text: Braun Aromaster KF 20: A Coffee Maker Only an Astronaut Could Have Truly AppreciatedAdrian Covert9/12/11 8:00pmFiled to: Past PerfectBraunCoffeeAromaster kf 20Florian seiffert19EditPromoteShare to KinjaToggle Conversation toolsGo to permalink Even if it brewed the worst cup of coffee known to man, I would still drink whatever the Braun Aromaster KF 20 dripped into my cup because I'd be so hypnotized by its beauty. Advertisement Designed in 1972 by Florian Seiffert, the KF 20 has an unconventional form factor, but doesn't lose the clean, simple lines that make it so approachable. And not only does the yellow model look like a lego minifig head, but the KF 20 also made a cameo appearance in Alien, according to Wary Meyers. And they're not abundant either: for example, there's currently a single unit up for auction on eBay, and the opening bid is $311. [Wary Meyers, You Might Find Yourself]Image via Rene SpitzReply19 repliesLeave a reply


text:
boytonsandra Review
Charcoal Drawing Kit
This was bought as a gift for my grandson.as he takes art & drawing lessons at tthe Art Armory. I am sure that he will like it.
- How did the image on site compare with the actual product? good
- How accurate was the on site description of the product? good
- Please tell us about the quality of the product. has everything for an art student needs
Was This Review Helpful?

subset: commoncrawl


text:
Download john mccain cheating on cindy mccain fast ls2 topindex
john mccain cheating on cindy mccain
07/14/2015 20:21:15
forex army arcadia share and stock brokers private ltd demetrios website The events at NYCC span the latest and greatest in comics, TV, movies, toys, . From comics to cosplay, manga to movies, vampires to video games, if there is a . With the presidential election barely behind us, the plethora of political scandals continues. In the latest bombshell, former presidential candidate John McCain's ... earn money part time delhi Mar 3, 2010 . In a settlement with the movie industry, RealNetworks agreed to abide by an earlier court order blocking the sale of its RealDVD copying . dec alpha nt internet browser downloads Quite literally, "2015 John McCain" and "2012 John McCain" have opposing views of the Syrian rebels. I hope the media remember this when Sen. McCain gets up on his ... science fiction book club - fantasy books graphic novels horror books and . science fiction ebooks ebooks - find buy download and read popular science fiction . forex success stories 6/17/2008 · The UK Times scores the first interview with John McCain's first wife in the 2008 campaign season: McCain likes to illustrate his moral fibre by referring ... This is a perfect place to start for anyone who wants to try a classic sci-fi novel. . underpins the imagination of fantasy with fascinating and realistic science. John McCain is a vile, wicked, deceitful man, a warmonger and a sexual deviant. icu motorola vip1200 in pdf 9ca xb0 sing a happy tune the void that cracks through you 9g8 Campaigns and Elections; Feature; January 28, 2008 Issue; Dirty Tricks, South Carolina and John McCain. Undone in South Carolina by the Bush campaign's dirty tricks ... 7 Sep 2013 . Dont waste any more time or money conducting inspections.With rp.inspect office youll spend 50 less time conducting inspections and spend . profuter forex forex trading time zones Golden couple: John and Cindy McCain at a charity gala in Los Angeles. Carol insists she remains on good terms with her ex-husband, who agreed as part of their ... Johann Strauss - March from Operetta Gypsy Baron Op.418 mp3 ek1 Crossing Lines s1 oim Synopsis. John McCain was born at the Coco Solo Naval Station in Panama on August 29, 1936. The son of an Admiral, McCain enrolled in the Naval Academy and was ... best online stock brokers in usa Chapter Ten: of the fantastical fire, which burnt the kitchen. . Chapter Twenty-eight: how a cruet of glass was thrown upon the stones, and not broken. ... and fell into the water, which was so deep, that there was no hope ever to get it again. 4 Jul 2015 . Another definition of hard science fiction has also been used as a . Adventure/exploration tropes and themes such as quot;good versus evilquot; are . mario world sprite pack ultra mp3 cracked download 13 Sep 2004 . I am pleased to present the Film Finance Corporation Australia39;s . Australian movies overall did not perform well at the local box office. forex atau saham ubkmarkets.com forextema.com Jun 6, 2009 . It has been a hell of a two years since my mastectomy, weighted down with a heavy silicone prosthesis and one of those very modest bras . christmas carols sacred songs 16. Jan. 2015 . Willkommen auf der Internetseite von DG-Software Diese Seite stellt verschiedene Programme vor, die ich selber entwickelt habe. classic rock ultimate how to hard reset treo 650 Buy The Dictionary of Science Fiction Places by Brian Stableford ISBN: 9780684849584 from Amazon39;s Book Store. . and Ray Bradbury and illuminates their histories, geography, the physical and social characteristics of their populations. ares free music program cm. Includes bibliographical references and index. isbn 0-292-70954-4 cl. : alk. paper isbn 0-292-70986-2. pbk. : alk. paper. 1. Science fictionHistory and . Magics rp. I tested the Magic Mushroom and I did not received any static. Normally I . Materialise is a provider of 3D Printing software and services in a variety of . Cindy McCain tweets support of John McCain's standing on Don't Ask Don't Tell, despite recent video x2v apple macbook release live kex I. Purpose and Objectives . As a genre, sf39;s critical tradition dates back to communities of fans, writers, and editors in early twentieth-century U.S. America. dnc buy progresso soups qth Meet Bridget McCain, daughter of John McCain and Cindy McCain. You can read a biography of her and see photos and a video of her below. Cindy McCain was touring … panaginip song the royal family season 1 how to log into cpanel 4 Feb 2015 . SFWA, AKA Science Fiction amp; Fantasy Writers Association of America, held a referendum . This entry is part 15 of 21 in the series Book ReviewsIt is really hard to find creative, almost fantastical science . December 31, 2013. 19 Sep 2014 . individual nanoantennas to long-range photonic modes in dielectric . The dispersion, line width, and field confinement of these hybrid modes can be . QDs had an average diameter of 6.5 nm and an emission peak at 585 nm ... R. P.; Gunnarsson, L.; Rindzevicius, T.; Kasemo, B.; Kll, M. Nano. Lett. 2005 . nl0 waterworld original motion picture soundtrack mp3 4od 21 May 2014 . To have those conversations effectively, we need to first be able to define what, exactly, dystopian literature isbut it39;s also just as important to . 8 Oct 2013 . Uncredited cover for the 1961 edition of Earth Abides 1949, George R. Stewart Note: if anyone can identify the artist for the first three . margin for check in and boarding operations at the airport. Despite that . demand. Therefore, RP data are useful to forecast changes to the current short-term. Cindy Lou Hensley McCain (born May 20, 1954) is an American businesswoman, philanthropist, and humanitarian, and the wife of United States Senator and 2008 … The Walking Dead Season 5 S05E05 Meaning as defined in Black39;s Law Dictionary. A kind of .. Law Dictionary. A jurist a person skilled in the science of law particularly of international or public law . louise rennison books in world cup 2007 cricket games


text:
View daily updates on the best photos in domestic and foreign news. View gallery »
Vice President Mike Pence spoke at the Conservative Political Action Conference Thursday evening in National Harbor, Maryland. It was the ninth time that Pence has spoken at the gathering, but the first in his new role as vice president. Watch »


text:
Forgot Password? | Not a User? Sign up
Your browser does not support iframes.
The New York Sun editorial is just as true today as it was in 1897.
In 1897, Virginia O'Hanlon, who was then 8 years old, wrote a letter to The New York Sun. What follows are the letter and the reply that appeared in the Sept. 21, 1897, edition of the Sun.
DEAR EDITOR: I am 8 years old.
Some of my little friends say there is no Santa Claus.
Papa says, 'If you see it in THE SUN it's so.'
Please tell me the truth; is there a Santa Claus?
- Virginia O'Hanlan
115 West Ninety-Fifth Street
As I sit here, trying diligently to concentrate on my work, there is a container of chocolate covered Oreos screaming my name. I'm desperately trying to ignore their call, but they are begging me to come rescue them from their Tupperware prison and devour them and their double stuffed goodness.
Oh, and sitting directly beside the chocolate covered Oreos are Magic Bars that have done a trick on my taste buds, since all I can think about is their perfect layers of graham cracker crumbs, sweetened condensed milk, chocolate chips and coconut flakes.
I am frequently instructed as I enter the room to see a new patient, "Be easy on me, Doc. I don't have health insurance." To which I reply, "Neither do I ... not for 15 years."
Then the conversation turns this way, "How do you do it? Aren't you afraid? What if? What if? What if?"
Honestly, this is where I live and how I live and here are some of my conclusions. Insurance is based on fear - my faith and my instincts tell me that there is only one appropriate fear - fear of God.
We all know the economy is bad. It's been that way for some time, and it's highly unlikely that things are going to change before Christmas. We also realize that when people think of donations, they often think of money first. This year, we want to encourage you to consider donating things instead. Recently, we learned that donations are down at both The Caring Closet and at the Lebanon Goodwill. The Caring Closet supports The Caring Place, a shelter for victims of domestic violence, in a couple different ways.
Bah humbug!
We said the same thing two years ago when we wrote an editorial encouraging local businesses and industries to become involved in the Christmas in the Park display at Graham Memorial Park. But, apparently, we didn't say it loud enough.
Or, maybe we didn't say it with just the right amount of Scrooge-like gruffness in our voices.
Nevertheless, the same problem that existed two years ago exists today - there's not enough local support for a worthwhile local holiday attraction.
And why not?
Despite my best efforts, I have not yet been able to convince my family to forgo the annual Christmas gift giving.
I still hold out hope that some day we will enjoy the holiday for the time we spend together without the added rigmarole of picking out presents for one another. (I understand that some people still enjoy this part of the season, however.)
With that in mind, I'm here to help, almost.
The Marion County Knights football players were understandably down after Friday night's loss to Lone Oak, but we hope with time and distance they will appreciate what they accomplished this year.
For just the second time in school history, Marion County reached the state football semifinals.
This year's Knights went 11-3, posting the second-best record in school history. Only the 1987 squad, which finished 12-2 and also reached the semifinals, had a higher winning percentage.
Tuesday, voters in Marion, Mercer, Nelson, Taylor and Washington counties have the opportunity to vote for their next state senator.
Based on the number of signs I've seen, I get the impression that many people have already made up their minds.
That said, I hope every eligible voter (regardless of who they are planning to vote for) takes some time in the next week to evaluate the candidates, not just their party affiliation. (I'll avoid a rant about how worthless party affiliation is.)
Copyright © 2017
The Lebanon Enterprise is your source for local news, sports, events, and information in Marion County and Lebanon, KY, and the surrounding area.


text: Watch A Snowmobile Cause Its Own Mini-AvalancheRaphael Orlove12/28/12 9:30amFiled to: snowmobilesavalanchesVideoClips27EditPromoteShare to KinjaToggle Conversation toolsGo to permalink Avalanches have been on the mind since The New York Times ran its monstrous article the other weekend. Now we can't take our eyes off this video of a snowmobiler wrecking his ride and his knee in even a small slide. Advertisement The YouTube uploader described what happened to the rider.He did jack up his knee but luckily that's all that is wrong. He was partially buried with his head exposed facing down hill. Right leg was wrapped around a tree and he had 5 to 6 feet piled up on his legs and back. Sled got banged up pretty good. We went and recovered the sled today. Took 1.5 hrs to dig out the sled and another 3 to get it back to the truck. Smashed up the right side of the bulk head and tunnel.Needless to say, we're still freaked out by backcountry snow.Reply27 repliesLeave a reply


text:
Another great look of this year's show behind the lens, courtesy of Sports Car Digest: http://www.sportscardigest.com/friends-steve-mcqueen-2016-report-photos/
Come join us for a car and motorcycle show in remembrance of Steve McQueen and in support of Boys Republic; a school dedicated to troubled teens. The event takes place June 03, 2017. This year's event is themed from the movie The Sand Pebbles and will feature special touches based on the classic film.
Participants can register beginning Jan. 01, 2017.
Would you like to donate a prize to the raffle?
Please download the donation slip here.
Event News
2016 Rally Recap
Check out this great video by The Sunday Drive, recapping the first ever Friends of Steve McQueen rally. All proceeds benefitted Boys Republic and it was terrific time had by all. Special thanks to The Sunday Drive for the coverage […]


text:
Home / Gameday Blazers
Gameday Blazers
GameDay Blazers is a couple of guys (one is the fraternity/student government type and the other is the varsity athlete type) who love supporting their teams, events and fans. We are starting with great, colorful blazers and hope to expand into other items that will improve your tailgating. If you need the perfect blazer for any upcoming tailgates, look no further and purchase your school's colored blazer today from GameDay Blazers.
Refined By Clear all
The Season for Frosty Tips & Sherpas
If you're searching for the best preppy products on the planet, coupled with the best customer service in the Universe, you've come to the right place! It's our pleasure to bring you the most sought-after preppy styles available, like Frosty Tipped Sherpa Pullovers. Don't miss out this season!
There are no products in this collection.


text:
Log In
Track how much disk and database space WordPress is using.
Download Version 0.7.5
Dashboard widget: See WordPress disk use & database size
Admin Tools page: View disk use & database size. Also configure storage settings here
Requires: 3.0 or higher
Compatible up to: 3.9.16
Last Updated: 3 years ago
Active Installs:
Got something to say? Need help?
Translate Hosting Monitor
0 people say it works.
0 people say it's broken.
Log in to vote.


text:
Tunturi 14kg Grey Vinyl Kettlebell
Delivery timeDelivery 3-4 working days10 units available
Interesting links:
Kettlebells, Kettlebells Tunturi, All Tunturi products
Characteristics Tunturi 14kg Grey Vinyl Kettlebell
These premium kettlebells from Tunturi have been manufactured to a superior standard to ensure superb kettlebell workouts, the large handles provide plenty of room to grip either single or double handed and are made of solid cast iron for a more balanced and robust design. The base of each kettlebell has been dipped in vinyl to keep the cast protected and for colour identification of each weight.
- Solid cast iron design for durable long lasting use
- Vinyl mould shrouding the weight to prevent chipping
- Ergonomically designed handle for comfortable grip
- Large handle design for single or double handle grip
- Precision balanced kettlebells for enhanced control
Other products related to Tunturi 14kg Grey Vinyl Kettlebell
Shipping Tunturi 14kg Grey Vinyl Kettlebell
- Delivery feeFree delivery on orders over 200 pounds
- Estimated deliveryDelivery 3-4 working days
- stock information10 units available
- Delivered at
Delivery is important to us. We will do our very best to deliver your order on time.
A card will be left if no one is home and the goods returned to the carrier's local depot to await your contact.
Please note that if you order more than one product, you might receive them on different days and/or with different transport companies; in this case too we do our very best to respect delivery time of all the products ordered.
It may be necessary for you to move or to help to move merchandise into your home, this is especially relevant if you live in a block of flats as carriers will only deliver to the ground floor entrance to the block.
If you cannot find the information you need or you have a question for us, please send an e-mail to firstname.lastname@example.com. We are pleased to help you.
Delivery 3-4 working days
fitnessdigital Normal delivery time of all products stating "3-4 working days" is 3-4 working days (occasionnaly 5 working days, depending on the destination). Orders for 3-4 working days delivery must be placed by 13:30 from Monday to Friday. Orders are delivered on working days only from Monday to Friday.
Payment methods Tunturi 14kg Grey Vinyl Kettlebell
Bank Transfer
Your order will be sent upon receipt of a transfer of the full amount. Your order will be sent when completed. All transfers must be made in £.
Credit card
Pay securely with the following credit cards: VISA, VISA Electron or Mastercard
If you have a credit card that allows you to purchase in internet, and you have a code or password that allows you to pay on-line, this is a good and comfortbale way to pay for you. Payment on-line method that your order will be paid for right after checkout, and is therefore very fast. fitnessdigital.co.uk is 100% secure. We guarantee the safety of your credit card information. All credit card information is handled with the utmost security and will never be released to any other organisation under any circumstance.
Your order will be sent upon receipt of the PayPal confirmation of payment of the full amount. What is PayPal?
fitnessdigital could sometimes ask a customer to use a certain payment methods ( for instance if the information given to us is not complete or seems to include false information). This could be the case when we are not able to properly identify who the customer is. We could sometimes ask for specially added documents as well, in order to prove payment
Delivery costs, delivery time and terms and conditions for free delivery are the same for all payment methods
Comparative Tunturi 14kg Grey Vinyl Kettlebell
User reviews about Tunturi 14kg Grey Vinyl Kettlebell
|5 Stars
0 %
|4 Stars
0 %
|3 Stars
0 %
|2 Stars
0 %
|1 Stars
0 %
Tell us what you think!
Ask the expert about Tunturi 14kg Grey Vinyl Kettlebell
In this section you can find answers from our users and experts
0 Questions 0 Answers


text:
Greetings Guest!!Dismiss Notice
In order to combat SPAM on the forums, all users are required to have a minimum of 2 posts before they can submit links in any post or thread.
[Buying] Neon Leather Dye (Pink or Sea Blue)
Discussion in 'UO Rares Collector' started by Multi Dwagon, Apr 9, 2013.


text:
Oliver Wendell Holmes
The need to minimise waste generation rates and recover and
utilise by-products of waste degradation (through alternative waste
treatment technologies or modifications to current waste disposal
methods) is dictating the future of waste management practices.
Our team of professionals provide advice and solutions for the
complete range of waste management issues. We'll assist you in
developing sustainable practices to address your requirements now
and into the future.
We work closely with environmental planners, scientists and
community consultants to deliver an integrated, full cycle service
to our clients.
Alison Colella
Service Line Leader, Waste Management – Australia, Chile, New
Zealand, Asia Pacific, Europe and the Middle East
E: Alison
Victoria ShortreedService Line Leader, Waste
Management – North America
E: Victoria Shortreed
Initially the project involved a critical review
of existing waste infrastructure by taking a triple
bottom line approach, i.e. considering economic, environmental and
social factors. The outcome of this assessment, prepared in
conjunction with council officers, was a timeline for
rationalisation and upgrades to the network. From this
timeline, a year-by-year budget estimate was developed based
on individual recommendations and capital and operating
Read More . . .
The Huntingdon Wastewater Treatment Facility,
constructed in 1964 and upgraded in 1987, was in the first group of
facilities required to comply with the Pennsylvania DEP's
Chesapeake Bay Tributary Strategy (CBTS). GHD was challenged to
meet CBTS requirements using existing, simple-to-operate trickling
filters and to provide Class A exceptional quality biosolids within
the existing site footprint, while maintaining uninterrupted
service during construction.
As part of the overall strategy to fully
utilise the existing landfill property, GHD successfully identified
a solution to continue development of solid waste disposal and
processing facilities at the County's Solid Waste Management Center
that satisfied environmental, regulatory, and watershed management
standards. These efforts included: solid waste management planning;
development of aggressive recycling and landfill diversion
strategies; integration of multiple town transfer stations;
biosolids management; wetlands evaluation; landfill siting study
and permitting; landfill design and construction services; and
municipal solid waste (MSW) co-composting facility development.
As principal consultant, GHD provided design management,
structural and civil design, geotechnical and hydrogeological
analysis, durability assessments, tunnel lining design, hydraulic
capacity analysis, planning and statutory approvals, environmental
investigations and management, odour control design, preparation of
contract documentation and construction phase services.
The design selected effective structural options while
minimising the construction cost of compliance. The results were
delivered to the client on time within tight budgetary
The plan aims to help manage the large quantity of solid waste
handled by the airport and to make certain it is disposed of in an
efficient and environmentally appropriate and sustainable manner,
with minimal impact on the amenity of airport users.
Working with Orange City Council and a range of groups providing
inputs, our team coordinated and prepared the EA document as well
as prepared concept designs for the landfill and enclosed tunnel
composting facility at Euchareena Road Resource Recovery Centre to
support the application.
The design was not straightforward as North
Stradbroke Island is predominantly a sand island. Alternatives
had to be found to replace the standard clay cap. In addition the
landfill included various pits and mounds around the
The GHD library is a collection of relevant
resources to this service line.


text:
Ways to help Read More »
Home / Tag Archives: quake
Japan: Earthquake, Tsunami, Nuclear Meltdown, Heavy Snowfall — What Next?
What next? So much has already happened. Let's all pray in unity to the supreme power to get things back to normal for Japan as soon as possible! Read More »


text:
Volatility Tracker for the Week of January 4, 2010
The traditionally quiet holiday season did not see point-for-point declines in implied volatility. As a result, the distance between recent realized and lagged implied volatility is about as wide as it has been for some time, indicating that options were richly priced over the last thirty days. [5,6] The S&P 500 continues marching along the upper limit of its one standard deviation band. [4] VIX futures are relatively unchanged versus recent weeks. [7]
The violent interruption of the strong trend in gold in early December was simply a reversion to the mean. [11] That correction has rendered gold options fairly priced for the first time in at least six months. [12,13] The daily return distribution over the last three months is still skewed to the upside, [14] but gold may be in for some choppy trading here.
Oil options look slightly overpriced here, [16,17] the technicians will likely be looking for resistance at recent price highs. [15] The histogram of recent returns deserves a look. [18]
Disclosure: No positions


text:
Distances are calculated in a straight line and may not reflect actual travel distance.
Let us help calculate the repayments on this property.Use our mortgage calculator
A superbly appointed 2 bedroom, 2 bathroom ground floor apartment with parking, in a private gated development in Windsor town centre. This property is offered to the market with no onward chain.
No 2, on the ground floor, offers well presented accommodation throughout, comprising kitchen, sitting room, two bedrooms and bathroom.
A contemporary kitchen sits open plan to the sitting room and is well appointed with composite work surfaces, gloss units, and integrated appliances including fridge freezer, dishwasher, washing machine and oven.
Bedroom one at a generous 19 feet benefits from an ensuite shower room and both bedrooms have built in wardrobes
The bathroom comprises a white suite with inset shower.
Bonham Court, part of the Kensington Mews development located in the 'Boltons' area of Windsor, is near to the varied range of shops, bars and restaurants that the town has to offer.
Two train stations provide links to London Paddington (via Slough) and Waterloo (mainline approx 50 mins).
Sporting and leisure facilities are varied with horse racing at both Windsor and Ascot, polo and horse riding in Windsor Great Park, golf at Sunningdale and Wentworth and rowing/boating on the River Thames.
An excellent range of schools are available in both the Independent and State sectors, including St George's, Upton House, Brigidine, Eton College, St. John's Beaumont, Papplewick and Lambrook-Haileybury, Trinity St Stephen, Trevelyan, St Edwards Catholic Middle School and Windsor Boys' and Girls' Secondary Schools to name a few.
Residents of Bonham Court benefit from gated parking.
Contact branch for relevant Energy Performance Certificate


text:
A collection of photos from Gold Beach 2013 can be found if you click on my website below. ------------------------------------------------- - I'm lucky enough to live by the beach on the central Oregon Coast. I love to take pictures and always have my camera in my pocket, just in case. I'm totally impressed by all the awesome pictures posted here and it's fun with all the comments and rating. The feedback on my photos are also very much appreciated.-Kristina
WunderPhotos Upload
Scenic Route
Uploaded by: kristinarinell
Sunday May 1, 2011
Depoe Bay, OR (Current Weather Conditions)
Caption: Between Depoe Bay and Cape Foulweather. While US-101 runs straight up the hill, this road (Otter Crest Loop) hugs the mountain side and is a much prettier drive.
Image Description:
Manufacturer: Canon
Model: Canon PowerShot SX210 IS
Orientation: top - left
x-Resolution: 72.00
y-Resolution: 72.00
Resolution Unit: Inch
Software: Adobe Photoshop CS2 Windows
YCbCr Positioning: co-sited
RelatedImageWidth: 4320
RelatedImageLength: 3240
XP Title:
XP Keywords: canon
XP Subject:
Compression: JPEG compression
Exposure Time: 1/1000 sec.
FNumber: f/3.1
ISO Speed Ratings: 200
Exif Version: Exif Version 2.21
Components Configuration: Y Cb Cr -
Compressed Bits per Pixel: 3.00
Shutter speed:
Aperture: 3.25 EV (f/3.1)
Exposure Bias:
MaxApertureValue: 3.25 EV (f/3.1)
Metering Mode: Pattern
Flash: Flash did not fire, auto mode.
Focal Length: 5.0 mm
User Comment:
SubSecTimeOriginal: 00
SubSecTimeDigitized: 00
FlashPixVersion: FlashPix Version 1.0
Color Space: sRGB
PixelXDimension: 4318
PixelYDimension: 2878
Focal Plane x-Resolution: 17777.78
Focal Plane y-Resolution: 17802.20
Focal Plane Resolution Unit: Inch
Sensing Method: One-chip color area sensor
File Source: DSC
Custom Rendered: Normal process
Exposure Mode: Auto exposure
White Balance: Auto white balance
Digital Zoom Ratio: 1.00
Scene Capture Type: Standard
May 1, 2011 Photo Series
Viewer Comments
You must be signed in to leave comments.
Members can leave comments, upload and share photos in our Wunder Photos section and participate on the WunderBlogs.
About kristinarinell
Recent Comments for kristinarinell
(2 days ago)view 1 comment »
(2 days ago)view 2 comments »
(12 days ago)view 8 comments »
(35 days ago)view 1 comment »
(35 days ago)view 1 comment »
(35 days ago)view 1 comment »
(48 days ago)view 3 comments »
(48 days ago)view 4 comments »
(48 days ago)view 1 comment »
(51 days ago)view 1 comment »


text:
Video Editor wanted
This project was successfully completed by nsroller for $42 USD in a day.Get free quotes for a project like this
Project Budget$30 - $250 USD
Completed In1 day
Total Bids14
Project Description
Video Editor wanted
I will be filming a show and then sending you all the following stuff
- I will send you all the raw video files (.mov files)
- I will send you all the MP3 audio files
- You will create all graphics for show
- You will mix all the video and audio to make sure it is sync
- You will have creative freedom , as long as it looks good
I will be sending you files and you will be putting the whole thing together… if you look at [url removed, login to view] you will see how the show was done before, it's your job to make it even BETTER!
12 shows but please provide a price PER show…
So what is your bid per show!
Once I send all the files you will have 7 Days to put the show together......
So please bid on price Per show…
Completed by:
Skills Required
Looking to make some money?
- Set your budget and the timeframe
- Outline your proposal
- Get paid for your work
Hire Freelancers who also bid on this project
Looking for work?
Work on projects like this and make money from home!Sign Up Now
- Forbes
- The New York Times
- Time
- Wall Street Journal
- Times Online


text:
|Thread Tools
post #1 of 2 Old 09-11-2011, 05:35 PM Thread Starter
Seachem Stress Guard
anyone familar with this product? I wanted to find a good product when adding new fish. I know about stress coat but have heard bad things about the aloe vera that is in it. Is stress guard worth buying or is there something better
post #2 of 2 Old 09-12-2011, 11:02 AM
This is best if added to the shipping water when transporting fish long distances. I would not use it in an established aquarium. Fish have a remarkable ability to heal themselves in clean stable water. Most water conditioners contain some form of slime coat protection anyway, which is more than adequate.
Byron Hosking, BMus, MA
Vancouver, BC, Canada
The aquarist is one who must learn the ways of the biologist, the chemist, and the veterinarian. [unknown source]
Something we all need to remember: The fish you've acquired was quite happy not being owned by you, minding its own business. If youre going to take it under your wing then youre responsible for it. Every aspect of its life is under your control, from water quality and temperature to swimming space. [Nathan Hill in PFK]
|Thread Tools
Show Printable Version Show Printable Version
Email this Page Email this Page
|Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts
BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On


text:
Saccharomyces cerevisiae adapts to changing nutrient conditions by regulating its genome-wide transcription profile and cell-wide protein complement in correlation with the reigning nutrient conditions. The target of rapamycin (TOR) signalling pathway is one of the major control mechanisms within the cell that facilitates these changes. The transcription, intracellular trafficking, and protein turnover of nutrient transporters, including the hexose transporter proteins (Hxts), are regulated in response to nutrient conditions. The Vid and Gid proteins facilitate the nutrient-dependent degradation of the gluconeogenic enzymes FBPase and Mdh2p when glucose-starved cells are replenished with glucose. Three members of the VID and GID gene families, VID30/GID1, GID2, and VID28/GID5 are needed for the rapamycin or nitrogen starvation-induced degradation of the high-affinity hexose transporter Hxt7p is shown here. In addition, evidence that the functions of several Vid and Gid proteins are in close relation to the TOR signalling pathway is provided.
Saccharomyces cerevisiae senses and adapts to its changing nutrient environment by altering its transcriptional profile and modifying the protein complement within the cell. For example, yeast cells are starved of glucose when grown with either ethanol or acetate as a carbon source. When these cells are replenished with glucose, gluconeogenic enzymes such as fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase (Mdh2p) are degraded (Horak & Wolf, 1997; Huang & Chiang, 1997; Chiang & Chiang, 1998). The degradation of FBPase can occur in the vacuole or the proteasome. Upon glucose replenishment of 3 days glucose-starved cells, FBPase is targeted to novel vacuolar import and degradation (vid) vesicles, which in turn fuse with vacuoles to result in the vacuolar degradation of FBPase (Brown et al., 2003; Regelmann et al., 2003; Hung et al., 2004). A group of Vacuolar Import and Degradation (VID) genes, including VID22, VID24, VID27, VID28, and VID30, are needed for this function (Hoffman & Chiang, 1996; Huang & Chiang, 1997). Alternatively, upon glucose replenishment of one day glucose-starved cells, FBPase is ubiquitinated and targeted to proteasomes for degradation in a manner dependent on nine Glucose Induced Degradation (GID) genes (GID1 to GID9) (Schork et al., 1995; Regelmann et al., 2003). Some GID and VID genes (GID1/VID30, GID4/VID24 and GID5/VID28) encode the same protein product and therefore function in the proteasomal and vacuolar degradation of FBPase (Brown et al., 2003; Regelmann et al., 2003; Hung et al., 2004).
A systematic proteomics approach to identify protein–protein interactions in S. cerevisiae led to the identification of multiple in vivo protein complexes of which one complex contained seven Vid or Gid proteins (Ho et al., 2002). This complex, consisting of Vid30p, Gid2p, Vid24p, Vid28p, Gid7p, Gid8p, Gid9p, and Moh1p, was recovered from GID7 and MOH1 tandem affinity purification (TAP)-tagged strains (Ho et al., 2002). Independently, Regelmann et al., (2003) identified Gid2p as a member of a large cytoplasmic complex (c. ∼600 kDa), termed the Gid protein complex (GPC) and proposed that it contained those components identified in the systematic protein complex identification performed by Ho et al., (2002). All the components of the GPC, except Moh1p, are needed for FBPase degradation (Chiang & Chiang, 1998; Brown et al., 2003; Regelmann et al., 2003). Consistently, Vid30-TAP was used to identify the Vid30p protein complex (Vid30c) (Pitre et al., 2006), which contained all the interacting Vid and Gid proteins identified by Ho et al., (2002) with the exception of Gid7p and Moh1p, but included the uncharacterized protein Ydl176wp. Using a protein–protein interaction prediction algorithm to investigate interactions between components of the Vid30c, Pitre et al., (2006) proposed that the strongest interaction within the Vid30c existed between Vid30p and Vid28p to form the core components of the proposed Vid30c.
Several lines of evidence specific to individual components of the Vid30c indicate its involvement in the nutrient-related adaptation of S. cerevisiae. As stated, all the components of the Vid30c are needed for the degradation of FBPase when yeast cells adapt to glucose replenishment following glucose starvation (Chiang & Chiang, 1998; Brown et al., 2003; Regelmann et al., 2003). In addition, VID30 is needed for the effective transcription of nitrogen-regulated genes (van der Merwe et al., 2001), and Gid2p functions in the carbon-related ubiquitination and subsequent proteasomal degradation of FBPase (Regelmann et al., 2003). Also, Vid24p was identified as a peripheral membrane protein that participates in the fusion of vid vesicles with the vacuole to enable the degradation of FBPase (Chiang & Chiang, 1998; Brown et al., 2003). Very little is known about the specific role(s) of the remaining members of the Vid30c in nutrient-mediated adaptation of yeast.
Yeast cells use well-defined signalling pathways to respond to nutrient conditions. The target of rapamycin (TOR) pathway controls a variety of cellular processes, including cell cycle progression, ribosome biogenesis and tRNA synthesis, translation, nitrogen- and carbon-regulated gene expression, and autophagy (Pedruzzi et al., 2000, 2003; Schmelzle & Hall, 2000; Raught et al., 2001; Lorberg & Hall, 2004; Powers et al., 2004; Roosen et al., 2005). Rich nutrient conditions activate the pivotal TOR kinases while nutrient limitation or treatment of the yeast with the macrolide antibiotic rapamycin renders TOR inactive (Heitman et al., 1991). TOR regulates the activities of a series of intracellular phosphatases and kinases, such as PP2A, Sit4p and Npr1p, which in turn control a wide range of intracellular processes. For example, the nucleocytoplasmic translocation of many transcription factors, including the redundant stress response transcription factors Msn2/Msn4p, the activators Gln3p and Gat1p that are needed for nitrogen- and carbon-related gene activation, and Rgt1/3p that activates the transcription of respiratory genes, are controlled by TOR (Cooper, 2002; Crespo et al., 2002; Powers et al., 2004). In addition, the ubiquitination and turnover of several nutrient-regulated permeases, including Gap1p, Tat2p, Hip1p, and Hxt1p, are controlled by TOR via Npr1p and the E3 ubiquitin ligase Rsp5p (Schmidt et al., 1998; Beck et al., 1999; De Craene et al., 2001).
Hexose transporters mediate the uptake of hexose sugars in S. cerevisiae. Low-affinity hexose transporters, like Hxt1p and Hxt3p, are active when fermentable sugars such as glucose and fructose are abundant, while high-affinity transporters, such as Hxt6p and Hxt7p, are active when these sugars are limiting. The transcription of the HXT genes and the abundance of their protein products are regulated in response to the reigning nutrient conditions. The transcription of HXT7 is repressed by the presence of high concentrations of glucose and nitrogen starvation, but induced by nonfermentable carbon sources such as raffinose and ethanol (DeRisi et al., 1997; Gasch et al., 2000). Also, the presence of high concentrations of glucose and a limiting amount of nitrogen leads to the rapid internalization of Hxt7p from the plasma membrane and its targeting to the vacuole for degradation (Krampe et al., 1998; Krampe &Boles, 2002). Hxt7p contains three ubiquitination sites (Peng et al., 2003) and its internalization occurs in a mechanism dependent on Rsp5p, Doa4p and the endocytic pathway protein End4p (Krampe et al., 1998; Krampe & Boles, 2002). It therefore seems feasible to suggest that the endocytosis of Hxt7p occurs in an ubiquitin-dependent manner.
Hxt7p was identified as a binding partner of Gid7p (Ho et al., 2002). Also, the gid7Δ mutant has an increased ability to consume glucose in nitrogen-limited growth conditions (Gardner et al., 2005). At least one component of the Vid30c, Gid2p, is needed for the ubiquitination of a target protein, FBPase, and its subsequent degradation (Regelmann et al., 2003). It was therefore hypothesized that the GPC/Vid30c or individual members of this complex are needed for the regulation of Hxt7p turnover in response to changing nutrient conditions. In this study, the involvement of TOR and the GPC/Vid30c was investigated in Hxt7p turnover by analysing the degradation of Hxt7p in cells lacking Vid28p, Vid30p, Gid2p, or Gid7p.
Materials and methods
Strains and growth conditions
All the yeast strains used in this study are listed in Table 1. Yeast cells were grown in YPD medium (1% yeast extract, 2% Bacto peptone, 2% glucose; 2% agar for solid media) for preculturing and transformation purposes and were always incubated at 30 °C.
|Yeast strain
|BY4742 VID30::KanMX4
|BY4742 GID2::KanMX4
|BY4742 GID3::KanMX4
|BY4742 VID24::KanMX4
|BY4742 VID28::KanMX4
|BY4742 GID6::KanMX4
|BY4742 GID7::KanMX4
|BY4742 GID8::KanMX4
|BY4742 GID9::KanMX4
|BY4742 YDL176w::KanMX4
|BY4742 MOH1::KanMX4
|BYvid30 HXT7-GFP
|BYvid30 HXT7-GFP::His3MX6
|BYgid2 HXT7-GFP
|BYgid2 HXT7-GFP:: His3MX6
|BYvid28 HXT7-GFP
|BYvid28 HXT7-GFP:: His3MX6
|BYgid7 HXT7-GFP
|BYgid7 HXT7-GFP:: His3MX6
|BYvid28 HXT7-3HA
|BYvid28 HXT7-3HA:: His3MX6
|BYvid30 HXT7-3HA
|BYvid30 HXT7-3HA:: His3MX6
|BYgid2 HXT7-3HA
|BYgid2 HXT7-3HA:: His3MX6
|BYgid2vid30 HXT7-GFP
|BYgid2 VID30::natMX HXT7-GFP::His3MX6
|BYvid28vid30 HXT7-GFP
|BYvid28 VID30::natMX HXT7-GFP::His3MX6
|BY4742 URE2::KanMX4
|BY4742 GLN3::KanMX4
|Yeast strain
|BY4742 VID30::KanMX4
|BY4742 GID2::KanMX4
|BY4742 GID3::KanMX4
|BY4742 VID24::KanMX4
|BY4742 VID28::KanMX4
|BY4742 GID6::KanMX4
|BY4742 GID7::KanMX4
|BY4742 GID8::KanMX4
|BY4742 GID9::KanMX4
|BY4742 YDL176w::KanMX4
|BY4742 MOH1::KanMX4
|BYvid30 HXT7-GFP
|BYvid30 HXT7-GFP::His3MX6
|BYgid2 HXT7-GFP
|BYgid2 HXT7-GFP:: His3MX6
|BYvid28 HXT7-GFP
|BYvid28 HXT7-GFP:: His3MX6
|BYgid7 HXT7-GFP
|BYgid7 HXT7-GFP:: His3MX6
|BYvid28 HXT7-3HA
|BYvid28 HXT7-3HA:: His3MX6
|BYvid30 HXT7-3HA
|BYvid30 HXT7-3HA:: His3MX6
|BYgid2 HXT7-3HA
|BYgid2 HXT7-3HA:: His3MX6
|BYgid2vid30 HXT7-GFP
|BYgid2 VID30::natMX HXT7-GFP::His3MX6
|BYvid28vid30 HXT7-GFP
|BYvid28 VID30::natMX HXT7-GFP::His3MX6
|BY4742 URE2::KanMX4
|BY4742 GLN3::KanMX4
Open Biosystems.
This study.
For growth analyses yeast cells were precultured in YPD to exponential growth phase, washed with sterile water and cell densities adjusted to an initial OD600 nm of 1.0. Tenfold dilutions were transferred to solid YPD media containing either rapamycin [1 mg mL−1 in drug vehicle (90% Ethanol and 10% Tween-20)] at final concentrations of 20 or 200 ng mL−1, or drug vehicle (DV) as a control. YPD+DV plates were incubated for 3 days and the YPD+rapamycin plates for 4 days.
Yeast strains used to monitor Hxt7-GFP localization and Hxt7-3HA degradation were precultured in YPD to generate biomass. Cells were washed and transferred to synthetic media [0.17% (w/v) Yeast Nitrogen Base without amino acids and ammonium sulphate] containing 3% (w/v) raffinose and 0.5% (w/v) ammonium sulphate (hereafter raffinose with ammonium). Amino acids were added to complement auxotrophic requirements. Following a 4 h incubation to stimulate HXT7 expression, cultures were divided in two equal volumes. Samples were collected for either fluorescence microscopy or protein extraction (time 0). Rapamycin was added to one volume to a final concentration of 200 ng mL−1 and drug vehicle added to the other. Samples were subsequently collected at the indicated times and used for fluorescence microscopy or protein extraction.
The nutrient-dependent turnover of Hxt7-GFP was performed by shifting strains cultured in raffinose with ammonium media for 4 h to YNB-based media containing either 5% glucose or 3% raffinose as carbon sources. In addition, these media either contained ammonium sulphate at 0.5% (w/v) (glucose with ammonium or raffinose with ammonium) as a nitrogen source or were devoid of ammonium sulphate (glucose without ammonium or raffinose without ammonium). Samples were collected for fluorescence microscopy before the shift (time 0) and at the indicated time intervals following the shift to fresh media.
The analysis of Hxt1-3HA degradation was performed by growing transformants of pTB380 (gift from Mike Hall) in glucose with ammonium media to exponential phase. Cells were washed and shifted to fresh glucose with ammonium media for 4 h before rapamycin was added to a final concentration of 200 ng mL−1. Samples for protein extraction were collected before (time 0) and 3 and 6 h following rapamycin addition.
Strain construction
The chromosomally tagged strains used in this study were created using the PCR-based integrative transformation procedure described previously (Longtine et al., 1998). The primers used contained 100 nt at the 5′ ends homologous to the native chromosomal locus and 20 nt at the 3′ ends homologous to the specific plasmid used to amplify the respective integration cassettes. For chromosomal tagging of HXT7, the forward and reverse primers were designed with homologous sequences upstream and downstream of the stop codon, respectively. Plasmids pFA6a-3HA-His3MX6 and pFA6a-GFP(S65T)-His3MX6 were used to generate the respective integration cassettes (Longtine et al., 1998). Following transformation, the correct integration events were verified by PCR.
The BYvid28 HXT7-GFP and BYgid2 HXT7-GFP strains were used to construct the BYvid28vid30 HXT7-GFP and BYgid2vid30 HXT7-GFP double mutants, respectively. The regions of the primers homologous to the yeast genome were 80 nt in length and were designed to replace the entire coding region of VID30 with the natMX6 cassette of pFA6a-natMX6 (Van Driessche et al., 2005). Using a similar approach, the BY4742 strain served as the parent strain to construct the BYure2 mutant. Homologous primers were designed to replace the URE2 gene with the kanMX6 cassette amplified from pFA6a-kanMX6 (Longtine et al., 1998).
RNA extraction and Northern analysis
Yeast strains were cultured in the indicated media before RNA extraction. The phenol-based RNA extraction procedure, electrophoresis and RNA transfer to nylon membrane were described previously (van der Merwe et al., 2001). A PCR digoxigenin Probe Synthesis Kit (Roche) was used according to the manufacturer's recommendations to generate digoxigenin-labelled probes with primers VID28F2 (TGGGAGACCAGTTGGCTAAG) and VID28R2 (AATGGATCAAACCACCAAGG) to detect VID28, ACT1F6 (ACCAACTGGGACGATATGGA) and ACT1R6 (TAATACGACTCACTATAG GGCCACCAATCCAGACGGAGTA) to detect ACT1, and GFP-FP (GAGAAGAACTTTTCACTGGAG) and GFP-RP (TAGTTCATCCATGCCATGTG) to detect HXT7-GFP transcripts. Prehybridization was performed at 50 °C using 10 mL digoxigenin Easy Hyb solution (Roche) for 30 min. Denatured probes were added to 5 mL digoxigenin Easy Hyb solution and hybridized for 18 h. Membranes were washed for 2 × 5 min in 2 × SSC/0.1 × sodium dodecyl sulphate (SDS) at room temperature and 2 × 15 min in 0.1 × SSC/0.1 × SDS at 50 °C. Membranes were blocked for 30 min, followed by a 30 min incubation with anti-digoxigenin antibodies (1 : 10 000 in blocking solution; Roche) and detected with CDP-Star (Roche) for 5 min. Membranes were exposed to autoradiography film for visualization.
Fluorescence microscopy
The monitoring of the subcellular localizations of Hxt7-GFP were performed by preparing slides directly from the indicated cell cultures followed by immediate analysis using the × 100 objective lens of a Nikon Eclipse E600 microscope. Images were recorded using a Coolsnapfx monochrome CCD digital camera (Roper Scientific) and processed using Metamorph (Universal Imaging, Version 5.0).
FM4-64 staining was used to confirm Hxt7-GFP is internalized via the endocytic pathway. Wild type and vid28 mutant strains were incubated in raffinose with ammonium media and incubated for 210 min to induce HXT7 expression. The cells were concentrated (OD600 nm=1.25) and FM4-64 (16 mM stock in DMSO) was added to a final concentration of 80 μM. The cells were incubated with FM4-64 for 15 min, harvested and washed, and resuspended in fresh raffinose and ammonium medium. Rapamycin was added to a final concentration of 200 ng mL−1 followed by a 6 h incubation after which the cells were collected for analysis and imaging as described.
Protein extraction and Western blotting
Harvested cells were resuspended in a lysis buffer (0.7 M Sorbitol, 50 mM Tris pH 7.5, 2 mM phenylmethylsulphonyl fluoride, 5% SDS; plus protease inhibitor cocktail tablets, Roche), added to 0.3 g glass beads and vortexed for 2 min. Lysates were centrifuged at 16 000 g for 20 min to remove cell debris and the supernatants collected. Protein concentrations were determined using the DC Protein Assay (Biorad) according to the manufacturer's recommendations. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis (PAGE) using a 7.5% acrylamide gel and transferred to a nitrocellulose membrane for 1 h. The ECL Detection kit (GE) was used to detect the Hxt7-3HA or Hxt1-3HA protein according to the manufacturer's recommendations. Rabbit antihemagluttinin (HA) antibody (Sigma) was used as a primary antibody and donkey anti-rabbit immunoglobulin G conjugated to horseradish peroxidase (GE) as a secondary antibody. Equal amounts of protein in each lane were confirmed with rabbit antialdehyde dehydrogenase (ADH) (200-4144, Rockland) primary antibody as described previously (Onodera & Ohsumi, 2004). Membranes were exposed to autoradiography film for visualization.
Several gid and vid mutants have altered sensitivity to rapamycin
VID30 is needed for the turnover of FBPase in response to a carbon shift (Hoffman & Chiang, 1996; Huang & Chiang, 1997; Regelmann et al., 2003) and the vid30Δ mutant is hypersensitive to rapamycin (van der Merwe et al., 2001). In combination these lines of evidence suggested that the function(s) of the VID/GID genes were controlled by a nutrient-sensing pathway such as the TOR pathway. The growth of eleven mutants associated with either the GPC or VID30c in the presence of increasing concentrations of rapamycin was consequently analysed. The cells of the mutant (vid30Δ, gid2Δ, gid3Δ, vid24Δ, vid28Δ, gid6Δ to gid9Δ, ydl176wΔ, and moh1Δ) and wild-type strains were precultured as described and transferred to YPD plates containing either the drug vehicle or rapamycin. The isogenic gln3Δ and ure2Δ mutant strains were included as controls as they are known to be rapamycin resistant and hypersensitive, respectively. The experiment was performed in the presence of 20, 50, 100, and 200 nM rapamycin, but only the data of the lowest and highest rapamycin concentrations are shown. Although the wild-type and all the mutant strains showed normal growth in the presence of the drug vehicle, it was evident that seven of the mutant strains were hypersensitive to rapamycin treatment (Fig. 1). The vid30Δ, gid2Δ, gid3Δ, vid24Δ, vid28Δ, gid8Δ, and gid9Δ mutants were all hypersensitive to rapamycin treatment as these strains showed a growth pattern more similar to that of the ure2Δ mutant than the wild type strain. In contrast, the gid6Δ, gid7Δ, moh1Δ, and ydl176wΔ mutants showed a growth phenotype similar to the wild type strain. These strains were also tested for rapamycin hypersensitivity in YP media containing 3% (w/v) raffinose as the sole carbon source. Although all the strains tested were less affected by rapamycin in the presence of raffinose, the patterns of hypersensitivity to rapamycin were similar to that observed for all the strains with glucose as a carbon source (data not shown). These growth characteristics were consistent at 20, 50, 100, and 200 nM rapamycin, indicating that the mutants were affected similarly by low and high concentrations of the drug. These findings suggest that all the VID/GID genes analysed, with the possible exception of GID6, GID7, MOH1, and YDL176w, have a function associated with the TOR signalling pathway.
It is known that the transcription of VID30 is regulated by TOR as rapamycin treatment increases its expression in YPD (van der Merwe et al., 2001). Similarly, rapamycin treatment of the wild type cells in YPD activated the transcription of GID2, GID3, GID7, GID8, and GID9 (Huang et al., 2004). Interestingly, the transcription of VID24 and GID6 were down-regulated in response to rapamycin treatment (Huang et al., 2004). No data could be found that described VID28 transcription in response to rapamycin treatment. The rapamycin hypersensitivity of the vid28Δ mutant (Fig. 1) along with the presence of two potential Msn2/Msn4p DNA-binding sites (STREs) in the VID28 promoter, prompted us to investigate the effect of rapamycin on VID28 expression. Wild type cells were cultured in YPD to exponential growth phase and subjected to rapamycin treatment. Samples were collected for RNA extraction directly before (time 0), and as indicated following rapamycin addition. Rapamycin treatment clearly induced the transcription of VID28 (Fig. 2). After 30 min of treatment the expression of VID28 was induced and this level of expression remained consistent after 60 and 90 min of rapamycin treatment. The increased expression of VID28 was therefore not transient. These lines of evidence indicate that the transcriptional regulation of VID28, like many of the other VID/GID genes, is controlled by the TOR pathway.
Rapamycin-induced Hxt7p turnover is dependent on Vid28p
Gid7p directly interacts with the high-affinity hexose transporter Hxt7p (Ho et al., 2002), which led to the hypothesis that GID7 is involved in Hxt7p turnover. The glucose utilization of wild type, vid28Δ, and gid7Δ strains grown in YPD media containing 20% (w/v) glucose was anslysed. Compared with the wild type, both mutant strains have increased abilities to consume glucose (data not shown). TOR is one of the major nutrient signalling pathways in yeast and vid28Δ is hypersensitive to rapamycin treatment. In combination these observations suggested that Vid28p, or other members of the Vid30c, were involved in hexose transporter turnover.
Three copies of hemagluttinin-encoding DNA (3HA) or the GFP gene was fused to HXT7 in both the wild type and vid28Δ strains. These strains were precultured in YPD to generate biomass. The expression of HXT7 is glucose-repressed and is therefore not activated in YPD. Cells were washed and shifted to fresh raffinose with ammonium media. Following a 4 h incubation to induce HXT7 expression, the cells were treated with rapamycin (see 'Materials and methods'). Hxt7-GFP was highly expressed in raffinose with ammonium media and was clearly present in the plasma membranes of both the wild-type and vid28Δ mutant strains (Fig. 3a, time 0). Rapamycin treatment of the wild-type cells resulted in the internalization of Hxt7-GFP and ultimately its degradation with time. Loss of Hxt7-GFP from the plasma membrane, as seen in the decreased fluorescence of the plasma membrane, started after 3 h and continued through 6 h until its absence after 12 h of rapamycin treatment (Fig. 3a). Hxt7-GFP localized to internal, vesicle-like structures reminiscent of multivesicular bodies (MVBs) (Nikko et al., 2003; Luhtala & Odorizzi, 2004). A stain of wild type and vid28Δ mutant cells with FM4-64, a dye used to identify the vacuole and components of the endocytic pathway, showed the colocalization of Hxt7-GFP and FM4-64 confirming these structures as components of the endocytic pathway (Fig. 3c).
In contrast, wild-type cells treated with drug vehicle still showed fluorescence in the plasma membrane after 24 h (Fig. 3b). Although the internalization of Hxt7p could be observed with the drug vehicle treatment alone as MVBs were formed, Hxt7-GFP was clearly present in the plasma membrane at each time point (Fig. 3b). In combination these data showed that the internalization and degradation of Hxt7-GFP is accelerated in the wild-type strain in response to rapamycin treatment, indicating that TOR inactivation contributed to Hxt7p internalization and ultimately its turnover in the cell. Thus, active TOR can potentially serve to stabilize Hxt7p in the plasma membrane.
The vid28ΔHXT7-GFP strain was analysed in parallel and it was found that Hxt7-GFP clearly remained associated with the plasma membrane for longer following rapamycin treatment. Internal structures started appearing after 6 h of rapamycin treatment and fluorescence was still clearly present in the plasma membrane following 24 h of rapamycin treatment (Fig. 3a). Analysis of the drug vehicle-treated strains showed similar fluorescence intensities in the plasma membranes of the wild-type and vid28Δ strains after 12 h of growth (Fig. 3b). Clear intracellular structures could be observed in the wild type strain after 18 and 24 h of growth. These structures were largely absent from the vid28Δ strain (Fig. 3b). In combination these results show that Hxt7p is partially stabilized in the plasma membrane in the vid28Δ mutant in response to rapamycin treatment.
To confirm these data, the same experiment was performed with wild-type and vid28Δ strains containing HXT7 chromosomally tagged with 3HA. Western analysis performed with these samples confirmed that the levels of Hxt7-3HA detected in the vid28Δ mutant were higher than that of the wild-type strain in response to rapamycin treatment (Fig. 4a). The amount of Hxt7-3HA detected in the wild-type strain was abundant in the absence of rapamycin, but decreased after 6 h and were very low after 12 h of rapamycin treatment. In contrast, the vid28Δ mutant still had clearly detectable levels of Hxt7-3HA present after 18 h of treatment (Fig. 4a). The levels of Hxt7-3HA did therefore decrease in the mutant, but the turnover was markedly delayed in comparison to the wild-type strain.
The persisting fluorescence of Hxt7-GFP in the plasma membrane of the vid28Δ cells could be due to differences in the transcription of HXT7-GFP between the vid28Δ mutant and wild type strains in response to rapamycin treatment. To investigate this possibility wild type (VID28 HXT7-GFP) and vid28ΔHXT7-GFP strains were grown in raffinose with ammonium and RNA samples were collected before and 12 h following rapamycin addition. Northern blot analysis showed that the levels of HXT7-GFP transcript were similar in wild type and vid28Δ cells, both before and following rapamycin treatment (Fig. 4b). This data indicated that the transcription of HXT7-GFP was not regulated in a manner dependent on VID28. Collectively, these data indicate that Vid28p plays a role in the internalization of Hxt7p in response to rapamycin treatment.
Vid30p and Gid2p are involved in Hxt7p turnover
The clear involvement of Vid28p in rapamycin-induced Hxt7p internalization prompted us to investigate the roles of other Vid and Gid proteins in this process. The internalization and degradation of Hxt7-GFP in the vid30Δ, gid2Δ, and gid7Δ mutants compared with that of the wild type strain in response to rapamycin treatment. Hxt7-GFP internalization and turnover was delayed in the vid30Δ and gid2Δ strains, but not in the gid7Δ mutant. Following 12 h of rapamycin treatment Hxt7-GFP was still clearly associated with the plasma membranes of the vid30Δ and gid2Δ strains, but largely absent from the plasma membranes of the wild type and gid7Δ strains (Fig. 5). Western analysis of Hxt7-3HA degradation in the vid30Δ and gid2Δ strains showed the transporter was more stable in these mutant strains compared with the wild type (Fig. 6). In contrast, Hxt7-3HA was internalized and degraded similarly in both the wild type and gid7Δ strains (Fig. 6). In combination these data indicate that in addition to Vid28p, Vid30p and Gid2p are also involved in the degradation of Hxt7p in response to rapamycin treatment.
Core components of the Vid30c are partially redundant in Hxt7p internalization
An increased stability of Hxt7p exists in both the vid28Δ and vid30Δ mutants. These observations suggested that Vid28p had a slightly larger role in the turnover of Hxt7p than Vid30p as Hxt7p was more stable in the vid28Δ mutant than the vid30Δ mutant. Vid30p and Vid28p were proposed to be the core components of the Vid30c (Pitre et al., 2006) and could be partially redundant in the functioning of this complex. This potential functional redundancy in the turnover of Hxt7-GFP was analysed using the vid28Δ, vid30Δ, and vid28Δvid30Δ strains. Fluorescence microscopy clearly indicated the increased stability of Hxt7-GFP in the vid28Δvid30Δ double mutant compared with either of the two single mutants (Fig. 7). These observations suggest that the functions of Vid28p and Vid30p overlap in Hxt7p internalization and turnover.
Gid2p was needed for the ubiquitination of FBPase targeted for degradation (Regelmann et al., 2003). Also, Gid2p was predicted not to be part of the Vid30c core complex, but rather a member of the subcomplex due to its predicted weak interaction with the core components (Pitre et al., 2006). Analysis of Hxt7-GFP localization in the vid30Δ, gid2Δ, and gid2Δvid30Δ mutants following rapamycin treatment clearly showed an increased stability of Hxt7-GFP in the double mutant vs. either of the single mutants (Fig. 7). However, this increased stability was not as pronounced as that observed in the vid28Δvid30Δ double mutant. These observations indicate that at least three components of the Vid30c overlap functionally in the turnover of Hxt7p in response to TOR signalling.
Rapamycin-mediated degradation of Hxt1p is independent of the Vid30c
Hxt1p is degraded rapidly in response to rapamycin treatment (Schmelzle et al., 2004). To determine if both Hxt1p and Hxt7p were degraded in a Vid30c-dependent manner in response to rapamycin treatment the wild type and vid30Δ, gid2Δ, vid28Δ, and gid7Δ strains were transformed with pTB380 which contains HXT1 fused with 3HA at its 3′ end (Schmelzle et al., 2004). Proteins were extracted from transformants grown in glucose with ammonium media before and following rapamycin treatment. Similar levels of Hxt1p-3HA were detected in the wild type and respective vid/gidΔ mutant strains following 3 h of rapamycin treatment (Fig. 8). Hxt1p turnover is rapid and samples analysed following 6 h of rapamycin treatment showed no detectable Hxt1p-3HA (data not shown). These observations suggest that Hxt1p is degraded in a mechanism that is independent of the Vid30c.
Vid30c function in the nutrient-related turnover of Hxt7p
The rapamycin treatment of yeast cells is known to simulate nutrient starvation (Heitman et al., 1991). Previous studies identified specific nutrient conditions needed for the degradation of Hxt7p. The presence of high concentrations of glucose combined with nitrogen starvation resulted in the rapid degradation of Hxt7p (Krampe et al., 1998; Krampe & Boles, 2002). These findings, in combination with the present results showing that components of the Vid30c are needed for the rapamycin-induced internalization of Hxt7-GFP, implied a role for the Vid30c in the nutrient-dependent internalization and degradation of Hxt7p.
Wild-type HXT7-GFP and vid28Δvid30ΔHXT7-GFP strains were cultured in raffinose with ammonium media to induce Hxt7-GFP expression and shifted these cells to abundant glucose with or without ammonium sulphate and abundant raffinose with or without ammonium sulphate media. Samples were collected at 6 h intervals following the respective media shifts and the internalization of Hxt7-GFP was monitored by fluorescence microscopy. When cells were shifted to glucose-based media, with or without ammonium sulphate, Hxt7-GFP was rapidly internalized from the plasma membranes and degraded at similar rates in both the vid28Δvid30Δ and wild type strains (data not shown).
Next the link between nitrogen availability and the stability of Hxt7-GFP in the plasma membrane was investigated using raffinose as a carbon source to prevent glucose-mediated regulation of Hxt7p. When wild-type HXT7-GFP and vid28Δvid30ΔHXT7-GFP cells were grown in raffinose with ammonium media (time 0), a comparable presence of Hxt7-GFP in the plasma membranes of both wild type and vid28Δvid30Δ double mutant strains (Fig. 9a) was found. Following a shift of these cells into fresh raffinose with ammonium media, the fluorescence intensities remained similar for the first 12 h followed by a slow but gradually decrease in intensity as internal structures became visible. This observation is similar to that seen in the drug-vehicle treated cells (Fig. 3b) and was thought to be a result of nitrogen depletion. This possibility was investigated by analysing the presence of ammonia in the growth media at times 0, 6, 12, and 24 h and found significant levels (1.14±0.03 g L−1) of ammonia were still present in the media after 24 h of growth. The reason for this gradual loss of Hxt7p from the plasma membrane is therefore not due to the depletion of ammonia.
In contrast, Hxt7-GFP was less stable in the plasma membrane of the wild-type strain when cells were shifted to raffinose without ammonium media. Internalization of Hxt7-GFP started occurring within 6 h postshift with a concurrent decrease in fluorescence from the plasma membrane (Fig. 9b). The vid28Δvid30Δ double mutant, however, showed a delayed turnover of Hxt7-GFP in these conditions. Hxt7-GFP was still clearly present with high intensity in the plasma membrane of the vid28Δvid30Δ double mutant after 12 h in the raffinose without ammonium media. In addition, 24 h postshift Hxt7-GFP was still present in the plasma membrane of the vid28Δvid30Δ cells while it was not detectable in the wild type strain. These results mimic the rapamycin-induced internalization of Hxt7p (compare Figs 7 and 9b). Rapamycin treatment might therefore mimic nitrogen starvation-induced Hxt7p turnover. In combination these findings suggest that the nitrogen-mediated internalization and ultimate degradation of Hxt7p is dependent on the Vid30c.
The roles of individual components of the GPC/Vid30c in carbon-related protein turnover is known (Brown et al., 2003; Regelmann et al., 2003; Hung et al., 2004). However, the nutrient-related roles of the VID and GID genes are not necessarily restricted to adaptation to preferred carbon sources. To this end, VID30 is needed for nitrogen-regulated activation of gene expression, it is transcriptionally regulated by TOR and the vid30Δ mutant is hypersensitive to rapamycin treatment (van der Merwe et al., 2001). Here, the nutrient response in yeast is better understood by providing evidence that the functions of the VID/GID genes are related to the TOR signalling pathway. The involvement of the TOR signalling pathway in the regulation of Hxt7p presence in the plasma membrane was investigated and it was found that rapamycin treatment induced the internalization and degradation of this high-affinity hexose transporter. Linking the TOR signalling pathway to the regulation of Hxt7p is consistent with the known role of TOR in the turnover of other nutrient permeases, including Gap1p, Tat2p, and Hip1p (Schmidt et al., 1998; Beck et al., 1999; De Craene et al., 2001; Schmelzle et al., 2004).
It was shown that at least three components of the Vid30c, Vid30p, Gid2p, and Vid28p were needed for the efficient internalization of Hxt7p in response to rapamycin treatment. To the authors' knowledge this is the first report in which components of the Vid30c have been linked to the internalization and degradation of a plasma membrane protein. The colocalization of Hxt7-GFP and FM4-64 indicates that the Vid and Gid proteins not only function in the turnover of proteins via the vid or proteasomal pathways as described previously (Brown et al., 2003; Regelmann et al., 2003; Hung et al., 2004), but also have a role in protein turnover via the endocytic pathway. In combination these observations predict a role for the Vid30c that is pivotal to a variety of protein turnover mechanisms within the cell.
Vid28p and Vid30p were proposed to be the core components of the Vid30c, while Gid2p was suggested to form part of a subcomplex of the Vid30c that interacts weakly with the core complex (Pitre et al., 2006). The loss of Vid28p function delayed Hxt7p turnover more severely than the loss of either Vid30p or Gid2p. A double mutant lacking both VID28 and VID30 showed significant delay in Hxt7p internalization, indicating that these two proteins have some degree of functional redundancy. These data support the proposed model in which all the components of the Vid30c interacts with both Vid28p and Vid30p (Pitre et al., 2006). Thus, deleting either VID28 or VID30 could still support the existence of a complex containing most of the components of the Vid30c. However, the vid28Δvid30Δ double mutant should prevent the formation of the Vid30c. It is important to note that turnover of Hxt7p still occurred in the vid28Δvid30Δ double mutant, albeit at a significantly delayed rate. This indicates that either the remaining components of the Vid30c can still support the degradation observed in the vid28Δvid30Δ double mutant or another pathway that functions in parallel to the Vid30c still enables the internalization and degradation of Hxt7p.
Gid2p has previously been shown to be involved in the ubiquitination of an enzyme targeted for degradation following a nutrient shift (Regelmann et al., 2003). Hxt7p contains multiple ubiquitination sites and is degraded in response to either glucose replenishment or nitrogen starvation or both. These findings show a prolonged presence of Hxt7p in the plasma membrane following rapamycin treatment in cells lacking Gid2p, but internalization and degradation of Hxt7p did occur. The delayed turnover was prolonged in cells lacking both Gid2p and Vid30p, further supporting the model that individual components of the Vid30c function in concert to mediate the turnover of Hxt7p.
The internalization of Hxt7-GFP in the vid28Δ (Fig. 3a and b) and vid28Δvid30Δ mutants (Fig. 7) revealed that the intracellular trafficking of Hxt7-GFP to the vacuole might be affected by the Vid30c. Similar to the wild type strain, these mutant strains have clear intracellular structures, possibly MVBs, visible after 12 h. However, these structures disappear in the mutant strains with a concomitant increase in fluorescence in the plasma membranes. This effect is more pronounced in the vid28Δvid30Δ double than the vid28Δ single mutant. These observations indicate that the targeting of Hxt7-GFP from the MVB to the vacuole might be dependent on the Vid30c. Understanding this phenomenon will require further investigation.
Gardner (2005) showed a gid7Δ mutant ferments glucose and fructose more effectively than its wild-type strain in nitrogen-limited broths. The fermentation characteristics of a gid7Δ mutant were independently studied and had an increased ability to metabolize glucose (data not shown), consistent with the findings of Gardner (2005). The Gid7p-Hxt7p interaction provided a potential explanation for the increased sugar utilization and suggested Gid7p could be involved in the nutrient-dependent degradation of Hxt7p. However, investigations of this study showed that TOR-regulated Hxt7p turnover was not dependent on Gid7p. The specific function of Gid7p remains unclear.
In contrast to HXT7, the low affinity transporter HXT1 is highly expressed in the presence of glucose (DeRisi et al., 1997; Gasch et al., 2000). Upon rapamycin treatment, Hxt1p is degraded in a mechanism dependent on End4p (a component of the endocytic pathway) and Rsp5p (an E3 ubiquitin ligase) (Schmelzle et al., 2004), both of which have been shown to be involved in Hxt7p degradation (Krampe et al., 1998; Krampe & Boles, 2002). The stability of Hxt1p in the vid/gid mutants after 90 min (data not shown) and 3 h of rapamycin treatment (Fig. 8) was tested and it was found that Hxt1p degradation occurred similarly in the mutant and wild-type strains. Although Hxt7p and Hxt1p are needed in the plasma membranes of yeast cells in different carbon conditions, the internalization and degradation of both these permeases are regulated by the TOR pathway and require the proper functioning of similar components of cellular ubiquitination and endocytosis machineries. It is anticipated that the signalling mechanisms downstream of TOR resulting in these two endocytic events would be different. Here it was shown that components of the Vid30c are needed for the TOR-mediated degradation of Hxt7p, but not for Hxt1p degradation.
The investigation of the nitrogen starvation-dependent internalization and degradation of Hxt7p clearly showed that the Vid30c is needed for this process to occur in the absence of glucose. Hxt7p remained present at similar levels in the plasma membranes of both wild type and vid28Δvid30Δ cells grown in raffinose with ammonia media, indicating that the signalling cascade needed for the activation of Hxt7p internalization was not activated in these growth conditions. However, Hxt7p endocytosis was activated in raffinose media lacking nitrogen and this event was dependent on Vid28p and Vid30p. Interestingly, no impact of Vid28p and Vid30p was found on the internalization of Hxt7p in abundant glucose conditions, irrespective of the nitrogen availability. The combination of these two nutrient conditions, high glucose and nitrogen starvation, leads to the rapid degradation of Hxt7p and appears to occur independent of the Vid30c. These findings suggest that Hxt7p degradation occurs in both Vid30c-dependent and Vid30c-independent mechanisms. The precise signalling mechanism that governs the nitrogen-induced Vid30c-dependent internalization of Hxt7p is currently unclear.
The precise molecular mechanism by which Vid28p, and ultimately the Vid30c, function in the TOR-mediated internalization of Hxt7p is currently unclear. However, there are multiple stages in the process of Hxt7p internalization and degradation in response to nutrient conditions where Vid28p and other components of the Vid30c could potentially exert their function. These include (1) regulating the activities of specific phosphatases and kinases, potentially Sit4p or Npr1p, in response to nutrient signals, and (2) activating components of the ubiquitination machinery, potentially Rsp5p, to enable the ubiquitination of Hxt7p, (3) initiating the endocytosis of Hxt7p, and (4) trafficking Hxt7p to the vacuole. Elucidating the precise molecular function of the Vid30c will increase the understanding of nutrient adaptation of S. cerevisiae.
We thank Dr Mark Longtine for supplying plasmids pFAa-kanMX6, pFA6a-3HA-His3MX6 and pFA6a-GFP(S65 T)-His3MX6, Dr Mike Hall for supplying pTB380, and Dr Dev Mangroo for the use of the fluorescence microscope. This research was supported by a National Science and Engineering Research Council (NSERC) Discovery Grant to GvdM.


text:
Together, we are stronger.
Cancer cells can spread from the anus to other parts of the body. This spread is called metastasis.
Understanding how a type of cancer usually grows and spreads helps your healthcare team plan your treatment and future care. If anal cancer spreads, it can spread to the following:
Celebrating cancer survivors at Relay For Life
For cancer survivors, the Canadian Cancer Society provides a unique opportunity to celebrate their courage in the fight against cancer. During hundreds of Relay For Life events across the country, thousands of survivors join together for the Survivors' Victory Lap.


text:
The Arden Oak
Arden Oak Road, Sheldon, Birmingham, West Midlands, B26 3LU
Arden Oak Road, Sheldon, Birmingham, West Midlands, B26 3LU
Tommy's Baby-Friendly Awards are run in association with parenting group Bounty and celebrate individuals and organisations which make a difference to the lives of families all over the UK. Harvester has now won the Best Family-Friendly Restaurant category in 2013, 2014 and 2015.
You voted for us because...
You know The Arden Oak in Sheldon goes the extra mile when it comes to young guests with specially sized portions of our famous grills, sauces and sides along with our unlimited salad bar and friendly, welcoming atmosphere.
Your vote helped support life-saving research into pregnancy problems and providing crucial information to parents. For each vote, Bounty pledged to donate £1* to maternity charity Tommy's.
Founded in 1992, Tommy's helps to raise funds for research into stillbirth, premature birth and miscarriage, and to provide vital information to parents. The charity believes every parent has the right to a healthy pregnancy and baby, and seeks to halve the number of babies that die during pregnancy or birth by 2030.
*For the first 5,000 votes


text:
3D Juump Infinite is the only software marketed by REAL FUSIO, designed to facilitate the transition from a service economy to a product economy.
Development of 3D Juump Infinite drew on the team's perfect knowledge of the manufacturer's expectations in exploiting digital mock-ups and more than 10 years of experience in R&D.
3D Juump Infinite allows clients to rapidly create and update project reviews, technical documentation and training support materials.
The software performs the 3D rendering of complex digital mock-ups enriched with engineering data and job-related information.
It provides users with easy access to up-to-date data, using their own computer.
Thanks to user authentication mechanisms, 3D Juump Infinite secures data exchanges and encourages collaborative work.


text:
Posted in
Business, News, Norwich, Politics, Rest of Norfolk, Rob Setchell
Norfolk County Council Leader George Nobbs speaks to Rob Setchell about Chancellor George Osborne's eighth budget, including the devolution plans which would see one elected mayor for Norfolk, Suffolk and Cambridgeshire.
You must be logged in to post a comment.
Mustard TV is Archant's new broadcasting division, which was awarded a local TV licence in 2012.
© 2017 Mustard TV Ltd. An Archant Community Media Ltd company.


text:
TV host Giuliana Rancic says two years after her breast cancer diagnosis, she has finally come to a point where the thought of cancer "doesn't take over my life every day."
Speaking to CBS News in the midst of breast cancer awareness month, Rancic — who underwent a double mastectomy — says, "I'm not as sad and, kind of, rattled by it as I used to be. Now it's more about empowering other women, and talking about it in more positive way."
Rancic explains the mastectomy at the age of 36 was "a personal decision to not have to look over my shoulder for the rest of my life. I just wanted to move on." Despite her job at E! News, which has her covering red carpets and dissecting look on "Fashion Police," Rancic says "Vanity is just not something that really exists in my life. I'm actually a very simple person, and I didn't care what I looked like. I just wanted to get the cancer out."
Breast cancer awareness month, Rancic says, should be a reminder to women to stay in touch with their doctors and keep the conversation going about monthly self exams and mammograms.
"I hear a lot of women come up to me and go, 'My mom had breast cancer, I don't have the gene, but maybe I'll get the surgery,'" Ranic says. "I don't know what sort of message people are getting, but it's so important to know the facts."
And she has a message for women who are considering preemptive mastectomy: "If you have breast cancer, it runs in your family or you have the gene and maybe you are thinking of having the surgery, talk to your doctor," Rancic says. "Maybe it actually might make sense for you. But there are a lot of scenarios where a mastectomy would not make sense. So you have to make sure you're going to a good doctor, getting a second opinion, which was a big deal in my case, and just that you know what you're getting yourself into. It's not a glamorous surgery by any means."
In May, Angelina Jolie revealed she underwent double mastectomy to decrease her risk of breast cancer, sparking a nationwide conversation about the BRCA1 and BRCA2 breast cancer genes and prophylactic mastectomy.


text:
News By Tag
Industry News
News By Place
Industry News
Making a Difference in Today's Economy
Allied Foundation, a company that specializes in foundation repair, in Houston, Texas has taken a different approach to this struggling economy.
Richard Pacheco, owner of Allied Foundation, www.crackedslab.com, began to realize that he needed to do something in order to save the jobs of his employees. The company has been family owned since 1982.
"My employees are like my family. I had to do something to make sure that they were taken care of in this time of need across America. I didn't want to have to let anyone go because every one of them plays such a vital role in this company," said Pacheco.
Pacheco started looking at his business plan and what he could do to make sure that none of his 75 employees were laid off. He changed the work week from Monday thru Friday to Tuesday thru Friday. This little family owned business also needed a way to save money on marketing in such a big city. Just by being proactive in his search to make a profit and save his employees jobs, he still has all his employees and is still going strong. The business has received Awards of Excellence from Angies List and the Better Business Bureau.
"When times got tough, everyone got a little nervous. You hear it everyday that someone is being laid off, but Richard is different. He truly treats us like family and takes care of his employees. He takes care of us and we take care of the customers. I'm proud to work for a man like Richard," said Nelson Reyes, supervisor at Allied Foundation.
For more information about Allied Foundation and what we can do for you, please visit us at www.crackedslab.com. Also if you believe in what we're trying to do, become a fan of our fan page at www.facebook.com/
Allied Foundation is a foundation repair company that serves the Greater Houston area. We are family owned and have been in the business since 1982. Our services include:
Under slab leak detection, plumbing repair, root barriers, foundation irrigation, and sprinklers.Most of our jobs are completed in one day. We take pride in providing excellent service at a reasonable cost.
# # #
Gielow Marketing, established in 2010, is growing rapidly to meet your marketing and public relations needs with social, online and traditional media. Our diverse staff have a combined 30+ years of marketing expertise.
Account Email Address Account Phone Number Disclaimer Report Abuse


text:
Category : Beaches & Bay
Operating time : 06.00 - 18.00
Ao Thian Ao Thian
Tambon Ko Tao, Amphoe Ko Pha-ngan, Surat Thani
Send Inquiry
Part of Ko Tao, Ao Thian can be seen from a viewing point located on the hilltop of Ta To Cape.
Tourism Authority of Thailand
1600 New Phetchaburi Road, Makkasan, Ratchathevi, Bangkok 10400, THAILAND
Tel: 662 250 5500, TAT Contact Center: 1672
All the travel information regarding products, services and travel deals on the website is provided by the suppliers of the particular products and services. The information may change without notice; therefore, check the accuracy of the information with the relevant supplier before making use of it.


text:
Marijuana might cause pain and vomiting in the people who value the drug the most. Doctors should learn more about cannabinoid hyperemesis syndrome.
I recently read a CBS news story about CHS, or Cannabinoid Hyperemesis Syndrome, describing a 100% increase in cases in Colorado since the legalization of marijuana there. A search for 'THC' and 'CHS' pulls stories from a range of sources including High Times, Wikipedia, Fusion.net, and Current Psychiatry. A broader search reveals articles calling the disorder 'fake news'.
Most articles about CHS describe the condition as rare, but becoming less rare as the legalization movement takes root and grows. The syndrome occurs in heavy, long-time users of marijuana who first notice reduced appetite, mild nausea, and sometimes weight loss. Those symptoms, and the symptoms that follow, are relieved by smoking marijuana, leading those with the condition to become heavier users who come to see marijuana as beneficial to their health.


text:
Have you ever purchased from PartSelect.com before?
Thank you for helping make our site better.
Below is a list of all parts for the Amana Refrigerator AFI2538AEB - Bottom Freezer. To find the repair part you need, you can browse the list below or you can narrow your choices by searching for a part by name or description.
This assembly includes the bin, auger and blades.
Mounting screws not included.
This part is used to divide your pantry drawer.
This wheel is sold individually - purchase quantity needed.
Sold individually.
Sold individually.
This is the glass ONLY. Shelf frame sold separately.
All brand logos are trademarks of their respective owners.
The PartSelect logo is a Registered Trademark of Atlantic Laundry Centres, Ltd.
Copyright © 1999-2017 , Eldis Group Partnership. All rights reserved.


text:
St. John Collection
Shimmer Bouclette Dress
Product not available
Have questions?
Chat with a designer specialist or call 1.877.543.7463.
HideDetails & Care
A metallic-speckled knit evokes moonlit sparkle for a curvaceous, short-sleeve dress. Front trim braided with delicate chain links defines an Empire waist.
- Back zip closure.
- Approx. length from shoulder to hem: 39 1/2".
- Unlined.
- Wool/rayon/polyester; dry clean.
- By St. John Collection; made in the USA of imported fabric.


text:
You Are Here :
Home > Fragrances > Valentino > Valentino Valentina Acqua Floreale Eau De Toilette For Women (50 ml./1.7 ml.)
© 2005, 2016- All Rights Reserved.
Warning: you must be of legal age to purchase tobacco (18 years old in most countries)
products. In any case of purchasing tobacco products be sure to check the restrictions
and regulations in your country/state. Cigarettes smoking is hazardous to your health.


text:
You missed it This great deal for Kasheta BodyFit is over. But don't despair, we've got more great deals every day.
Related Deals That You May Like
MetaBody Yoga and Fitness
30 Class Pass
Orlando - East$350$19.99
Pro Sport Fitness
Month of Unlimited Adult Fitness Classes
Orlando - East$70$34.99
Body20 Personal Training Studio
Three 20-Minute Personal Training Sessions
Orlando - East$147$69.99
Hotshots Athletics
8 Group Basketball Training Classes
Orlando - East$150$99.99
back by popular demand:
Kasheta BodyFit
Personal Training Customized to Your Goals
- Save 75%
- 5 purchased
100% recommendedin LivingSocial customer ratings
"I'm thrilled with... the way my experience has been totally tailored to my individual needs, and the fact that I started to feel results right away," says one happy client of Kasheta BodyFit:
• $39 ($160 value) for four 30-minute personal training sessions
• $79 ($360 value) for four one-hour personal training sessions for two people
• Let Jason Kasheta, a certified personal trainer, personalize each session
• Or, bring a friend or significant other with you for workouts with double the motivation
• Testimonials speak for themselves
• Located on North Orange Avenue inside Winter Park Health and Fitness
Why We Think Jason is Great
Jason holds bachelor's degrees in Exercise Science and Nutritional Chemistry; is a former NCAA college football player and a current North American Strongman competitor; and has a client list that ranges from children and senior citizens to professional athletes and MMA fighters.
Want more? Check out Kasheta BodyFit on Facebook.
PAID VALUE EXPIRES ON February 28, 2022
PROMOTIONAL VALUE EXPIRES ON January 27, 2014
(Inside Winter Park Health and Fitness)
1560 North Orange Avenue, Winter Park, FL 32789
407-920-0939 | get directions


text:
Many countries would think twice about choosing a profoundly anti-establishment artist, let alone one whose work would be described by some as downright unpatriotic, to represent them at an international show such as the Venice Biennale. Britain, we are happy to say, is not among them. In promoting Jeremy Deller, whose exhibition is called English Magic, the British Council and its selection committee knew exactly what they were likely to be getting. Nor will they be disappointed. The Royal Family, Tony Blair, and revered setpieces of national life are all taken to task.
Some might say the choice of Deller reflects a pervasive British negativity about itself and so represents the worst of British. We beg to differ. Deller's work shows pluralism, democracy and a healthy iconoclasm at work, and a society that is strong enough to take it. Other people's dictators and censors should take note.Reuse content


text:
Cocktail Party Invitations
Cocktail party invitations for inspirational delightful party invitation ideas create your own design 2
Need inspiration for an awesome ideas for your invitation? In this article, I'm going to tell you about awesome cocktail party invitations! Lets see how to apply this idea in your invitation card. Cocktail party invitations will inspire you with awesome ideas for your invitation with a various styles and trends.
Choosing the right color is very important! Learn how to match colors and use fabrics, accessories, artwork, and more as inspiration for crafting a color scheme that fits your invitation and your style.Not only choosing the colors, but choosing of the materials used is also important.
This cocktail party invitations is being packed with cool design gallery. So, we hope this cocktail party invitations will give you an extra ideas to make your own invitation! Get more creative ideas below and get inspired!


text:
Robert Pattinson has further fueled speculation he and "Twilight" girlfriend Kristen Stewart are dating for real by confessing it's becoming easier to kiss the actress. The couple has refused to entertain rumors of a real-life romance away from the cameras, but Pattinson and Stewart constantly poke fun at the dating reports - most recently sharing an awkward lip-lock at the MTV Movie Awards.
And now Pattinson admits kissing Stewart isn't as strange as it was in the first two "Twilight" films. He tells Parade magazine, "Kissing felt a little bit more natural in Eclipse, even if there was plenty of it. In New Moon and Twilight, it was always like, 'Oh, I'm gonna kill you if I kiss you' or something. It was always kind of odd."
"But, in this one, they (characters Bella and Edward) both understand their situation a lot more, which makes it easier. It's the first time Kristen and I have been able to just act like a normal couple."


text:
My Account
Sign in
This brand was recommended by my Vet since they use less metals during processing. It's rather large size pills but you can easily cut it and pour over food for smaller dogs to eat. Does have a strong "fishy" smell to it but doesn't seem to bother my dog at all.
Copyright 2017 © EntirelyPets. All Rights Reserved.
Sign into your EntirelyPets Account for the best experience!


text: My husband and I stopped for a quick lunch while traveling through on I-75. Don't think I could recommend anything there. The trash cans were overflowing. Food and trash on the floor throughout the restaurant. Barely any tables clean. Don't know why there wasn't a staff assigned to the dining area because we saw 3 or 4 people in the... More


text:
Adam Hurcewicz
Adam Hurcewicz's website (Google translation) Unmannedspaceflight.com member Adam Hurcewicz
Latest Processed Space Images
Posted 2013/10/09 | 0 comments
Juno captured the images for this view of the Moon on October 9, 2013 at 11:07 UTC.
Posted 2012/04/10 | 0 comments
Cassini was about 71,500 kilometers from Mimas on February 13, 2010 when it captured the images for this false-color composite. Images taken through infrared, blue, and ultraviolet filters produce a view of the moon that emphasizes the interesting bluish color splotch on its leading hemisphere, centered on the giant crater herschel. The alignment of the color frames is not perfect because when it is as close to Saturn as Mimas' orbit, Cassini is moving very rapidly, so Mimas appeared to rotate from color frame to color frame. The background is not perfectly dark because Mimas was poised in front of Saturn at the time that these photos were taken. (Here is a simulated view from JPL's Solar System Simulator.)
Planetary Defense
An asteroid or comet headed for Earth is the only large-scale natural disaster we can prevent. Working together to fund our Shoemaker NEO Grants for astronomers, we can help save the world.
Featured Video
Intro Astronomy 2017. Class 5: Venus & Mars
Space in Images
Pretty pictures and
awe-inspiring science.
Join The Planetary Society
Let's explore the cosmos together!


text:
The Collected Writings of Zelda Fitzgerald
Sign Up Now!
Already a Member? Log In
You must be logged into Bookshare to access this title. Learn about membership options, or view our freely available titles.
- Brief Synopsis:
- This comprehensive collection of Zelda Fitzgerald's work puts the Jazz-Age heroine in illuminating literary perspective.
- Long Synopsis:
- This comprehensive collection of Zelda Fitzgerald's work puts the Jazz-Age heroine in illuminating literary perspective. The volume includes Zelda's only published novel, Save Me the Waltz, an autobiographical account of the Fitzgeralds' adventures in Paris and on the Riviera; her celebrated farce, Scandalabra, eleven short stories; twelve articles; and the letters she wrote to her husband over the span of their courtship and marriage, revealing the couple's loving and turbulent relationship. The Collected Writings affirms Zelda Fitzgerald's place as a writer and as a symbol not only of the Lost Generation but of all generations as she struggled to define herself through her art.
- Book Quality:
- Publisher Quality
- 9781476758923
- Publisher:
- The University of Alabama Press
- Date of Addition:
- 04/29/14
- Copyright Date:
- 1997
- Copyrighted By:
- Zelda Fitzgerald
- Adult content:
- No
- Language:
- English
- Categories:
- Literature and Fiction
- Submitted By:
- Bookshare Staff
- Usage Restrictions:
- This is a copyrighted book.
Reviews of The Collected Writings of Zelda Fitzgerald (0 reviews)
0 - No Rating Yet


text:
Bill Clinton, the former United States president, has paid tribute to the courage Muhammad Ali has shown throughout his entire life, not just during his boxing career.
Ali celebrated his 70th birthday yesterday and is known as one of the greatest fighters the world has seen, but Clinton feels Ali has fought his greatest battle over the past 18 years since being diagnosed with Parkinson's disease.
"He made it [boxing] part theatre, part dance and all power," Clinton told BBC Sport. "And then when he risked it all to oppose the Vietnam war, in a funny way it could have destroyed him. But it didn't because people realised he had been very forthright and he was prepared to pay the price for his convictions."
Clinton added: "Struggling with Parkinson's he has shown a different kind of, and perhaps a greater, courage than he showed as a young man risking having his body, face and brain battered by the shock of boxing.
"And it's taken a lot of courage to continue to go out, to be seen, he wasn't self-conscious. He's something special."Reuse content


text:
Centre bounce howlers
11:55am Feb 22, 2017
Jack's spot lost, Giant's future, more
3:43pm Feb 21, 2017
Injuries galore, MRP divides, more
5:30pm Feb 20, 2017
11:55am Feb 22, 2017
3:43pm Feb 21, 2017
5:30pm Feb 20, 2017
Power coach responds to your Facebook questions
3:30pm Feb 22, 2017
Magpie recruit believes he still has four to six years of good football left
3:14pm Feb 22, 2017
Giant Docker on track to play in Freo's final pre-season contest
2:39pm Feb 22, 2017
AFL legend and respected commentator Leigh Matthews gives his unique take on how the weekend of football will unfold. Lethal pulls no punches as he covers the big games
Blues skipper Marc Murphy has been tagged into mediocrity this year, Leigh Matthews says
10:00am Aug 31, 2013
Jordan Lewis is the perfect match-up for Nick Malceski on Friday night, Leigh Matthews says
5:53pm Aug 29, 2013
Quick centre clearances are the key to Geelong beating Fremantle on Saturday night, Leigh Matthews says
10:00am Jun 29, 2013
Returning pair Travis Boak and Jackson Trengove will add significantly to Port on Saturday, says coach Hinkley
11:30am Jun 28, 2013
Collingwood has become too reliant on Travis Cloke and needs Quinten Lynch to provide another option, Leigh Matthews says
11:15am May 31, 2013
Leigh Matthews previews round eight's big clash and picks his winners for the weekend
7:00am May 17, 2013
AFL great Leigh Matthews gives a surprising assessment of the Tiger skipper
8:24am Apr 26, 2013
Leigh Matthews says Scott Gumbleton is the key Anzac Day player
6:57am Apr 25, 2013
Can Cats comeback again? Can Pies stop Deledio? Leigh Matthews has his say
6:52pm Apr 18, 2013
Can the Pies shut down Hawthorn's forward line?
6:20pm Apr 11, 2013
7:50pm Sep 26, 2013
9:00am Sep 21, 2013
8:11pm Sep 19, 2013
8:00am Sep 14, 2013
7:43pm Sep 12, 2013
9:00am Sep 8, 2013
8:11pm Sep 5, 2013
7:56pm Aug 22, 2013
7:00am Aug 17, 2013
7:18pm Aug 15, 2013
7:58pm Aug 8, 2013


text:
So my mum and I dont talk. The only time we do is if theres an emergency and the other day she sent me a txt reminding me of something I had said about 2 years ago (back when we did talk)
2 years ago my sister and I lived together for awhile. And I noticed she would be getting what she called migraines everyday and so was taking these tablets called mersyndol (sorry spelling?) at first i didnt think anything of it. After about a month I noticed that she really was taking these tablets every single day without fail, combined with panadol or ibuprofen and tablets for hayfever that contained codine. I said something to mum at the time, and tried to get her to go to a dr about her migraines (though she never had any issues with light and noise with those migraines so i suspected they were played up a bit) After talking to mum about it (useless as a a kick in the head) she told my sister what I had said and she flew off the handles and moved out.
She then moved around from boyfriends house to the next (another story) and mum said the other day that she was still taking the tablets the whole time but didnt think anything of it. Now my sister is back living at home with my parents, shes still taking the mersyndol everyday plus celebrex (given to her by her new boyfriend that lives with them) and hayfever tablets. As soon as she doesnt have them her moods are quote 'totally unbearable' she still refuses to see the family GP or any doctor about her 'migraines' Shes only going on 20 and is VERY naive about consequences and expects everything being handed to her and nothing bad will ever happen to her.
The fact my mother braved a potential 'why the heck are you contacting me' ear full to ask for my help indicates to me that things are pretty bad. But I have no idea what to do. I cant sit down and talk to her as I live over 1000km away from them, and even if I could I cant see her reaction being one of consideration or thought. For 2 years she's been clearly abusing over the counter pain killers and that has to be doing some serious damage!
If you've gotten this far thanks for taking the time to read.


text:
Wego.com recently found 77 hotels in Furano, Japan. Book last minute hotel or find great hotel deals such as Furano Tsuru Apartments for US$175 per night. Save more on cheap hotels with rates from as low as US$35 per night.
Filters help you save time when choosing where to stay. Try clicking on a few now...
Furano Hotels 4 Star Hotels Furano 3 Star Hotels Furano Cheap Hotels Furano Cheap Accommodation Furano
Flights to Japan Flights from Japan Japan Airports Japan Airlines