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You may have heard a simple but hopelessly catchy song about heartbreak over the past couple weeks called "Somebody That I Used To Know," by Gotye (Gaw-TEE-ay). It's intense, intimate and achingly relatable to anyone who's taken part in the death of a relationship, with a video depicting symbolic paint shading on the naked bodies of the two people in the clip. It also happens to be far and away the most successful music video of 2012 so far.
Australian musician Gotye is the man behind the music – it's his face and naked body which is slowly painted with geometric shapes in pastel shades until he blends into the wall pattern behind him.
"I don't even need your love, but you treat me like a stranger and that feels so rough," he laments before the verse is taken over by New Zealand artist Kimbra Johnson. As she loses her own paint as a symbol of the pair's growing distance, they sing at each other with a level of authenticity that evokes the painful disconnect of an ending affair.
It's simple, it's dark, it's beautiful – and it's also a staggering success. Forget about current YouTube champions Jessie J (her "Domino" video has been seen 16 million times) and Lady Gaga ("Marry The Night" has 30 million hits). "Somebody That I Used To Know" is now about to surpass 50 million views.
The song has been in rotation since last July, where Australians picked up on it right away, making it the longest-running chart-topper by a local artist in 15 years. It has since exploded worldwide. An impressive cover of the song by Walk Off The Earth has on its own gained nearly 30 million views.
As for Gotye, the man behind it is a 31-year-old musician known as Wouter De Backer (Wally to most), who has been releasing records with Rock trio The Basics, as well as the sample-based electronic solo act Gotye for around a decade. Small-potatoes local success was all the man knew until the release of "Somebody That I Used To Know," which is changing everything in his life.
The album the song is on, Making Mirrors, is quite a bit more abstract and challenging than the lone single, but it's worth an exploration. Dig into Gotye's work at his official site.


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A few days ago, Ed Sheeran announced that he had new music coming out on Friday. Keep in mind, we haven't seen anything new from Sheeran since his last album, 'X,' which came out in 2014 (there were a couple of tracks he guested on in 2015, but nothing major), so this is HUGE news.
Hello 2017… pic.twitter.com/1UyjpgoFbo
— Ed Sheeran (@edsheeran) January 1, 2017
And we got not one, but TWO stellar tracks – check 'em out:

subset: commoncrawl


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The Play Clan is one of India's most successful fashion designers store from India that offers exclusive range of designer wear from top Indian designers. Browse best fashion store India that offers a wide quality of designer clothes and home décor products such as Mens graphic tees, women's tees, women sarees, tops, tunics, jacket, waistcoat, caftan, shorts, scarfs, Kurta, wallets, coasters, cushion covers, handbags, aprons, stationery and a largest variety of Indian inspired dresses for women from top Indian designers. The playclan.com has a great collection of designer clothing and home décor products. Buy Now.
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What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
I can't download submissionsAsked by SQ1Printing on November 02, 2016 at 11:14 AM
I can't download submissions. I either get an error that jotform.com is redirecting too many times and chrome suggests I clear my cookies, which I did. Still no luck. OR I get an error that the Bandwidth limit was exceded… see image attached.
JotForm Support
I've checked your account in order to solve your problem.
Firstly, which format do you want to download? Because I've tried to download your submission list as an excel format and it took just a second as you can see the screen below:
Secondly, I've used Mozilla to download your submissions. Try in the different browser and please clear the caches.
If the issue still persist, please let us know.
Thanks in advance.
Hi! I just uploaded the same problem I am having here- I have cleared everything and tried different browsers- still the same issue. It is not working. Thanks
Nope. Still not working. I've used Firefox, Safari, and Chrome. All of them give me the same error message… see attached screen shots.
Also, I don't need to download my submission list. I honestly couldn't care less about that. I need to be able to download my customers' files that they're uploading to me. PDFs, JPGs, etc., etc. Those are what I can't download.
JotForm Support
The issue seems to be fixed now, I just tried downloading the same file, and it worked, please try it: https://files.jotform.com/jufs/SQ1Printing/43128510889965/353900957452938068/Makail's%20Bridal%20shower%20invite.pdf?md5=XU29_iK40Pg9pXjUJ0VFrQ&expires=1478114606
I am having a similar issue today. Getting an error 404 and then nginx error when trying to view an uploaded file. Downloading all uploaded files gives me a blank .zip file. Is there a known issue right now?
It's working now for me. Elton Chris on another thread said there was a bandwidth cap set that has been cleared. Thanks!
Uh-oh… I may have replied too soon. It WAS working. Now I'm getting the same error message that goldstaradmin is. The 404 nginx error screen.
JotForm Support
Please try again, the link to that file downloads without an issue:


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Posted by Disappointed
He's cut/dry & to the point which I don't mind but has a horrible temper/attitude. He never gave me complete copies of all my bk discharge papers and when I asked for them he got very angry. Recently I had to contact him because I had an issue with 1 of the mortgage companies included in the bk and he did not reply. One gets what one pays for; his price are very competitive but the service is bad.


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Honda Civic BTCC Race Car Revealed
After recently announcing plans that it would once again return to challenge for the title in the British Touring Car Championship, Honda has now released the first photos of the car it intends to do so in.
The Next Generation Touring Car (NGTC) Civic is based on the European Civic hatchback model (not on our new 2012 Civic model) and is hitting the track for its first public test today at Brands Hatch in the UK.
"This is the first opportunity we have had to get behind the wheel to really see what the car has got. The team are really pleased with the results from the test at MIRA last week and I can't wait to finally start putting the car through its paces," said triple-BTCC champion Matt Neal. "It will be interesting to see how the car performs, but I'm optimistic that we'll walk away from today confident we have a championship winning car."
The first race of the British Touring Car Championship will take place at Brand's Hatch on April 1st.
GALLERY: 2012 Honda Civic BTCC Race Car


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Legacy Boxer Rescue
www.savetheboxers.com | firstname.lastname@example.com
Bell is a sweet young female that loves to play fetch as long as you throw the ball. She gives lots of kisses and loves to cuddle. She needs to be an only dog, as she becomes food and toy aggressive with other dogs. Because of this it's recommended that she go to a home without young children. I'm a very smart girl and I know how to sit, shake,and leave it. I love to be right beside my humans and sometimes lay on them, I'm 63 lbs of pure love. I love pulling toys from the toy box just to mix things up. I love going on walks and I enjoy running around in the back yard. I love shadowing my humans when we are in the house, because I am a part of your family. I stay in the crate during the days when my foster parents are at work. I'm not a big fan but I'll do it for them. I'd really love someone that could stay home all the time with me, as I love getting lots of attention from my humans and I love getting good ear rubs. I'm 11 months old, so I'm still learning, but I pick up quickly. I need clear boundaries, so I can be the best dog for you. I do an amazing boxer wiggle, and I'm house trained by hitting my bells hanging on the back door. We are continuing to let her settle in and decompress so we can make sure we can find her a great forever home.
If you are interested in adopting me, please fill out our adoption application to begin the process.
If you aren't able to adopt at this time, please consider making a much needed, tax-deductible donation to Legacy Boxer Rescue by clicking here.
We, the founder and volunteers of Legacy Boxer Rescue, have made it our mission to seek out these Boxers and create a legacy they deserve, a loving family and home of their own.
We firmly believe that a Boxer's future should never be uncertain, so we will help to create better, happier legacies for them; one Boxer at a time.
A legacy that replaces despair with hope, sorrow with happiness, fear with security, and pain with love.
* Applications will only be accepted within the Dallas/Ft Worth Metroplex. Please email us if you are outside of this area so that we can assess your situation individually.
* Any other pets that you have must be fully vetted, this includes that they be spayed/neutered and up to date on ALL vaccinations. No exceptions will be made to this rule, so please do not apply if your pets are not spayed/neutered and current on vaccinations. This also includes cats! All dogs and cats (since it was only recently discovered that cats are susceptible to heartworms some exceptions may be made for indoor cats in this regard) must also be tested for heartworms and on some form of heartworm prevention. If you purchase heartworm prevention and/or flea/tick products through an online pharmacy like www.petcarerx.com we will need copies of your purchase history which can be obtained from the online pharmacy.
* All applicants must be 21 years of age and living on your own. If you are under 21 and living at home with your parents, please have them fill out the adoption application for you.
* If you have children under the age of ten, certain criteria will need to be met before a boxer is placed in your home. LBR will be happy to discuss this with each family as the need arises.
* All domesticated animals (with the exception of feral cats) must be kept as indoor residents of your home. We do not place our boxers in homes with or as outdoor companion animals.
* We will not place two female dogs in the same home. With the dominance issues commonly seen in females, we feel we can increase the odds for a successful adoption by not placing females in the same home together.
Once we have received your completed application, here is a list of what you can expect. We realize that this seems like a lot, and it may even seem invasive to some, but we want you to adopt a boxer if you meet the criteria described herein, so please bear with us. Most, if not all, of our volunteers work full time and have families of their own to care for, and we do our best to ensure that all applications are processed in a timely fashion but we do ask that you please have patience.
* An LBR volunteer will check the personal and veterinary references listed on your application. Since most vets require their clients permission to release any records, please contact your vet so that we are able to complete the vet check for your application. Once the personal and veterinary references are checked, and deemed acceptable, an LBR volunteer will contact you to schedule a home visit. We conduct the home visit to ensure that our boxers have a safe and loving environment to call home.
* On the application, you are asked to list any boxers that you have seen on LBR's website that you may be interested in adopting. But please be aware that often times we have more than one applicant interested in the same boxer. In that case, the home best suited for the boxer will be chosen for placement. LBR does not do adoptions on a "first come, first served" basis, and in many cases we may make a recommendation depending on your circumstances.
* After finding a boxer that you and LBR are in agreement would be a good match for you, we ask that you make arrangements to meet the boxer. Generally, this involves attending one of our Meet Our Boxer events, or we may ask the foster family to contact you directly. When you visit a boxer, we ask that all family members be present, as well as any current pets if possible.
* In some cases, we may feel that a particular boxer is not a good match for you and your family, please do not be offended by this. LBR does not take the placement of a boxer lightly, and we strive to do our best to ensure that each placement is forever, as many of these wonderful boxers have led very unstable lives before coming into rescue and we only want what is best for them. We will be happy to disclose our reasons for the decisions that we make in this regard.
Once your application has been approved and you have found the boxer of your dreams, there are only a few items remaining in this process.
* Our adoption donation's range from $125.00 to $300.00 depending on the dog being adopted. This determination is based on many factors, to include, but not limited to the dog's age and any past adoption history with LBR. Our boxers are examined for any health issues, spayed/neutered, fully vaccinated, heartworm tested, and are protected from heartworms, fleas and ticks while in foster care. LBR uses Revolution, an all-inclusive topical agent for heartworm, flea and tick prevention, or Heartgard HW Prevention and Frontline Flea/Tick Prevention. While in foster care, LBR volunteers work with the boxers on basic obedience commands, but we cannot guarantee that every dog will learn basic obedience commands while in foster care. LBR always recommends that the new owners find a training center that utilizes only positive training techniques for their new family member to help him or her be the best possible companion animal for you and your family.
* Upon adoption LBR will have you sign a legally binding adoption agreement. If there ever comes a time when you wish to give up your boxer he/she MUST be returned to LBR as stated in the adoption agreement. Adopter also agrees that the provided Legacy Boxer Rescue tag will remain on the boxer at all times, and LBR must be notified if the boxer is ever lost or stolen, we have many resources in the area that could help in locating your boxer should such an event ever take place.
* Be prepared for the arrival of your new boxer, please click here for a list of items that LBR recommends that you have on hand when he/she comes home.
Thank You!
Your message has been sent to Legacy Boxer Rescue.
You'll receive a copy, too, at to help you keep track of which pets you've inquired about, and which shelters and rescues you've emailed.
NOTE: Some shelters have physical locations you can visit; some of these shelters may only have pets for a limited time, so please do not wait for a reply—just go visit the shelter! Other organizations are rescue groups run by busy volunteers who may take a while to reply. You can find information about the shelter or rescue group caring for this pet, and their adoption procedures, on the pet's details page on Adopt-a-Pet.com.


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Stephan Lewandowsky
Can it be moral to kill? Can we sacrifice a life to save many others? Or is killing always wrong? This fundamental question looms large in the philosophy of ethics. On the one hand, deontologists such as Immanuel Kant, argue that the morality of an action is not judged by its consequences, but by the nature of that action. According to Kant, some actions—such as killing or lying—are intrinsically wrong and hence can never be justified. In a nutshell, deontological morality can be expressed as "Do the right thing. Do it because it's the right thing to do."
As we recently noted on this blog, this moral absolutism can yield disturbing outcomes: The prohibition against lying would make it deontologically moral to tell the Gestapo the truth about the Jewish family being sheltered by your neighbors.
Those disturbing implications of deontological moral imperatives are overcome by utilitarian (or "consequentialist") ethics, which considers the consequences of one's actions as one important benchmark for morality. On a utilitarian account, lying to the Gestapo to save a family would be considered moral because it avoids horrendous suffering. Indeed, on a utilitarian account, it would be moral to sacrifice a few people to save the lives of many others.
How do people navigate this ethical landscape? Are they following Kant and the deontologists or the consequentialists?
A large body of research has established that people are quite utilitarian in their moral judgments, but that they place considerable importance on the type of one's actions in addition to their consequences. Thus, as we noted on this blog earlier, people judge identical harm to be "less wrong" if it arises as a side effect of a justifiable action rather than as a direct material consequence. In the classic trolley problem, two actions that have an identical outcome—namely, sacrificing one life to save 5 others, are judged very differently depending on the nature of the action:
We are reluctant to push someone to their death, even if it saves 5 lives, whereas we find it easier to operate a switch that ultimately entails the same consequences but without personal involvement.
A recent article in the Psychonomic Bulletin & Review revisited some of the classic moral dilemmas, such as the trolley problem in the above video, using a new and more balanced framing. The team of researchers led by Petko Kusev ensured that allconsequences of all possible actions were presented in the problem framing and decision question. For example:
Sacrifice one workman in order to save five workmen
Sacrifice five workmen in order to save one workman."
The italicized sentence is the critical addition compared to previous versions of the trolley problem, which focused on saving the 5 workmen without providing a symmetrically-stated alternative about saving 1 (large) workman. Moreover, in contrast to previous version of the moral problems using a binary yes/no question on only one of the decision alternatives, Kusev and colleagues offered a choice between both utilitarian alternatives and their consequences.
The results of the study are shown in the figure below, which shows the number of participants who chose to save 5 lives (as opposed to 1) across a number of conditions:
First consider the two panels of the figure, ignoring the other variables for now: The panel on the right refers to the novel framing of the problem, as in the vignette above with the italicized sentence present. The panel on the left refers to the more conventional presentation, in which the italicized sentence was absent. It is very clear that people are more likely to save 5 lives at the expense of 1 life, if the options are presented with all consequences of all possible actions. In fact, statistically the odds of that choice were nearly 20 times larger when a dilemma was presented with full information than when it was presented with only partial information.
Next consider the differences between the light and dark gray bars, which refer to the type of involvement. In the personal involvement condition, you are asked to push a large workman into the path of the trolley (either off a bridge or from beside the track: the experimenters presented two slightly different versions of the dilemma, labeled Trolley and Footbridge, that we need not be concerned with here). In the impersonal condition, you flick a switch that results in the same downstream consequences but without the need to get personal with your victim. It is clear from the figure that people are reluctant to kill close-up and personal, regardless of how the problem is presented. Statistically, the odds are 5 times greater that people will save 5 lives if they can do so in an impersonal manner than if they have to personally push the life-saver-victim onto the tracks.
Kusev and colleagues conclude that when people are presented with the full implications of their actions, they are more likely to weigh their choices in a manner that is consistent with utilitarian ethics.
Intriguingly, Kusev and colleagues also found that people took the least time to make their decision when they were given the full information and when they chose to save 5 lives at the expense of 1, irrespective of whether the involvement was personal or impersonal. In striking contrast, when only partial information was provided (i.e., the italicized sentence in the vignette above was absent), people were much slower to decide, and the decision to save 5 lives took a particularly long time when it involved personal contact. Clearly, when the utilitarian options are not fully spelled out, people take a long time to decide that they should, after all, personally take one life to save 5 others. This dilemma is far less conflicting—and hence quicker to resolve—when all options are spelled out.
Article focused on in this post:
Kusev, P., van Schaik, P., Alzahrani, S., Lonigro, S., & Purser, H. (2016). Judging the morality of utilitarian actions: How poor utilitarian accessibility makes judges irrational. Psychonomic Bulletin & Review. DOI: 10.3758/s13423-016-1029-2.
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GAFFNEY, S.C. – The No. 15 Saint Leo men's lacrosse team fell to No. 2 Limestone in non-conference action on Saturday afternoon, 17-10, in an offensive battle in Gaffney, S.C.
Saint Leo scored the opening goal, but Limestone out jump to a 3-1 lead, and they would not relinquish that lead despite efforts by the Lions.
Junior Kyle Pauwels scored six goals in the game to keep the Lions in it, and fellow junior Jake Gilmour added three assists. His three assists give him 58 for his career, tying him with George Proechel atop the Saint Leo Career Assists record board.
Sophomore Charlie Kurtenbach added a hat trick in the game, while junior defender Jake Bye scored his first goal of the season.
The Basics
#15 Saint Leo fell to #2 Limestone, 17-10
Location: Saints Field | Gaffney, S.C.
Records: Saint Leo 3-1 | Limestone 5-0
How it happened
1Q| On the opening face, Mitch Wilson snagged the ground ball, and the Lions were off running. Junior Bruce Preeschl found a wide open Kyle Pauwels to give the Lions the first score of the game. Limestone would win the next face, a tallying the tying goal, 1-1. Limestone would tack on their second goal off a costly turnover by the Lions. The Saints scored their third just seconds after the face. Charlie Kurtenbach would catch the ball behind the cage, and wrap around to the front for the score. Limestone would sneak in two more before Kurtenbach would tally his second of the quarter. | Limestone 5-3
2Q| Limestone put in the opening goal of the second quarter almost three minutes into action and would tally their seventh goal just a moment later. A third-straight goal by the Saints gave them an 8-3 lead. Jake Gilmour caught a pass behind the cage and dished it to Pauwels who flashed in front of the net for the score. | Limestone 8-4
3Q |The Saints scored three early goals to bring it to 11-4 with 12:19 to play in the third. The duo of Gilmour and Pauwels would team up again, as Gilmour found Pauwels cutting to the cage, for the first score of the second half. The Lions would score their second-straight as this time Kurtenbach would find Pauwels for the score. Pauwels continued to have a day as the junior would score off the ensuing face to make it 11-7, Limestone, with 4:47 to play in the third. Limestone would score their 12th goal to end the Pauwels' run. Kurtenbach would score his third of the game to make it 12-8. A Limestone attackman was able to snake his way around the cage and score the final goal of the quarter. | Limestone 13-8
4Q | Junior Jake Bye, a d-pole, took the Lions' clear attempt all the way up the sideline, into Limestone's box, and scored his first career goal for the green and gold. Limestone would snag their first goal of the fourth quarter with just 7:57 left to play, and they would follow it with their second just seconds later. Pauwels would score his sixth goal of the game, off another pass from Gilmour, making it 15-10 with 4:25 left in the match. Seconds late, Limestone scored another. The Saint would add their 17th goal with 1:16 left in the game. | Limestone 17-10
Points to Note
Jake Gilmour finished the game with three assists, giving him 58 for his career, which ties him for first in the Saint Leo record books.
Kyle Pauwels' six goals is a season-high for any Lion so far this year. Pauwels now has 16 goals in just four games.
Thomas Tatarian finished with 11 saves in the game.
The Lions went 15 for 16 on clear attempts but lost the ground ball battle, 53-30.
Up Next
Saint Leo heads to Emmanuel (Ga.) at 1 p.m. on Monday, March 6.
For the latest news, updates, and information on the Saint Leo men's lacrosse team visit SaintLeoLions.com. Fans can also stay up-to-date on the latest Lions news by following Saint Leo Athletics on Facebook, Twitter, Instagram, and YouTube.


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Avocado Basil Pasta
READY IN: 35mins
Recipe by Barb G.
This looks so good, I belive it could be served as a salad or as a one-one dish meal. The recipe comes from Better Homes & Gardens Magazine.
Top Review by FlyingCook
This was fantastic! DH asked why we had never used avocado in pasta before since it was so delicious. I made sauce as written and added a diced red bell pepper and cherry tomatoes for color and to make it a main course. Fresh, flavorful, and easy. Will definitely make again.
See Discussion:16 Reviews 2 Tweaks
Ingredients Nutrition
- 8 ounces bow tie pasta or 8 ounces wagon wheel macaroni
- 2 medium avocados, halved,seeded,peeled and coarsely chopped
- 6 slices bacon, crisp cooked,drained and crumbled
- 2⁄3 cup chopped fresh basil
- 2 tablespoons lemon juice
- 1 tablespoon olive oil
- 3 cloves garlic, minced
- 1⁄4 teaspoon ground black pepper
- 1⁄8 teaspoon salt
- 1⁄2 cup finely shredded pecorino romano cheese
Directions See How It's Made
- Cook pasta according to package directions.
- Drain.
- Meanwhile, in a large bowl combine the avocados, bacon, basil, lemon juice,olive oil, garlic, pepper, and salt.
- Add the hot pasta and toss to combine.
- Transfer to a serving bowl.
- Sprinkle with cheese.


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"Poor service"
Posted by Anonymous
I came as a carer with a resident who is 91 years old. She came to have her dressing changed on her leg and it took over 40 minutes for her to be seen. When i asked the receptionist they said 'the nurse isn't ready yet' .. Well im not being funny or anything but why book me in knowing the nurses are busy!? I think it's disgusting making an elderly lady sit there for over 40 minutes for a job that simply took 5 minutes!
Response from Barbara Castle Way Health Centre 13 months ago
Thank you for your comments regarding your experience of
Barbara Castle Way Health Centre and thank you for taking the time to do so. I am sorry for your experience and in order for me to investigate further I would need to understand a bit more about which service you were visiting.
I would appreciate if you could contact me (Helen Ritchie) on 01254 734471 or you can email me on email@example.com with further details.
Again I am very sorry for your experience
Kind regards
Helen Ritchie
Senior patient experience facilitator.
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Thank you for your feedback.
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The majority of real-life lasers are productive members of society, contrary to their negative Hollywood portrayals as family shrinkers and stealers of guns' jobs. Discover the truth with today's Groupon to Arbor Lakes Lazer Chiropractic and Wellness in Maple Grove. Choose between the following options:
- For $99, you get six laser hair-removal treatments on one small area (up to a $699 value).
- For $299, you get six laser hair-removal treatments on one large area (up to a $2,999 value).<p>
Click here for a list of applicable treatment areas.
At Arbor Lakes Lazer Chiropractic and Wellness, Dr. Don Jewell and his staff enlist Cutera CoolGlide laser hair-removal technology to fleece customers of unwanted fur. During a brief consultation, patrons single out 1 of 12 bodily areas for laser treatment. An optimal regimen of six sessions can evict squatting tendrils while also slowing hair's regrowth, helping patrons maintain a bodily husk smoother than a billiards ball smothered in Crisco. Constant cooling mitigates the heat of intense pulsed light as it strobes over flesh of any complexion.
Arbor Lakes Lazer Chiropractic and Wellness
Dr. Don Jewell oversees an assortment of alternative health treatments and laser services intended to bolster overall wellness and self-image. Chiropractic care aims to straighten misaligned spines through precise adjustments, while holistic services—including lifestyle advice and nutritional counseling—empower clients to make more informed decisions about diet, exercise, and nutritional supplements. To cultivate surface-level beauty, the clinic also performs a range of laser-aided services, such as Zerona circumference-slimming treatments.


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Today we are happy to announce the launch of our latest investment: Marble Solar.
Marble Solar is a new business dedicated to the design and build of commercial and residential solar power panel systems. It is a collaboration between Fubra Limited and the directors of the Marble Group, a construction firm we have invested in.
Working with our friends at Bytewire over the past 2 months we have built, what I believe to be, one of the best solar PV estimation tools available online.
Step 1a – Enter your postcode
Step 1b – Draw your roof
Step 2 – View estimated income
Step 3 – Book a survey appointment
The estimator allows you to draw an outline of your roof on Google Maps, and select the orientation. It will then propose a suitably sized system for your roof, and uses either SAP 2005, or NASA solar irradiation data along with panel efficiency information to estimate the amount of electricity you could generate from the system over the course of the year. This data is combined with financial data such as Feed In Tariff rates and some assumptions on future price inflation to give you a projection of the amount of income and savings the system may generate over it's lifetime.
For me, it's great to see what our network of small companies can achieve when we work together.


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So what do Accountants do and why do you need others like bookkeepers? Well there are a few types of Accountants and the definition of bookkeepers can be wide and varied, but who do you need to help you with your business?
Tax Accountant (definitely need one) will assist with:
- Business setup
- Taxation compliance
- Just about anything tax related
A Management Accountant (that's me) will help you analyse your business results and work with you to understand your business, the drivers and how to improve them.
A bookkeeper will help keep all of your bookwork organised and recorded accurately.
Sometimes you will find a bookkeeper who can do what a management accountant does, an sometimes you will find a tax accountant who will work with you to understand your business. But in my experience this is a rarity rather than the norm.
I wanted to distinguish these roles to highlight what expectations you should have when engaging their services.
As I mentioned before a tax accountant will handle your taxation requirements and although some will assist you with understanding your business, most are so busy "doing" compliance work that they are unable to spend the time with you (and it can be mighty expensive) to discuss your business. So most tax accountants can really only help you out with tax in relation to your business.
A management accountant on the other hand, can help you to understand your business because they work with you on the operational side to see what is happening. They will not understand the taxation implications of things and should in all cases refer you to your tax accountant.
A great bookkeeper is a godsend and can be worth their weight in gold. How do you find one? Well I find that if they love to be organised and love paperwork, then they are passionate about bookkeeping. That's what you want,someone that is passionate about your bookwork.
So who do you need in your business? Definately a tax accountant and perhaps you want to understand the numbers in your business, then you need a management accountant. A bookkeeper is a must if you hate paperwork, give the task to someone who truly enjoys it.


text: Don't panic yet! Do as someone else said and take the spark plugs out. If you can turn the engine you are probably ok. If you can't turn it, then you may have a recoil start problem. This is fixable. If the recoil start is ok, then you probably have a big problem.


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What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
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Attendance Areas & Designated Schools
Designated schools are determined by a child's residence. Parkland School Division, with the authority of the School Act, has created an attendance boundary map system which identifies the designated school based on the child's municipal address.
Parkland School Division serves only families who reside in the County of Parkland, the town of Stony Plain and the City of Spruce Grove. We currently have some out-of-jurisdiction students who are permitted to complete the program at their current school. No further out-of-jurisdiction students are being registered in PSD. If your family is not a resident of Parkland School Division, your resident school board is responsible for meeting your educational needs.
Once you identify your designated school, we encourage you to contact the school for a tour. A tour is a great way to give both you and your child an opportunity to visit, ask questions, and meet the school principal and some of the staff.
2017 - 2018 Spruce Grove Attendance Areas
January Update
Since Parkland School Division's Board of Trustees determined the new school attendance areas in February of 2016, school administrators, senior executive staff and the Transportation Services Department have been working to transition students, staff and families into their new schools.
Here is what has been happening:
- Construction at Spruce Grove's second new K-9 School is now tentatively scheduled to open in time for the 2018 - 2019 school year. United Construction Company Inc. has been chosen as the builder and construction will begin in the spring of 2017.
- Kindergarten Open Houses are being planned. (See below)
- Kindergarten Registrations officially open Wednesday, February 1, 2017
- Grade 4 transitions to Middle School are always coordinated between schools and will continue this year
- School Principals are working as a team to ensure successful transitioning
- Prescott Learning Centre will add Grade 9 for the 2017 - 2018 school year.
- We continue to appreciate the support of so many parents who recognize the care and concern that is going into every level of detail in working through the logistics of transition for every student when it comes to the two year implementation plan since the new attendance areas were introduced in September of 2016. The goal of right-sizing our Spruce Grove Schools is a process and was always expected to take a couple of years. Some schools will continue to be very full until the opening of new School #2.
- New School #2 will open Kindergarten to Grade 8.
- Individual opportunities for cross attendance registrations are very limited. If you wish to explore attending at a school of choice please contact the school to see if they are accepting cross attendance registration at your grade level. Cross attendance opportunities may become available in specific grades and at specific schools depending on space and resources. This will be determined by the Principals at a later date.
- Until such time and the opening of Spruce Grove's second new K-9 school on the city's west end, some schools will continue to be over-populated.
Who Goes Where?
- Students currently enrolled in Kindergarten to Grade 3 in Spruce Grove and would be designated for New School #2 will remain at their current school for the 2017 - 2018 school year.
- Grade 4 students from Brookwood School and Millgrove School who would be designated for New School #2 will attend Woodhaven School for the 2017 - 2018 school year.
- Incoming Kindergarten students who would be designated for New School #2 will attend Brookwood School for the 2017 - 2018 school year.
- Grade 8 students currently attending Woodhaven School or Greystone Centennial Middle School who would be designated for New School #2 will remain at their current schools and transition to High School for the 2019 - 2020 school year.
Upcoming Transition Events
Tuesday, February 21st - (6-7pm) French Immersion Kindergarten Open House for École Broxton Park School in Spruce Grove and École Meridian Heights in Stony Plain.
Monday, March 20th - (6-7pm) Kindergarten Open House for all Spruce Grove and area schools. This excludes École Broxton Park School and Graminia School but does include Parkland Village School.
Wednesday, April 12th - (6-7pm) Kindergarten Open House for Graminia School.
Wednesday, April 12th - (6-7pm) Kindergarten Open House for Muir Lake School.
Wednesday, April 19th - (Time TBD) Stony Plain schools Kindergarten Open Houses.
Parents with questions are encouraged to call their current Principal. Please ask your questions and let us provide accurate information and assistance. We anticipate an exciting and smooth start up in the fall!
If you are unsure of your child's designated school, please contact Transportation Services at 780-963-8452 with your child's street or municipal address.
Note: Our attendance boundary maps are intended to give you a good idea as to your designated Parkland School. To confirm your designated school please contact Transportation Services at 780-963-8452.
For more information contact:
Transportation Services
4603-48 Street
Stony Plain, AB
Phone: 780-963-8452
Email: firstname.lastname@example.com
How to Locate a School
Step 1: Filter our list of schools by area and grade level.
Step 2: Choose any school attendance area on the legend just right of the map.
Step 3: Enter the address you are staying at in the field marked 'Do you live within this boundary?'
Step 4: See if your pinned address is found within a school's attendance area.


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Rimu Schematic is an electrical and electronic schematic capture software.
Main features:
- Easy to use, Rimu Schematic follows concepts you will be familiar with from other Windows based graphical applications.
- Online context-sensitive help system.
- Presentation quality schematics - make a good impression on your employer or customers.
Schematic Design is a practice program with tutorials and practice vignettes and schemes.
Contains one passing and one failing solution for two sample vignettes and a list of references. Updated July 2011. Before you begin, it helps to toggle between the Program and the work screen, looking at each in the
context of the other.
PC|SCHEMATIC Automation is an electrical CAD software.
Main features:
- The entire electrical documentation is collected in one single document.
- Tables of contents – at all levels – are automatically updated.
- You always have a general view of all parts of every single component.
BoardMaker3 is an integrated environment for the design of Printed circuit boards, offering a comprehensive set of tools that allow schematic entry, Spice simulation, 3D viewing and an Autorouter Interface.
BoardMaker3 provides powerful features and a comprehensive toolset within a user configurable windows interface, allowing engineers to maximize their productivity.
FilterLab allows you to design low pass, high pass, and band pass filters, showing the schematic diagram of the filter circuit with component values and displaying the frequency response. It has a filter wizard to design a filter step by step by selecting: type of filter, frequency (Hz), attenuation (dB) and approximation. Also you can design analog to digital converters.
Now you can get a Complete Flow Diagram Schematic with all Process and Thermo-Physical properties with One-Button-Click!
- New Auto Flow Chart Diagram
- New Individual Process Line Color Control
- New Individual Point Color, Shape and Size Control
- New Winter "V" Air Mixing Capability with Condensation
- New Fog Region Property Display
-Browse files and folders on your Nokia 3230, Nokia 6600, Nokia 7610.
-Cut, copy, paste files and folders on your Nokia 3230, Nokia 6600, Nokia 7610.
-Rename and delete files and folders on your Nokia 3230, Nokia 6600, Nokia 7610.
-Create new files and folders on your Nokia 3230, Nokia 6600, Nokia 7610.
RTflow is a free, lightweight dataflow modelling tool for real-time systems, somewhat similar to tools like MathWorks Simulink, National Instruments LabVIEW and SystemBuild. In RTflow, you build your system in a graphical language that is both parallel and deterministic, so that threading and timing problems are completely avoided.
Build circuits with resistors, light bulbs, batteries, and switches. Take measurements with the realistic ammeter and voltmeter. View the circuit as a schematic diagram, or switch to a life-like view.
Sample Learning Goals are : discuss basic electricity relationships, build circuits from schematic drawings, use an ammeter and voltmeter to take readings in circuits and more
Configuration Calculator is an app that allows you to determine the number of fixtures each circuit in your installation can support, based on type of fixture, power source (Data Enablers for color and iW fixtures, line power for eW fixtures), line voltage, circuit load and cable lengths. Also, it calculates your configuration's total power consumption in both watts and amps.
Print Layout Designer is professional in designing and dynamic printing label, business card, bar code, check, ticket, coupon, flowchart, interface, cover, schematic diagram, customer sticker, CD label, Paper package and customized invitation.
This driver will let you establish a cable connection between your Phone and your PC.
The USB port will let your Nokia Phone to interact with your PC, opening the Internet world at your hands, so you could manage the games, the music, the ringtones and photos.
Nokia Suite is a program that allows you to transfer photos, videos, music and contacts between the phone and the computer. You can also get access and download the latest applications for your phone, download music or free street maps and back up important content.
Windows Driver Package - Nokia pccsmcfd 7.1.34
Nokia PCsuite 126.96.36.199 is a program that connects a Nokia phone to a computer. With a great appearance and ease of use, it comes packaged with Nokia phones. This version comes with many features like backup, synchronizing, etc .. and new features like the 'Video Manager' , 'Image Store' etc.
Nokia Software Updater provides you with an easy-to-use tool for updating Nokia phone software. Free software updates from Nokia make sure you always have the best performance and the latest features. Just like a computer, your Nokia phone uses software to run.
Nokia Lifeblog helps the user to create a photo album with the pictures taken with mobile phone or any other means.
This software will organize all the pictures on the computer and mobile phone (cellular) for viewing, editing, sharing and search purposes.
You can move pictures from and to PC and mobile phone (both ways) and make changes to the images that will be kept in a safe place.
Nokia Ovi Player 2.1.11020.2


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Bob Birch, the longtime bassist for Elton John's backing band, died this morning (August 15), John's official website confirms. He was 56.
A statement reads: "We are very sad to report the sudden death today of Bob Birch, Elton's bass player and backing vocalist, in Los Angeles. Bob had been a member of the Elton John band since 1992, the longest-serving bassist in Elton's touring band."
John's site did not reveal the cause of death. Authorities at the Los Angeles County Police Department confirmed Birch's death to CBS Local, but could not confirm the cause of death until a coroner's report has been completed. Many outlets are reporting a self-inflicted gunshot wound in an apparent suicide.
Birch had played with John on the road and in the studio since 1992, as well as a myriad of other superstar acts including Tina Turner, Stevie Wonder, Eric Clapton, Cher, Sting and many more. Birch, also a saxophonist, performed on John's most recent tours, and as recently as last year, appeared alongside Elton and Leon Russell on the Late Show With David Letterman.
Birch, born Robert Wayne Birch in Detroit, is survived by his wife, Michelle, and his son, Jonathan.
– Jillian Mapes, CBS Local
More Elton John News On Fresh1027.com
Read More On Fresh1027.com:
- Billy Joel Wears Star of David in Rare Political Statement
- Taylor Swift Reveals Second Serpentine Teaser Ahead of New Music
- My Name Is Prince Exhibit to Include Clothes, Guitars & More
- Mariah Carey's Favorite Vegas Hot Spot Is… The Dentist?
- Freddie Mercury Biopic 'Bohemian Rhapsody' Cast Revealed
- New R. Kelly Accuser Says She Had Underage Sex with the Singer


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Pan Bagna
- Servings: 8
- 1 loaf Italian or country bread with crust that is not too heavy
- 1 large clove garlic, halved
- 1/4 cup extra-virgin olive oil
- 1 1/2 tablespoons white-wine vinegar
- 1 small red onion, very thinly sliced
- 1 large ripe tomato, thinly sliced
- 8 anchovy fillets, drained
- 1 small roasted red bell pepper, cut into strips
- Oil Poached Tuna
- 12 basil leaves
- 12 oil-cured black olives, pitted and minced
Halve bread horizontally. Pull out the soft interior, to within 1-inch of the crust; discard interior or reserve for another use. Rub the inside of bread all over with garlic, then drizzle with oil and vinegar.
Cover the bottom half of the bread with onion and tomato slices. Top with anchovies and pepper strips. Evenly distribute tuna and basil.
Spread the top half of bread with minced olives, and place it olive-side down over filled bottom half. Wrap in plastic wrap. Place on a large plate, and top with a second plate. Weight with several heavy cans. Refrigerate for 2 to 3 hours. Just before serving, cut into 6 to 8 wedges or slices.


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I know what you're thinking... It feels like summer just started, why am I already talking about Back to School shopping right?! Well, before you know it it'll be the end of August and school will be starting up again so why not get a head start on Back to School shopping.
One of my all-time favorite stores to shop for my boys, whether it's the beginning of summer or the end of it, is The Children's Place. Not only do they offer an amazing selection of clothes for my boys, but the prices are great too. I know when I shop at The Children's Place I can get everything my boys need without breaking the bank.
This year Trevor will be starting preschool so I'm not only shopping for school clothes for Brayden but for Trevor too so I like that I can go to one store and shop for both of them since The Children's Place carries infant sizes all the way up to size 14. Not only do they carry a variety of sizes, but they carry a variety of styles too... Whether you have a little sports fanatic or a little rockstar, a little diva or a little boho princess, The Children's Place is sure to have outfits your kids will WANT to wear on the first day of school.
We were recently provided with a $50 gift card to do a little Back to School shopping at The Children's Place and it couldn't have come at a better time! You see, The Children's Place has had all kinds of great change-of-season sales over the past few weeks so we were able to head to our local store and pick up some great deals including graphics tees for $3.99, jeans for $12, and even some nice button downs for only $2.99... Crazy!
Needless to say we ended up stocking up for the first few weeks of school and will definitely be heading back to do some more shopping before summer ends! Want to score some awesome Back to School clothes for your kids at The Children's Place? Be sure to come back between July 17th and 31st for your chance to win a $50 gift card for The Children's Place as well as $50 worth of Tiny Tillia products for the little ones during the Back 2 School Bloggie Boogie!
Disclosure: I was provided with the product(s) mentioned above free of charge by the company or PR representative for review purposes. I was not compensated in any other way for this post. All opinions are my own.


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The pharmacological activation of the cystic fibrosis gene protein cystic fibrosis transmembrane conductance regulator (CFTR) was studied in human airway epithelial Calu-3 cells, which express a high level of CFTR protein as assessed by Western blot and in vitro phosphorylation. Immunolocalization shows that CFTR is located in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novel synthesized xanthine derivative 3,7-dimethyl-1-isobutylxanthine (X-33) is an activator of the CFTR channel in Calu-3 cells. Whole cell current activated by X-33 or IBMX is linear, inhibited by glibenclamide and diphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene. Intracellular cAMP was not affected by X-33. An outwardly rectifying Cl− current was recorded in the absence of cAMP and X-33 stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the use of short-circuit recordings, X-33 and IBMX were able to stimulate a large concentration-dependent CFTR transport that was blocked by glibenclamide but not by DIDS. Our results show that manipulating the chemical structure of xanthine derivatives offers an opportunity to identify further specific activators of CFTR in airway cells.
- cystic fibrosis transmembrane conductance regulator
- chloride conductance
- pharmacology
cystic fibrosis (CF), the most common lethal autosomal recessive genetic disease, is caused by mutations of the CF gene, which normally encodes a multifunctional and integral membrane protein, the CF transmembrane conductance regulator (CFTR) (35). The native protein is a Cl− channel located in the apical membrane of epithelial cells, where it mediates Cl− transepithelial transport (for review see Ref. 25). CFTR mutations lead to an impaired or absent Cl− conductance (13, 25), which generates defective Cl− transport across the epithelium and perturbs the quantity and composition of epithelial fluids. This results in the manifestations of the disease, which include airway obstruction and infection, pancreatic failure, male infertility, and elevated levels of salt in sweat (for review see Ref.23).
When the CFTR channel binds ATP at nucleotide binding domains and is phosphorylated at multiple sites within the R domain by cAMP-dependent kinases, it opens and generates a Cl− flux (25). CFTR, mainly physiologically regulated by processes increasing intracellular cAMP, can also be stimulated by cAMP-independent pathways. Compounds such asp-bromotetramisole or levamisole (4, 6), genistein (29), 5-chloro-2-hydroxyphenyl-1,3-dihydro-2H-benzimidazol-2-one (NS-004) (15, 22), 1-ethyl-2-benzimidazolinone (1-EBIO) (15), 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), and 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) (5) have been proposed as new CFTR activators. All these molecules activate CFTR without altering the cell cAMP content. These results suggest other mechanisms of activation, such as the inhibition of endogenous CFTR-associated phosphatases (3,4) or perhaps the direct binding to nucleotide binding fold (NBF) 1 or/and 2. Indeed, genistein has been shown to directly bind to NBF-2 and to compete with ATP binding on that site (34).
One goal of our group is to design pharmacological tools for research study and therapeutic application in CF. We recently showed that the use of synthetic xanthines may be important in discovering potent activators of the wild-type CFTR in Chinese hamster ovary (CHO) transfected cells by using iodide efflux and cell-attached patch-clamp experiments (9). Calu-3 cells, which are derived from a pulmonary adenocarcinoma, express high levels of CFTR mRNA and protein (21, 38) and secrete a cAMP-dependent Cl−(38) and bicarbonate (16) fluid. The preponderance of CFTR channels in Calu-3 cells allowed further examination of channel properties in a well-differentiated epithelial cell. Moreover, Calu-3 cells resemble serous gland cells (21,38), which have been identified as critical components in mucosal defenses and are implicated in CF lung disease (20,43) and, therefore, are a relevant model to test the efficacy of CFTR activators.
We first describe in vitro phosphorylation and immunolocalization of CFTR on Calu-3 cells and report experiments using iodide efflux, whole cell recordings, and short-circuit techniques showing the effects of xanthine derivatives, including a novel compound obtained by chemical synthesis, 3,7-dimethyl-1-isobutylxanthine (X-33).
Cell culture.
Native CHO cells [CFTR(−) CHO] or CHO cells stably transfected with pNUT vector containing wild-type CFTR [CFTR(+) CHO] were provided by J. R. Riordan and X.-B. Chang (Scottsdale, AZ) (40). Cells cultured at 37°C in 5% CO2 were maintained in α-MEM containing 7% fetal bovine serum, 0.5%l-glutamine, 0.5% antibiotics (50 IU/ml penicillin and 50 μg/ml streptomycin), and 100 μM methotrexate, as described previously in detail (4, 40). Calu-3, a cell line of human pulmonary origin (38), was cultured at 37°C in 5% CO2 and maintained in DMEM-Ham's F-12 (1:1) nutritive mix supplemented by 10% FCS and 1% antibiotics (50 IU/ml penicillin and 50 μg/ml streptomycin).
Cell membrane preparation.
Calu-3, CFTR(−) CHO, and CFTR(+) CHO cells were removed from cell culture flasks by EDTA treatment and washed several times in Ca2+- and Mg2+-free PBS. Cell membranes were obtained as previously described (36) by lysing whole cells in Tris buffer (20 mM Tris · HCl, pH 7.45, 10 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 1 mM dithiotreitol, 0.2 mg/ml benzamidine, 1 μg/ml pepstatin A, 0.1 mM phenylmethylsulfonyl fluoride, 20 μg/ml aprotinin, and 20 μg/ml leupeptin), and cell homogenates were centrifuged at 25,000 g for 30 min. The pellets containing crude membranes were resuspended in Tris buffer and precipitated with 4% TCA (final concentration). After centrifugation, pellets of membrane proteins were solubilized in SDS-PAGE sample buffer (62 mM Tris · HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.001% bromphenol blue) to obtain a protein concentration of 2–4 mg/ml.
For immunoblotting, a monoclonal antibody raised against the COOH terminus of CFTR was obtained from Genzyme (Cambridge, MA). For immunoprecipitation and immunolocalization studies, an antibody was raised against a peptide consisting of the 23 COOH-terminal amino acids of CFTR, as previously described for a CFTR antibody directed at the first nucleotide binding domain region (33). Briefly, the peptide was synthesized and coupled to keyhole limpet hemagglutinin (10 mg peptide/8 mg keyhole limpet hemagglutinin; Cambridge Research Biochemicals, Northwich, Cheshire, UK), and antisera were prepared by injection of conjugates (100–200 μg/ml), emulsified in Freund's adjuvant, intradermally into rabbits. The antisera were affinity purified using peptide coupled to CH-Sepharose 4B (Pharmacia), with elution of antibody fractions in 0.1 M glycine-HCl, pH 2.5. The antibody to CD59 (BRIC229) was obtained from the National Blood Service (Bristol, UK).
Electrophoresis and immunoblotting.
SDS-PAGE of solubilized membrane proteins (50 μg) was performed by the method of Laemmli (32), with a 7% separating gel. After electrophoresis, proteins were electrotransferred to a polyvinylidene difluoride (PVDF) membrane at 50 V for 90 min at 4°C in a 25 mM Tris · HCl-0.7 M glycine buffer. Unreactive sites on the PVDF membrane were blocked with nonfat dry milk. The PVDF membrane was then incubated for 1 h at room temperature with a primary antibody raised against the COOH terminus of CFTR (Genzyme) at 0.4 μg/ml. After several washes, the membrane was incubated with a secondary antibody, horseradish peroxidase-labeled sheep anti-mouse IgG. Immunoreactive bands were visualized with a commercial enhanced chemiluminescence system (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Immunoprecipitation and phosphorylation of CFTR.
Calu-3, CFTR(−) CHO, and CFTR(+) CHO cells were lysed in ice-cold RIPA buffer (50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mM iodoacetate, 1 mM phenylmethylsulfonyl fluoride, and 10 μg/ml each of chymostatin, pepstatin A, antipain, aprotinin, and leupeptin) for 30 min on ice. Nuclear and cell debris were removed by microcentrifugation (13,000g) for 15 min at 4°C. The supernatant (50 μl, ∼4 mg protein/ml) was incubated with affinity-purified CFTR antibody (80 μg/ml) for 90 min at 4°C, and the antibody-CFTR complex was precipitated with Pansorbin (10% suspension of Staphylococcus aureus cells prewashed in RIPA buffer). The precipitate was washed, resuspended in phosphorylation buffer (50 mM Tris · HCl, pH 7.5, 10 mM MgCl2, 0.1 mg/ml BSA), and phosphorylated in vitro (60 min at 37°C) using the catalytic subunit of protein kinase A (PKA, 75 nM) and 10 μCi of [γ-32P]ATP. Phosphorylation was terminated by addition of RIPA buffer, and after several washes the immune complex was dissociated by solubilization in electrophoresis sample buffer (0.125 M Tris · HCl, pH 6.8, 5% SDS, 25% sucrose, 5% 2-mercaptoethanol) for 15 min at 37°C. Samples were subjected to SDS-PAGE on 7% separating gel and then Coomassie blue staining. Gels were dried and autoradiographed overnight using Hyperfilm-MP (Amersham). Lanes from the autographs were scanned by densitometry (model GS-670, Bio-Rad) using Molecular Analyst software.
Immunolocalization of CFTR.
Calu-3 cells, grown on coverslips to ∼80% confluence, were fixed for 5 min at −20°C in 5% acetic acid in ethanol. All subsequent steps were carried out in PBS containing 0.1% fish gelatin and 1% BSA at room temperature. Cells were permeabilized with 0.2% (vol/vol) Triton X-100 for 20 min, and nonspecific protein binding sites were blocked by incubation first with 50 mM glycine for 30 min and then with 10% normal goat serum for 1 h. Cells were incubation with primary antibody (1:100 dilution) for 18 h at 4°C, washed three times for 15 min each, and incubated with secondary antibody conjugated with FITC (anti-rabbit IgG for CFTR; anti-mouse IgG for CD59) for 1 h and then washed three times. Slides were then mounted in Fluorosave Reagent (Calbiochem, La Jolla, CA). Fluorescence was detected using confocal laser scanning microscopy on a Leitz Fluovert FU microscope (Leica, Germany) fitted with a TCS4D scanner (Leica). Confocal images were collected at a magnification of ×100 under oil immersion and displayed as Kalman averages on a 512 × 512-pixel 72-dpi screen. Images were processed using Scanware software (Leica), and image manipulation was performed using Corel Photopaint.
cAMP measurement.
Calu-3 cells were incubated in the presence or absence of test compounds. After a 5-min incubation period at 37°C, the reaction was stopped by addition of 250 μl of 12% TCA at 4°C. An RIA kit (RIANEN, NEN Life Science Products) was used to determine cAMP levels.
Iodide efflux experiments.
CFTR Cl− channel activity was assayed by measuring the rate of iodide (125I) efflux, as previously described (5). All experiments were performed at 37°C. Calu-3 cells grown in 12-well plates were washed twice with 2 ml of efflux buffer containing (in mM) 137 NaCl, 4.4 KCl, 0.3 KH2PO4, 0.3 NaH2PO4, 4.2 NaHCO3, 1.3 CaCl2, 0.5 MgCl2, 0.4 MgSO4, 5.6 glucose, and 10 HEPES, pH 7.5. Cells were then incubated in efflux medium containing 1 μM KI (1 μCi Na125I/ml; NEN, Boston, MA) for 1 h at 37°C to permit the iodide to reach equilibrium. Cells were washed with efflux medium. After 1 min, the medium was removed to be counted and quickly replaced by 1 ml of the same medium. This procedure was repeated every 1 min for 11 min. The first three aliquots were used to establish a stable baseline in efflux buffer alone. Efflux medium containing the appropriate drug was used for the remaining aliquots. At the end of the incubation, the medium was recovered and cells were solubilized in 1 ml of 1 N NaOH. The radioactivity was determined using a gamma counter (LKB). The fraction of initial intracellular 125I lost during each time point was also determined, and time-dependent rates of125I efflux were calculated from ln(125It 1/125It 2)/(t 1− t 2), where125It is the intracellular125I at time t, and t 1and t 2 are successive time points (41). Curves were constructed by plotting rates of125I vs. time. All comparisons were based on maximal values for the time-dependent rates (peak rates), with exclusion of the points used to establish the baseline. Values are means ± SE ofn separate experiments. Differences were considered statistically significant using the Student's t-test whenP < 0.05.
Patch-clamp experiments.
Whole cell recordings were performed on Calu-3 cells plated on 35-mm petri dishes and cultured at 37°C in 5% CO2. Currents were recorded with a List EPC-7 patch-clamp amplifier. The membrane potential was clamped to −40 mV and pulsed from −80 to +80 mV by 20-mV steps. Pipettes with resistance of 2–3 MΩ were pulled from borosilicate glass capillary tubing (GL150-T10, Clark Electromedical, Reading, UK) using a two-step vertical puller (Narishige). They were connected to the head stage of the amplifier through an Ag-AgCl pellet. Seal resistances of 10–30 GΩ were obtained. The pipette solution contained (in mM) 113 l-aspartic acid, 113 CsOH, 27 CsCl, 1 NaCl, 1 MgCl2, 1 EGTA, 3 MgATP, and 10 TES, pH 7.2; osmolality was 285 mosM. The external solution consisted of (in mM) 145 NaCl, 4 CsCl, 1 MgCl2, 1 CaCl2, 5 glucose, and 10 TES, pH 7.4; osmolality was 315 mosM. Results were analyzed with the pCLAMP6 package software (pCLAMP, Axon Instruments). All experiments were performed at room temperature. Cells were stimulated with xanthines or appropriate compounds at the concentration indicated (dissolved in DMSO; final DMSO concentration 0.1%). In control experiments, the currents were not altered by DMSO.
Synthesis of X-33.
The starting material for X-33 was theobromine prepared in a solution of dry dimethylformamide (75 ml), which was stirred under nitrogen before the addition of NaH (5.5 mM). The mixture was then brought to 75°C for 30 min before the addition of the isobutyl bromide (5.5 mM). After 4–10 h at 75°C, the reaction mixture was cooled and hydrolyzed. The solvent was evaporated, and the dry residue was extracted with methylene chloride. The solution was dried over Na2SO4 and purified by column chromatography on silica gel (elution with ethyl acetate). The product was further purified by sublimation. 1H-NMR spectra, infrared spectra, and elemental analysis were consistent with the assigned structures: X-33, white powder, 0.75 g (63%), melting point 145°C (9).
Short-circuit current measurements.
Calu-3 cells were seeded on Snapwell permeable supports (Corning Costar) at a density of 5 × 105cells/cm2. The media on the apical (0.5 ml) and basolateral (2 ml) sides were changed daily. Short-circuit current (I sc) was measured after 8 days of culture on Snapwell inserts. The inserts were mounted in a modified Ussing chamber (Corning Costar) filled on both sides with 5 ml of a Krebs bicarbonate solution containing (in mM) 126 NaCl, 0.4 KH2PO4, 2.1 K2HPO4, 1 MgSO4, 1 CaCl2, 24 NaHCO3, 10 glucose, and 0.04 phenol red. During the experiments, this solution was kept at 37°C and continuously bubbled with 5% CO2-95% air. The epithelium was short-circuited with a voltage clamp (model 558-C5, Dept. of Bioengineering, University of Iowa) connected to apical and basolateral chambers with Ag-AgCl electrodes. The potential difference and the fluid resistance between potential sensing electrodes were compensated. The I sc was recorded on a chart recorder (model L6512, Linseis). The Calu-3 cells occasionally showed some amiloride-sensitive current. To remove Na+ transport, all experiments were performed in the presence of 10 μM amiloride in the apical solution.
All products were obtained from Sigma Chemical (St. Louis, MO), except α-MEM and DMEM-Ham's F-12 nutritive mix, which were acquired from Fisher and GIBCO BRL. TS-TM calix[4]arene (5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]arene) was a generous gift of Drs. Singh and Bridges (University of Alabama at Birmingham).
Expression and in vitro phosphorylation of CFTR in CHO and Calu-3 cells.
Calu-3 is an airway epithelial cell line that has retained most of the characteristics of serous gland stem cells (38). CFTR expression was analyzed using SDS-PAGE and immunoblotting. Results from Calu-3, CFTR(+) CHO, and CFTR(−) CHO cells are shown in Fig.1 A. In Calu-3 cells, the major CFTR form was a 175-kDa protein, as determined by molecular mass standard, which appreciatively comigrates with permanently expressed CFTR protein in CHO cells (Fig. 1 A, lane 2). The smaller 145-kDa protein expressed in CFTR(+) CHO cells was visually absent in Calu-3 cells. No immunoreactivity was detected in CFTR(−) CHO cells (Fig. 1 A, lane 3). As previously described (10), the 175-kDa protein represents the mature, fully glycosylated CFTR and the 145-kDa protein represents the immature or core-glycosylated CFTR. In addition, the upper band migrating at 175 kDa coincided with the phosphorylated band obtained by CFTR immunoprecipitation followed by phosphorylation of immunoprecipitates by the catalytic subunit of PKA in Calu-3 and CFTR(+) CHO cells (Fig.1 B, lanes 1 and 2).
CFTR is in the apical membrane of Calu-3 cells.
CFTR was shown to have a predominantly apical location (Fig.2 A). It showed the same pattern of localization (Fig. 2 B) as the GPI-anchored apical membrane protein CD59 (14). This was seen inx-y scans as a patch of immunofluorescence in the first confocal section (<1 μm from the apical surface). The labeling appeared as a ring of fluorescence at 3 μm from the apical surface and was not detectable 6–8 μm into the cell, thus having characteristics of an apical location. Specificity of CFTR immunofluorescence was shown by the demonstration that it was abolished by preabsorption of antibody with COOH-terminal peptide (Fig.2 C). Figure 2, A,d and B,d, show the side view (x-z scans) of CFTR and CD59 immunofluorescence, which also clearly indicates an apical location for both proteins.
Stimulation of CFTR-mediated iodide efflux by xanthine derivatives in Calu-3 cells.
We examined the effects of IBMX and X-33 on Calu-3 cells. IBMX and X-33 were able to generate an iodide efflux significantly different (P < 0.0001) from the control conditions, i.e., in the absence of agonist (Fig. 3 A). Addition of 250 μM IBMX or 250 μM X-33 increased the peak rate of iodide efflux from 0.09 ± 0.01 (n = 12) to 0.19 ± 0.01 (n = 24) and 0.15 ± 0.01 (n = 16), respectively (Fig. 3 A). Caffeine (1,3,7-trimethylxanthine) at 250 μM was ineffective in stimulating an efflux from Calu-3 cells (data not shown). 8-Cyclopentyl-1,3-dipropylxanthine (CPX) was also applied at three different concentrations (10 nM, 10 μM, and 250 μM), but no significant iodide efflux was observed (data not shown). Forskolin (5 μM, n = 4) stimulates CFTR, as shown by the increase of peak rates (Fig. 3 B). This forskolin-induced iodide efflux was strongly inhibited by 100 μM glibenclamide (n = 4; Fig. 3 B) but was not affected by the outwardly rectifying Cl− channel inhibitor (39) TS-TM calix[4]arene (200 nM, n = 4; Fig. 3 B). The average dose-response relationships for IBMX and X-33 are presented in Fig. 3, C and D, respectively. These curves were obtained by normalizing the mean peak rates at various xanthine concentrations to that obtained with the saturating concentration. The EC50 values for IBMX and X-33 were 45 and 100 μM, respectively.
cAMP levels.
We tested the possibility that activation by X-33 might be due to elevation of cAMP. In resting Calu-3 cells, the cellular cAMP content was 0.55 ± 0.05 pmol cAMP/well (n = 6; Fig.3 E). As expected, forskolin (5 μM) increased the cAMP level measured after 5 min (7.14 ± 0.71 pmol cAMP/well,n = 6; Fig. 3 E). In contrast, the corresponding cAMP level determined in the presence of xanthine derivatives X-33 (250 μM, n = 6), CPX (10 nM,n = 3), and caffeine (250 μM, n = 3) was not increased compared with the basal level (Fig. 3 E). These results argue against a role of cAMP in mediating the effect of X-33 on CFTR. IBMX (250 μM, n = 6) induced a weak elevation of cAMP content, significantly different from the basal level (P < 0.05; Fig. 3 E).
Activation of CFTR current by the xanthine derivative X-33.
It is established that Calu-3 cells are well polarized in culture (39), and we showed in Fig. 2 that CFTR is apically located. The iodide efflux data were completed by whole cell recordings to characterize the currents in Calu-3 cells. Figure4 presents typical whole cell currents and associated current-voltage (I-V) plots in the presence or absence of activator in the bath. In control patch-clamp experiments on Calu-3 cells (Fig. 4, A and C;n = 20), i.e., in the absence of any activator, no current was recorded. We first used forskolin to test for the presence of cAMP-dependent Cl− current. The addition of 5 μM forskolin (Fig. 4 B) stimulated a time-independent, nonrectifying conductance in 11 of 17 (65%) Calu-3 cells, indicating the presence of a linear Cl−-selective current typical of functional CFTR. The forskolin-activated Cl− conductance (Fig. 4 C) had a current density of 17 ± 3.9 pA/pF (n = 11) when measured at +60 mV. The increase was statistically significant from the basal current (P < 0.001).
To further prove that the forskolin-activated current was due to CFTR activity, we performed ion-substitution experiments. Replacing all except 8 mM of the Cl− in the bath solution with an equimolar amount of iodide led to a shift of the reversal potential toward positive potentials, from −37 ± 0.3 to +28 ± 2 mV (n = 3) in the presence or absence of forskolin, indicating a larger permeability for Cl−, i.e., a selectivity of Cl− ≫ I−, consistent with a CFTR channel (26, 40).
We then tested the effects of the two xanthine derivatives IBMX and X-33 (Fig. 4, B and C). Addition of 250 μM IBMX to the bath caused a statistically significant increase in CFTR current amplitude compared with basal current [when measured at +60 mV, basal = 2.4 ± 0.3 pA/pF (n = 20) and IBMX = 20.4 ± 4.2 pA/pF (n = 4); Fig.4 C]. In CFTR(−) CHO cells, forskolin, IBMX, or X-33 failed to activate any current (n = 5 for each, data not shown). Addition to the bath of 250 μM X-33 caused the activation of a time-independent, nonrectifying conductance in 11 of 20 (55%) Calu-3 cells (Fig. 4, B and C). These increases were significantly different from basal current [16.2 ± 2.5 pA/pF (n = 11) in the presence of X-33 and 2.4 ± 0.3 pA/pF (n = 20) in control conditions at +60 mV]. Overall, X-33 activates a Cl− conductance with CFTR-like kinetics (time- and voltage-independent, linear I-Vrelationship) in Calu-3 cells similar to that obtained for CFTR(+) CHO cells (not shown).
We then quantified the relationship between the X-33 concentration and the activated current in whole cell membranes from Calu-3 cells. The effects of increasing concentrations of X-33 were observed in the presence of 200 nM TS-TM calix[4]arene, a specific blocker of outwardly rectifying Cl− current (39). A time course of the X-33 concentration-dependent activation of current in a Calu-3 cell is shown in Fig.5 A. Figure 5 B shows averaged I-V curves obtained for different concentrations of X-33 applied to the bath. A complete dose-response relationship (Fig.5 C) was obtained by normalizing mean current amplitudes at various X-33 concentrations to that obtained with 250 μM X-33 (n = 4 for each concentration). This concentration was chosen because it is likely to maximally activate the X-33-dependent current. Elevating the X-33 concentration from 50 to 100 μM increased significantly the CFTR current. A concentration >250 μM generated a saturating X-33-dependent current. The EC50 was consistent with the iodide efflux data.
Effects of Cl− current inhibitors on X-33-activated current.
The experiments described above show that CFTR activated by X-33 (Fig.5) or forskolin (Fig. 3 B) was not sensitive to the outwardly rectifying Cl− current blocker calixarene. To confirm that this Cl− current was mediated by the CFTR Cl−channel, we examined the effects of two other Cl− channel blockers, the sulfonylurea glibenclamide and the arylaminobenzoate diphenylamine-2-carboxylate (DPC), both known as CFTR inhibitors (37). Results of such experiments are illustrated in Fig.6, which shows representative current traces, time course of current, and I-V relationships in Calu-3 cells. The linear and nonrectifying X-33-activated Cl− current was reduced to the basal level in the presence of 100 μM glibenclamide [16.2 ± 2.5 pA/pF with X-33 (n = 11) and 2.7 ± 0.4 pA/pF with glibenclamide (n = 3) at +60 mV; Fig. 6, A andC]. A time course of the activation and inhibition of current from the same whole cell recording is shown in Fig.6 A,c. Addition of X-33 (250 μM) after 3 min of recording increased current within 6 min. In the presence of X-33, addition of glibenclamide (100 μM) at 7 min inhibited the majority of the current in 3 min. Overall, glibenclamide inhibited ∼83% of the current (Fig.6 D). A similar result was obtained in iodide efflux experiments with glibenclamide (Fig. 3 B). We also examined the effects of DPC. As shown in Fig. 6, B and C, 500 μM DPC induced a rapid and voltage-independent inhibition of X-33-activated current (0.55 ± 0.15 pA/pF, n = 3, at +60 mV). The time course (Fig. 6 B,c) shows that, in the presence of X-33, DPC completely inhibited the current in 2 min. The effect of DPC is partially and slowly reversed (Fig. 6 B,c). The maximal inhibition was 97% (Fig. 6 D). The fact that DPC inhibition was voltage-independent is rather surprising but could be explained by the high concentration (500 μM). Flufenamic acid (FFA, 500 μM, n = 3) was also found to inhibit forskolin- or X-33-activated Cl− current (not shown).
Isc measurement.
We have measured the effects of X-33 on transepithelial ion transport under I sc conditions. Indeed, in this cell preparation, increases in I sc are an indication of anion secretion (16). Application of X-33 (10–250 μM) in apical and basolateral solutions determined a fast increase ofI sc. The response consisted in an initial peak that was followed by a sustained phase (Fig.7 A). At 100 μM, the peak and the sustained phase (measured 15 min after X-33 application) were 14.7 ± 0.6 and 10.4 ± 1.1 μA/cm2(n = 4), respectively. The current activated by X-33 was not blocked by DIDS (Fig. 7 B). Similar results were obtained using iodide efflux and whole cell recordings (data not shown). On the contrary, glibenclamide strongly blocked (Fig.7 A) or prevented (Fig. 7 C) the effect of X-33. The sensitivity to glibenclamide and not to DIDS again suggested that X-33 activates the cAMP-dependent Cl− channel, i.e., CFTR, as observed using iodide efflux and whole cell patch-clamp techniques. Consistent with this assumption, X-33 was ineffective when applied after stimulation with 5 μM forskolin (Fig. 7 D), a concentration at which the cAMP-dependent current is maximally activated.
We have tested three other xanthines, IBMX, CPX, and caffeine, to compare their effectiveness with that of X-33. On a qualitative basis, the biphasic time course of the I sc activated by IBMX or caffeine was very similar to that of X-33 and forskolin (Fig.8 A). However, IBMX was more potent, since at 10 μM, a concentration at which X-33 and caffeine had a small effect, this xanthine elicited almost a maximal current increase (Fig. 8 B). On the contrary, caffeine was less effective, since only a weak effect was obtained at 250 μM. CPX was also found to have no effect (10 nM and 10 μM) or a weak effect (250 μM), as observed in iodide efflux (data not shown).
Outwardly rectifying Cl− current in Calu-3 cells.
It is now clear that the activity of CFTR in Calu-3 cells is not affected by DIDS or TS-TM calix[4]arene, as reported here. During whole cell patch-clamp experiments, we sometimes recorded a DIDS- and TS-TM calix[4]arene-sensitive Cl− current. Figure9 shows representative whole cell current traces and corresponding I-V curves (Fig. 9 E) observed in 10 of 43 (23%) Calu-3 cells. In those cells, for high positive pulses (i.e., greater than +60 mV), a small outwardly rectifying Cl− current, which deactivates with time, was observed in the absence of forskolin (Fig. 9 A). Addition of 5 μM forskolin to the bath leads to the activation of a weakly rectifying linear Cl− current (Fig. 9 B), which was partly inhibited by 100 μM glibenclamide (Fig.9 C). The remaining glibenclamide-insensitive current was outwardly rectifying, deactivates with time, and was abolished in the presence of external 200 μM DIDS (Fig. 9 D). These data indicate that an outwardly rectifying Cl− channel is expressed in Calu-3 cells, which could be activated by a cAMP-independent mechanism. Haws et al. (26), indeed, described at a single-channel level the properties of an outwardly rectifying Cl− channel in Calu-3 cells. This channel may be responsible for the outwardly rectifying whole cell Cl−current we recorded here. Because DIDS also inhibits several Cl− transporters, we tested the effect of TS-TM calix[4]arene, a compound related to DIDS and a more potent blocker of outwardly rectifying Cl− channels (37,39). In the experiment described in Fig.10, an outwardly rectifying Cl− current was spontaneously activated (Fig.10 A). The addition of 200 nM TS-TM calix[4]arene completely and rapidly inhibited this outwardly rectifying Cl− current (Fig. 10 B; n = 5). Then, in the continuous presence of TS-TM calix[4]arene, 5 μM forskolin activated a linear CFTR current (Fig. 10 C). This experiment reveals that TS-TM calix[4]arene inhibited outwardly rectifying Cl−, but not CFTR, current in Calu-3 cells and further demonstrates that the cAMP-activated Cl− current in this cell is mainly, if not exclusively, due to CFTR. In the 20 cells tested for activation by X-33, 11 responded by a linear CFTR Cl− current but none by an outwardly rectifying Cl− current, which clearly indicates the specificity of the xanthine derivative for CFTR.
Since the first electrophysiological characterization of the Cl− channel CFTR, the protein product of the CF gene (35), studying its pharmacology has became possible and appears to be of high importance not only to understand its regulation and role in normal and CF epithelia but because our knowledge of the pharmacology of CFTR (for review see Ref. 37) may serve as a model for the general pharmacology of Cl− channels.
Calu-3 cells, which are derived from a pulmonary adenocarcinoma, express a high level of CFTR mRNA and protein (21, 38), show cAMP-dependent Cl− (38) and bicarbonate (16) secretions, and resemble serous gland cells (26), the major pulmonary cells implicated in CF lung disease (20). In this study we showed that CFTR is in the apical membrane and is phosphorylated by PKA. Another type of outwardly rectifying Cl− current was also identified but was unlikely to contribute to the cAMP-dependent Cl− current in this cell.
CFTR and non-CFTR Cl− currents in Calu-3 cells.
Despite the description of the basic properties of CFTR channels in this cell line (26, 42), little was known about their whole cell characteristics and pharmacology. At a single-channel level, Haws et al. (26) characterized two types of Cl− channels in Calu-3 cells: CFTR and outwardly rectifying depolarization-induced Cl− channels. In cell-attached recordings, CFTR has a linear I-V relationship with a single conductance of 7.1 pS, is voltage independent, and is activated by cAMP-elevating agents. We showed in this report that the cAMP-activated iodide efflux and CFTR Cl− current were inhibited by glibenclamide, DPC, and FFA but not by DIDS and TS-TM calix[4]arene.
Non-CFTR channels were observed in 17% of unstimulated cell-attached patches and 8% of stimulated excised patches in Calu-3 cells (26). An outwardly rectifying depolarization-induced Cl− channel was observed in unstimulated patches of Calu-3 cells but not in cell-attached patches (26, 42). In 13% of excised patches exposed to ATP plus PKA, Haws et al. (26) recorded outwardly rectifying depolarization-induced plus CFTR channels. In good agreement with these data, we recorded outwardly rectifying Cl− current in only 23% of the unstimulated Calu-3 cells but in 0% of stimulated cells studied here. Since this current is activated mainly in the absence of cAMP, this suggests that the outwardly rectifying Cl− current is not directly under the control of a cAMP pathway. The pharmacology of outwardly rectifying Cl− current is based on the inhibitory effect of stilbene derivatives such as DIDS, SITS, or 4,4′-dinitrosostilbene-2,2′-disulfonate (39). Recently, TS-TM calix[4]arene, a compound related to DIDS and a more potent blocker of outwardly rectifying Cl− channels, was developed (39). We have also shown here that DIDS or TS-TM calix[4]arene inhibited the outwardly rectifying Cl−current.
Xanthine as CFTR activator.
The story of xanthine derivatives as modulators of CFTR began in 1991, when Drumm et al. (17) demonstrated that wild-type CFTR, ΔF508 CFTR, and other mutants expressed in Xenopus oocytes could be activated by IBMX in a concentration-dependent manner, but in the presence of forskolin (17). Then, in the absence of forskolin, the methylxanthine drug IBMX by itself was shown to activate normal and mutated CFTR Cl− channels in epithelia (3, 25, 26) and transfected cells (4, 37). IBMX, 1,3-dipropyl-7-methylxanthine, and the synthesized xanthines X-33 and X-32 stimulate CFTR in transfected CHO cells without altering ATP contents and with little effect on intracellular cAMP level (9; this study). Nanomolar concentrations of CPX were reported to increase36Cl efflux in CFPAC-1 pancreatic cells (homozygous for the ΔF508 allele) (19). Moreover, Arispe et al. (1) found that CPX could activate wild-type CFTR in planar lipid bilayer after a modest exposure to PKA and ATP. However, CPX seems to be more selective for ΔF508 CFTR than for the wild-type. Cohen et al. (11) proposed that this agent activates CFTR by a direct interaction on NBF-1. However, some authors have not observed any acute activation of CFTR by CPX (9, 27, 31), whereas others have reported a potentiation of the response to forskolin on ΔF508 CFTR-expressing cells consistent with that obtained with IBMX (27, 28).
Xanthine as CFTR activator in Calu-3 cells.
Having demonstrated, along with others, that CFTR Cl−channel is the main cAMP-dependent apical Cl− pathway in Calu-3 cells (26, 38; this study), we have investigated the effect of xanthine derivatives on CFTR. As expected, IBMX elicited a significant and concentration-dependent (EC50 ∼45 μM) iodide efflux and whole cell current with all the characteristics of CFTR-mediated Cl− transport. The synthetic xanthine X-33 was found to be effective in Calu-3 cells by the use of three different techniques (iodide efflux, whole cell patch-clamp, and short-circuit measurements). The I-V relationship of the activation of Cl− transport was linear and time independent in whole cell recordings, with an EC50 of ∼100 μM. The X-33-activated iodide efflux, whole cell current, andI sc were remarkably DIDS insensitive and glibenclamide sensitive. More importantly, the effect of X-33 on Calu-3 cells was insensitive to calix[4]arene. This demonstrates that the outwardly rectifying Cl− current found in this preparation is not affected by this agent or by forskolin. The other xanthines tested, caffeine and CPX, were poorly effective in activating a Cl− current.
Mechanisms of xanthine-dependent activation of CFTR.
Xanthine derivatives, depending on their structure, may act as inhibitors of phosphodiesterases (PDEs) (2) or phosphatases (3, 12) or as antagonists of adenosine receptors (7). Activation of CFTR channels using xanthine derivatives was initially attributed to inhibition of PDE activity, which would elevate cAMP by inhibiting its degradation. However, IBMX increased the maximal forskolin-dependent ΔF508-CFTR activity without increasing cellular cAMP, suggesting a cAMP-independent mechanism (28). A similar conclusion was drawn previously by Chappe et al. (9). They showed that, among the nonspecific PDE inhibitors IBMX, theophylline (1,3-dimethylxanthine), caffeine, and 8-cyclopentyltheophylline, only IBMX and theophylline were able to open CFTR (9). In this study, the xanthine derivative X-33 did not alter the intracellular cAMP content, as previously shown in CFTR(+) CHO cells (9). Moreover, the specific PDE inhibitors rolipram and milrinone failed to open CFTR channels in CFTR(+) CHO cells (9). For these reasons, it is reasonable to think that xanthine derivatives activate CFTR through a cAMP-independent mechanism. In particular, substituted xanthines IBMX and theophylline inhibit alkaline phosphatase (3, 12). Becq et al. (4) showed that these two compounds slowed the rundown of CFTR channel activity in excised membrane patches. Xanthine derivatives are also known to be antagonists of adenosine receptor A (7). Using pancreatic duct cells expressing the ΔF508 mutation (CFPAC cells), Eidelman et al. (19) initially reported the activation of Cl− current by CPX, a potent A1 adenosine-receptor antagonist (19). No human A1 adenosine-receptor mRNA was later detected in these cells (30), excluding this receptor as a mediator of CPX-elicited Cl− efflux. The authors then suggested that the action of CPX on ΔF508-CFPAC cells represents a novel site of action apparently unrelated to a known adenosine receptor. Casavola et al. (8) reported that CPX decreased intracellular pH, which would be linked to the inhibition of Na+/H+ exchange. It is not known, however, whether the action of CPX on the Cl− efflux may be correlated with the variation of intracellular pH or with some other intracellular mechanisms. More recently, Kunzelman et al. (31) reported that the CPX-induced Cl− efflux observed in CF cells could not be attributed to a direct activation of ΔF508 but, on the contrary, may be due to a pH-dependent mechanism. Finally, a controversy appeared when other groups failed to reproduce the effect of CPX (27, 31). Some authors showed a biphasic response to CPX, i.e., a stimulation for low concentrations (10–30 nM), whereas higher concentrations (100 nM–10 μM) caused inhibition of 36Cl efflux (19, 24,30). In our experience (4, 9; this study), stimulation of CFTR by CPX has not been observed in CHO cells stably transfected with wild-type CFTR and in Calu-3 cells, whatever the concentration used (10 nM, 10 μM, and 250 μM).
Structure-function study.
We are presently using a series of alkyl-substituted xanthine derivatives to study the effects on CFTR of chemical modifications of the xanthine skeleton (9). Previously, we showed a correlation between the potency of a series of 1,3,7-trialkylxanthine derivatives and the opening of the CFTR Cl− channel (9). To understand the substitutions implicated in the activation of CFTR, caffeine was chosen in this study, because it differs from X-33 only at N-1 (methyl and isobutyl, respectively). This modification leads to a potent activator (X-33) and a far less active one (caffeine). The alkyl substitution at N-1 thus appears to be important for CFTR activation. However, the same modification (isobutyl) at N-3 produces a more potent activator (IBMX). So we can hypothesize that the presence of a long alkyl bond at N-1 generates agents with high potency on CFTR activation, but a second important site seems to be located at N-3. Although X-33 is about one-half as potent as IBMX, X-33 has no effect on the cell cAMP levels, and it is extremely important to increase the spectrum of compounds capable of elevating specifically the CFTR activity. Our data suggested that xanthines substituted at C-8 (CPX) failed to open CFTR in Calu-3 cells, which suggests that the general mechanism of CFTR opening by xanthines required alkyl substitution (at N-1 or N-3) and vacancy at C-8 in CHO and Calu-3 cells. The role of CFTR in the Cl− conductance of several tissues (e.g., heart, nephron, or exocrine pancreas) may be clarified by the use of specific activators. For example, the cardiac CFTR channel has been recently shown to be activated by levamisole (18), a compound we have identified by its effects in pancreatic duct cells (3) and in CHO cells (4).
In conclusion, the pharmacology of CFTR is at the point where numerous families of chemicals of interest have been discovered, which opens a very exciting avenue toward the development of agents of high importance for the knowledge of the physiology of epithelia and other tissues, including the heart, and of the pathophysiology, including CF. Solving the structure-activity relationships of these chemicals will help build further steps toward those goals in the future.
The authors thank Ashvani Singh and Robert Bridges for the generous gift of calixarene.
↵* L. Bulteau and R. Dérand contributed equally to these results.
This work was supported by postdoctoral fellowships to L. Bulteau and a thesis grant to R. Dérand from the Association Française de Lutte contre la Mucoviscidose, by institutional grants from Centre National de la Recherche Scientifique, and by Association Française de Lutte contre la Mucoviscidose Grant P-97003.
Address for reprint requests and other correspondence: F. Becq, Laboratoire de physiologie des régulations cellulaires, UMR 6558, 40 ave. du recteur Pineau, 86022 Poitiers, France (E-mail:frederic.becq{at}univ-poitiers.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2000 the American Physiological Society


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scare the crap out of
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informal + impolite
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Question: Elizabeth Irwin s design team has proposed the following system
Elizabeth Irwin's design team has proposed the following system with component reliabilities as indicated:
What is the reliability of thesystem?
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Another reason to write on walls.Tags: interior design, home decor, vinyl, graphic, lucite, laundry, wall decals, decals, wall decorAdded 11 Years Ago from Wonderfulgraffiti


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The Sri Lanka Economic Summit 2012 will be held from July 10 to 12 under the theme Positioning Sri Lanka in the Global Economy.
This forum is the key initiative to identify and address future economic and commercial issues to develop appropriate strategic plans to meet these challenges.
This year's event will comprise of technical sessions titled Sri Lankan Economy in Perspective, Moving Beyond Conflict, Opportunities in Emerging Markets, Branding Sri Lanka, Skills and Productivity to Compete in the Global Market, Ease of Doing Business, Sri Lanka Ranked 89. Fact or Fiction? Investing in Sri Lanka and Positioning Sri Lanka in the Global Economy.