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Attention all the expecting parents out there or those looking to upgrade their stroller!! It is that time of year again!! Yep, time for the Britax Free Ride Event. This time the Free Ride Event is with Britax's new luxury stroller, the Affinity! You remember the customizable stroller that we introduced you to back in March… Read More
Bugaboo Bee3 Review (VIDEO)
Bugaboo recently debuted their 3rd generation Bee stroller with some pretty major changes and we have the skinny for you. The Bugaboo Bee3 has a new Bee bassinet, a modified basket, buckle, and an improved chassis and canopy. Is it the best Bee yet? I guess you'll have to watch the Baby Gizmo video below… Read More
Urbini Touri Travel System Review (VIDEO)
We know that not everyone wants to spend a fortune on a stroller travel system but they still want something great! It shouldn't have to cost you more than a mortgage payment for a great stroller, right?!? Today, we are taking a look at the budget-friendly, adorable Urbini Touri Travel System. Sure, you can purchase… Read More
Mamas & Papas Armadillo Flip XT
Last week, we introduced you to the Mamas & Papas Armadillo Flip and today we want to let you know about the XT version. The Armadillo Flip XT is an upgraded version of the Flip earning it the name "XT". It's just a little more extra special! To see the upgraded features such as the… Read More
Mamas and Papas Armadillo City Sneak Peek (VIDEO)
If you're looking for a lightweight, easy-to-fold stroller these days (and who isn't, right?!), you might want to check out the newest addition to the Mamas & Papas line of strollers. Mamas & Papas debuted the travel-friendly, ultra-compact Armadillo City this month! With a true, one-handed fold, the Armadillo City is great for urban living!… Read More
Nuna Mixx Stroller Sneak Peek (VIDEO)
After the excitement of EPISODE ONE: At Home with Baby Gizmo this morning, we need to get back to ABC Kids Expo 2014 coverage! This time we are talking about the very popular baby company, Nuna, and their new Mixx stroller coming out next month. This luxury stroller is stocked with features such as a… Read More
Britax Free Ride Event Giveaway
It is that time of year again! Yep, almost time for the Britax Free Ride Event. This time the Free Ride Event is with Britax's new luxury stroller, the Affinity! We introduced you to this customizable stroller back in March and now the price has dropped and you have the opportunity to get a FREE… Read More
Cybex Priam 2015 Stroller (VIDEO)
Cybex debuted their new luxury Priam stroller at the ABC Kids Expo this week. This top-of-the-line customizable stroller has a modern look, reversible seat, one-handed, deep recline and smooth push. There are three types of wheels to choose from when ordering this stroller (this makes it a "customizable stroller") so that you can have the… Read More
Summer Infant 3D Flip (VIDEO)
There is a new reversible seat, umbrella stroller in town and this one is super budget-friendly! The Summer Infant 3D Flip is a lightweight, umbrella stroller that features a reversible seat, deep recline, a large canopy, taller handles, and a shoulder strap for travel. While all those features normally will set you back quite a… Read More
Baby Jogger City Mini Zip Stroller (VIDEO)
The Baby Jogger City Mini Zip stroller was released in Jun 2014, and we have an exclusive video of it just for you! With the capability of carrying a child from birth through to 55lbs, and weighing only 16lbs, this is one you should see. It boasts the smallest Baby Jogger stroller fold, that you… Read More
Summer Infant Zyre Stroller Sneak Peek (VIDEO)
Summer Infant just debuted the new lightweight Zyre Stroller at the ABC Kids Expo 2014 this week. This "upgraded 3D Lite" stroller has a larger canopy, thicker and sturdier fabric, bigger wheels, an easier, one-handed recline and other features that you need to see to believe. Best part? It will only retail for $119. Take… Read More
BOB Revolution PRO Stroller Review + Giveaway!
Giveaway Closed! Congrats to our winners – Kim Kuiper and her friend Gina Hiskes The BOB Revolution Flex review and giveaway was so popular last week that we had to do it again! This time all the fun is with the new BOB Revolution PRO! If you are a sporty parent or parent-to-be, chances are you… Read More
BOB Revolution Flex Stroller Review + Giveaway!
BOB makes one of the most popular jogging strollers in North America. They have been dominating the parent running space for years. The robust jogging tires, the roomy seat, the ginormous canopy and the easy, quick fold have sporty parents swooning for a BOB the second they are expecting. While they are one of the… Read More
The Amazing Journey with the Vue (GIVEAWAY)
GIVEAWAY CLOSED! Congrats to our winner – Jayme H. Dear Baby Gizmo Family, In the last 21 days, I had no idea how much fun that I would have with you on your big journey from Illinois to North Carolina. To my surprise, the Southern Caribbean just happened to be on the route but I'm… Read More
Britax Debuts the B-Agile 4
There is a new four-wheel stroller from Britax hitting the streets soon! The B-Agile 4 is the beloved B-Agile stroller but with the four wheels that some parents feel that they MUST have! It has the same lightweight, aluminum frame, one-handed fold, easy push, large canopy and all-wheel suspension, but the B-Agile 4 rolls on… Read More
Journey with a Vue #VueJourney
Summer travel is here and we are taking you along for an exciting journey with a Vue. The Baby Jogger Vue is a perfect travel stroller and over the next 3 weeks it will be taking the trip of a lifetime…a stroller lifetime that is! The Vue will be hitting the road and exploring the… Read More
Baby Gizmo Spotlight Review: Inglesina Quad Stroller
Today we are continuing our coverage of the new Inglesina gear that has hit the market in 2014. Last week we showed you the ins and outs of the brand new Trilogy stroller and today we are tackling the luxury Inglesina Quad Stroller. This full-size stroller, for newborns and children up to 55 lbs, features… Read More
Which Affinity Are You? GIVEAWAY!
Maybe the end of the school year is making us crazy (or crazier than normal!) but we are announcing another giveaway! That's right, our third giveaway for the month and I'm not promising that it isn't our last! This one is all about the new Britax Affinity stroller! We reviewed the Affinity back in March… Read More
Baby Gizmo Spotlight Product Review: Inglesina Trilogy
Inglesina was quiet for the past couple years but they are back with a vengeance for 2014. With the introduction of two new strollers and a new high chair, Inglesina is planning to take the baby gear world by storm. Today we are talking about the new Inglesina Trilogy. This full-size stroller, for newborns and children… Read More
Spotlight Product Review: Safety 1st Lift LX Travel System (VIDEO)
Not everyone is able (or wants!) to spend a fortune on a stroller and car seat for their baby. They want a set that can safely get their little one from A to B but don't want to take out a second mortgage to afford it. Good thing that companies still make budget-friendly strollers that… Read More
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All Schools
(19) All Schools
(19) High Schools
(6) High Schools
(6) Elementary Schools
(16) Elementary Schools
(16) Preschools
(2) Preschools
(19) All Schools
(19) High Schools
(6) High Schools
(6) Elementary Schools
(16) Elementary Schools
(16) Preschools
(2) Preschools
There are 19 private schools in Tennessee belonging to the General Conference of the Seventh-Day Adventist Church (GCSDAC), serving 1,457 students.
General Conference of the Seventh-Day Adventist Church (GCSDAC) Private Schools
- School Location Students Grades
CollegedaleA.W. Spalding Elementary School
PO Box 568
Collegedale, TN, 37315
(423)396-2122(Seventh-Day Adventist)280 students | Gr. PK-5
CalhounAdvent Home Learning Center
900 County Road 950
Calhoun, TN, 37309
(423)336-5052n/a students | Gr. 6-12
All-boys | Special Program Emphasis (Seventh-Day Adventist)
CollegedaleCollegedale Academy
Po Box 628
Collegedale, TN, 37315
(423)396-2124(Seventh-Day Adventist)357 students | Gr. 9-12
DicksonDickson Adventist School
746 Highway 46 S
Dickson, TN, 37055
(615)446-7030(Seventh-Day Adventist)8 students | Gr. KG-7
DunlapDunlap SDA School
105 Apache Lane
Dunlap, TN, 37327
(423)949-2920(Seventh-Day Adventist)16 students | Gr. K-8
NashvilleFh Jenkins SDA Elementary School
814 Youngs Lane
Nashville, TN, 37207
(615)227-8992(Seventh-Day Adventist)95 students | Gr. K-8
GreenevilleGreeneville Adventist Academy
305 Takoma Ave
Greeneville, TN, 37743
(423)639-2011(Seventh-Day Adventist)77 students | Gr. KG-10
MontereyHeritage Academy
23100 Clarkrange Highway
Monterey, TN, 38574
(931)839-6675(Seventh-Day Adventist)54 students | Gr. 9-12
PortlandHighland Academy
211 Highland Circle Dr
Portland, TN, 37148
(615)325-2036(Seventh-Day Adventist)92 students | Gr. 9-12
JellicoJellico Sda School
170 Adventist Ln
Jellico, TN, 37762
(423)784-9355(Seventh-Day Adventist)16 students | Gr. 2-8
KnoxvilleKnoxville Adventist School
3615 Kingston Pike
Knoxville, TN, 37919
(865)522-9929(Seventh-Day Adventist)56 students | Gr. K-9
MadisonMadison Campus Elementary School
1515 Sutherland Dr
Madison, TN, 37115
(615)865-4575(Seventh-Day Adventist)155 students | Gr. KG-8
Deer LodgeMeister Memorial Sda School
1145 Meister Hills Rd
Deer Lodge, TN, 37726
(931)863-4944(Seventh-Day Adventist)8 students | Gr. 2-7
MemphisMemphis Junior Academy
50 N Mendenhall Rd
Memphis, TN, 38117
(901)683-1061(Seventh-Day Adventist)66 students | Gr. PK-10
OoltewahOoltewah Adventist School
9209 Amos Rd
Ooltewah, TN, 37363
(423)238-4449(Seventh-Day Adventist)97 students | Gr. K-8
RidgetopRidgetop Adventist Elementary School
Po Box 829
Ridgetop, TN, 37152
(615)859-0259(Seventh-Day Adventist)27 students | Gr. KG-8
Johnson CityTri City Sda School
314 Delmer Salts Rd
Johnson City, TN, 37615
(423)477-8421(Seventh-Day Adventist)19 students | Gr. K-9
TullahomaTullahoma Sda School
231 Turkey Creek Dr
Tullahoma, TN, 37388
(931)455-1924(Seventh-Day Adventist)15 students | Gr. 1-6
KnoxvilleUniversity Seventh-day Adventist School
1837 Brandau St
Knoxville, TN, 37921
(865)524-1424(Seventh-Day Adventist)19 students | Gr. KG-8
State Statistics
|About This State (TN)
|(TN) State Statistics
|Nationwide Statistics
|Population (Approximate)
|6,238,518 people
|304,788,332 people
|Age 25+ w/College Degree
|Median Household Income
Recent Articles
Updated October 26, 2014
What do you do when you are dissatisfied with your local public schools? Homeschool your children? Send them to private school? Let's look at your options.
Your religion matters to you. You want your child to attend a school which will combine academics and religious education. Here is an overview of what is available together with some links to denominational web sites.
Add things up and you'll quickly find the answer- math is crucial both in academics and the real world. By following these strategies and following personal training programs, students can uncover their weaknesses and conquer math.
More ArticlesPre-elementary, Paying For It, High School Issues, Why Private School, Opinion

subset: commoncrawl


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LITTLE ROCK, Arkansas — Republicans have locked up control of the Arkansas Legislature for 2015.
The GOP on Tuesday won at least 62 seats in the 100-member House, while Democrats took at least 34. Results are pending on four races.
In the 35-member Senate, Republicans will hold a 24-11 margin after winning all four races decided Tuesday.
Republicans also won all seven constitutional offices.
In 2012, Republicans won control of the Legislature for the first time since Reconstruction, clinching 51 seats. Afterward, lawmakers passed one of the strictest abortion laws in the country and approved a voter ID law — but both laws were later found unconstitutional.
Legislators also passed a bipartisan plan to expand health coverage to more than 200,000 people by using Medicaid money to buy private health insurance for poorer Arkansans.


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activate walls, DONT USE SPEED HACKS, INSTANT DEFUSE, BOXES, INSTANT .
Go into your Crossfire folder and go to rez
(May be different for others)
Ex. C:/Program Files/Z8Games/CrossFire/rez/
after this make a copy of RF002.REZ and RF016.REZ into a different folder
onto your desktop...
then extract the two modified REZ files provided and put them into your crossfire rez folder
go into the game and pwn some people
*Note* Make sure you have followed these steps or you may need to reinstall crossfire all over again...
These hacks contain:
- No reload for deagle, AK47/74, and some others guns (Not all guns)
- Chams/Weapon Chams (color black - don t complain, or just don t bother using the rez files)
- Flashbang removal (YAYYYYY no more crappy flashs from teamates that make you die)
Download Link:


text:
Ideo in una die venient plagae eius,
mors et luctus et fames et igne comburetur.
Vae, vae civitas illa magna Babylon, civitas illa fortis,
quoniam in una hora veniet iudicium tuum.
Cecedit Babylon magna
et facta est habitatitio daemoniorum,
quia de vino irae
fornicationis eius biberent omnes gentes
et reges terrae cum illa scortati sunt
et mercatores terrae de virtute eius
deliciarum divites facti sunt.
Vae, vae civitas illa magna
quae amicta erat bysso
et purpura et deaurata erat aura.
Post hac vox citharoedorum et musicorum,
tibia atque tuba canentium
non audietur in te amplius,
quia mercatores tui erant principes terrae
et in veneficiis tuis erraverunt omnes gentes.
Et sanguis Prophetarum
et Sanctorum in te inventus est.
Revelation 18: 7-9, 2, 3, 15, 21-24
So in a single day shall her plagues come,
death and mourning and famine and she shall be burned with fire.
Woe, woe to that great city Babylon, that strong city,
since in one hour shall your judgement come.
Fallen is Babylon the great
and become a dwelling place of demons,
because there shall all peoples drink of the wine
of the anger of her fornication,
and the kings of the earth have committed fornication with her
and the merchants of the earth have grown rich
with the wealth of her favours.
Woe, woe to that great city
that was clothed in fine linen
and purple and was decked about with gold.
After this there shall be heard no more in you
the sound of the harpers and minstrels,
of flute players and trumpeters,
because your merchants were the great men of the earth
and all nations were deceived by your sorcery.
And the blood of the prophets
and saints was found in you.
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text:
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text:
Earlier this year actor Saif Ali Khan and producer Ramesh Taurani had a big fall out. It was reported that for a new film with Taurani, Saif demanded Rs. 16 crores as fee. However Taurani did not agree and instead offered 10 crores and a percentage of the profit upon realization. The difference b
Sunil Shetty's store R House Launch
Ramesh s taurani News
The Government has come up with the decision of levying a service tax of 12.36% on television and film actors. However, Bollywood is expected to call a strike on the issue. Mukesh Bhatt who is the president of the 'Film and Television Producers Guild of India' said in an interview, "We are
Producer Pahlaj Nihalani's son's wedding reception took place in Mumbai on October 26, 2012 and many noted Bollywood celebrities attended the wedding reception. Abbas
Latest Bollywood News
- Suzanne-Mehr's friendship ends due to Arjun-Suzanne's affair
- Richa Chadha, Rajkumar Rao to host IFFI closing ceremony
- Tiger Shroff replaces Shahid Kapoor in 'Heer and Ranjha'
- Rajinikanth skips 'Kochadaiyaan' screening at the IFFI
- Shilpa Shetty meets Arnold Schwarzenegger in Macau


text:
When you think of exercise and fitness, what is the first thing that comes to mind? For me, it has always been running, probably because I marvel at folks who can hit the pavement and just go. Perhaps because it's something that I've never been comfortable doing (the jarring impact is too much for my joints), or perhaps it's the sleek runner's body that I so jealously admire.
The Four Types of Fitness
3 Reasons to Boost Your Protein Intake
As a Registered Dietician I get a lot of questions from friends about what they should be eating, and I've heard some pretty crazy things. One friend recently told me that in an effort to lose weight he's been eating fruits and vegetables exclusively for several weeks. Macronutrients are a vital part of a healthy diet; they alone will not sustain your body. While every body is different in its needs; however, a major component to all balanced diets is protein.
Understanding Stress and Managing Your Health
Do work, family or other circumstances have you feeling stressed? Does it bring inconsistency to your mood and energy levels? The root of the issue begins with stress hormones which can sabotage your health efforts when you're stressed.
The Question of Kale – Cooked or Raw?
Kale is one hot menu item these days. Everyone from A-list celebrities to athletes and politicians are raving about the health benefits of this dark, leafy, "king of the greens."
Are You Sabotaging Your Own Sleep?
Does it sometimes feel like every little thing interrupts your dream of a perfect night's sleep? While we know that having a lot on your mind and stress can keep you from getting all of the zzz's your body needs, did you know that you might be sabotaging your OWN dreamtime?
Meet Copper, the Micromineral with Big Impact
What do you know about copper? When you think of copper do you think of a shiny penny or perhaps the pretty green patina of the Statue of Liberty? Perhaps you think of electrical wiring or plumbing. Copper is so much more than an industrial metal or a medium for coins and art. Copper is an essential trace mineral that is imperative to life.
VIDEO: Getting Started With Interval Training
Fitness expert and Team BSN athlete Scott Herman* partners with Puritan's Pride to demonstrate a beginner's Interval Training routine.
VIDEO: Getting Started With A Bodyweight Routine
Fitness expert and Team BSN athlete Scott Herman* partners with Puritan's Pride to demonstrate a beginner's bodyweight exercise routine.
Eating for a Better Body Composition
Weight loss is a hot topic this time of year as people are donning their swimsuits and shedding layers to beat the heat by the pool and at the beach. Why is 'weight loss' the important topic of conversation rather than body composition? My personal training clients rarely see dramatic weight loss up front because the focus of my training and nutrition is to build lean muscle mass and create a healthy lifestyle.
Protein 101
Maybe it's time to reach your fitness goals once and for all, or perhaps you're just trying to maintain a healthy body. If your wellness goals include losing weight, building lean muscle mass is the best way to keep a strong and healthy body that will be prepared for any and all of life's challenges.


text:
Home » News
Judge reinstates suspended Rockingham County attorney
The decision will not immediately restore Reams to office. Judge Richard McNamara agreed to a 30 day stay of his decision so that the matter can be taken up by the state Supreme Court.
- Comedian Joan Rivers dead at age 81 says daughter - 0
- U.S. says Islamic State video of journalist's killing is authentic - 0
- No paper on Monday; check UnionLeader.com for updated, breaking news - 0
- Concord attorney Leahy dies - 0
Britain's Prince William and wife, Kate, expecting second babyREADER COMMENTS: 0
- Plaistow town planner arrested on forgery, fraud charges - 0
- Nashua man charged as drug enterprise leader; three others also arrested - 0
- Unlicensed employees cost Concord financial firm $100,000 - 0
- Woman killed in Nashua crash - 0
- Wednesday night Derry blaze under investigation - 0
- Weare residents turn out to discuss police department concerns - 0
- Manchester, Uber headed for a collision over taxi regulations? - 14
- Dave D'Onofrio's Patriots Notebook: Defense is Lions' weapon - 0
- UNH Notebook: Outright league title on UNH's agenda - 1
Claremont police investigate false report that locked down elementary schoolREADER COMMENTS: 1
Former mayor makes pitch for commuter rail
Shaheen, Ayotte split on Keystone vote
More details, photos emerge on man accused of murdering parents and burning down their home
Premiums drop, so do subsidies for Obamacare
VA wait times: They are still bad
Shaheen, Ayotte split on Keystone vote
King Obama: Creating a constitutional crisis
Premiums drop, so do subsidies for Obamacare
Former mayor makes pitch for commuter rail
VA wait times: They are still bad


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Indoor Pumps
Outdoor/Indoor Pumps
Fountain Pump and Light Combos
Lion's Head Peaktop Fountain Pump Parts
Lights For Fountains/Ponds
Mister Foggers
Pond and Outdoor Foggers
Misting Fountains with Lights
Tabletop Water Fountains
Hand Knit Accessories
Tupperware Products
Wholesale Fountain Supplies
Customer Testimonials
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©2009 Artistic Delights
Shop Our Huge Selection of Indoor and Outdoor Fountain Foggers, Lights & Pumps
Beautiful Misting Lighted Tabletop and Floor Fountains
OCTOBER SALES SPECIALS--NOW THROUGH NOVEMBER 30th.
Mist Maker single disk fogger with no light
--only $18.99 each, 2 for $36, 3 for $52!
WA65-S Mini Pump with On/off Switch
Regular price: $16.95
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AP75 (75 GPH) Indoor Fountain Pump
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Jebao JP900 145gph Outdoor Fountain Pump
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$18.95, 2/$36.00, 3/$51.00
Misting Floor Fountain in Frosted Blue
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(PT-505/606/HI-1000 Substitute)
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Sale price:
$24.95, 2/$48.00
Halogen Lights with
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Regular price: $19.95
Sale price:
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JP900L (145 GPH) Fountain Pump
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Regular price: $26.95
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offers the
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lowest prices
on indoor and outdoor fountain supplies. We carry submersible fountain pumps, all sizes of fogger/misters including outdoor models, as well as halogen fountain lights.
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We do NOT ship to CANADA.
E-bridge design


text:
The third Monday in January is popularly known in Hollywood circles as "hungover-as-shit Monday," as it immediately follows the Golden Globes, the award season's drunker, more badly-behaved little cousin. Among all the questions raised by the Golden Globes last night (What was the point of asking a ...Read More »
Michel Hazanavicius's "The Artist" and Alexander Payne's "The Descendants" led the winners 69th Golden Globe Awards, each winning a best picture prize and a best actor prize.Read More »
Okay, a few comments until our deeper analysis tomorrow. First thing, "The Descendants" -- while taking two awards -- dosen't jump to immediate frontrunner status. "The Help," with its civil rights theme, was never a movie that was going to connect in a big way with the ...Read More »
The Golden Globes went very much the way we all expected. The Weinsteins came out ahead thanks to the musical comedy category. They had six wins total, including "The Artist," best actor Jean Dujardin, and score, as well as actress Michelle Williams for "My Week with Marilyn" and...Read More »
Yes, indeed we are in the thick of awards season, kicking things off with one of our favorite awards shows, the wild and woolly Golden Globes. What's not to love about the Globes? Everyone's drunk, wandering from table to table, in a festive and jovial mood, Ricky Gervais is offending every...Read More »
Angelina Jolie has the name, but Pedro Almodovar and Asghar Farhadi were the panel's stars.Read More »
The Palm Springs International Film Festival announced this year's award winners today, chosen from a pool of 188 films from 73 countries.Read More »
As distributors continue the tradition of playing off the Golden Globes to publicize a range of films, the specialized and awards business still shows the positive trends that were seen during the holidays. A couple of significant expansions ("Pina" and "The Iron Lady") lead ...Read More »
The Golden Globes weekend is well under way. I love roaming a crowded party packed with Hollywood powerbrokers and talent to watch how they interact with one another. I am the ultimate voyeur. Of course I do talk to some of them occasionally.Read More »
Yet again, there's much to learn from the Golden Globe nominees for Best Foreign Language Film. Screen International's Mike Goodridge moderated the Cinematheque panel: Angelina Jolie ("In The Land of Blood and Honey," USA), Pedro Almodovar ("The Skin I Live In," Spain), A...Read More »


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Celebrity Archive
The Rock (As Hercules) Plays Patty Cake With A 2 Year Old GirlPosted on July 21, 2014The Rock on the set of Hercules plays patty cake with a 2 year old girl
Doug Benson The Trailer Park Boys And Snoop DoggPosted on July 21, 2014Doug Benson The Trailer Park Boys and Snoop Dogg – The highest photo ever taken
Steven Spielberg Must Be Stopped!Posted on July 20, 2014Steven Spielberg must be stopped!
20 Pics And Gifs Of Iggy Azalea BootyPosted on July 20, 2014So fancy! Everyone is now talking about Iggy Azalea's booty. Here are 20 pictures and gifs of Iggy Azalea's epic butt.
Brad Picked The Wrong GirlPosted on July 19, 2014Brad picked the wrong girl!
What Do Lawyers Wear To Court?Posted on July 18, 2014What do lawyers wear to court?
Drake Blows Into Lance Stephenson's Ear – GifPosted on July 17, 2014Drake blows into Lance Stephenson's ear – gif
Weird Al Is So Hot Right NowPosted on July 17, 2014Weird Al is so hot right now
Anything Is Possible For GodPosted on July 17, 2014Anything is possible for God – Dan Savage
Joaquin Phoenix's Forehead… When You See ItPosted on July 17, 2014Joaquin Phoenix's forehead… when you see it
Probably The Best Response To Steven Spielberg's Dinosaur 'Hunting' PhotoPosted on July 16, 2014Here's probably the best response to Steve Spielberg's dinosaur 'hunting' photo
90′s RockPosted on July 16, 2014I love 90′s Rock
The Different Types Of CommasPosted on July 16, 2014The different types of commas
Kid Takes Selfie With Warren Buffet And Paul McCartney (Sitting On A Bench In The BackgroundPosted on July 15, 2014Kid takes selfie with Warren Buffet and Paul McCartney (sitting on a bench in the background)
And Y'all Mad At LeBronPosted on July 13, 2014and y'all mad at LeBron
LeBron Is Going Back To Cleveland! Every Cleveland Fan Right Now…Posted on July 12, 2014LeBron is going back to Cleveland… every Cleveland fan right now…
GabeN's Famous One World EmailsPosted on July 11, 2014GabeN's famous one word emails
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Ļoti interesanta - ne nu gluži malnbalta, ne krāsaina.
A very beautiful light on these reflections,Ainars.Ciao Daniela
To Daniela: well, I get myself surprised too how this simple street looks under night light.
Exactly as Simka said, picture - not in full colour and not in pure b/w!
At the end of this street are car parking, with lot of light and motion. This are why that point looks little overexposed and yellow.
This view inspired me do more like this, when I will have time! :) Just need a lot of free time, big patience and seek for interesting landscape.
Cheers, Ainars.
Awesome shot!!
Hi, Slance1 thanks for spending time on browse my collection! Cheers, Ainars
Hey by the way, i thought this place kinda looked familiar :D i live SORTA nearby :D
Taking this photo for me was one of my experimental nights and now I see also - my successfull one too. :D Can I uncover one detail about this photo? I remeber, overall darkness at night was very deep, and it was not easy to set up camera parameters and put tripod enough accurate. So, I made whole serie of captures till finally get this one! :D
Photo taken in Uzvaras Parks, Zemgales priekšpilsēta, Riga, Latvia


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Films By Dena Curtis
Ethel and her friend Mary resort to an unlikely occupation at night to top up their pensions. Ethel's daughter is horrified when she discovers they are not really 'playing cards'. ... --- ... D/S Dena Curtis P Kath Shelper TD video/col/2007/6mins ... Read more
The joy of a young indigenous boy's birth disintegrates as it reveals a much darker truth. ... --- D Dena Curtis P Darren Dale S Rhea Stephenson WS Blackfella Films L Warrumungu w/English subtitles TD digibeta/2009 ... Read more


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Posted by ButterBall Devil on April 22, 11 at 11:52:22:
not only does the Baseball team pull out the extra inning win in Berkley, but the Softball team wins vs. au on a grand slam by Boyd in the top of the 7 inning.
Dallas Escobar - true frosh from (phx) St. Mary's - pitches a 1 hit shut out, with 11 K's in 7 innings.
impressive, to say the least.
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Party-goer didn't notice he'd been shot in head. For five years
He's not sure when it happened. Or how. But he does clearly remember that heading a soccer ball hurt for quite some time.
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There's drunk. And then there's "so hammered I didn't notice I'd been shot in the head" drunk.
A Polish man living in Germany was the latter. In either "2004 or 2005." He's not sure which.
The unnamed man was due in hospital this week to remove what he thought was a cyst on the crown of his head. Turns out, it was a .22-calibre bullet.
Shown the pea-sized projectile, the unnamed 35-year-old told police that he vaguely recalled a sharp blow to the head at a New Year's party some time before. He wasn't sure exactly what year, but told them it was about five years ago.
He did recall that he was extremely intoxicated at the time.
After being shot, the man staggered home. And did nothing.
"He told us he remembered having a sore head, but that he wasn't really one for going to the doctor," a police spokesperson told Reuters.
So the toughest man in western Europe went about his business. The wound healed, but he suffered from recurring pain.
That led him to hospital and his sobering discovery. He's expected to make a full recovery.
Police speculated that the bullet may have fallen into the man's head after being fired in the air in celebration.
They are not treating the shooting as suspicious. Mainly because the prime suspect is described as being very, very blurry.
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Nags Head House Rental Photos and Description
7 Bedroom, 7.5 Bath Room Comfortably Sleeps 18 (Jun 1-8 $2500)
Rocky's seven generous bedrooms each feature private baths and TVs. With 6 Queen beds, a group of couples can enjoy a wonderful vacation together without losing privacy and autonomy!
As you would expect in a vacation home of this caliber, Rocky features a generously sized private pool and hot tub, but the exterior of the home has been crafted to create an extension of the living space. Here, you'll find not just a grill, but an entire barbecue area complete with seating. This is a place to gather around and rekindle friendship and romance together.
You will also enjoy plenty of games to bring out the kid in everyone including horseshoes to keep everyone hoopin' and hollerin' for a while! When you're all worn out and have to seek shelter, you will find a recreation room complete with a pool table and kitchenette waiting for you on the lower level. Challenge everyone to a friendly game - last one inside fixes the margaritas!! The living room is VERY generously sized and extensively furnished, you will all be able to gather around and watch a movie on the home theater system together!
Not only is Rocky kid-friendly, it is also pet friendly! Your dog is welcome to join you in the Coastal Cove subdivision and enjoy his own vacation! There is plenty for everyone at Rocky, including a great Nags Head location, close to all the Outer Banks shops and restaurants you have come to know and love over the years. So, come on down and enjoy a spectacular vacation at Rocky!
The pool is 12x28. This house is located at mile post 14.5
Level 1: 1 Master Bedroom with Queen, 1 Bedroom with 2 Pyramids and Full Bath, Laundry, Rec Room with Pool Table and Kitchenette
Level 2: 4 Master Bedrooms with Queens
Level 3: 1 Master Bedroom with Queen and Jacuzzi Tub, Dining and Kitchen Area, Living Room, and 1 Half Bath
Bedding: 6 Queen, 2 Pyramid Bunk
NO LINEN, BOOKING OR CLEANING FEE..!! Convenient KEY LESS entry.
BEACH VOLLEY BALL COURT IS ADDED in 2013.
Available dates and rate
September 15 - September 20 - $1200 / 5 nights (minimum stay 3 nights $358 per night)
- Suitability:
- Events Allowed
- Minimum Age Limit for Renters
- children welcome
- non smoking only
- pets considered
Guestbook comments from the owner
Coordination of the rental was very smooth and seamless; check-in was the best with the keyless entry ... all was set up exactly as we expected with beds already made and linens provided. The house was awesome and a grand time was had by all!


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Added To Cart
Subtotal: $26.45
Items in Cart: 5
Give your bedroom the update it needs with this floral bedding set from North Shore Linens™! This set includes all the pieces you need to give your bedroom a fresh makeover. Featuring a floral and butterfly appliqué and embroidery, the set adds a lightness to your bedroom in light blue, green, red, yellow and white.
Full Size:
Comforter: 76" x 86"
Bed skirt: 54" x 75" + 15"
Pillow shams: 20" x 26" + 2"
One square decorative pillow: 16" x 16"
One breakfast decorative pillow: 14" x 14"
One neckroll decorative pillow: 6" x 14"
Queen Size:
Comforter: 88" x 88"
Bed skirt: 60" x 80" + 15"
Pillow shams: 20" x 26" + 2"
One square decorative pillow: 16" x 16"
One breakfast decorative pillow: 14" x 14"
One neckroll decorative pillow: 6" x 14"
King Size:
Comforter: 101" x 86"
Bed skirt: 78" x 80" + 15"
Pillow shams: 20" x 26" + 2"
One square decorative pillow: 16" x 16"
One breakfast decorative pillow: 14" x 14"
One neckroll decorative pillow: 6" x 14"
Machine wash cold on gentle cycle with mild detergent. Do not bleach. Line dry or tumble dry low using air fluff. Do not dry clean. Decorative pillows are spot clean only. Made from 100% polyester. Made in China.
PLEASE NOTE: We make every effort to ensure colors are represented accurately online. Due to possible variances in manufacturer dye lots, however, colors may vary slightly from what is depicted.
Quality bedding is often defined by the thread count of the fabric, whether it's a sheet, a comforter or a blanket. Thread count is the number of woven threads in one square inch of fabric. Higher thread counts create higher-quality weaves that generally feel smoother and softer. Lower thread counts can pill and feel rough, but are affordable and serve their purposes just fine.
Common thread counts range from 180 to 320, but thread counts as high as 700 also exist. The thread count alone doesn't determine how a fabric performs and feels, though. You'll also want to look at the yarn size and ply when determining the level of quality you desire.
Yarn Size: Yarn size refers to the thickness of the individual threads that make up a woven fabric. The higher the yarn size, the finer the thread. Quality sheets usually have a yarn size ranging from 40 to 100. Finer yarns can only be made from high-quality, long staple cottons such as Supima. Yarn size is important to thread count because finer yarns take up less space than thicker yarns, thus the highest thread count sheets are generally made from the finest yarns.
Ply: The other important component to thread count is ply. Single ply fabrics are made from individually woven threads of yarn, while two-ply fabrics are made from two threads of yarn that are first twisted together and then woven. Since two-ply fabrics have essentially double the thread count of their single ply counterparts, two-ply fabrics must be made from extremely fine yarns, otherwise the end product might feel too bulky and heavy.
*Free standard shipping offer available on purchases made with your ShopHQ Credit Card until 11:59pm ET, December 1, 2014. Excludes shipments to Alaska or Hawaii.May not be applied to previous purchases. Your discount will be applied at checkout. Subject to credit approval.


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2015 NCA National Specialty
"Turn the Tide"
2015 Newfoundland Club of America National Specialty
Crowne Plaza, Warwick, Rhode Island
May 5 – May 9, 2015
The Newfoundland Club of New England is honored to be your host for NCA's 2015 National, as we return to Warwick, RI.
Thanks to many volunteers, nearby regional clubs and NCA Committees, an event-filled week has been planned for you! There will be two draft tests, two obedience trials, two rally trials, four days of conformation judging, and many informative and educational events. We have also made sure to allow time to share a memorable week with friends old and new, and to celebrate our wonderful Newfoundland dogs!
The Crowne Plaza is our home away from home for the week. The experienced staff at the Crowne have been most helpful with our Specialty planning, and are looking forward to the return of the Newfs for a fourth National Specialty!
All events are held on the grounds of the hotel. All judging will take place outdoors and subject to the usual weather changes of New England, so please plan accordingly.
T.F. Green Airport, located just minutes of the hotel, is serviced by major airlines. Easy access to Interstate 95 makes an easy drive from locations up and down the east coast. Our overflow hotel is located an easy 5 minute drive from the show site. Many malls, restaurants, shops and a public golf course are nearby. Warwick is 10 minutes south of Providence and located within an hour or less from Boston, Plymouth Rock, Connecticut's casinos, Mystic Seaport, Rhode Island's beautiful beaches, and the mansions of Newport.
Our logo store will be up and running in time for your holiday shopping! Check back often for updates as we look forward to 2015!
Donna Thibault, Chair


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Good location; atmoshere is similar like fast food restaurant, but service is in perfect level; nice and helpfull staff; tasty chenese food; good value for money;
The price is very clean and the hairy crab meat dumpling is tasty, but the price is little bit high for me.
Don't miss the xialongbao here but other dishes also are great. We had the shrimp shu mai which was great. Service was good once we ordered which took a while but this was at the noon hour when it was hectic.
Enjoyed a great meal here... my friends took me here and told me that this is one of the best places to eat in Shanghai. And they were right!!!
Had some amazing dumplings and enjoyed great beer with some authentic food preparations.
Highly recommended.
Delicious Xiao Long Bao! This is what you come here for! Located in the super brand mall the second we walked up you are greeted by the little xiaolongbao mascot they have, cute little statue if you ask me! It was full of people but the wait was not too long! We ordered the xiao long bao and a couple...
I never had Dim sum before. What a treat. This restaurant was delicious. The green beans were wonderful. All of the dim sum was wonderful. Highly recommend!
I love Dim Sum, and especially Shanghai dumplings. I actually went here twice, it was so good. The menu is in both English and Chinese, which is great, and I had the pork dumplings, the chicken dumplings, and the pork and crab. Pork was my favorite.
The first thing you will notice is the cleanliness of the place with an open kitchen. The second thing will be the real kindness of the team! Very nice and smiley people! Then it is also about the food which is delicious! I really enjoyed the dim sum! The best is the black truffle pork or chicken dumpling! Try this...
Service is great. dumplings are authentic and yummy, with many other items on the menu that our western family loves. Views of the Bund from SuperBrand mall location are pretty. We usually try to go at off times because waits for a table can be long on holidays and peak dining hours, but they really try respect the customer. We...
Din Tai Fung in various locations in Beijing and Shanghai has been one of my favorite Chinese restaurants for 10 years. Their dumplings are wonderful. Our typical dinner consist of marinated cucumber, malantou (shepherd's purse?) in dry tofu skin, dumplings of all kinds, fried rice with shrimp and a glutinous rice dumpling filled with pork. There are many more interesting...
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3 days in Shanghai
Shanghai for young kids
Shanghai for free


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Biosketch / Results /
Kevin L Du, M.D., Ph.D.Assistant Professor;
Department of Radiation Oncology (Radiation Oncology )
Clinical AddressesPerlmutter Cancer Center
Handicap Access: yes
Phone: 212-731-5003
Medical SpecialtiesRadiation Oncology
Clinical ResponsibilitiesGastrointestinal Cancers, Metastatic Cancers to the Brain and Spine, Lung Cancers, Head and Neck Cancers, Prostate Cancers
InsuranceAETNA HMO, AETNA INDEMNITY, AETNA MEDICARE, AETNA POS, AETNA PPO/EPO, AFFINITY, AFFINITY EXCHANGE- ESSENTIAL, CIGNA EPO/POS, Cigna PPO, EBCBS EPO, EBCBS HLTHY NY, EBCBS HMO, EBCBS INDEMNITY, EBCBS MEDIBLUE, EBCBS PATHWAYS / PATHWAYS ENHANCED, EBCBS POS, EBCBS PPO, ELDERPLAN, FIDELIS CHLD HLTH, FIDELIS EXCHANGE, FIDELIS FAM HLTH, FIDELIS MEDICARE, Fidelis Medicaid, GHI CBP, HEALTHPLUS CHLD HLTH (AMERIGROUP), HEALTHPLUS FAM HLTH (AMERIGROUP), HEALTHPLUS MEDICAID (AMERIGROUP), HEALTHREPUBLIC, HIP ACCESS I, HIP ACCESS II, HIP CHLD HLTH, HIP EPO/PPO, HIP HMO, HIP MEDICAID, HIP MEDICARE, HIP POS, LOCAL 1199 PPO, MAGNACARE PPO, METROPLUS CHLD HLTH, METROPLUS EXCHANGE PLANS, METROPLUS FAM HLTH, MULTIPLAN/PHCS PPO, Medicare, MetroPlus Medicaid, NY MEDICAID, NYS EMPIRE PLAN, OSCAR, OXFORD EXCHANGE, OXFORD FREEDOM, Oxford Liberty Oxford Medicare, Tricare, UHC COMMUNITY & STATE PLAN, UHC EPO, UHC HMO, UHC MEDICARE, UHC POS, UHC PPO, UHC TOP TIER, UNITED EXCHANGE- COMPASS
Insurance Disclaimer: Insurance listed above may not be accepted at all office locations. Please confirm prior to each visit. The information presented here may not be complete or may have changed.
Board Certification2012 — Ab Radiology - Radiation Oncology
Education1996-2006 — University of Pennsylvania School of Medicine, Medical Education
2006-2007 — Hospital of the University of Penn. (Internal Medicine), Internship
2007-2011 — Hospital of University of Pennsylvania (Radiation Oncology), Residency Training


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The present invention provides an FK506 binding protein gene containing a base sequence coding for the amino acid sequence shown under SEQ ID NO:1, in particular such a FK506 binding protein gene containing the base sequence shown under SEQ ID NO:2, a method of producing a recombinant FK506 binding protein through expression of such gene, and the recombinant protein thus produced.
The use of the gene of the present invention enables FK506 binding protein expression, and the protein is useful particularly in elucidating the mechanism of immunosuppression in living bodies or in developing or screening out therapeutic agents for autoimmune diseases (e.g. rheumatism, SLE, etc.), among others.
1. An isolated purified FK506 binding protein gene which comprises bases 70-393 of SEQ ID NO:3 .
2. A method of producing a recombinant FK506 binding protein which comprises introducing a base sequence containing the FK506 binding protein gene of claim 1 into a host to thereby transform said host, cultivating the thus-obtained transformant, and recovering the recombinant FK506 binding protein thus produced.
3. An expression vector which contains a gene as claimed in claim 1.
4. A host cell which is transformed with a vector as claimed in claim 3.
5. A recombinant FK506 binding protein produced by the method as claimed in claim 2.
6. An isolated and purified FK506 binding protein gene which comprises a base sqeuence coding for the amino acid sequence of SEQ ID NO:2.
7. An expression vector which contains a gene as claimed in claim 6.
8. A host cell which is transformed with a vector as claimed in claim 7.
9. A method of producing a recombinant FK506 binding protein which comprises introducing a base sequence containing the FK506 binding protein gene of claim 6 into a host to thereby transform the host, cultivating the thus-obtained transformant, and recovering the recombinant FK506 binding protein thus produced.
10. A recombinant FK506 binding protein which has the amino acid sequence of SEQ ID NO:2.
The present invention relates to an FK506 binding protein gene.
FK506 is a potent immunosuppressant and, like cyclosporin A [Borel, J. G. et al., Agents and Actions, 6, 468-471 (1976)], it is often used for preventing allograft rejection [Thomson, A. W., Immunol. Today, 10, 6-9 (1989)]. It is considered that the mechanism of its immunosuppresant action is in focus on the suppression of translation, during T cell activation [Tocci, M. et al., J. Immunol., 143, 718-726 (1989)], of a series of lymphokine genes which is critical to early immune responses. However, the precise mechanism remains unclear.
Recently, an AF506 binding protein was found in human T cells. It was identified as the 12 kDa cytosolic receptor of FK506 and shown to have peptidylprolyl cistrans isomerase activity (PPIase activity) [Siekier, J. et al., J. Immunol., 143, 1580-1583 (1989)].
Subsequently, on the basis of its amino acid sequence, a number of FK506 binding proteins (FKBPs) and corresponding cDNA clones have been isolated and the nucleotide sequences of said clones have been determined [for example, human FKBPs such as FKBP-12 (Standaert, R. F. et al., Nature, 346, 671-674 (1990); Maki, N, et al., Proc. Natl. Acad. Sci. USA, 87, 5440-5443 (1990)), FKBP-13 (Jin, Y. et al., Proc. Natl. Acad. Sci. USA, 88, 6677-6681 (1991)), FKBP-25 (Wiederrecht, G. et al., Biochem. Biophys. Res. Comm., 185, 298-303 (1992)) and FKBP-52 (Peattie, D. A. et al., Proc. Natl. Acad. Sci. USA, 89, 10974-10978 (1992)) as well as murine FKBP (Nelson, P. A. et al., Gene, 109, 255-258 (1991)), bovine FKBP (Mozier, N. M. et al., Eur. J. Biochem., 194, 19-23 (1990)), yeast FKBP (Heitman, J. et al., Proc. Natl. Acad. Sci. USA, 88, 1948-1952 (1991)), Neurospora crassa FKBP (Tropschug, M. et al., Nature, 346, 674-677 (1990)), Neisseria meningitis FKBP (Sampson, B. A. et al., Proc. Natl. Acad. Sci. USA, 89, 1164-1168 (1992)), etc.].
The above-mentioned FKBPs all have PPIase activity, which is presumably essential for protein folding during protein synthesis in cells [Ficher, G. et al., Biomed. Biochim. Acta, 43, 1101-1111 (1984)]. Although no clear evidence is available to prove that said PPIase activity is necessary for T cell activation, binding of FK506 to FKBPs is believed to bring about inhibition of PPIase activity and thereby inactivate T cells [Tropschug, M. et al., Nature, 346, 674-677 (1990)].
Accordingly, it is an object of the present invention to provide a novel FK506 binding protein (FKBP) gene, more particularly a novel FKBP gene which is highly homologous to the above-mentioned human FKBP-12 and whose expression product protein has high PPIase activity.
As a result of their intensive investigations, the present inventors succeeded in isolating, from a human fetal brain cDNA library, a novel gene coding for a protein highly homologous to the human FK506 binding protein 12 kD (FKBP-12) and in determining its full-length cDNA sequence. Based on these results, they have now attained the present invention.
Thus, the present invention is directed to an FK506 binding protein gene coding for an amino acid sequence defined by the sequence shown under SEQ ID NO:1 or SEQ ID NO:2 in the sequence listing.
The abbreviations for amino acids, peptides, base sequences, nucleic acids and so forth as used herein in the present specification are those recommended by the International Union of Pure and Applied Chemistry (IUPAC) and the International Union of Biochemistry (IUB) and in the "Guidelines for drafting patent specifications relative to base sequences and/or amino acid sequences" edited by the Japanese Patent Office or those commonly used in the relevant field of art.
The gene of the present invention includes genes characterized in that they comprise an open reading frame consisting of a 324 nucleic acid sequence coding for the 108 amino acid residues as shown under the above-mentioned SEQ ID NO:1.
The gene of the present invention also includes genes characterized in that they comprise an open reading frame consisting of a 240 nucleic acid sequence coding for the 80 amino acid residues as shown under the above-mentioned SEQ ID NO:2.
The novel FK506 binding proteins encoded by the genes of the present invention are characterized by high PPIase activity.
Although the gene of the present invention is represented by a single-stranded DNA sequence, as shown under, for example, SEQ ID NO:3, the present invention also includes the DNA sequence complementary to such a single-stranded DNA sequence as well as a component comprising both of these. The DNA sequence representing the gene of the present invention shown in the above-mentioned SEQ ID NO:3 is an example of the codon combination coding for the respective amino acid residues according to the amino acid sequence shown under the above-mentioned SEQ ID NO:1. The gene of the present invention is not limited to the above-mentioned one but may, of course, have any other DNA base sequence comprising a combination of codons arbitrarily selected for the respective amino acid residues without altering the above-mentioned amino acid sequence. Selection of said codons can be carried out by the conventional method in which the codon usage or codon choice in the host to be used for gene recombination is taken into consideration [Nucl. Acids Res., 9, 43-74 (1981)], and these codons can be produced, for example by chemical synthesis, etc.
The gene of the present invention further includes DNA sequences coding for those equivalents to the above-mentioned amino acid sequence that are derived from the latter by deletion, addition or like modification of one or more amino acid residues or part of the amino acid sequence and have similar PPIase activity to that of the FK 506 binding protein. While production, alteration (mutation) or the like of these polypeptides may occur spontaneously, they can also be produced by posttranslational modification. Furthermore, any desired gene can be produced by gene engineering techniques such as the site-specific mutagenesis technique in which the natural gene (gene of the present invention) is altered, by a chemical synthesis technique such as the phosphite triester method in which mutant DNAs are synthesized or by combining both procedures.
By utilizing the gene of the present invention, namely by incorporating the same into a vector for use with a microorganism, for instance, and cultivating the transformant microorganism, the FK506 binding protein can be expressed readily and in large quantities, and said protein can be isolated and provided. Since said protein has PPIase activity, it is effective for various pharmacological purposes, not to speak of T cell activation, and it is also useful, among others, in elucidating the pathogenesises, the pathologies or the like of various diseases. More specifically, since the recombinant FK506 binding protein obtained by utilizing the gene of the present invention is an immunosuppressant binding protein, it can effectively be used, for example, in elucidating the mechanism of immunosuppression in living bodies, developing or screening out therapeutic agents for autoimmune diseases (e.g. rheumatism, SLE (systemic lupus erythematodes), etc.), searching for endogenous ligands to the novel binding protein and developing therapeutic agents therefor.
In the following, the gene of the present invention will be described in more detail. The gene of the present invention can be isolated by general genetic engineering techniques, for example, by selecting an appropriate clone from among a human fetal brain cDNA library (cDNA synthesized in the conventional manner from mRNA isolated and purified from total RNA obtained in turn from appropriate origin cells containing a gene coding for the FK506 binding protein) using appropriate probes, purifying said clone, and determining the base sequence thereof. In this way, the gene of the present invention can be obtained.
In the above procedure, the origin cells may be any animal cells or tissues where the occurrence of an FK506 binding protein is known, or soluble fractions of cultured cells derived therefrom. This can be isolated and purified for the culture supernatant by various chromatographic processes.
Said origin cells capable of FK506 binding protein expression can be cultured by the conventional culture method using an appropriate cell culture medium. Examples of the medium that can be used in this case are RPMI 1640 medium, CEM medium, CMRL-1066 medium, Dulbecco's modified Eagle's minimum essential medium (Eagle's MEM), Fisher's medium, F-10 medium and the like. Where appropriate, serum, such as fetal calf serum (FCS), and/or serum components, such as albumin, may adequately be added to these medium. Cultivation can be carried out in the conventional manner, for example by the carbon dioxide incubator method, generally at about 30 to 40� C., preferably at about 37� C., for about 5 to 17 days, preferably for about 8 to 11 days.
Separation of total RNA from said cultured cells or tissues can be effected by a conventional extraction method. This extraction procedure is preferably carried out at a due time when the FK506 binding protein production and accumulation in the culture supernatant resulting from the above-mentioned culture is maximal. Total RNA extraction from said origin tissue or cultured cells can be carried out as follows. When an origin tissue is used, the tissue is disrupted in an appropriate buffer solution, such as potassium phosphate buffer, supplemented with EDTA, DTT (dithiothreitol) or the like, under ice cooling, then partially or completely disrupted and solubilized by using a guanidine-isocyanate mixed solution or an appropriate surfactant such as SDS, NP-40, Triton X-100 or deoxycholic acid, or by physical means such as a homogenizer or freezing and thawing and, thereafter, chromosomal DNA is cut to a certain extent using a Polytron mixer (Kinematica, Switzerland) or a like mixer or a syringe, followed by separation into a protein fraction and a nucleic acid fraction. For the last-mentioned procedure, in particular, phenol-chloroform extraction or the cesium chloride density gradient method using ultracentrifugation at about 100,000�g [Chirgwin, J. M., et al., Biochemistry, 18, 5294 (1979)], among others, can be generally employed. In each of the processes mentioned above, it is advisable to prevent RNA decomposition otherwise caused by RNase by adding an RNase inhibitor, such as heparin, polyvinylsulfuric acid, diethyl pyrocarbonate, vanadium complex, bentonite, macaloid or the like.
Separation and purification of mRNA from the RNA obtained by the above-mentioned extraction process can be carried out, for example by subjecting the extract to an adsorption column, such as an oligo(dT)-cellulose (Collaborative Research Inc.), poly(U)-Sepharose 2B (Pharmacia) or Sepharose (Pharmacia) column, or by a batch method.
The purified mRNA obtained as mentioned above is generally unstable. Therefore, it is reversely transcribed to the stable complementary DNA (cDNA) form and the latter is joined to a replicon of the microorganism origin for enabling amplification of the desired gene. The above-mentioned in vitro transcription of mRNA to cDNA, namely cDNA synthesis, can be carried out generally in the following manner.
First, using oligo(dT) (either free oligo(dT) or oligo(dT) already joined to a vector primer) as primer, and the mRNA as template, single-stranded CDNA complementary to the mRNA is synthesized based on the latter using reverse transcriptase in the presence of dNTPs (DATP, dGTP, dCTP and dTTP). The next step differs depending on whether free oligo(dT) is used or oligo(dT) joined to a vector primer is used, as follows.
In the former case, the mRNA used as template is removed by decomposition with alkali treatment, for instance, and, then, double-stranded DNA is produced using the single-stranded DNA as template, together with reverse transcriptase or DNA polymerase. Both ends of the thus-obtained double-stranded DNA are treated with exonuclease, and an appropriate linker DNA or a plurality of bases whose combination allows annealing is joined to each, followed by insertion into an appropriate vector. This can be carried out, according to the vector used, by a conventional method, for example by the Gubler-Hoffman method. For the above-mentioned cDNA synthesis, a commercially available cDNA synthesis kit may also be used. The use of such a kit is advantageous in that the procedure becomes simple and easy. The vector to be used here is not limited to any particular species. It is recommendable, however, to select it suitably from among λgt phage vectors, EK plasmid vectors and the like, either singly or in combination, according to the host to be used. As the λgt phage vectors, there may be mentioned λgt10, λgt11, etc. The process dealing with these λgt phage vectors can be carried out by the method of Young et al. [Young, R. A., et al., in DNA Cloning, 1, 49 (1985)].
In the latter case, while the mRNA used as template is retained, the mRNA-cDNA hybrid, a linearized plasmid with a plurality of bases combined so as to enable annealing as mentioned above, and a linker DNA (often used is a DNA fragment containing a region capable of autonomous replication in animal cells and a transcription promoter region of mRNA) are together subjected to annealing to give a circularized product. Then, the mRNA is replaced with a DNA chain in the presence of dNTPs, RNase H and DNA polymerase I, to give a completed plasmid DNA.
The DNA obtained in the above manner can be introduced into an appropriate host of the vector, for example Escherichia coli, Bacillus subtilis, Saccharomvces cerevisiae or the like, to thereby transform the host. Employable for this DNA introduction into host for transformation are those methods that are generally used, for example the method comprising collecting cells mostly at the logarithmic growth phase, treating them with CaCl2 to place in a condition suited for ready spontaneous taking up of the DNA, and allowing the plasmid to be taken up. In the above process, Mgcl2 or RbCl mayor RbCl may be caused to be present so that the transformation efficiency can be further improved, as is generally known. It is also possible to employ the method comprising converting microbial cells to spheroplasts or protoplasts, followed by subjecting these to transformation. Details of these methods are described by Gubler and Hoffman [Gubler, U. and Hoffman, B. J., Gene, 25, 263 (1983)]. In cases where λ phage, which is generally and quite often used as a phage vector, is used, a cDNA library in X phage can be constructed by in vitro packaging. Commercially available cDNA libraries, for example various cDNA libraries available from Clontech, can also be used as said CDNA library.
Screening of the thus-obtained cDNA library for the gene of the present invention can be performed in the conventional manner. Thus, as the screening method, there may be mentioned, for example, the method comprising selecting the corresponding cDNA clone by Western blotting using an antibody specific to the FK506 binding protein against the protein produced by cDNA, the Southern blotting method which uses a probe selectively binding to the desired DNA sequence, the Northern blotting method, and the combination of these. As regards the probe to be used here, DNA sequences or the like chemically synthesized based on the information concerning the desired DNA or RNA sequence or the amino acid sequence encoded thereby are generally used, among others. DNA or RNA derived from the nature can also be used as such a probe.
In more detail, the above-mentioned probe is prepared in the following manner. Thus, using an oligo(dT)-cellulose column, poly(A)+ RNA is selected from among RNA obtained from a tissue or cultured cells containing the FK506 binding protein, single-stranded cDNA is synthesized by the method mentioned above and, after the reaction is terminated, the single-stranded cDNA is amplified by the PCR method [Saiki, R. K., et al., Science, 230, 1350-1354 (1985)] using primers estimably corresponding to information about the amino acid sequence of parts of the FK506 binding protein using an automatic oligonucleotide synthesizer.
Then, the amplified cDNA fragment is isolated and purified by 1.0% agarose gel electrophoresis. The base sequence of the thus-obtained DNA fragment can be determined in the conventional manner. For example, after the DNA fragment obtained is digested with an appropriate restriction enzyme, sequencing can be performed by the dideoxy method [Sanger, F., Nicklen, S. and Coulson, A. R., DNA sequencing with chain-terminating inhibitors, Proc. Natl. Acad. Sci. USA, 74, 5463-5467 (1977)] or the Maxam-Gilbert method [Maxam, A. M. and Gilbert, W., Methods in Enzymology, 65, 499 (1980)], for instance. Furthermore, said base sequence determination may also be readily carried out using commercially available sequencing kits or the like.
The thus-determined full-length base sequence of a DNA containing the gene of the present invention is as shown in the sequence listing under SEQ ID NO:3.
In the practice of the present invention, it is also possible to use a part of the DNA fragment sequenced in the above manner as a probe, label this using a random prime DNA labeling kit (available from Takara Shuzo, Amersham, etc.) in accordance with the random prime DNA labeling method [Feinberg, A. P., et al., Anal. Biochem., 137, 266-267 (1984)], for instance, and use the thus-obtained labeled probe in screening out the desired FK506 binding protein gene.
Using the above-mentioned labeled probe, for instance, the desired DNA can be screened out by the laque hybridization technique developed by Benton and Davis [Benton, W. and Davis, R., Science, 196, 383-394 (1977)].
The gene of the present invention as obtained in the above manner can be cloned in various plasmids in the conventional manner. For instance, after cleavage with an appropriate restriction enzyme and purification, the gene of the present invention can be inserted into a cloning vector (e.g. plasmid) cleaved with the same restriction enzyme and purified, at the cleavage site thereof, whereby a recombinant plasmid can be obtained. By introducing said recombinant into an appropriate host (e.g. Escherichia coli) for transformation, a restriction enzyme map of the clone containing said gene can be drawn using the transformant by a conventional known method, for example the method described in Molecular Cloning (A Laboratory Manual), T. Maniatis, E. F. Fritsch and J. Sambrook, Cold Spring Harbor Laboratory (1982), pages 104-106. After digestion of the above clone with an appropriate restriction enzyme, the base sequence of said clone can be determined by the above-mentioned dideoxy method or the Maxam-Gilbert method, for instance. The base sequence determination mentioned above may also be readily performed using a commercially available kit or the like.
The thus-determined DNA base sequence of the FK506 binding protein gene of the present invention and the corresponding amino acid sequence encoded thereby are as shown in the sequence listing under SEQ ID NO:1 and SEQ ID NO:2.
Using the above-mentioned gene (DNA) of the present invention, the recombinant FK506 binding protein can be obtained by various known gene recombination techniques [cf. for example Science, 224, 1431 (1984); Biochem. Biophys. Res. Comm., 130, 692 (1985); Proc. Natl. Acad. Sci. USA, 80, 5990 (1983)].
Said FK506 binding protein is produced, in more detail, by constructing a recombinant DNA allowing expression of the gene of the present invention in host cells, introducing this into host cells for transformation thereof, and cultivating the transformant strain. The host cells may be either eukaryotic or prokaryotic. As an expression vector for use with vertebrate cells, it is possible to use one containing a promoter generally located upstream of the gene to be expressed, an RNA splicing site, a polyadenylation site and a transcription termination sequence and so on. This may further have a replication origin, as necessary. Yeasts are often and generally used as eukaryotic microorganisms and, among them, yeasts belonging to the genus Saccharomyces are advantageously used. Usable as expression vectors for use with said yeasts and other eukaryotic microorganisms are pAM82 [A. Miyanohara et al., Proc. Natl. Acad. Sci. USA, 80, 1-5 (1983)] containing a promoter for the acid phosphatase gene, and like vectors. Escherichia coli and Bacilus subtilis are generally and very often used as prokaryotic host cells. When these are used as hosts in the practice of the present invention, an expression plasmid is preferably used which is derived, for instance, from a plasmid vector capable of replication in said host microorganisms and provided with a promoter, the SD (Shine and Dalgarno) base sequence and further an initiation codon (e.g. ATG) necessary for the initiation of protein synthesis, upstream from the gene of the present invention so that said gene can be expressed. As the hose Escherichia coli mentioned above, the strain Escherichia coli K12 and the like are often used and, as the vector, pBR322 is generally and often used. However, the host and vector are not limited thereto, but other various known microbial strains and vectors can also be used. As regards the promoter, the tryptophan (trp) promoter, 1 pp promoter, lac promoter and PL promoter, for instance, can be used.
The thus-obtained desired recombinant DNA can be introduced into host cells for transformation thereof by various conventional methods. The transformant obtained can be cultivated in the conventional manner, leading to production and accumulation of the desired FK506 binding protein encoded by the gene of the present invention. The medium to be used in said cultivation can adequately be selected, according to the host cells employed, from among various media in common use. When Escherichia coli or like cells are used as host cells, for instance, transformant cultivation can be conducted using LB medium, E medium, M9 medium, M63 medium or the like. To these media, there may be added, as necessary, generally known various carbon sources, nitrogen sources, inorganic salts, vitamins, nature-derived extracts, physiologically active substances, etc. The above-mentioned transformant cultivation can be carried out under conditions suited for the growth of the host cells. In the case of Escherichia coli, such conditions can be employed, for instance, as a pH of about 5 to 8, preferably 7 or thereabout, and a temperature of about 20 to 43� C., preferably 37� C. or thereabout. In the above manner, the transformant cells produce and accumulate intracellularly or secrete extracellularly the desired recombinant FK506 binding protein.
Said desired protein can be isolated and purified by various separation techniques utilizing its physical, chemical and other properties [cf. for example "Seikagaku (Biochemistry) Data Book II", pages 1175-1259, 1st edition, 1st printing, published Jun. 23, 1980 by Kabushiki Kaisha Tokyo Kagaku Dojin; Biochemistry, vol. 25, No. 25, 8274-8277 (1986); Eur. J. Biocehm., 163, 313-321 (1987)]. As specific examples of said techniques, there may be mentioned conventional reconstitution treatment, treatment with a pretein precipitating agent (salting out), centrifugation, osmotic pressure shock treatment, ultrasonic disruption, ultrafiltration, various liquid chromatographic processes such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography and high performance liquid chromatography (HPLC), dialysis, and combinations of these. In the above manner, the desired recombinant protein can be produced on an industrial scale with ease and with high efficiency.
FIG. 1 schematically shows the base sequence of OTK4(6-1) (top) and that of OTK4(4-1) (bottom).
FIG. 2 shows the results of analysis of the OTK4 protein by SDS-PAGE.
FIG. 3 shows the PPIase activity of the OTK4 protein.
FIG. 4 shows the results of Northern blot analysis of various tissues using the OTK4 gene.
FIG. 5 shows the results of RT-PCR analysis of various tissues to investigate the expression of OTK4(4-1) and OTK4(6-1).
Examples will be described below to illustrate the present invention in more detail.
(1) Cloning and sequencing
From a human fetal brain cDNA library (Clontec, CA; Uni-ZapII vector), a 1.0 kb clone highly homologous to the human FK506 binding protein 12 kDa was selected in the conventional manner and designated OTK4.
Using the above-mentioned OTK4 as probe, the same CDNA library (about 1 million plaques) was screened, and two further cDNA clones designated OTK4(6-1) and OTK4(4-1), respectively, were obtained. The DNA sequence of each of these three cDNA clones was determined by the dideoxy termination method using 32 S-dTTP.
As a result, it was revealed that OTK4 and OTK4(6-1) have one and the same nucleic acid sequence. The full nucleic acid sequence of OTK4(6-1) is as shown in the sequence listing under SEQ ID NO:3.
This clone has an open reading frame consisting of a sequence of 324 nucleic acid residues coding for the 108 amino acid residues shown under SEQ ID NO:1. Its 5' noncoding region comprising a sequence of 72 bases contains a GC-rich sequence and its 3' noncoding region comprising a sequence of 470 bases contains the poly-adenylation signal AATAAA, followed by a poly(A) tail.
On the other hand, the other clone OTK4(4-1) encodes the 80 amino acid residues represented by SEQ ID NO:2 and this is supposed to be a form derived from OTK4(6-1) by splicing. Said OTK4(4-1) has a sequence of 45 bases coding for 14 amino acid residues and the termination codon as inserted in the coding region of OTK4(6-1) at a site of the 199th base (behind Ala at position 66) and, as a result, codes for the above-mentioned 80 amino acid residues.
Comparison based on the structural analysis of FKBP-12 [Van Duyne, G. D. et al., Science, 252, 839-842 (1991)] revealed that OTK4(4-1), as compared with OTK4(6-1) having five beta-sheets, is lacking, in its C-terminal region, two beta-sheets and one loop between said sheets.
Both of the above clones are schematically shown in FIG. 1. In the figure, arrows indicate the PCR primers FK-1, FK-2 and FK-3 to be later mentioned herein, and "EX" indicates the insertion region of the above-mentioned sequence of 45 bases.
(2) Homology with other FKBPs
OKT4(6-1) showed the highest homology with human FKBP-12. Thus, its homology with human FKBP-12 was found to be 76% in terms of nucleic acid sequence and 88% in terms of amino acid sequence.
Furthermore, it showed 49.5% (97 amino acid residues) homology, 44.6% (92 amino acid residues) homology and 38.0% (108 amino acid residues) homology with human FKBP-52 [Peattie, D. A. et al., Proc. Natl. Acad. Sci. USA, 89, 10974-10978 (1992)], FKBP-13 [Jin, Y. et al., Proc. Natl. Acad. Sci. USA, 88, 6677-6681 (1991)] and FKBP-25 [Wiederrecht, G. et al., Biochem. Biophys. Res. Comm., 185, 298-303 (1992)], resepctively.
(3) Recombinant protein production
The full coding redion of the OTK4(6-1) cDNA was amplified by the PCR method using, as primers, FK-1 and FK-2 shown below, which have a BamHI site.
TABLE 1______________________________________Primer Base sequence______________________________________FK-1 (SEQ ID NO:4) 5'-GTGGATCCGCTATGGGCGTGGAGAT-3' - FK-2 (SEQ ID NO:5) 5'-AAGGATCCGTCCCAGTGGCAGACAG-3' - FK-3 (SEQ ID NO:6) 5'-TGATTCATCCAGAGACAGAA-3'______________________________________
The PCR product was digested with BamHI and cloned in the PGEX2T expression vector (Pharmacia) at its BamHI site.
The recdombinant FK506 binding protein (OTK4 protein) of the present invention was produced and purified by causing it to be expressed as a GST-fused protein in Escherichia coli DH5 according to the method described in the literature [Ayer, D. E. et al., Cell, 72, 211-222 (1993)].
Thus, a colony of the Escherichia coli strain transformed with the PGEX2T expression vector obtained as mentioned above was inoculated into 20 ml of LB/ampicillin medium and incubated at 37� C. for 12 to 15 hours.
The culture was diluted (1:10) with 200 ml of fresh LB/ampicillin medium. After 1 hour of incubation, IPTG was added (1 mM) and incubation was further continued for 3 to 5 hours. Cells were pelleted at 3,000 rpm, resuspended in 5 ml of cold lysis buffer, lysed by sonication and then incubated on ice for 1 hour. The cell lysate was centrifuged at 3,000 rpm, and the supernatant was added to Glutathion Sepharose 4B (Pharmacia) and incubated at 4� C. for 1 hour. The mixture was centrifuged at 300 rpm for 5 minutes and the supernatant was discarded. The Glutathione Sepharose 4B was washed three times with PBS and once with PBS-T (PBS containing 1% Triton X).
To obtain the full length OTK4 protein without the GST tag, the GST-fused protein was cleaved with thrombin. The Glutathione Sepharose 4B was washed with washing buffer (50 mM Tris-HCl (pH 7.5)/150 mM NaCl), 900 ml of thrombin cleavage buffer (washing buffer containing 2.5 mM CaCl2) and 100 ml of thrombin (Sigma) were added to the Glutathione Sepharose 4B, and the mixture was incubated at 25� C. for 1 hour and then centrifuged at 300 rpm. The thus-obtained OTK4 protein (supernatant) was stored at -80� C.
(4) SDS-PAGE, Coomassie blue staining and protein assay The OTK4 protein was electrophoresed on a 15% polyacrylamide gel and then analyzed by Coomassie brilliant blue staining [Wilson, C. M. et al., Methods Enzymol., 91, 236-247 (1983)].
The results are shown in FIG. 2. In FIG. 2, lane 1 is for the total cell protein obtained without the above-mentioned IPTG induction; lane 2 is for the total cell protein obtained with said IPTG induction; lane 3 is for the GST-OTK4 fusion protein after purification treatment; and lane 4 is for the OTK4 protein after cleavage treatment.
The OTK4 protein purified in the above manner was quantitated by the Lowry method [Lowry, O.H. et al., Nature, 337, 476-478 (1989)] to be 500 mg protein per ml.
(5) Demonstration of PPIase activity
The enzyme activity of the purified protein was determined by the method described in the literature [Fisher, G. et al., Nature, 337, 476-487 (1989)] as modified to measure the isomerization (cis to trans) of the proline-alanine peptide bond in the peptide (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide).
The trans form of the above peptide is readily cleaved with chymotrypsin, releasing p-nitroanilide which can be quantitated by absorbance measurement at 405 nm.
The reaction mixture (1.0 ml) contained the above-mentioned peptide substrate (30 μl taken from a 2.1 mM stock solution; final concentration 0.1 mM), 100 mM Tris-HCl (pH 7.8) and 1.0 μg/ml or 10 μg/ml of the OTK4 protein, and the reaction was carried out at 25� C.
One minute later, 30 μl of 100 mM Tris-HCl (pH 7.8) containing 2 mg/ml of chymotrypsin (Sigma) was added and mixed. Ten seconds after the mixing and thereafter, the absorbance at 405 nm was measured on a spectrophoto-meter at 10-second intervals.
The results obtained are shown in FIG. 3 [ordinate =absorbance (405 nm); abscissa=time (�10 seconds)]. In FIG. 3, the curve containing open circles indicates the results obtained with 1.0 gg/ml of OTK4 protein; the curve containing closed circles indicates the results obtained with 10 μg/ml of OTK4 protein; and the curve containing closed triangles indicates the results obtained with a control without OTK4 protein.
As seen in said figure, a marked increase in absorbance were observed in the OTK4 protein groups as compared with the control, and activity levels dependent on the dose of OTK4 protein were observed.
(6) Northern Blot Analysis of Various Organs Levels of expression of the OTK4 gene in various tissues were examined using Clontec's mRNA (human multiple tissue Northern blot system).
Thus, 4 hours of prehybridization and 18 hours of hybridization were carried out at 50� C. in a solution containing 50% formamide, 10�Denhardt's solution, 5�SSPE, 2% SDS and 100 μg of denatured salmon sperm DNA.
The probe used was 32 P-labeled OTK4 cDNA. Washing was performed with 2�SSC/0.05% SDS at room temperature for 10 minutes (three times) and then with 0.1�SSC/0.1% SDS at 50� C. for 15 minutes (two times), followed by 60 hours of exposure at -80� C.
The results are shown in FIG. 4. In the figure, lane 1 and the succeeding lanes correspond, in that order, heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon and peripheral blood leukocyte. On the utmost left, there are shown mRNA size markers (in kb).
From said figure, it is evident that OTK4 gene mRNA expression is observable in various tissues.
(7) RT (reverse transciptase)-PCR Analysis of Various Organs
Using the random primer p(dN)6 (Boehringer Mannheim GmbH), 10 μg of each organ RNA was reverse-transcribed, followed by amplification by PCR using the primers FK-1 and FK-2 (cf. Table 1). The PCR reaction conditions were as follows: 40 cycles with the regime: 94� C. for 0.5 minute, 54� C. for 1 minute, and 72� C. for 1 minute per cycle.
In the conventional manner, each product was then subjected to 2% agarose gel electrophoresis, transferred to a nylon membrane, and hybridized at 50� C. with terminally 32 P-labeled FK-3 (cf. Table 1). After washing with 6�SSC at room temperature for 10 minutes and further at 50� C. for 10 minutes, the membrane was autoradiographed for detection, with acting as control.
The results thus obtained are shown in FIG. 5.
The following or agns were used:
liver, kidney,
skeletal muscle,
ileum and
From said figure, it is evident that both OTK4(4-1) and OTK4(6-1) are expressed ubiquitously although the expression of OTK4(4-1) is superior to that of OTK4(6-1).
The present invention provides an FK506 binding protein gened. The use of said gene makes it possisble to produce the FK506 binding protein with ease and in large quantities, and said protein, which has PPIase activity, can be used in developing therapeutic agents for various diseases.
__________________________________________________________________________# SEQUENCE LISTING - - - - (1) GENERAL INFORMATION: - - (iii) NUMBER OF SEQUENCES: 6 - - - - (2) INFORMATION FOR SEQ ID NO:1: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 108 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: - - Met Gly Val Glu Ile Glu Thr Ile Ser Pro Gl - #y Asp Gly Arg ThrPhe 1 5 - # 10 - # 15 - - Pro Lys Lys Gly Gln Thr Cys Val Val His Ty - #r Thr Gly Met Leu Gln 20 - # 25 - # 30 - - Asn Gly Lys Lys Phe Asp Ser Ser Arg Asp Ar - #g Asn Lys Pro Phe Lys 35 - # 40 - # 45 - - Phe Arg Ile Gly Lys Gln Glu Val Ile Lys Gl - #y Phe Glu Glu Gly Ala 50 - # 55 - # 60 - - Ala Gln Met Ser Leu Gly Gln Arg Ala Lys Le - #u Thr Cys Thr Pro Asp 65 - # 70 - # 75 - # 80 - - Val Ala Tyr Gly Ala Thr Gly His Pro Gly Va - #l Ile Pro Pro Asn Ala 85 - # 90 - # 95 - - Thr Leu Ile Phe Asp Val Glu Leu Leu Asn Le - #u Glu 100 - # 105 - - - - (2) INFORMATION FOR SEQ ID NO:2: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 80 amino - #acids (B) TYPE: amino acid (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: protein - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: - - Met Gly Val Glu Ile Glu Thr Ile Ser Pro Gl - #y Asp Gly Arg Thr Phe 1 5 - # 10 - # 15 - - Pro Lys Lys Gly Gln Thr Cys Val Val His Ty - #r Thr Gly Met Leu Gln 20 - # 25 - # 30 - - Asn Gly Lys Lys Phe Asp Ser Ser Arg Asp Ar - #g Asn Lys Pro Phe Lys 35 - # 40 - # 45 - - Phe Arg Ile Gly Lys Gln Glu Val Ile Lys Gl - #y Phe Glu Glu Gly Ala 50 - # 55 - # 60 - - Ala Gln Leu Gly Pro Leu Ser Pro Leu Pro Il - #e Cys Pro His Pro Cys 65 - # 70 - # 75 - # 80 - - - - (2) INFORMATION FOR SEQ ID NO:3: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 879 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (iii) HYPOTHETICAL: NO - - (iv) ANTI-SENSE: NO - - (vi) ORIGINAL SOURCE: (A) ORGANISM: Human fet - #al brain cDNA library - - (vii) IMMEDIATE SOURCE: (B) CLONE: OTK4(6-1) - - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 70..393 - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: - - CGCTGGGCCG GAGCCGAGCC GGGGTCGGGC AGCAGCAGGA CCCCCAGAGG CG -#GGGCCTGT 60 - - GGGACCGCT ATG GGC GTG GAG ATC GAG ACC ATC TCC - # CCC GGA GAC GGA 108 Met Gly Val Glu Ile - #Glu Thr Ile Ser Pro Gly Asp Gly 1 - # 5 - # 10 - - AGG ACA TTC CCC AAG AAG GGC CAA ACG TGT GT - #G GTG CAC TAC ACA GGA156 Arg Thr Phe Pro Lys Lys Gly Gln Thr Cys Va - #l Val His Tyr Thr Gly 15 - # 20 - # 25 - - ATG CTC CAA AAT GGG AAG AAG TTT GAT TCA TC - #C AGA GAC AGA AAC AAA204 Met Leu Gln Asn Gly Lys Lys Phe Asp Ser Se - #r Arg Asp Arg Asn Lys 30 - # 35 - # 40 - # 45 - - CCT TTC AAG TTC AGA ATT GGC AAA CAG GAA GT - #C ATC AAA GGT TTT GAA252 Pro Phe Lys Phe Arg Ile Gly Lys Gln Glu Va - #l Ile Lys Gly Phe Glu 50 - # 55 - # 60 - - GAG GGT GCA GCC CAG ATG AGC TTG GGG CAG AG - #G GCG AAG CTG ACC TGC300 Glu Gly Ala Ala Gln Met Ser Leu Gly Gln Ar - #g Ala Lys Leu Thr Cys 65 - # 70 - # 75 - - ACC CCT GAT GTG GCA TAT GGA GCC ACG GGC CA - #C CCC GGT GTC ATC CCT348 Thr Pro Asp Val Ala Tyr Gly Ala Thr Gly Hi - #s Pro Gly Val Ile Pro 80 - # 85 - # 90 - - CCC AAT GCC ACC CTC ATC TTT GAC GTG GAG CT - #G CTC AAC TTA GAG39 - #3 Pro Asn Ala Thr Leu Ile Phe Asp Val Glu Le - #u Leu Asn Leu Glu 95 - # 100 - # 105 - - TGAAGGCAGG AAGGAACTCA AGGTGGTGGC TGGAGATGGC TGCTGCTCAC CC -#TCCTAGCC 453 - - TGCTCTGCCA CTGGGACGGC TCCTTGCTTT TGGGGCTCTT GATCAGTGTG CT -#AACCTCAC 513 - - TGCCTCATGG CATCATCCAT TCTCTCTGCC CAAGTTGCTC TGTATGTGTT CG -#TCAGTGTT 573 - - CATGCGATTC TTGCTTGAGG AAACTTCGTG CAGATTAAGC ATTCAAGGTT GT -#GCATTTTG 633 - - TGTGATGCAG TAGTAGCCTT TCCTGATAAC AGAACACAGA TCTCTTGTTC GC -#ACAATCTA 693 - - CACTAGCCAT TACCTTCACA TTAAACCACA CACAAGGTGC TCAGACATGA AA -#TGTACATG 753 - - GCGTACCGTA CACAGAGGGA CTTGAGCCAG TTACCTTTGC TGTCACTTTC TC -#TCTTATAA 813 - - ATTCTGTTAG CTGCTCACTT AAACAATGTC CTCTTTGAGA AAATGTAAAA TA -#AAGGCTCT 873 - - GAGCTT - # - # -# 879 - - - - (2) INFORMATION FOR SEQ ID NO:4: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: - - GTGGATCCGC TATGGGCGTG GAGAT - # - # 25 - - - - (2) INFORMATION FOR SEQ ID NO:5: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: - - AAGGATCCGT CCCAGTGGCA GACAG - # - # 25 - - - - (2) INFORMATION FOR SEQ ID NO:6: - - (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base - #pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - - (ii) MOLECULE TYPE: cDNA - - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: - - TGATTCATCC AGAGACAGAA - # - # - # 20__________________________________________________________________________
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The Star Poker Summer Series is back from November 27th - December 8th at The Star Casino.
Crown Resorts Ltd. announces proposed $60 million Sydney Arts Fund.
Crown Resorts Ltd. have been granted permission to build a casino in Sydney, Australia.
Jackie Glazier has won the WSOP Europe €1,100 Ladies Event for €21,850 and her first WSOP gold bracelet.
The 2013 Adelaide Casino Poker Championships have been announced ...
The Australia and New Zealand Player of the Year has been awarded to Japan's Iori Yogo ...
Billy "The Croc" Argyros took down the 2013 APPT Melbourne Main Event after a three-way deal at the final table.
The UK's Ashley Mason leads the final table of the 2013 APPT Melbourne Main Event.
David Yan leads 42 survivors after Day 2 of the 2013 APPT Melbourne Main Event.
Yanji Ge leads after Day 1b of the 2013 APPT Melbourne Main Event.
John Lombardo leads after Day 1 of the 2013 APPT Melbourne Main Event. Joe Hachem among 65 survivors.
The World Poker Tour announces that Lynn Gilmartin will replace Kimberly Lansing as anchor on FOX Sports Net.
The 2013 APPT Melbourne Main Event begins on Friday 6 September.
PokerNetwork takes a look at same facts and figures from the PokerStars World Championship Of Online Poker.
The 2013 Federal Election is around the corner and it could spell bad news for online poker in Australia.
Following a successful 2013 pre-season tour, Arsenal Football Club welcomed Bodog as Official Asian Betting Partner in a 3...
Daniel Laidlaw won the 2013 Sydney Poker Champs Main Event his weekend.
Crown Casino in Melbourne, Australia has announced the dates and schedule for the 2014 Aussie Millions.
Jonathan Bredin defeated a field of 126 players to be crowned the ANZPT Queenstown Main Event champion.
The ANZPT Queenstown Main Event reaches its final table. Jonathan Bredin leads the last nine players.
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Artpolitic, q-tip, ears, brain, thoughts, intelligence, autistic, crazy, insane, stab, kill, death, head, smart, freak, Cool, funny, stylish, genius
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Archive for February, 2012
// 3 Ways to Make Thanksgiving Less About You
byon November 24, 2014
Thanksgiving is fast approaching. More than likely you've already started making plans. As you begin to finalize those plans, let me challenge you to expand your vision of what Thanksgiving could be. Here are three ideas to get you thinking: Open up the guest list. For many of us, Thanksgiving is a family-focused holiday. We look […]
// Consider the Steadfast Love of God // Psalm 107
byon November 23, 2014
// Missional Community is All of the Above
byon November 19, 2014
What in the world is a Missional Community?! For those exploring the Missional Community world, or slowly learning to live in it (like our Remedy people), this is a great post. It gives a very good fly-by of the breadth and depth of what a strong, healthy Missional Community can be. Be sure to read […]
// Engaging the Mission Pt. 2 // Matthew 13
byon November 16, 2014
// Engaging the Mission Pt. 1 // Acts 4.18-20
byon November 9, 2014
// 5 Practices to Help Busy People Make Time for Mission
byon October 22, 2014
This post was written by Stephanie Williams and taken from the 3DM leadership blog. It has been adapted slightly in the formatting, but all content is original and unchanged. Have you ever had something dawn on you in a way that hit you like a ton of bricks? This was my experience just a few […]
// Our Family Has A Mission // John 17.17-24
byon October 19, 2014
// We are a Spiritual Family // Ephesians 2.11-22
byon October 12, 2014
// Growing in Spiritual Maturity, pt. 2 // Colossians 1.28-29
byon October 5, 2014
// Responsibilities of Shepherds and Sheep // 1 Peter 5.1-5
byon September 28, 2014