A refusal to compromise. There is always something so unbearable that it must be righted with no more delay and by any means, by even the last means, necessary. That final means is war.Ever since mankind became able to produce and possess more than necessary for survival, there has been a surplus of resources. It is the treasure that has built our civilization and shaped the special magic and glory of what it is to be a human being.The struggle for control and division of that treasure sometimes infects men's minds with a unique
The Little MermaidShare|ArchiveThe Little MermaidBy spottedSomeday by alicexzForeword by techgnoticElisa (aka spotted) steps out from behind the scenes as one of our intrepid Today Page senior editors to contribute her appreciation of "The Little Mermaid." We hope it will be the first of many contributions to come, another hidden talent having been revealed by this longtime DeviantArt special projects manager.It was 25 years ago that a redheaded mermaid made a splash in theaters ushering in a new era for the identity of the Disney princess.
Collection: The LeonidsShare|ArchiveThe LeonidsThe year's best naturally-occurring light show, the Leonids meteor shower, is due overhead on the nights of November 15 - 20. The passing comet "Tempel-Tuttle" leaves a debris field of meteoroid particles in its wake each year that the Earth will spin through on those nights. Larger "Leonids," about 10 mm across, have a mass of half a gram and are known for their brightness. The Leonid shower may deposit 13 tons of particles across our planet. The Leonid meteor storm of 1833 may have been the biggest, brightest ever, producing an estimated 240,000 meteoroids entering our atmosphere over 9 hours. Both Nati
Space & Sci-Fi Features #55 :thumb205072932: :thumb112073507:Like the Pheonix by TylerCreatesWorlds The Okuda Factor by Casperium The Haunted Station by Shue13Off Road by FacundoDiaz Ecstasy by Reiku-Rilona Japan monument by UlricLeprovostAphrodite by goodforn0thing :thumb208870888: Industry by HideyoshiPortfolio Slug by Fealasy Gaudi city by neisbeis Million Years Sunset by Shimmering-SwordPoisonous by Vyter Nagrand by Athena-Erocith Calamity by BLPHanother nebula by MilaySVK Vertigo by goodforn0thing emerald by jesper-ullbingCGHUB Creature planets Comp by semiconductor Fortress by Hideyoshi room by JimHatamaVoyager by manwesulemo GLaDOS: You Can Haz Caek by Julian-Faylona Cataclysm by Grim962space docks by JimHatama Triumph of light by AlexNIKO Maelstrom - Ghost Ship by jrmaloneSeason of the Seeds by ArthurBlue Full of Stars by TK769 battlefield detail 900 by max4everFly Softly by fmacmanus Sci-Fi Study by UlricLeprovost :thumb210357413:Takemura Nebula by Casperium Earth Wallpaper by Hameed LAST STAND * returning to this painting in 2014 by StarrZEENeon Manga by JonasDeRo Source by Vyter :thumb208696230:


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Bennie Rosato is a maverick lawyer, and business at her law firm has never been better. Then, without warning, a savage murder tears the firm apart. All evidence points to Bennie, who has motive aplenty and an unconfirmable alibi. Her world turns upside-down as the lawyer becomes the client, and the law is now after her. When another killing takes place, Bennie runs for her life, a fugitive armed only with her wits and courage. She is determined to find the real killer -- or die trying.
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Owner cited after shooting dog at Cincinnati park
Police were called to a dog park after receiving a call that an owner shot another dog.
Cincinnati police responded to the Doris Day Dog Park on Westwood Northern Boulevard.
Officers said one owner shot another dog after it began attacking his dog.
Police said Robert Marx, 74, used a 9 millimeter handgun to shoot Katlynn Hornsby's dog.
"I'm heartbroken. I've been crying all day," Hornsby said.
Hornsby's dog, a 1-year-old Pit bull named Bruski, had to be euthanized after being shot.
"We took him to the dog park all the time, at least twice a week and he never ever got into fights with dogs. He was perfectly good with dogs. He loved to play with them. He was so excited every time we pulled up to the dog park. And today it was just different," Hornsby said.
Police said Bruski and the other dog got into a fight Tuesday afternoon.
"As soon as it happened the dude ran up and threatened to shoot my dog and before we even had a chance to break them apart he shot him," Hornsby said.
Hornsby said the park was crowded with other dogs and children at the time of the shooting.
"There was kids there and he pulled out a gun and shot. A kid could have been hurt. I could have been hurt. I was holding my dog as he shot him," Hornsby said.
Officers said witnesses told them Bruski attacked Marx's dog but there were no signs of injuries.
Police said the owner who shot the dog had a concealed-carry permit and was allowed to have a gun.
Police cited the owner for discharging a firearm within the city limits and criminal damaging.
Copyright 2013 by WLWT.com All rights reserved. This material may not be published, broadcast, rewritten or redistributed.
Troopers ID woman killed in crash on SR 132 in Clermont County
Family vacation to Disney World turns to tragedy after 5 killed in late-night crash in Louisiana
First-grader denied bag lunch at school
Girl attacked by dogs continues long recovery
Bengals' Marvin Lewis: Former player's ex-wife should 'be more truthful'


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Word: lumpish
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Wear It Now
Gulden Touches: The Best Mustard Accessories for Under $40
The season's meanest color, mustard, is everywhere, and any guy can get in on the trend for less than two Jacksons
7. The easiest way to get acquainted with our favorite fall shade is around your waist. A slim nylon D-ring belt like this is ridiculously affordable, and pops even on basic khakis.
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- GQ Style: Wes Anderson's Highly Influential, Slightly Tweaked Take on Reality
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- Under the Influence: How Def Jam's Early Days Inspired this Season's Look
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- David Chang's Kitchen: Don't Get All "Fresh" with Me
- David Chang's Kitchen: Your Idea of Bologna is Total Baloney
- Chart: The Family Tree of Bourbon Whiskey
- Recipe: Semi-Summer Salad - Chef Jeff Taibe of Oak + Almond
- The Great Franco-Italian Pasta Experiment
- How to Pick from an Overpriced Restaurant Wine List


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It's Mc. Donalds, Burger King, and King Kong mixed together as seen in Red Alert 2.
GI:"We'll Garrison that Mc. Burger Kong over there to protect our selves from those flak troopers."
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High School Sports
Winslow is tired of state runner-up endings
Winslow High's softball program's streak of losing a state championship game sits at three and it also lost another title game in the past five seasons, so there's no need to tiptoe around what exactly the program wants this season — a state championship victory.
"This is the year we are going to send the seniors on a high note," Winslow's second-year coach Kelli Fifelski said.
Taylor Baca. Katonya Begay. Renee Bollin. Halle Hayes. Jelesia Hayes. Megan Lymer.
Those are the six Winslow seniors who've seen another team savor the spoils of winning a title three times. Fed up probably doesn't even begin to describe how those seniors feel about the past.
But so far they and their teammates are playing like champions this season, winning their first 16 games of the season before the sectional portion of their schedule begins. The pitching is good and so is the defense for Winslow, which received even more good news recently with the return of Bollin, who tweaked a shoulder sliding into home during a game early this year.
That injury forced her to sit out some games, but Bollin picked up where she left off, hitting a double in her first at-bat after returning from her injury. At third base, she showed off her strong arm and cat-quick reactions after bunts.
Bollin had a .468 average and 33 RBI's last season, but last year's hitting leader was Halle Hayes (.685 average in 2013), who also finished with a team-high 64 innings and 76 strikeouts in the circle. Back problems have slowed Hayes this season in the circle, however, but not at the plate, where Hayes was hitting .455 this season.
Winslow is blessed with depth and versatility this year, and it was easy for Fifelski to move Baca (7-0, 1.13 ERA), the team's cleanup hitter, into the role of ace. Baca's velocity has improved, coach said.
Winslow also is using another dual threat, Richelyn Villanueva (2.96 ERA), in the circle. Villanueva is one of six juniors who are also contributing this year along with the seniors.
"Mentally, they are ready to take it (title) this year," Fifelski said.
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A Battle of Beauties: Megan Fox vs. Angelina Jolieby Mischalova at . Comments
Megan Fox is starring in the movie Transformers this summer.
But it looks to us as though this gorgeous actress is trying to transform herself in the Angelina Jolie in the meantime.
The seductive look. The shoulder blade tattoo. The attraction to Brad Pitt (what, Megan Fox is female, right?).
Megan Fox is an up and coming actress... one that's trying to resemble Angelina Jolie in every way possible.
Does this mean Fox will be adopting a brood of foreign kids any time soon? Or shedding her clothes like the Angelina of yesteryear (and the Tila Tequila of this year) in every film? It's hard to say for sure.
But Paul Sculfor wanna hide in the closet for a bit. If Fox is truly trying to emulate Jolie, no boyfriend of Jennifer Aniston is safe.
I think Megan fox is pretty but lacks substance. Angelina Jolie is very different from your typical hollywood starlet, she is confident enough to be different. Megan is way to similar to Angelina. Angelina has had her moments of acting out but it came from a more real place, I belive. In Megans case it seems all too orchestrated. She projects this bad girl image and at moments makes her self look and sound bad. If she grows up in the near future she just might really regret who she was at a younger age. Angelina Jolie is a deep,intelligent (Woman) and Megan well she is a (girl) trying to market an image, it reaks of an actress trying to be like someone else.
Angelina Jolie is by FAR MORE SEXIER than Megan Fox will ever be. Megan Fox is just another Angelina Jolie copy cat who doesnt pull it off very well. I realy hope Megan Fox doesnt start taking Angelina Jolie's roles in films,Angelina is still young and got alot of years left in her yet, look at the film (Wanted) only from last year,Angelina was perfect in that.
I think Angelina is so naturally beautiful..I love megans look but beauty wise angelina is a godess.
A blowjb from them both at the same time is what makes them both hot to me.
i also gotta say is that girls are just jealouse towards megan, cuz she is very pretty and yeah haha they say she wears alot of make up but then we have younger bitches now wearing more makeup or even the same lyk them so why even talk all the shyt bout megan...angelina at can i say shes still pretty but her personality makes me hate her, yeah she addopted kids aliret everyone does dat now a days,..i swera wat a 2face anjie is..................megan is beautiful, she has everythng mayeb a girl wants..but angie shes getting older shes lik wat in her 40's ..megan just started soo fuck u all lil haterz ass bitches who talk botu megan
i think they are both beautiful but then again after ive heard all that shyt about angelina jolie being a slut and this and that makes me just dislike her cuz i hates sluts and bitches who also take away men from their marriage lyk angie did...now megan fox shes still young and i thnk shes so gorgeous, her eyes her lips, and she may be this and that but i choose megan fox, more than angie..yea angie is hot to but its stupid why people try to say that megan is trying to copy angie.hahah i kno i wouldnt cuz sluts will always be know as sluts..
I don't think that Megan is copying Angelina. It just so happens that they have some similar traits. But so what? There are probably a million other women out there that look similar to, or have similar tattoo's to Angelina. But they are not famous, so we don't see them. I think Megan is beautiful, she has class style and charisma, and so does Angelina. But no body is ever going to like a new-comer are they?
There's nothing to compare, we all know that angelina jolie is the most Gorgeous women in hollywood, Megan fox stop trying to look like jolie because you never will, angelina jolie has this exotic natural classy look and no one could be or atleast try to be like her, why would people call megan fox the next angelina jolie is so obvious that she's never going to look as gorgeous as jolie does, Plus jolie knows how to act so much better than fox does.
megan fox is a wannabe, angelia was here WAY before that lil skank, angelia was more than 'arm candy' like how megan was in transformers, no she kicked ASS. Shes a ACTUAL actor and has more personality.


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AmerasianSyllabification: Am·er·a·sian
Pronunciation: /ˌamərˈāZHən /
Definition of Amerasian in English:
Having one American and one Asian parent.
Example sentences
- She turned her attention and resources especially toward Amerasian children.
- They were not among the few thousand Amerasian children who came to the United States before the war's end.
- Last year this 29 year-old Amerasian woman was desperately trying to come to the United States with her husband and two children
nounBack to top
A person with one American and one Asian parent.
Example sentences
- And then in 1996, after a three year search, with the help of an American writer researching Amerasians, she located her father, who came to Vietnam to meet her.
- For the first time in their lives, they learn to be proud of themselves as Amerasians.
- Fifty-eight more Amerasians were flown out of Vietnam today.
Definition of Amerasian in:
- British & World English dictionary
What do you find interesting about this word or phrase?
Get more from Oxford Dictionaries
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Word of the day peart
Pronunciation: pərt
lively; cheerful


text: ESPN Feed: kevin arnovitz http://assets.espn.go.com/i/tvlistings/tv_espn_original.gif ESPN en-us 10 ESPN Inc. http://espn.go.com/ Copyright 2010 ESPN Inc. Stephen Dunn/NBAE/Getty ImagesThe Lakers and Rockets played one of Wednesday night's few meaningful games. LOS ANGELES -- Give the Los Angeles Lakers...]]> April 17, 2013 23:35:17 PDT 5-on-5 roundtable: Our experts pick the opening round of the Western Conference playoffs.]]> April 17, 2013 14:47:58 PDT NBAE/Getty ImagesJust as their teams were hitting their strides, Marc Gasol and Ty Lawson went down.For a while, the Memphis Grizzlies were the Wester...]]> March 26, 2013 14:31:56 PDT Russell Westbrook | Thabo Sefolosha | Kevin Durant | Serge Ibaka | Kendrick Perkins Minutes Played: 1,024 Offensive Rating: 110.1 points per 100 pos...]]> March 13, 2013 09:13:24 PDT The Spurs came back from 24-point deficit with pick-and-roll precision.]]> May 19, 2012 14:13:23 PDT The final night of the NBA regular season had an odd feeling to it.]]> April 26, 2012 17:31:11 PDT San Antonio Spurs point guard Tony Parker sat out Friday night's game against the Los Angeles Clippers with a strain of the right quadricep.]]> March 09, 2012 14:10:26 PST Andrew D. Bernstein/NBAE/Getty Images In case you haven't noticed, the Spurs haven't lost a game since Jan. 29. The least profitable death pool in h...]]> February 18, 2012 17:30:33 PST NBA fandom usually begins with the love of a team. My love of the NBA, for instance, springs directly from a childhood spent listening to Blazer games...]]> August 21, 2009 08:28:00 PDT D. Clarke Evans/Getty Images The Clippers' Eric Gordon, who scored 21 points, has emerged as one of the top rookies in the league. Glenn James/N...]]> January 08, 2009 17:11:14 PST


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Best place for Spices online?
Ok I'm in a very rural are and or local grocery store have and extremely limited supply of spices and of what they do have its pretty expensive. So Ive been looking for a place online that sells spices, especially mixes. Ive found a few but they all seem really expensive. I have found one that seems reasonable, but its also a lot less than the others, so before I get anything id like to know if anyone's dealt with them before. Its called myspicer.com.
Also does anyone know any other online places to get good spices in general?
What's your idea of "expensive"? Before they opened a store in my city I regularly ordered spices from Penzey's and I always found them very reasonable. Others have mentioned the Spice House, which is also run by a Penzey, and they're reasonable as well.
re: woodleyparkhound
I 2nd Penzey's - awesome quality.
I was excited for Penzey's when it opened in my city (Los Angeles), but this place absolutely blows Penzey's out of the water. I will say that I feel like Penzey's strong point, to me, is more their baking spices. Still, I think Spice Station is cheaper and the product is superior.
re: Littleman
i've used this company many times.
In addition to Penzey's, The Spice House and The Spice Barn, I have good luck with spices from the Savory Spice Shop: http://www.savoryspiceshop.com/
The Spice Barn seems to lean more toward the commercial/mass market where the others are more small consumer oriented.
If cost is an concern, definitely go for the resealable bag option rather than jars. The usual suspects will sometimes give you twice as much spice for the same price point that way.


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The cloning of the heparinase gene from Flavobacterium Heparinum using the polymerase chain reaction is described. The Open Reading Frame (ORF) corresponded to 1152 base pairs encoding a precursor protein of MW 43,800 daltons. The amino acid sequence reveals a 20-residue leader peptide. The gene was expressed in two expression systems in E. coli.
1. A method for obtaining a nucleic acid molecule encoding a modified heparinase I from Flavobacterium heparinum comprising
mutating a nucleic acid molecule encoding the sequence in SEQ ID No. 2,
expressing the mutated sequence in an appropriate host, and
screening the expressed heparinase for enzymatic activity.
2. The method of claim 1 further comprising screening the heparinase expressed from the mutated nucleic acid molecule for modified binding activity.
3. The method of claim 1 further comprising screening for increased specific activity.
This is a divisional of U.S. Ser. No. 07/783,706 filed on Oct. 23, 1991, now U.S. Pat. No. 5,714,376 by Ramnath Sasisekharan, Kelley Moremen, Charles L. Cooney, Joseph J. Zimmermann, and Robert S. Langer entitled "Heparinase Gene from Flavobacterium heparinum."
This invention is generally in the area of heparinases and is specifically directed to the gene encoding heparinase I, expressed in Flavobacterium heparinum.
The United States government has rights in this invention by virtue of grant number 25810 from the National Institutes of Health.
Heparin is an anticoagulant that activates serine protease inhibitors (serpins), which play a key role in the blood clotting cascade, as described by Damus et al., Nature 246:355-357 (1973). According to Lindahl et al., Trends Biochem. Sci. 11:221-225 (1986), heparin is the most acidic natural polymer known to date. It consists of a major 1,4-linked disaccharide repeating unit of D-uronic acid 1,4-β-D-glucosamine, and has an average of four negative charges (three sulfate groups and one carboxylate group) per monosaccharide unit. Heparin is both polydisperse, having an average molecular weight between 3,000 and 45,000 daltons, and heterogenous due to partial epimerization of D-glucuronic acid to L-iduronic acid and incomplete N- and O- sulfation, as reported by Kusche et al., Proc. Natl. Acad. Sci., 77:6551-6555 (1980) and Comper, Polymer Monograph 7, 1981.
In addition, proteoglycans like heparin have a wide range of biological influences, including in blood chemistry, growth factor interaction and wound healing, interaction with basic structural proteins in the extracellular matrix and in cellular mediated immune responses. The basic nature of protein/peptide--heparin/complex carbohydrate interaction is important. Although heparin seems fairly heterogenous, it is now quite clear that different heparin fractions exhibit distinct and unique properties indicating some compositional and possibly structural specificity for its biological role, as reviewed by Cardin, A. D. and H. J. R. Weintraub, Arteriosclerosis 9, 21-32 (1989).
Heparinase, also referred to as heparin lyase, is the only known enzyme capable of degrading heparin that has been extensively characterized. It has been designated EC 22.214.171.124 by the Enzyme Commission. According to Galliher, et al., Eur. J. Appl. Microbiol. 15:252 (1982), the enzyme is a polysaccharide lyase found in the periplasmic space of Flavobacterium heparinum, a Gram-negative soil isolate. F. heparinum utilizes heparin as its sole source of carbon and nitrogen, as described by Hoving and Linker, J. Biol. Chem. 245:6170 (1970). Heparinase is the initial enzyme of heparin catabolism. Although constitutively expressed in low amounts, Galliher, et al., App. Environ. Microbiol. 41:360 (1981), have discovered that enzyme expression is induced by heparin and reversibly repressed by sulfate in the medium. Lindhardt, et al., Appl. Biochem. Biotechnol. 9:41 (1984), have shown that heparinase is inhibited by other polyanionic polysaccharides.
Heparinase has been purified by standard chromatographic techniques and its enzymatic properties characterized extensively, as described by scientists including Yang, et al., J. Biol. Chem. 260:1849 (1985). The enzyme is a 44,000 dalton monomeric protein with a pI of approximately 9.
Heparinase acts as an eliminase, leaving behind an unsaturated double bond at the non-reducing end group. This double bond is exploited in an assay for heparinase activity by the absorbance of the unsaturated product at 232 nm. The enzyme is marginally tolerant to salts and is very specific for heparin, having a kd of 30 nM. Heparinase has an activation energy of 4.5 kcal/mol, a km of 8�10-6 and a Vmax of 4�10-7 M/min.
Heparin is often used in surgery to prevent blood clotting and to increase the compatibility of extracorporeal devices such as heart-lung and kidney dialysis machines. The enzymatic degradation of heparin by heparinase is sufficient to eliminate the anticoagulation properties of heparin in surgery. As described by Langer, et al. in Biomaterials: Inter-facial Phenomenon and Applications, Adv. in Chem. Symposium Series, Chap. 13, pp. 493-509 (1982), this property has led to the use of heparinase as an immobilized bioreactor in conjunction with heart-lung or kidney dialysis machines to deheparinize blood. Commercial application of the heparinase bioreactor is pending clinical trials.
A principal problem in the use of the heparinase bioreactor is the availability of sufficient amounts of pure heparinase to be immobilized onto a surface. This is primarily because the amount of heparinase constitutively expressed in F. heparinum is very low. Inducing expression of heparinase in F. heparinum with heparin is very expensive due to the amounts of heparin needed and the size of the fermentation to produce reasonable amounts of heparinase for any practical applications.
Cloning and expression of the heparinase gene is important in several ways. First, the only enzyme cloned and characterized to date which acts to depolymerise proteoglycans is heparinase. Second, heparin is the only anticoagulant commonly used in surgery so deheparinizing blood is an important medical problem. Moreover, heparinase catalyzed degradation of heparin into lower molecular weight heparin molecules can be used to yield products with specific anticoagulant activity, as discussed by Rosenfeld and Danishefsky, Biochem. J. 237:639-646 (1986).
Designing recombinant heparinases with altered activitie(s) would be interesting academically, as well as commercially. For example, heparinase can be used to deheparinize blood because the enzyme cleaves right at the AT-III binding oligomer. On the other hand, by further understanding the mechanism of the enzyme binding and depolymerizing heparin, recombinant heparinases with altered specificity could be designed, i.e. an AT-III binding heparin fragment not cleaved by the recombinant enzyme. This would be a very useful way of generating an AT-III binding heparin oligosaccharide, which currently is not available in large amounts, for use as an anticoagulant. Producing heparinases which could help and or improve in the enzyme purification or immobilization would also be quite valuable. For example, a tag (a particular peptide sequence) could be added at a region which does not alter the activity of the enzyme but makes the immobilization chemistry very efficient. This would help in improving enzyme loading onto the immobilization matrix.
It is therefore an object of the present invention to provide the gene encoding heparinase and a system for expression to facilitate the production of large amounts of heparinase.
It is another object of the present invention to provide methods and means for modifying the gene to produce recombinant heparinases having altered specificity and other desirable properties.
It is another object of the present invention to provide pure heparinase for use in the area of cytokine-proteoglycan interactions, as a tool or diagnostic as exemplified by fibroblast growth factor--heparin interactions.
The cloning of the heparinase gene from Flavobacterium Heparinum using the polymerase chain reaction is described. Two degenerate oligonucleotides, based on amino acid sequence derived from tryptic peptides of purified heparinase were used in the PCR with Flavobacterium genomic DNA as the template to generate a 600 base pairs probe. This probe was used to screen a pUC 18 Flavobacterium genomic library. The Open Reading Frame (ORF) corresponded to 1152 base pairs encoding a precursor protein of MW 43,800 daltons. Eleven different tryptic peptides (approximately 48% of the total amino acids) mapped into the ORF. The amino acid sequence reveals a 20-residue leader peptide.
Heparinase can be expressed from the gene. Additionally, the gene can be modified to produce heparinase with altered enzymatic activity, specificity, or binding properties. The sequence can also be used as a probe in the isolation of genes encoding other related enzymes.
FIG. 1 is a schematic representation of the PCR products Yl:C and D:C which are 600 and 160 basepairs, respectively. The 600 basepair PCR product was used as a template with D and C as primers to generate the 160 basepair D:C product.
FIG. 2 is the restriction map of the genomic DNA pUC 18 plasmid, pRS.HEP51, having an insert containing the heparinase gene. The plasmid is 5631 bases long and has approximately 2300 bases of insert. The heparinase gene is in the KpnI--KpnI fragment.
FIG. 3 is a KpnI--KpnI fragment map showing the heparinase gene structure with the different tryptic peptides mapping into the open reading frame. Six different peptides mapped into the heparinase gene translation region.
The gene encoding heparinase in F. heparinum has been cloned, wherein the coding and non-coding sequences are set forth in Sequence ID No. 1. The nucleotide and amino acid sequences are shown below:
The following sequence encodes a leader peptide, shown at positions 1 through 172 in Sequence ID No. 1: ##STR1##
The following sequence encodes the heparinase, shown at positions 173 through 1327 of Sequence ID No. 1: ##STR2##
The following is the amino acid sequence of heparinase, set forth in Sequence ID No. 2: ##STR3##
Because preliminary cloning attempts by others utilizing 1) antibody screening, 2) screening for functionally active heparinase in E. coli and 3) screening for the heparinase gene using probes derived from protein sequences regenerated by cyanogen bromine (CNBr) chemical digest were unsuccessful, the polymerase chain reaction was used to clone the heparinase gene. The reverse phase purified heparinase was reduced, alkylated and digested with trypsin to obtain approximately 60 peptide peaks which were separated and collected by reverse phase HPLC monitored at 210 nm and at 277 nm (for tyrosine and tryptophan), as described below.
Tryptic Digest and Protein Sequence Analyses
Heparinase was purified as described by Dietrich et al., J. Biol. Chem. 248:6408 (1973), Otatani et al., Carbohyd. Res. 88:291 (1981), and Yang et al., J. Biol. Chem. 260:1849 (1985), which are incorporated by reference herein. A final purification step was carried out by High Performance Liquid Chromatography (HPLC) using a reverse phase column that exploits the hydrophobic residues of the protein. A nanomole (approximately 45 μg) of the purified enzyme was denatured in 50 μl of an 8M urea, 0.4M ammonium carbonate solution, reduced with 5 mM dithiothreitol (DTT) at 50� C., cooled to room temperature, and alkylated with 10 mM iodoacetamide for 15 minutes in the dark. The total reaction volume was 200 μl. To this reaction mixture, 1/25th w/w of trypsin was added and digestion carried out at 37� C. for 24 hour. The reaction was terminated by heating the sample at 65� C. for 2 minutes. The digest was separated by reverse phase HPLC using a gradient of 0 to 80% acetonitrile. The tryptic peptides were monitored at 210 and 277 nm.
The tryptic peaks were collected in Eppendorff tubes. Based on the homogeneity of the peptide peak, eight different peaks were sequenced using an Applied Biosystems sequencer, model 477, with an on-line model 120 PTH amino acid analyzer located in the Biopolymers lab, Center for Cancer Research, MIT. The sequences are set forth in Table I below. The designation (K,R) is used in Table I to indicate that trypsin cuts at either lysine or arginine residues. The asterisks in Table I represent amino acids that could not be determined. The peptide designated td Lx is the longest peptide sequenced having 38 residues. Native undigested heparinase was also sequenced to determined the N-terminus amino acids.
TABLE I__________________________________________________________________________Sequences of Tryptic Peptides of HeparinasePeptideAmino Acid Sequence__________________________________________________________________________td 04, Sequence ID NO. 3(K,R)GICEQGSSRtd 09, Sequence ID NO. 4(K,R)TVYHYGKtd 09, Sequence ID NO. 5(K,R)TSTIAYKtd 21, Sequence ID NO. 6(K,R)FGIYRtd 33, Sequence ID NO. 7(K,R)ADIVNQQEILIGRDD*GYYFKtd 39, Sequence ID NO. 8(K,R)ITYVAGKPNGNKVEQGGYPTLAF*td 43, Sequence ID NO. 9(K,R)MPFAQFPKDCWITFDVAID*TKtd 40, Sequence ID NO. 10(K,R)NLSGYSETARtdm4, Sequence ID NO. 11KNIAHDKVEKKtd 72, Sequence ID NO. 12KTLSIEEFLALYDRtd Lx, Sequence ID NO. 13RSYTFSVYIPSSFPDNATTIFAQWHGAPSRTLVTPEIK__________________________________________________________________________
Three sets of primers were designed and synthesized, as shown in Table II. Primers were synthesized with an Applied Biosystems sequencer, model 477, with an on-line model 120 PTH amino acid analyzer located in the Biopolymers lab, Center for Cancer Research, MIT. These primer sets were used in the PCR amplification system for cloning the heparinase gene. The symbol "I" represents the nucleotide inosine. The amino acids of each peptide, depicted in boldface type, represent the residues chosen for the primer design. Two different sets of primers were constructed for tryptic peptide 33 to reduce the degree of inosine substitution at the 3' end of the primer.
TABLE II__________________________________________________________________________Heparinase Primer Design__________________________________________________________________________Peptide: td 04 Sequence ID NO. 3Amino Acid Sequence:KGICEQGSSRprimers:y1, Sequence ID NO. 145'-AAAGGIAT(T/C/A)TG(T/C)GA(A/G)CA(A/G)GG-3'y2 3'-CC(C/T)TG(C/T)TC(G/A)CA(T/G/A)ATICCTTT-5'Peptide: td 43Amino Acid Sequence: ##STR4##primers: ##STR5## ##STR6##Peptide: td 33Amino Acid Sequence:(K,R)ADIVNQQEILIGRDD*GYYFKAprimers: ##STR7## ##STR8## ##STR9##__________________________________________________________________________
Of the six RHPLC peaks initially sequenced (Table I), three were chosen for primer design. Three sets of primers were designed (Table II). The PCR product of the combination the primers td43 and td33 was about 150 base pairs in length. The combination of td4 and td33 primers were about 600 base pairs. Primer td43 was 5' to primer td33 and primer td4 was 5' to td43 primer. Using the PCR product of td4 and td33 as a template and td43 and td4 as primers the predicted 150 base pair product was obtained confirming that td43 was between td4 and td33.
The 600 basepair product shown in FIG. 1 represents about 51% of the approximated total 1170 base pairs for the heparinase gene, assuming 43,000 dalton for heparinase and a 110 dalton average amino acid with a molecular weight corresponding to about 390 amino acids times three which is 1170 bases.
The 600 base pair probe was chosen for screening a pUC 18 library by high stringency colony hybridization. Two positive clones were identified which were carried through for three rounds of colony purification.
Genomic DNA, RNA, and Plasmid Library
The F. heparinum genomic DNA was isolated by the A.S.A.P.� kit (Boehringer Mannheim, Indianapolis, Ind.) with the following modifications. The DNA was desalted over a Sephadex� G-50 column (Nick column, Pharmacia, Piscataway, N.J.) and concentrated using a Centricon� P-30 (Amicon Division, Beverly, Mass.) to a final volume of 100 l. From 1�109 cells, 105-115 g of DNA typically were obtained. Total cellular mRNA was isolated using the guanidine thiocyanate procedure set forth in the Promega technical information publication TB 087 12/89, Promega Corp. Madison, Wis. 53711. A pUC 18 plasmid was obtained from Dr. A. J. Sinskey, of the Department of Biology at the Massachusetts Institute of Technology. The library was constructed using the F. heparinum genomic DNA. The genomic DNA was sonicated and modified by adding EcoRI linkers and then ligated to the pUC 18 vector. DH5a was transformed with the pUC 18 genomic library.
Amplification of the PCR Product
Amplification of the heparinase tryptic digest primers was carried out in a 25 l reaction volume containing 50 mM KCl, 10 mM Tris HCl (pH 8.3), 1.5 mM MgCl2 and 0.01% gelatin plus the four deoxyribose nucleotide triphosphates(dNTPs) at 200M, using 0.5M primer and 3 l of the genomic DNA as the template, 2.5 units of the Taq polymerase (Cetus Corp., Emeryville, Calif.) and 25 l of mineral oil. The samples were placed on an automated heating block (DNA thermal cycler, Perkin Elmer Corp., Norwalk, Conn.) programmed for step cycles of temperatures 92� C. (2 minutes), 50� C. (1 minute) and 72� C. (3 minutes). This cycle was repeated 35 times. The final cycle had a 72� C. 10 minute extension. The PCR products were analysed on a 0.8% agarose gel containing 0.6 μg/ml ethidium bromide. The control reaction was provided by the Cetus kit.
Screening of the Flavobacterium heparinum pUC 18 genomic library
The pUC 18 library was titered to give approximately 1500 colonies to be tested by the probe generated by PCR. Each plate had approximately 100 colonies which were grown directly on nitrocellulose, to an appropriate small size, and then duplicated to be grown further overnight.
The PCR probe was labelled using the Random Hexanucleotide� kit (RHN) (IBI Biochemicals Ltd.) which is described briefly as follows. One microgram DNA from the PCR product run was isolated from a low melt agarose gel, denatured by boiling at 95� C. for 10 minutes, and then chilled on ice. To the denatured DNA were added 10 mM dNTPs (dATP, dGTP, dCTP, dTTP), random hexanucleotides in the reaction buffer, and 50 μCi of 32 PdCTP(3000 Ci/mmole). The reaction was carried with Klenow for 30 minutes at 37� C. and terminated using 0.2M EDTA. Following the labelling reaction, the labelled probe was purified from the free nucleotide by using a Sephadex G-50 column (Nick Column, Pharmacia, Piscataway, N.J.)
The colonies were screened with the labelled probe using standard colony hybridization procedures as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., incorporated herein by reference.
Two positive clones were isolated and the plasmids tested for their ability to generate the 600 basepair PCR product. Both of the clones tested positive and were further characterized by restriction mapping. Clone pRS Hep 51 is a 2.3 kb insert in pUC 18 (shown in FIG. 2) with a Kpn--Kpn fragment of about 1.6 kb. This fragment was a positive template for generating a 600 basepair PCR product. The KpnI--KpnI fragment of pRS 51 was subcloned into M13 and sequenced.
DNA Sequencing
DNA sequencing was performed using phage M13 and employing the dideoxyadenosine 5'-alpha-35 S-triphosphate and Sequenase (US Biochemical Corp, Cleveland, Ohio) as described by the manufacturer. The sequence data was obtained using successive nested deletions in M13 using T4 DNA polymerase as per Cyclone I Biosystems (International Biotechnologies Inc., New Haven, Conn.) or sequenced using synthetic oligonucleotide primers.
The sequence reveals a single, continuous open reading frame (ORF) of 1152 basepairs corresponding to 384 amino acids and a leader sequence of about 21 amino acids. The PCR product spans from 566 to 1216 bases from the start site and corresponds to about 57% of the total gene.
Initially six different tryptic peptides mapped into the ORF. Subsequently, five other peptides were sequenced for structural studies and all of them mapped into the ORF, for a total of about 48% of the total 367 amino acids. There are three cysteines in all, one associated with the signal peptide. The signal peptide is typical of prokaryotic sequences, having a charged N-terminal region, a core hydrophobic region and a cleavage region with a standard Ala.xxx.Ala site for cleavage.
Two different expression systems were selected for the expression of heparinase in E. coli: the Omp A expression system and the pKK hyper-expression system. The plasmid designs for both expression systems are shown in Table III.
Omp A expression system
The Omp A expression system secretes the protein of interest into the periplasmic space, as directed by the Omp A signal sequence, described by Ghrayeb, et al., EMBO J. 3:2437 (1984), incorporated herein by reference. This system was chosen since heparinase is naturally expressed into the periplasmic space of F. heparinum. The plasmid is under the control of the lac repressor and is induced by the addition of IPTG (isopropyl-β-D thiogalactoside) to the medium. The plasmid was inserted in the pIN-III omp A-3 vector.
The heparinase insert was generated by PCR utilizing the N terminal and the C terminal sequences of heparinase with two appropriate restriction sites suitable for cloning into the EcoRI-BamHI sites. Two primers were constructed as shown in Table II. The insert was amplified by 5 cycles of PCR and ligated to the Omp A pIN vector with the E. coli periplasmic leader sequence. DH5α was transformed and expression was induced with 1 mM IPTG for 3-5 hours.
As shown in Table III, the construct of the Omp A expression system results in two extra amino acids at the amino terminal of the heparinase gene, Gly and Ile. The heparinase sequence begins with a Gln.
The pKK expression system
The pKK expression system is used for over-expression of proteins in accordance with the methods of Brosius and Holy, Proc. Natl. Acad. Sci., 81:6929 (1984) and Jaffe et al., Biochem. 27:1869 (1988), incorporated by reference herein. This system contains a strong tac promoter which, in appropriate hosts, is regulated by the lac repressor and induced by the addition of IPTG, as in the Omp A system. The plasmid pKK223-3 has a pUC 8 multiple cloning site and a strong rrnB ribosomal terminator immediately following the tac promotor. The ribosomal binding site of the plasmid was utilized by cloning the heparinase gene into a SmaI site, which is about 12 bases from the start codon ATG. Like the Omp A construction, the heparinase insert is obtained by PCR with SmaI and HindIII restriction sites at the N and the C terminals of the protein. As shown in Table III, the native heparinase leader sequence was used for over-production into the periplasm.
Periplasmic proteins of E. coli were isolated by osmotic shock. Briefly, 1.5 ml of cells were centrifuged after induction and washed with 10 mM Tris pH 7.5. The cells were then suspended in 20% sucrose in 10 mM Tris pH 7.5 and 5 Al of 0.5M EDTA. After a five minute incubation on ice, the cells were centrifuged and osmotically shocked by adding approximately 150 μl water. The periplasmic extract was used to determine enzyme activity. Heparinase activity was determined by monitoring the wavelength at 232 nm and by the Azure A methods of Bernstein et al., Methods of Immunology 137:515 (1988), incorporated herein by reference.
The periplasmic extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using the method of Laemmli, Nature 227:690 (1974) and stained using Coumassie blue. In addition, a Western blot assay was performed to confirm the presence of heparinase using a heparinase monoclonal antibody. Heparinase was electrophoretically transferred from the SDS-PAGE gel onto nitrocellulose using the method of Gershoni and Palade, Analytical Biochem. 131:1 (1983), and then incubated with the monoclonal antibody. This antibody was stained using a secondary antibody conjugated to horseradish peroxidase.
TABLE III__________________________________________________________________________Design of OmpA and pKK plasmids for Expression of Recombinant Heparinasein E. coli__________________________________________________________________________Omp A secretion Expression system ##STR10##pKK over-Expression system ##STR11##__________________________________________________________________________
RNA Dot Blot Assay
The total cellular RNA was immobilized onto a Zeta probe� membrane (Biorad, Richmond, Calif.) by alkaline RNA denaturation and fixation, and probed using the 600 base PCR product, used in screening for the heparinase gene. The hybridization was carried out with dot blot apparatus in accordance with the method of Thomas, Proc. Natl. Acad Sci. 77:5201 (1980). The RNA signal under different growth conditions has been investigated by Galliher, et al., Eur. J. Appl. Microbiol. (1982). It was established by those studies that heparinase at the protein level is optimally expressed under low sulphur conditions, which removes the requirement of heparin for induction. Heparinase mRNA signal under low sulphur growth conditions was therefore studied with and without heparin induction.
Both the OmpA and the pKK systems expressed heparinase. The OmpA system did not efficiently transport heparinase to the periplasm. For reasons not known, a large fraction of recombinant heparinase was retained in the cytoplasmic region along with the Omp A signal sequence. At lower temperatures (25�-30� C.) of growth, there was some secretion into the periplasmic space.
The pKK overproduction system produced heparinase only in the periplasmic space. The pKK system used the native F. heparinum heparinase leader sequence in which there was no problem with the transport of the recombinant protein with a foreign leader sequence. The pKK system expressed heparinase without any aberrant processing, although the expression was again optimal at lower temperatures. The presence of heparinase in the periplasm was confirmed by western blotting and by comparing in situ tryptic digest of the recombinant heparinase with that of the native heparinase, in terms of the peak profiles and some peaks which were isolated and sequenced.
A positive signal was obtained for the isolated F. heparinum mRNA using the 600 basepair probe derived from the PCR which has been used for isolating the heparinase gene, confirming that the gene isolated was a F. heparinum gene cloned in E. coli.
The expressed heparinase appeared to have at least some heparinase activity.
The sequence can be modified to alter specific enzymatic activity or binding specificity or affinity by substitution of one or more amino acids, using site directed mutagenesis or substitution of oligomers into the sequence encoding the heparinase. Methods and materials to accomplish this are known to those skilled in the art. The modified gene is then expressed and the product routinely screened for the altered activity.
Although described with reference to two specific expression systems, other expression systems are well known and commercially available. The heparinase gene can be expressed in these systems, using similar vectors and signal peptides or leader sequences.
Modifications and variations of the present invention will be obvious to those skilled in the art. Such modifications and variations are intended to come within the scope of the following claims.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 21(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1379(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:CCTTTTGGGAGCAAAGGCAGAACCATCTCCGAACAAAGGCAGAACCAGCCTGTAAACAGA60CAGCAATTCATCCGCTTTCAACCAAAGTGAAAGCATTTAATACAATACCAGAATGTCGCA120TTTCCCTTTCAGCGTACTTTTTGGGTAAATAACCAATAAAAACTAAAGACGGATGAAAAA180ACAAATTCTATATCTGATTGTACTTCAGCAACTGTTCCTCTGTTCGGCTTACGCCCAGCA240AAAAAAATCCGGTAACATCCCTTACCGGGTAAATGTGCAGGCCGACAGTGCTAAGCAGAA300GGCGATTATTGACAACAAATGGGTGGCAGTAGGCATCAATAAACCTTATGCATTACAATA360TGACGATAAACTGCGCTTTAATGGAAAACCATCCTATCGCTTTGAGCTTAAAGCCGAAGA420CAATTCGCTTGAAGGTTATGCTGCAGGAGAAACAAAGGGCCGTACAGAATTGTCGTACAG480CTATGCAACCACCAATGATTTTAAGAAATTTCCCCCAAGCGTATACCAAAATGCGCAAAA540GCTAAAAACCGTTTATCATTACGGCAAAGGGATTTGTGAACAGGGGAGCTCCCGCAGCTA600TACCTTTTCAGTGTACATACCCTCCTCCTTCCCCGACAATGCGACTACTATTTTTGCCCA660ATGGCATGGTCGACCCAGCAGAACGCTTGTAGCTACACCAGAGGGAGAAATTAAAACACT720GAGCATAGAAGAGTTTTTGGCCTTATACGACCGCATGATCTTCAAAAAAAATATCGCCCA780TGATAAAGTTGAAAAAAAAGATAAGGACGGAAAAATTACTTATGTAGCCGGAAAGCCAAA840TGGCTGGAAGGTAGAACAAGGTGGTTATCCCACGCTGGCCTTTGGTTTTTCTAAAGGGTA900TTTTTACATCAAGGCAAACTCCGACCGGCAGTGGCTTACCGACAAAGCCGACCGTAACAA960TGCCAATCCCGAGAATAGTGAAGTAATGAAGCCCTATTCCTCGGAATACAAAACTTCAAC1020CATTGCCTATAAAATGCCCTTTGCCCAGTTCCCTAAAGATTGCTGGATTACTTTTGATGT1080CGCCATAGACTGGACGAAATATGGAAAAGAGGCCAATACAATTTTGAAACCCGGTAAGCT1140GGATGTGATGATGACTTATACCAAGAATAAGAAACCACAAAAAGCGCATATCGTAAACCA1200GCAGGAAATCCTGATCGGACGTAACGATGACGATGGCTATTACTTCAAATTTGGAATTTA1260CAGGGTCGGTAACAGCACGGTCCCGGTTACTTATAACCTGAGCGGGTACAGCGAAACTGC1320CAGATAGCAAAAGCCCTAAGCGCATCCGATAGGGCTTTTCTTATATTTACAATAAAATT1379(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 384 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:MetLysLysGlnIleLeuTyrLeuIleValLeuGlnGlnLeuPheLeu151015CysSerAlaTyrAlaGlnGlnLysLysSerGlyAsnIleProTyrArg202530ValAsnValGlnAlaAspSerAlaLysGlnLysAlaIleIleAspAsn354045LysTrpValAlaValGlyIleAsnLysProTyrAlaLeuGlnTyrAsp505560AspLysLeuArgPheAsnGlyLysProSerTyrArgPheGluLeuLys65707580AlaGluAspAsnSerLeuGluGlyTyrAlaAlaGlyGluThrLysGly859095ArgThrGluLeuSerTyrSerTyrAlaThrThrAsnAspPheLysLys100105110PheProProSerValTyrGlnAsnAlaGlnLysLeuLysThrValTyr115120125HisTyrGlyLysGlyIleCysGluGlnGlySerSerArgSerTyrThr130135140PheSerValTyrIleProSerSerPheProAspAsnAlaThrThrIle145150155160PheAlaGlnTrpHisGlyAlaProSerArgThrLeuValAlaThrPro165170175GluGlyGluIleLysThrLeuSerIleGluGluPheLeuAlaLeuTyr180185190AspArgMetIlePheLysLysAsnIleAlaHisAspLysValGluLys195200205LysAspLysAspGlyLysIleThrTyrValAlaGlyLysProAsnGly210215220TrpLysValGluGlnGlyGlyTyrProThrLeuAlaPheGlyPheSer225230235240LysGlyTyrPheTyrIleLysAlaAsnSerAspArgGlnTrpLeuThr245250255AspLysAlaAspArgAsnAsnAlaAsnProGluAsnSerGluValMet260265270LysProTyrSerSerGluTyrLysThrSerThrIleAlaTyrLysMet275280285ProPheAlaGlnPheProLysAspCysTrpIleThrPheAspValAla290295300IleAspTrpThrLysTyrGlyLysGluAlaAsnThrIleLeuLysPro305310315320GlyLysLeuAspValMetMetThrTyrThrLysAsnLysLysProGln325330335LysAlaHisIleValAsnGlnGlnGluIleLeuIleGlyArgAsnAsp340345350AspAspGlyTyrTyrPheLysPheGlyIleTyrArgValGlyAsnSer355340345ThrValProValThrTyrAsnLeuSerGlyTyrSerGluThrAlaArg370375380(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 10 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: "Xaa represents either lysine orarginine residues wherein trypsin cuts."(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:XaaGlyIleCysGluGlnGlySerSerArg1510(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: "Xaa represents either lysine orarginine residues wherein trypsin cuts."(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:XaaThrValTyrHisTyrGlyLys15(2) INFORMATION FOR SEQ ID NO:5:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 8 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: "Xaa represents either lysine orarginine residues wherein trypsin cuts."(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:XaaThrSerThrIleAlaTyrLys15(2) INFORMATION FOR SEQ ID NO:6:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: "Xaa represents either lysine orarginine residues wherein trypsin cuts."(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:XaaPheGlyIleTyrArg15(2) INFORMATION FOR SEQ ID NO:7:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 22 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: "Xaa at position 1 represents eitherlysine or arginine residues wherein trypsin cuts, andXaa at position 17 represents undetermined."(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:XaaAlaAspIleValAsnGlnGlnGluIleLeuIleGlyArgAspAsp151015XaaGlyTyrTyrPheLys20(2) INFORMATION FOR SEQ ID NO:8:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 25 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: "Xaa at position 1 represents eitherlysine or arginine residues wherein trypsin cuts, andXaa at position 25 represents undetermined."(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:XaaIleThrTyrValAlaGlyLysProAsnGlyAsnLysValGluGln151015GlyGlyTyrProThrLeuAlaPheXaa2025(2) INFORMATION FOR SEQ ID NO:9:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: "Xaa at position 1 represents eitherlysine or arginine residues wherein trypsin cuts, andXaa at position 21 represents undetermined."(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:XaaMetProPheAlaGlnPheProLysAspCysTrpIleThrPheAsp151015ValAlaIleAspXaaThrLys20(2) INFORMATION FOR SEQ ID NO:10:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 11 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: "Xaa at position 1 represents eitherlysine or arginine residues wherein trypsin cuts."(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:XaaAsnLeuSerGlyTyrSerGluThrAlaArg1510(2) INFORMATION FOR SEQ ID NO:11:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 11 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:LysAsnIleAlaHisAspLysValGluLysLys1510(2) INFORMATION FOR SEQ ID NO:12:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 14 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:LysThrLeuSerIleGluGluPheLeuAlaLeuTyrAspArg1510(2) INFORMATION FOR SEQ ID NO:13:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 38 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:ArgSerTyrThrPheSerValTyrIleProSerSerPheProAspAsn151015AlaThrThrIlePheAlaGlnTrpHisGlyAlaProSerArgThrLeu202530ValThrPheGluIleLys35(2) INFORMATION FOR SEQ ID NO:14:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 20 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: "N at position 6 is inosine, N atposition 12 is a T or a C, and N at positions 15 and18 are an A or a G."(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:AAAGGNATHTGNGANCANGG20(2) INFORMATION FOR SEQ ID NO:15:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 29 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: "N at position 6 and 21 is inosine,N at position 9, 18 and 27 is a T or a C, and N atpositions 15 and 24 are an A or a G."(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:ATGCCNTTNGCNCANTTNCCNAANGANGA29(2) INFORMATION FOR SEQ ID NO:16:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 23 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ix) FEATURE:(D) OTHER INFORMATION: "N at position 3, 15 and 18 isinosine, N at position 6 and 18 is a T or a C, and Nat positions 9 and 12 are an A or a G."(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:ATNAANCANGANATNNTNATHGG23(2) INFORMATION FOR SEQ ID NO:17:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 4 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:GlyIleGlnLys(2) INFORMATION FOR SEQ ID NO:18:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 12(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:GGAATTCAGAAA12(2) INFORMATION FOR SEQ ID NO:19:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:ACTGCCAGATAGGGATCC18(2) INFORMATION FOR SEQ ID NO:20:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:TAACCCGGGATGAAAAAA18(2) INFORMATION FOR SEQ ID NO:21:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 18(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:GCCAGATAGAAGCTTCCG18__________________________________________________________________________
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First of all, a little disclaimer: I am NOT at all an expert on running or running injuries. What I discuss here is purely through my own personal experience and knowledge. Take it or leave it but don't leave ME!
We have all experienced pain while running. Whether it's just those
Running is as much as a mental exercise as it is a physical exercise. Runners are often used to dealing with the pain and 'learn' to just run through it. But, how do you know when it is serious enough to STOP? Runners, especially long distance runners and beginning runners who are overtraining will run on serious injuries for months. Hoping the pain will go away… but it doesn't. It slowly gets worse and the risk for a more permanent and serious injury that will require a MUCH greater rest time (NO runner wants to rest for more than a week! I can barely handle a day off). Stress fractures for example are a very common injury in long distance runners, female runners, and runners that are overtraining. Runners have been known to run on stress fractures for months through the pain before they get it looked at and often risk ending up with a full fracture.
In any case, there are MANY different kinds of running injuries. Each and every one of you have unique physiology and some are more prone to certain injuries than others. The trick is to know WHAT is going on, when and IF you need to stop training and what the solution is. It is not an easy task. Listen to your body. The first step is the 'hop test'. If you cannot hop on the leg you are experiencing pain in, GET IT LOOKED AT. That is the #1 bad sign. Make sure you have range of motion in your ankle and knee. Is your leg visibly swollen, is it tender in certain areas to the touch? Does the pain start after you start running and does it increase? Does the pain go away when you run? Sounds crazy, but that is common. The pain I have goes away as the minutes go by. Are you healing? If you take a few rest days off from running and you are NOT feeling better, that is a warning sign. These are all things you need to be aware of.
I finally made my way to the orthopedic sports doctor at Boston Medical yesterday. I had my leg X-rayed and my leg examined by two doctors and an intern. The primary orthopedic doctor I saw is apparently known as the 'Boss-Man'. I definitely felt like I was in good hands! It was actually a very cool experience. I learned that I DON'T have a visible stress fracture from the looks of the X-ray… but that can only truly be ruled out after getting an MRI that will definitely show if I have a stress fracture. Because my pain goes AWAY while I run, that is indicative that it is not compartment syndrome which is what I was so so so scared of so YAY!
As of now, it may be a stress fracture or shin splints which is really just ambiguous muscle tearing around the shin bone from overuse. I won't know about the stress fracture until I get the MRI. But, in the meantime, here is the kicker:
He told me I should continue to run. He said there is no permanent damage being done and that if I could mentally deal with the pain that I should continue to train and that my brain will tell me if I should stop.
I had a difficult time wrapping my brain around this. Will I really know if I need to stop? Sure it hurts when I run but I CAN run. Will I always run with pain? I still feel like I do not have any answers. Maybe I overtrained in the beginning and was wearing the wrong sneakers and not stretching as much as I should have been and not allowing myself to recover. But the pain is here now. Will the new changes I have made now make a difference and will the pain slowly go away… or should I not run though the pain at all.
You may be asking now… what is WRONG with this girl!? Why does she continue to run?? If you are a runner, you will hopefully understand the urge and the NEED to run. The truth is my left leg has ALWAYS bothered me… on long walks, runs, you name it. I push through it and it dissipates, and now that I am running longer distances and loving it more and more the more I desire to push through it. Running is a major stress reliever in my life. No other exercise compares for me in terms of stress relief. I have asked myself, will it be possible now that I have new sneakers (not that my Mizunos were old… they were just too hard on my feet), I am stretching more, wearing a compression sleeve and so on… will these things allow my leg to get better WHILE I run if it is not doing damage? As the rest of my body and mind gets stronger… will my left leg not have a chance to catch up?
Honestly, I feel ridiculous saying that I want to and will continue to run. But if the doctor says it is okay… then why not just accept it for now?
I have a 5k, a 10k and two half marathons coming up within the next two months. I do not want to be in pain. Even if I CAN run, I don't want this pain. Is my brain just too stubborn to make me stop? YES.
So I ran 4 mikes after work yesterday in my new sneakers. My feet loved them. I had so much energy and my right leg was a rockstar. Why is it only my left leg? Isn't the definition of insanity doing the same thing over and over expecting different results? But honestly, it had been 9 days since my last run, and it never hurt so good.
As of now, ibuprofen has worked, icing it, stretching, rolling it and wearing a compression sleeve have all helped. But last night was the worst. It ached and burned deep in my mid-calf and kept me up last night. When I got up it wasn't much better. But once I got going it very quickly started to lessen. It waxes and wanes. Even after seeing one of the best specialists for this kind of thing I still feel left in the dark. Was I not describing my symptoms accurately? Perhaps, despite what he says, I AM doing damage? But if not, should I just accept the pain as a part of me?
I have also been taking Glutamine and MSM …. can't hurt right? I'll try anything (natural and not harmful) that may help Lefty out a little :)
If the MRI comes back showing that I have a stress fracture, I need to stay off my leg for 8 weeks so that it definitely heals. I am already trying to mentally prepare myself for that. I am prepared to plan a training schedule and races for LATE this year early next year. Until I need to cross that bridge… I will continue to listen to my body and run if I can. Please don't think I am crazy!
So on another crazy but quite hilarious note…. I also learned that it is probably not a good idea to eat GREEN overnight oats in a jar in a doctor's office waiting room. It will look gross. Guaranteed. No matter how delicious those oats are, it is guaranteed to gross people out. Good thing I have no shame! HaHA!
This morning I got to eat my green oats in private! Last night I conjured up a batch of strawberry banana overnight oats to bring to work with me today. I had a nearly empty jar of crunchy 365 peanut butter in the fridge at work I knew would make the perfect little oat jar.
Strawberry Banana Green overnight Oats in a Jar
- 1/3 cup old fashioned rolled oats
Mix it all up and store in the fridge until the AM! If you happen to have nearly empty nut butter jar you can soak the oats in the jar or add them the next morning.
last night I had a wine FAIL. I mean… okay, maybe it wasn't a complete fail. I mean, I did drink the wine after all. But I will never buy it again. Just like the label AND the synthetic cork told me… it was FUNKY.
Sorry… I do generally like Argentinean wines across the board. AND I appreciate cute marketing techniques. And I DO like a little funk in my vino from time to time.. especially if it is from South Africa or Chile where the wines are sure to be smokey, gamey and kinda funky… but this really did TASTE like a $5.99 bottle of wine. It seemed cheap to me vs. inexpensive. It wasn't crisp and fresh like Pinot Grigio should be… it was interpreted like an over-oaked Californian Chardonnay. Hard to explain and I've already talked your ears off enough… but don't buy this wine. There are plenty of other Bottles for under $10 that are FAR FAR better choices :)
How do you feel about eating gross looking food in public?
have you ever had a running injury or dealt with chronic running pain?
What is your go-to wine if you drink the juice!?
Thanks for reading through all my craziness… this was a lengthy one! <3
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November Featured Artists
First Wednesday Reception
November 5, 5-8 pm.
Novembers art exhibit, Group Dynamics, features artwork by the Salem Art Group (SAG). The members include: Tory Brokenshire, Dayna Collins, Nancy Eng, Kathy Shen, Celia Stapleton, and Katy Vigeland. The show reflects the womens different mediums and range of styles within the group. Salem Art Group (SAG) is composed of like minded artists who get together at least once a month to make art, play, support, challenge, and encourage each other in our art endeavors. If you are interested in forming your own art group, stop by the artists reception, First Wednesday, November 5th, 5:00 - 8:00pm and well tell you how. We even have hand-outs!
Ward Lofgren - Mini Canvas Show Peoples Choice Award!
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November 1-9, 2014 - Pick-up artwork.
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This page contains Cheats for Spirit Walkers: Curse Of The Cypress Witch organized by sections for Android. Spirit Walkers: Curse Of The Cypress Witch is a Adventure General game, made by G5 Entertainment, released on Jan 11, 2013 and published by G5 Entertainment. If you can't find a hint or secret in our list, then please check this page periodically for the latest updates.
- Developer:
Spirit Walkers: Curse Of The Cypress Witch Android Cheats
Inspired by the legend of a hiker-attacking ghost living in the forest, Maylynn and a group of friends decide to go on an entertaining, investigative trip. Almost immediately, things take turn for the worse when one of the friends gets seriously injured. In order to survive, they must travel between times and dimensions to overcome an ancient evil and save the souls of the innocent. Help these friends solve brain-twisting puzzles and explore the world of ghosts and lost souls in this heart-pounding Adventure game.G5 Entertainment
- Publisher: G5 Entertainment
- Genre: Adventure General
- Release Date: Jan 11, 2013
- ESRB: Not Set
Video Walkthroughs
Stuck on this game? Set a specific, clearly defined question and let others answer your question or view answers to previously asked questions. If you think you are an Spirit Walkers: Curse Of The Cypress Witch expert then please try to help others with their questions.
How do you solve the gem puzzle box...
Posted on: 09-27-2014
Hands keep going need to set time...
Posted on: 05-08-2014
Spirit Walkers: Curse Of The Cypress Witch - Official Launch Trailer
- Oct 31, 2013, Views: 64
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Mirallas also back, but Baines and Barry miss out.
Ian Snodin joins evertontv's Darren Griffiths for West Ham coverage.
Up-to-the-minute updates from the Club's official social channels.
Secure your seat for a host of thrilling Goodison games in 2015.
Available now. Order Today!
Shop for the latest Everton Autumn/Winter fashion today.
Check out the latest Everton Essentials fashion.
Catch up with the very latest press briefing from the boss.
A rundown of the latest injuries and suspensions affecting the senior squad.
There are a wide host of lounges here at Goodison to suit every taste and requirement - both on matchday and for events.
Holidays Are Coming! Okay, maybe it feels too soon - but if you don't book now you could miss out!
Take a step inside the world-renowned Everton Academy.
There are a range of products available offering you a chance to win big money prizes.
Details on our forthcoming coaching camps for youngsters.
An interactive journey through our illustrious history, from 1878 to present day.
Learn all you need to know about the men who have sat in the managerial hotseat.
Learn more about the charity that supports former Blues.
We have a wide array of exclusive signed items listed on our new Everton eBay Auction platform.
Find the nearest Supporters' Club to you on our interactive map.
Everton's array of partners deliver a plethora of offers and benefits for our fans.
West Ham (H) Sat 22 Nov 2014, 15:00, Barclays Premier League
by Daniel Alston
15 July 2013 09:39
Roberto Martinez says he wants Marouane Fellaini to have a 'massive influence' on his new-look Everton team. The Blues boss was speaking after making the Belgian skipper for the second half of Sunday's game against Austria Vienna. Newspaper speculation has linked Fellaini with a move away from the Club this summer but Martinez insists Goodison Park is the best place for the 25-year-old to progress his career. "Marouane is an international footballer and he's going to be involved, if all goes well, in a World Cup next year," Martinez explained."He's had a fantastic impact in the Premier League and hopefully now is the time he can carry on showing his massive influence in the game for us. "He's still a very young man but I am really looking forward to seeing Marouane working hard and making sure that he sets really good standards at this club."On his decision to hand Fellaini the armband, the Blues boss added: "We have a very good group of senior players in our dressing room, and some players need that extra bit of responsibility. "We have youngsters at the Club and they need to get opportunities but the senior players at the Club are massively important to start the season as strong as we can be."
Find out the latest information for Everton fixtures.
Barkley and Osman in Saturday's media.
MOTM: The winner of this afternoon's Man of the Match, as voted for by you, is... @Osman21Leon! Well played, Leon! http://t.co/C2MuW1odSi
REACTION: Martinez hails 'incredible' Osman, after the midfielder notched the winner on his 400th appearance: evertonfc.com/news/2014/11/2…
MAN OF THE MATCH: RT if James McCarthy got your vote today. #EFCvWHU http://t.co/ZMUhoq9LM2
MAN OF THE MATCH: RT if Leon Osman got your vote today. #EFCvWHU http://t.co/Jxffb2Vzym