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Dakota Gas Fireplace
The Dakota skillfully blends style, function and utility with outdoor living. A rugged design with stainless steel styling makes the Dakota an appealing outdoor gas fireplace customized for you to enjoy for years to come.
Key Features and Technologies
Learn more about key product features and technologies.
The IntelliFire™ Ignition System (IPI) is an advanced intermittent pilot ignition system. IPI constantly monitors ignition, ensures safe functioning and conserves up to $10/month in energy costs. IntelliFire™ Plus (IPI Plus) takes this technology to the next level with more safety and memory settings and a programmable wireless remote control.
Our refractory offers an authentic appearance with remarkable brick detail.
Traditional Refractory Pattern.


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Catalog Item
$ 7,675.00 $ 6,948.00
Discount from MSRP
Item No. 1393164
Your current wishlist is not saved. Please Log in to save it.
For further information or assistance on this item, call 203-847-1596 ext. 2206.
29 D x 33 H x 98 L
Custom Options
Also Available In Additional Fabrics And Finishes.

subset: commoncrawl


text:
Welcome to Adobe® ColdFusion® 9.0 Update 1 Read Me. This Updater release is a follow-up of ColdFusion 9 release. Adobe recommends that all ColdFusion 9 users install this free update.
The following table provides details of the Updater installation files:
|ColdFusion 9.0 Update 1
|ColdFusion Updater Installers
|ColdFusion 9.0 Update 1 Product Downloads
|ColdFusion 9.0 Update 1 Developer Tools and Additional Server Installers
The release highlights include:
- ORM support for multiple data sources
- Amazon S3 support
- Support for IIS 7
- New script functions implemented as CFCs
- Support for BlazeDS 4 and LCDS 3
- Configurable seed for password encryption
- Server monitoring enhancements to handle load conditions
The following table provides a list of documents that provide detailed information about this release. Click the appropriate link to access the information.
|What's new and changed in this release
|A quick summary of the new and enhanced features in this release.
|Installing the ColdFusion 9 Update
|Go through the document before you install ColdFusion 9 Update 1.
|Issues fixed in ColdFusion 9 Update 1
|Apart from various new features and enhancements, this release has fixed a lot of known issues. The document provides a complete list of issues fixed in this release.
|Known issues with ColdFusion 9 Update 1
|A list of known issues with this release.
|ColdFusion 9 Update 1 New Feature Notes
|The New Feature Notes document provides comprehensive information about the features that are newly added and enhanced in this release.
|FAQ about the ColdFusion 9 Update 1
|A set of frequently asked questions related to ColdFusion 9 Update 1.
|ColdFusion 9 documentation
|Your entry point to ColdFusion 9 documentation.
Twitter™ and Facebook posts are not covered under the terms of Creative Commons.


text:
FEED Projects has partnered with companies including Whole Foods Market, Barnes Noble, HSN, Disney, and pottery Barn.
This tea caddy is rather splendid Much of the pottery is gilded in 22 carat gold leaf.
Recently, however, he had left his job to focus on pottery making.
The fork end was used to lift sticky sweetmeats out of pottery jars; the spoon end was used to scoop up the luscious syrup.
As it has come down to us "on the borders of pottery and textiles, the meander resembles a maze or labyrinth."
For the Greek and Roman period, upon sculpture, pottery, and the written description of the more considerable authors.
It also includes the plants for the manufacture of machinery, cutlery, and pottery.
The pottery of the Iroquoian and Algonkian tribes of the north-east was, as a rule, rather crude and undeveloped.
Then the pottery and china and all that—those are the girls' presents to you, with their best love.'
But the time came when pottery was to accomplish another revolution, no less remarkable than the first.
late 15c., "a potter's workshop," from Old French poterie (13c.), from potier (see potter (n.)). Attested from 1727 as "the potter's art;" from 1785 as "potteryware."


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3D Product Design for Lipgloss/Lipstick Component
in Product Design held by Michelle Kim
Contest Ended, Winner(s) have been selected.
Description:I need a 3d product design for a lipstick and lipgloss component that i am having made custom so i need a 3d concept design for the manufacturers to make the mold for this packaging. The shape of the component will resemble a crystal and very geometric. I will attach a photo of what i envision but just in 3d. Also the technology of the component is a push button pop up style, with a spring on the inside im assuming to have it released out and also clicked back in. Chanel's allure lipgloss/lipstick technology is exactly what im looking for.
This is generally everything that i am looking for but feel free to be creative and go above and beyond in terms of the shape. Anything geometric and edgy looking i would love. I want this lipstick/lipgloss packaging to be very cutting edgy and modern. Very out there in style and eye catching.
Just a rough concept design of the exterior shapes.
I want a 3d design that you are able to spin and rotate around to see all edges and shape of it because the design i want will not be uniform all around.
Thank you. Have fun with designing!
Wants:3.5 inch tall + 1.75 cm in diameter preferred for lipgloss
3 inch tall + 2 cm in diameter preferred for lipstick
I would like the colors to be black and gunmetal and would like to see both options of the applicator to be gunmetal and also black. and the same with the lid and main part. Feel free to design the colors within black and gunmetal to how you think it would look good esthetically. Maybe with each edge to be different colors.
1st Winner
#30 Crystalline Lipstick Concept V1.1 by Edwardmanfre
Download Files
2016-04-13 09:02:47
#1 Lip Gloss 3D design by Victor Lau
Download Files
2016-03-30 06:50:04
#2 Amor by mink144
Download Files
2016-03-31 03:18:11
#3 Lip Gloss & Lip Stick 3D design by Victor Lau
Download Files
2016-03-31 07:11:24
#4 lipgloss by VOOKA
Download Files
2016-03-31 10:17:28
#5 Crystal Shaped Lipstick by Vladu11
Download Files
Hello, I made some 3D models and Renders of what I thought the lipstick should look like, it's not like the "concept1:, but it is edgy. In the "Flash Animation" folder, you can find 360 deg animation of the lipstick, to better visualize the edges. "3D Export" folder contains various 3D file types, including 3D PDF.
2016-03-31 16:05:38
#6 Lipgloss by Jakob
Download Files
2016-03-31 16:14:11
#7 Lipgloss 5 and Lipstick 2 by Scott_Tarcy
2016-04-01 02:42:21
#8 Lip Gloss & Lip Stick 3D design by Victor Lau
2016-04-01 06:31:27
#9 low poly lipstick by franznoise
Download Files
2016-04-04 00:42:35
#10 Lipstick/Lipgloss by Catalin
Download Files
2016-04-04 09:52:09
#11 Lip Gloss by Mahbub
Download Files
2016-04-07 12:12:42
#12 Lip gloss by Adam
2016-04-08 14:24:51
#13 : 3D Product Design for Lipstick Component by edward m barnes
2016-04-10 19:05:26
#14 lipstick by Rayeed
Download Files
2016-04-10 20:45:32
#15 Lipstick & Lipgloss - Style 1 by Crozycki
2016-04-10 22:17:49
#16 Lipstick & Lipgloss - Style 2 by Crozycki
2016-04-10 22:21:43
#17 Lipstick & Lipgloss V1 by Jorge Gomez
2016-04-11 10:55:40
#18 Lipstick & lipgloss V2 by Jorge Gomez
Download Files
2016-04-11 11:01:18
#19 Lipstick & lipgloss V3 by Jorge Gomez
2016-04-11 11:04:09
#20 Geometric Lipgloss. by Ethan Montague
2016-04-11 21:42:34
#21 twist by Ema Kovacevic
Download Files
2016-04-12 10:27:09
#22 twist by Ema Kovacevic
Download Files
2016-04-12 10:45:49
#23 3D Product Design for Lipgloss/Lipstick Component by Ivan Zerpa
2016-04-13 02:56:21
#24 3D Product Design for Lipgloss/Lipstick Component by Ivan Zerpa
2016-04-13 03:04:13
#25 3D Product Design for Lipgloss/Lipstick Component by Ivan Zerpa
2016-04-13 03:08:44
#26 Lipstick & Lipgloss V1.1 by Jorge Gomez
2016-04-13 08:15:29
#27 TWIST KAP by Ema Kovacevic
Download Files
2016-04-13 08:41:52
#28 twist kap 2 by Ema Kovacevic
2016-04-13 08:43:30
#29 twist kap 2 by Ema Kovacevic
2016-04-13 08:46:49
#31 Lipstick & Lipgloss V1.2 by Jorge Gomez
Download Files
2016-04-13 09:11:03
Fri, 01 Apr 2016 14:02:52 +0000
I am little confused, whether I should submit my design. I feel you were not very happy with my support on your earlier contest "Beauty Bullet". I don't know.
Reda, no it is not..
Thu, 31 Mar 2016 10:34:17 +0000
Is it for Chanel compagny ? "CC"
Is it for Chanel compagny ? "CC"
Wed, 30 Mar 2016 09:03:40 +0000
It means, Rayeed, that I'm asking the contest holder not to end the contest prematurely (before those remaining 4 days, not beyond them). Whether by choosing a winner to soon, before others can participate WITHIN the 4 days OR by canceling it because lack of participation BEFORE the 4 days have passed. Surely an experienced freelancer such as yourself has come across this particular situation once or twice... I think puts all sides at a disadvantage.
Kind regards,
Kind regards,
Vladu11, what does please wait for my entry mean ? The contest ends in 4 days. You make it or you dont.
Tue, 29 Mar 2016 20:18:17 +0000
Thank you for your feedback, please wait for my entry, I'm working on it. All the best!
Hi! I would love a 360 animation. Yes it covers all the way same with the lipgloss as well if you look up Chanel allure rouge lipstick and Chanel allure lipgloss it will show you exactly what im looking for. The push button release on the bottom where it is flat and the whole exterior shape is a "crystal" or any cool geometric edgy design.
Thank you.
Thank you.
Tue, 29 Mar 2016 15:49:23 +0000
I have a few questions. Do you need this for visualization purposes (renders of the "crystal" shape enclosing, 360 animation) or for straightforward production? I gathered the first, but let me know if otherwise. Also I understand the "allure" lipstick mechanism (it will cover the lipstick all the way), but what about the lipgloss? Is it supposed to behave the same or out of two parts of the same diameter sticking together? Cab you provide an example of the type of lipgloss if so?
I have a few questions. Do you need this for visualization purposes (renders of the "crystal" shape enclosing, 360 animation) or for straightforward production? I gathered the first, but let me know if otherwise. Also I understand the "allure" lipstick mechanism (it will cover the lipstick all the way), but what about the lipgloss? Is it supposed to behave the same or out of two parts of the same diameter sticking together? Cab you provide an example of the type of lipgloss if so?
Similar Contests on Cad Crowd
Modern and innovative cat tower
We are a small start up business focusing on bringing the outdoors inside for indoor cats. Indoor cats like to climb and feel safe when selecting a preferred level to perch at. Indoor cats like their outdoor cousins, need exercise, plants (especially grass) and access to view out of windows to keep them stimulated. If the cats requirements aren't enough of a challenge then the winning design must fit seamlessly with the ultra modern home interior. We invite you to submit your designs based upon shelves fitted to the ikea stolmen post, a floating shelf design or your own unique concept. Your design may be cat centric or multi purpose for both cat and pet parent.
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Titanium Nails Design
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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.
why is there no code for iWeb when I embed my form?Asked by nanush on August 28, 2015 at 01:02 AM
JotForm Developer
Our developers are working on it, it will be fixed soon.
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A child's (and a person's for that issue) means to read sets the quality of sound for energy occurrence. There is no tilt complete this speech. We have all seen the skill rate studies where the US waterfall behind many an other than countries. We have all heard in the region of the No Child Left Behind jolt from the US establishment and the redoubled constraint on our school systems to verbalize mensurable experiment change wads on uncomplicated acquisition. We all concord that Read to Succeed is more than a commercialism axiom. It is legitimacy in today's globally competing world for our brood. Yet we as parents motionless endure from study to publication myths, peradventure passed on from our parents, probably propagated by social group. If we as parents genuinely poorness our brood to read to succeed, we entail to prevail these tradition and assist our kids by biased what genuinely is established to donkey work for reading occurrence.
Hard-to-Believe Learning-to-Read Facts:
If we poverty to engender a quality for our children, we inevitability to cognize and accept the learning-to-read facts short activity our head's in the sand:Post ads:
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The shadowing facts furnish you an mental object of the austerity of our nation's reading problem:
Nearly 40% of Fourth Graders have not mastered chief linguistic process skills. It's well-nigh 60% in
California, and most fractional of these family before a live audience next to college-educated parents.
Source: - Council for Basic Education
If a youngster is a penniless reader at the end of First Grade at hand is a well-nigh 90% chance that
the juvenile will loiter a mediocre reader at the end of Fourth Grade.
Source: - The Public Library Association
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Experts say in the region of 5% of the nation's offspring learn to read next to ease, most intuitively.
An more 20% to 30% swot to publication near qualified easiness erstwhile they go into conservatory and
begin conventional content. However, the bulk of children (about 60%) have difficulty.
Source: - Council for Basic Education
Hard-to-Believe Learning-to-Read Myths:
Now, characterize the shadowing mythology that we as parents judge without intelligent as true, but are in information not so at all. These Learning-To-Read Myths abet perpetuate poverty-stricken reading skills and disqualify us from winning conduct to put together a change:
MYTH: Kids revise to publication by anyone publication to.
FACT: Reading to teenaged offspring will activity improve their involvement in reading. Many offspring swot up bits and pieces this way; however, "being publication to" does not tantamount "learning to read." And single 5% of offspring really acquire to read by person immersed in language. Learning to read is not similar research to speak, where on earth children accurately "soak up" a articulate idiom. Children must swot up the skills indispensable for reading, and for all but a few, this requires univocal coaching.
MYTH: Reading is a intuitive act that will crop up on its own when a tike is in position.
FACT: There must be a guaranteed horizontal of reading readiness, and peak two-year-olds, for example, are not all set to publication. At one time, language was thought to advance naturally, when a shaver was "mature" enough, but this is no longer the cognitive content. Research now indicates that the 4- to 6-year-old extent is the dessert blotch for law language. Beyond the age of 6 or 7, coaching a nipper to read is simply a crippled of fence in up. Most offspring do not acquire to publication "on their own" - and if a shaver is struggling to read at age nine, the Council for
Basic Education maintains there's a 75% break he or she will have difficulties next to language through full institution.
MYTH: All kids will acquire to publication in educational institution.
FACT: Not necessarily. The scope of entering skills among boyish family - unheeding of their situation - varies widely, and is really ambitious to negociate even for our longest teachers. Providing various focus and action supported on each child's inimitable capabilities, and managing an whole class at the said time, is a discouraging provoke. Yet reading experts say that brood necessitate large indefinite amount of procedure with reading fundamentals (phonics, looking out, blending, etc.). Given the "facts of life" in a schoolroom - overall trainee/teacher ratios and in general profession loading - best teachers do the first job they can. However, too heaps brood are human being left-handed aft. One situation is clear: Too oodles of our children cannot read or have predicament reading. And, if they don't read
fluently, the likelihood for a fulfilling time - in terms of job skills, pecuniary stability, or world success - are greatly vitiated.
What Parents Can Do With Your School:
As in all hitches parents have to solve, conception and characteristic relating the right facts and the "conventional wisdom" folklore is the key to fashioning improvements. This eldest maneuver is instruction. Take these facts and mythology to your kid's teacher, your kids school administrators, your institution territorial dominion and your friends. Ask them what they deem. Ask them to grudge these facts and folklore. Challenge them to frontage the proof in the order of what it takes to buccaneer a small fry to read, and to accustom to you how they are addressing these issues. Ask them if they are determined on on a winning streak their language programs? Ask them if they have a design... are they grasp technology; do they have individualised linguistic process programs? What are they active to do?
Get alive. Get thundery. Email this article, or enhanced yet, print it out and pocket if to you kid's educators in character. Make a lack of correspondence for your children beside your school group. Quite literally, your child's future, and the planned of our very good country depends on it.
What Parents Can Do On Your Own:
In quantity II of this article, we will present assist and suggestions as to what you as an idiosyncratic parent can do on your own to give a hand your boylike youngster to "Read to Succeed". Stay adjusted. The imminent of your children, and our nation is in your keeping.


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Let us know when your receive great advice so we can thank our lawyers and point others with similar questions in the right direction.Got it!
I have mortgage in my name only and property is homesteaded. Is this judgement lein valid? I did not sign any agreement or loan with my spouse. How can a judgement be valid against my homestead. Am wanting to sell and found the lein.
They cannot foreclosure so long as homestead, but it is valid. Depending on when the lien was perfected, you may need to get a release. Best to hire a consumer attorney in your regionAsk a similar question
As long at the loan did not involve purchase money for your homestead, the creditor does not have a valid lien against your homestead. I trust that the judgment was not against you as well. Either way, your homestead would not be subject to an ordinary creditor's judgment lien.
You should write a letter by certified mail to the lien claimant to demand that the lien be released, pointing out that the property is your homestead. If the lien claimant does not voluntarily release the lien, the creditor could be liable for a fraudulent lien under Chapter 12 of the Texas Civil Practice & Remedies Code. Chapter 12 can award damages of $10,000 or actual damages whichever is greater, plus attorney's fees. Point that out in the letter.
You can also demand that the lien be released under Section 53.160 of the Texas Property Code, which provides for a summary procedure (no trial necessary) for the removal of an invalid lien on someone's homestead.
I have a lot of mechanic's lien materials (the law, notice forms, deadline charts, etc.) at my web site, The Construction Report. The web address for The Construction Report is:
Good luck.Ask a similar question
You should file your own partial release of judgment lien. Here is an article I wrote about this specific procedure as well as many other articles about debt judgments in Texas. http://www.judgmentrelease.com/articles/Partial-Release-of-a-Judgment-Texas.htm
My comments are not legal advice and are for informational purposes only.Ask a similar question
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"The youth of our nation are the clearest mirror of our performance." ~Robert F. Kennedy
December 30, 2011 | By wonkum |
2011 has been a busy year for IMPACT. Whether volunteering to support the Congressional Black Caucus Job Initiative, or producing an annual gala with the National Bar Association designed to increase membership and optimize the network of African Americans working to advance progressive change through juris prudence, we worked diligently to ensure that young professionals had meaningful opportunities to obtain the information and to meet the individuals that can assist in professional development and philanthropic success. In what follows, Joshua Humbert, our December IMPACT Leader of the Month discusses the importance of developing a legacy of philanthropy—especially for young professionals. We also review several of IMPACT's initiatives from 2011 before previewing next year's programmatic focus on Political Engagement. Over the coming year we seek to ensure that all young professionals have the resources needed to be politically informed and engaged. Be sure to visit IMPACT for additional information on these initiatives and how you can get involved.
In whatever you do, ask yourself, are you making an IMPACT on your world? You should!


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Lund Abscess
The foremost reason why folks buy a rowing machine is to get into a habit of normal exercises to tone the muscle tissues and burn off calories. In other words, the mixture of low aerobic and muscular fitness enhanced diabetes risk far more than the sum of the two person dangers, Crump told Reuters Overall health by e-mail. What this signifies, is that your system will stick to that you can burn body fat hours as quickly as you have left the gym. Following the belief that the more calories you burn the more calories you can consume without gaining weight, trainers typically suggestions consumers to get in as a lot aerobic exercise as feasible. In contrast, lengthy-term functional reprogramming throughout educated immunity requires a metabolic shift toward aerobic glycolysis and is induced through a dec tin-1-Akt-mTOR-HIF-1α pathway. Playing singles tennis, with close to-continuous motion, is typically regarded as aerobic activity, while golf or doubles tennis, with their a lot more frequent breaks, may not be. Improvements can be produced with as small as 15 to 20 minutes of exercise three times a week.
Consequently, in spite of the skeptics and advertising mavins who claim otherwise, aerobic activity is helpful in burning fat, will not harm muscle mass when following an powerful diet program plan and weight instruction exercise technique, and will enhance metabolic price so that physique fat percentage can stay acceptable at all times. This is interval training at a low level but you still get benefits from it. You can comply with a related routine in your other aerobic exercising as effectively. Given that I place up the aerobic photos, nonetheless, items have taken a dramatic turn.
In one study that tested the correlation, researchers discovered in a study of 43 depressed female undergrads that a 10-week aerobic exercising treatment with no other medical remedy drastically decreased their symptoms. Exercise could help dampen the mental and physical hyperarousal" that appears to be a typical function in insomnia, with feelings of persistently elevated heart rate, mind racing and being unable to turn off your brain, said Christopher E. Kline, a researcher at the University of Pittsburgh Sleep and Chronobiology Center. Exercising can help stimulate the growth of such cells, which are vital to studying.
Morgan Hamilton provides specialist guidance and excellent guidelines relating to all aspects concerning Aerobic Exercises Check out our internet site for much more valuable info about Aerobic Workout routines and other equivalent topics. Too considerably extended duration low to medium intensity activity – the type that physicians advocate as the path to good overall health – can in fact harm your physique. Aerobic activity increases the heart price resulting in an expenditure of calories and weight education increases our metabolic rate, enabling our bodies can burn calories all the time. One of major disadvantage of opened or partially sealed system is sewage is broken down by bacteria and for the duration of this approach methane is released into campus or at ground floor level. … Read More


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The Pinzgau is one of the most manifold and impressing regions of the world - so is its capital city Zell am See! The smooth green mountains (Kitzbüheler Grasberge) with traditional mountain pastures, the mighty Alps Hohen Tauern decorated with everlasting glacier´s ice, the wide valley of the Salzach river with the biggest golf areal in austria, the lake, and the adorned alleis of the Old City are the highlights.
Amongst this alpine wunderworld resides the Sporthotel Alpin!
Our unique location right next to the skislope permits that you easy achieve the full batch of all winter an summer leisure activities in Zell am See.
Finally the most important factor: WE LIVE FOR OUR GUESTS and present AUSTRIAN "GEMÜTLICHKEIT"! Anticipate a candid smile everytime you enter our hotel!
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Elisabeth & Vroni Schwaninger & her "Alpine" Team


text:
End the South Korean Dog Meat Trade
2.5 Million dogs and thousands of cats are brutally tortured and killed in South Korea every year and this must end now!
2.5 Million dogs and thousands of cats are brutally tortured and killed in South Korea every year and this must end now!
There is nothing humane about dying inside a box as gas fills the space. Animals panic, cry, and often fight.
Sign this petition to show how much support exists for ending animal cruelty at the local law enforcement level
NSW Animal Pound's - Observe The Law & Get the Kill Rates Down! The general public need to become more aware of what
I want to ensure no pet owner ever has to put down their pet when they could have recieved the care their owners are
CCTV footage of the dog running through a car park while engulfed in flames was shown on national TV news channels and
Does an Animal Abuser Live in Your Neighborhood? Our communities have good reason to be concerned about the whereabouts
Petition Letter I just signed the following petition addressed to: Cristina Kirschner. ---------------- Prohibir la
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geting stronger laws . reguarding sex crimes and kidnaping We are a small group of people who are about pushing for
70 kids from Smardan school organized various contests to raise awareness about the Romanian dogs plight.
We want to inform these animal abusers' employers about their inappropriate actions with a live chicken
This shelter is the only no-kill animal shelter in the Bronx protecting pets in the neighborhood.
PepsiCo can help stop Conflict Palm Oil plantations from destroying the rainforest homes of the last wild orangutans.
SURPLUS - Giraffe KILLED on Sunday Feb 9th 2014!
Why must innocent animals be used in Human wars?
Taiji dolphin hunters have caught a real prize: A white albino dolphin calf who will die without his mother.
We are turning Mondays into No Kill Mondays, asking every kill shelter in the USA to not kill for one day a week!
Puppy Doe was brutally tortured and in her memory we want to help animals.
Dear friends, dear supporters, This is the latest
Find Cruelty Free Products! (http://www.peta.org/living/beauty-and-personal-care/companies/default.aspx)Pledge Cruelty
Join us in saving the Arctic from reckless oil drilling
Is killing animals entertainment? NBC is airing episodes of the NRA sponsored show promoting violence towards animals
Puppy Doe was rehomed through Craigslist, the people who took her sadistically tortured her.
"Re-homing" leads to sexual, physical, & emotional child abuse. This heinous practice must be investigated and stopped.
Never Be Silent (http://youtu.be/KEcSkAI45Rk)
All animals deserve legal protection from exploitation, cruelty, neglect, and abuse.
The Merced Police Dept. is killing injured kittens and dogs at their shooting range and it has to stop
Hello, i want everyone to make their own minds up about this place. So, in saying that please go to the links provided
Photo of emaciated dog in Florida creates internet
ELLA was a tame Deer and a well loved member of the local community.Ella lived within the Elmwood Cemetery grounds a
35 EUR costs one special, long lasting bowl like this one. Please make a donation at [email protected] When you choose
All the information about what is going on and why animal advocates are so concerned and demand change is in the link
Too many dogs and cats are being killed every day in California shelters and this practice needs to stop.
If you share more people will see the truth...
Claire & Gussie are safe in their new Loving home with Rachael! Thank you everyone who helped spread the word, its
Justice for Cabela.. Skamania County Sheriff's (http://skamaniasheriff.com/) deputies arrived to the home of
HELP CATCH THE KILLER! A group of pet-loving criminal lawyers has launched an online campaign to flush out whoever
The RSPCA Yagoona Shelter obviously like to kill the animals... not do what they can to save and rehome them! PLEASE
PLEASE read and then sign our petition. Brownie lived his days on a short, heavy chain, and fell victim to a callous,
Please support our campaign
PLEASE help make this a reality.WHY - well the picture and story of John and his amazing relationship with his
Dafna was an officer at Broward Animal Care. She was let go for not being able to kill dogs and cats that are not
A VERY BIG THANKYOU TO.. THE WONDERFUL ANIMAL ADVOCATE- SANDRA TEAGER FOR THIS BEAUTIFUL TRIBUTE xoxo.
Ag gag laws criminalize the collection of evidence of animal abuse on factory farms and are a threat to people across
PLEASE JOIN: worldwide support
41 People Get Their BRAVE On for the Dogs... Published on 16 Jul 2013 Song "Brave" by Sara Bereilles 41 people
George Zimmerman is a dangerous, violent person and should NOT be licensed to carry a weapon.
WARNING the images and videos posted by this man on facebook are GRAPHIC. HE LIVES IN - San Martín Texmelucan de
Violence to animals is a clear indicator of psychopathic behavior. Not to Judge Joe Smith, however, who released Sheila
This is an amazing video - please watch and share. "I AM HERE" An Urban Intervention To Make The Invisible
Seeking justice for this unspeakable crime
ANIMAL CRUELTY! Dog dies in LV Heat! The DA has promised to prosecute. PLEASE HELP US TO GET JUSTICE FOR THIS POOR
ARE YOU OUTRAGED OVER THE DEATH OF INNOCENT ROTTWEILER MAX? Then please - Call for Officer to be charged with animal
Elepahnt ( Elephant) being held hostage for ransom by Indonesian farmers. VIDEO - Published on 30th Jun 2013 The
Video: Dog slammed against wall at Orlando pet
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Maryland teenager accused of stabbing family puppy to death! PLEASE READ and share widely. News
Published on 23 Jun 2013 This video is being published to expose a teenager's heinous acts against defenseless animals
THIS IS IN REGARDS TO THE RSPCA IN THE HUNTER REGION OF NSW AUSTRALIA! ...their KILL RATES are pathetic!The RSPCA
WE NEED WORLDWIDE "polite" OUTRAGE VOICED TO THE PREMIER OF VICTORIA AUSTRALIA . BSL is way out of control in Victoria
BSL is out of control in so many countries worldwide. Have you got a Staffy? or any other breed that might resemble a
This is why should you sign and share this petition. Dog lured to its death by University of Limerick students in
From Australia: KERSER was seized because he went into a next door neighbour's backyard. They then called animal rescue
Please follow this cause for a state funeral for Drummer Lee Rigby
Billions of animals in factory farms are forced to live in cruel and inhumane conditions. From chickens that are
WHAT THE MILLION PAWS WALK IS REALLY ABOUT.. Link:
Please express support and sympathy for the families who lost loved ones in the Sandy Hook shooting, and those injured.
because what is the point of a social network when you can't be social? it should be up to the person to accept a
Dear Friends, To mark International Women's Day we are sharing with you a 12 min video on India's female genocide.
To spread the word about the Chinese fur-industry's barbaric animal-welfare violations and appeal for protection laws.
Help sick people in their fight for basic human rights!
Justice for Buddi - demand that Lake Macquarie Council take action on the OWNER of Dangerous dogs!. THIS IS WHY YOU
ANIMAL LOVING FRIENDS WORLDWIDE PLEASE HELP FIGHT FOR BATMAN. This poor old boy would have suffered greatly. His death
People need to sign this petition because Facebook refuses to do anything about this site. They state they don't see any
Wisconsin DNR Proposes Allowing Packs of Dogs to "Train" On Wolves During March Denning and Birthing Season. THIS
Bobo needs to be back with his family. It will be a year on March 20 since he was torn from his home. Do not let Bobo be
Dutch was accused of being a "vicious" or "dangerous" dog and is now in danger of euthanization by a Court Order. In
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Animal cruelty should be prohibited on Facebook in any shape or form.
Captain was a two year old German Shepherd dog who was discovered on July 18, 2012 wrapped in a bloody sheet and left in
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FACEBOOK PAGE.. Kincaid. Killed by Baltimore City
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Chino was shot and killed in Charlotte, NC on private property by a police officer D BRYAN on December 16, 2012. Ivy was
WHO KILLED PEARL? Trap Police Say Was Set 'Deliberately' Suffocates Indiana Pet .. Hebron Indiana ..the perpetrator
Officials in Chester County, Pennsylvania busted a dog fighting ring operating out of Shane Santiago and Laura
Communities have good reason to be concerned about the whereabouts of animal abusers. In story after heartbreaking
PLEASE SIGN AND SHARE..and join the facebook page. Protest Animal Cruelty at MCACC (Animal Control).. The abuses at
SIGN...SIGN....SIGN... WHAT IS AN ANIMAL CRUSH VIDEO? Answer.. The term "animal crush video" is defined under federal
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TELL THE PRESIDENT THAT ANIMAL CRUELTY NEEDS TO BE TAKEN SERIOUSLY....make the people who commit these acts of violence
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PLEASE SIGN AND SHARE WIDELY! The fate of CHUM AND CHAMP will be decided by Provincial Court -Judge Roderick Sutton on
Lawsuit filed on two-year anniversary of shooting death of Rosie the dog.. Please demand that action is taken and the
Hello and thankyou for taking action on this most brutal of Animal cruelty crimes.WE WANT JUSTICE FOR BUDDY...let the
PLEASE SIGN ...this woman is very DISTURBED! WAUSAU Wisconsin — A 20-year-old Wausau woman called -Sean D. Janas - was
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Animal cruelty and the case for harsher punishment.. Dear members of Let's turn Facebook Orange for Animal Cruelty
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JUSTICE FOR HARRIS! Callous owner dumps his dying cancer-stricken dog in freezing temperatures.. ---------------- This
Animal Testing is cruel, unethical and unscientific. We must stop animal testing. Please sign the petition against
Sapper Karl Boon lost his left leg in a Taliban rocket-propelled grenade attack in Afghanistan in 2010 and has been
Did you know?- Eukanuba and Purina Dog Chow are Major sponsors of the Eukanuba World Cup Dog Show in Belfast scheduled
We do know 22 suicides a day..waiting for care. We don't know how many should have received care under CURRENT Policies.
Each year millions of American cows, pigs, chickens, goats, sheep and other animals who are raised, bought and sold for
Please tell the EPA to stand by their decision that palm-based biofuels don't meet the greenhouse gas standards of the
To raise awareness about the illegal killings of animals in Romania and to punish those who harm animals. The European
On July 27th, protests and educational events will be held all over world in front of marine mammal parks and aquariums.


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THE PUBLISHERS' CABARET is back and brimming over with joy, drag and general ridiculousness. 20th Century Theatre, 291 Westbourne Grove, Notting Hill, London W11
April 13th & 14th 2007, doors open 7pm for 8pm start.
Tickets available now. Prices are £15 if paying by Paypal, £13.50 if paying by Cash or Cheque, and £10 per ticket for group purchases of 10 tickets or more. Please email Lucy Luck at email@example.com with ticket requests, or call 020 8992 6142.
Please note any similarity to professional shows of the same name or in fact any professional show of any name is entirely intended but most unlikely.


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Vitamin B-12 Complex Liquid 8 oz. by NOW Foods
Vitamin B-12 Complex Liquid 8 oz.
Vitamin B-12 Complex Liquid - 8 oz.
Item catalog number: 0465
- Nervous System Health
- Essential for Energy Production
- GMP Quality Assured
- A Dietary Supplement
- Vegetarian/Vegan
Suggested Use
Shake well before use. As a dietary supplement, in the morning, take a ¼ teaspoon, hold in mouth for 30 seconds, then swallow.
Refrigerate after opening to maximize freshness.
|Supplement Facts
|Serving Size: 1/4 Teaspoon (1.2 ml)
|Servings Per Container: about 198
|Amount Per Serving
|% Daily Value
|Vitamin C (as Ascorbic Acid)
|20 mg
|Thiamin (from Thiamin HCl)(Vitamin B-1)
|0.6 mg
|Riboflavin (Vitamin B-2)
|1.7 mg
|Niacin (as Niacinamide) (Flush-Free)
|20 mg
|Vitamin B-6 (from Pyridoxine HCl)
|2 mg
|Folic Acid
|200 mcg
|Vitamin B-12 (as Cyanocobalamin)**
|1 mg (1,000 mcg)
|Pantothenic Acid (from Calcium Pantothenate)
|30 mg
|Stevia Extract (Leaf)
|2 mg
|† Daily Value not established.
** This B-12 amount is equivalent to the normal injectable B-12 dose. There are no known toxic levels of B-12. Contains no additives or alcohol.
Other Ingredients
De-ionized water, vegetable glycerin, xylitol, malic acid, potassium sorbate (as preservative), natural vanilla flavor, natural peppermint flavor, ginger root, grapefruit fiber and cinnamon bark oil.
Not manufactured with wheat, gluten, soy, milk, egg, fish, shellfish or tree nut ingredients. Produced in a GMP facility that processes other ingredients containing allergens.
Caution: For adults only. Consult physician if pregnant/nursing, taking medication, or have a medical condition. Keep out of reach of children.
Contains Xylitol, do not feed to pets.
Not manufactured with wheat, gluten, soy, milk, egg, fish, shellfish or tree nut ingredients. Produced in a GMP facility that processes other ingredients containing these allergens. Caution: For adults only. Consult physician if pregnant/nursing, taking medication, or have a medical condition. Keep out of reach of children. Contains Xylitol, do not feed to pets. Contains no additives or alcohol. Made in the USA **This B-12 amount is equivalent to the normal injectable B-12 dose. There are no known toxic levels of B-12.
NOW's Mission
The NOW mission is: To provide value in products and services that empower people to lead healthier lives. NOW Foods is an award-winning and highly respected manufacturer of vitamins, minerals, dietary supplements and natural foods. In 1948, with the natural food and supplement industry in its infancy, entrepreneur Paul Richard paid $900 for the purchase of Fearn Soya Foods—a Chicago based manufacturer of grain and legume-based products. This began a six-decade legacy of providing health-seeking consumers with high-quality, affordable nutrition products.
History of NOW
In 1968, NOW Foods was founded under the belief that good health was not a luxury available only to the wealthy. For the past forty years, NOW has made it their life's work to offer health food and nutritional supplements of the highest quality, at prices that are fair and affordable to all those who seek them. Today, NOW Foods is one of the top-selling brands in health foods stores, an award-winning manufacturer, a respected advocate of the natural product industry, and a leader in the fields of nutritional science and methods development. And while NOW has grown considerably over the past four decades, one thing has never changed – NOW's commitment to providing products and services that empower people to lead healthier lives.
NOW Commitments
Customer Focused and Information Driven - NOW believes that their products, services, and the decisions they make should be primarily influenced by the desires and needs of NOW customers. The decisions that NOW makes should be based on accurate information and therefore NOW places a high value on knowledge and learning within their organization. Quality and Value Oriented - Acting in the best interest of NOW customers, NOW endeavors to produce the highest quality products at competitive prices. NOW's first priority is to maintain quality where it counts the most – in the products. NOW's exceptional cost-conscious team of employees then focuses their energies on driving costs down. Nurturing this competency of value drives NOW's ability to provide high quality products at the very best prices. Natural is Better - NOW is convinced that natural products are better than their synthetic counterparts and produce better results in human health. Therefore, wherever possible, NOW strives to provide products that contain natural ingredients because they are better for their customers.
NOW Science
NOW's experienced professional and technical staff formulates their products to be of the highest quality. NOW has a group of biochemists, chemists, nutritionists, and food technologists who review current science and nutritional parameters, and formulate our products to be effective for the intended use. NOW's structure/function claims are based on science for active ingredients, and on nutritional science for nutritional content. Serving sizes are based on doses fr:" ":als and laboratory studies. The heart of NOW science is third party independent research. NOW investigates and review clinical studies and other lab studies conducted on their ingredients and their formulas. Best science is used to support best formulations which lead to best quality. NOW products are constantly being tested in clinical trials conducted at top universities and research centers around the country and in various parts of the world. NOW products are being tested to determine such things as effectiveness for joint support, quality of life for cancer patients, cardiovascular support, and athletic endurance. Effective products mean health and wellness benefits, which mean quality.
GMP Quality Assured
NOW's GMP certification covers standard operating procedures, employee training, product specifications, expiration dating, vendor certifications and much more. Standard operating procedures include sampling and testing incoming materials according to NOW Foods specifications, inspecting manufacturing processes, and testing finished products to specifications. Tests include organoleptic evaluation (human senses such as sight, taste, smell), physical testing of tablets and capsules, chemical identity of ingredients, potency and contamination testing by the company's in-house state-of-the-art analytical testing lab, as well as microbiological testing by NOW Foods' in-house rapid analysis microbiological lab.


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I keep getting a rash under my left armpit, even when i don't shave for a few days. I changed deodorants to one with melaluca and it burns. I have had breast issues with my left breast for a long time and am still waiting to get answers. I also keep getting small red rash dots in the same area lower under my left armpit, closer to my breast. I have had various mammos/ultrasounds done due to ongoing breast pain and a thickening feeling I have had in my left armpit/breast area and they cannot see anything on the mammogram. Could this be a beginning sign of IBC? My nipple also is VERY tender whenever I touch it. I do check it every morning for leakage just because I am VERY afraid of getting Breast Cancer since it runs in my family. Any advice?READ MORE
Treating red rash under armpit?
Hi - It's a good sign the mammogram doesn't reveal anything; I'm glad you're diligent about noticing these changes in your breast, and addressing them.
If you've had the rash for longer than a few weeks, I'd suspect it's nothing to do with IBC; that would have develop into something more serious-looking by now. But what is it? That's the question. Could be a nerve issue; a hormonal issue...
Have you been to a breast specialist? I'd try a breast specialist before a dermatologist, because then you could ask about ALL the issues that have been bothering you. And if the breast specialist rules out cancer, then perhaps it's time to see a dermatologist? Hope you get some relief soon - PJH
Hi, I'm sorry the rash hasn't gotten better since we corresponded last week. If you read the link I sent you then about rashes and IBC, you've learned about some of the most likely causes for your rash. I've had a rash similar to yours under my left arm, and it is very different from what I experienced with my IBC. Mine clears up when I use an anti-fungal cream and when I am very careful to dry completely before getting dressed. Now I use the hair dryer on cool to make sure that my underarm is completely dry. However, your rash might be from a different cause, so a visit to the dermatologist might well be called for at this point.
Try not to touch the sore nipple area. It will probably get better sooner if left alone.
If I remember correctly, you have been concerned because of your grandmothers' breast cancers. My doctors (and others I've read about) recommend that the daughter or sister of a breast cancer patient start getting breast cancer screening when they are ten years younger than her primary relative was when her cancer was diagnosed. Since I got my cancer at age 50, my sisters and daughter should get checked out at age 40 even though we probably don't have either of the known breast cancer genes in our family. Breast cancers that occur after menopause are not likely to be genetic.
Based on your description of your rash, I really don't think you need to be scared about IBC. I wonder what there is about this rare form of cancer that has made you so worried. Most causes of breast pain or rashes are not cancer. I'm glad that you've had the imaging tests that can help rule out cancer for that area of thickening. Trust your doctor's assessment of the situation, and continue getting regular check-ups.
hi there im 37 soon to be 38 im worried the last few months or so i had some issues with my right breast. feels swollen and feels pain and burning feeling under arm. i did have mamogram where they found califcations said see you nexted year, but im so worried it ibc i also had a ultra sound should that rule ibc out! please help with any info im a nervous wreck can stress do this???READ MORE
what happened with this? I have it right now under my left arm pit as well....thanks for your post!READ MOREYou should know Answers to your question are meant to provide general health information but should not replace medical advice you receive from a doctor. No answers should be viewed as a diagnosis or recommended treatment for a condition. Content posted by community members does not necessarily reflect the views of Remedy Health Media, which also reserves the right to remove material deemed inappropriate.DISPLAY TEXTLINK URL


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Download PDF version: Situation Report Cyclone Phailin End Report
|What Happened
|Assessments in the affected region are still going on. Although there are severe damages, the preparations for the relief work seem also very well and teams are already working on re-installing infrastructure. So far the death toll is at 17 persons, most of them killed by falling trees before the landfall.
|National & Regional Links
|National Disaster Management Authority: www.ndma.gov.in
Odisha DM Authority: http://www.osdma.org/
Andhrapradesh DM authority: http://disastermanagement.ap.gov.in/
Meteorological Centre Bhubaneswar http://www.imdorissa.gov.in/
|Emergency Numbers
|Odisha State Emergency Operation Centre: 0674-2534177.
District control room numbers:
Ganjam 06811-263978,
Puri 06752-223237,
Kendrapara 06727-232803,
Jagatsinghpur 06724-220368,
Balasore 06782-262674,
Bhadrak 06784-251881,
Mayurbhanj 06792-252759,
Jajpur 06728-222648,
Gajapati 06815-222943,
Dhenkanal 06762-221376,
Khurda 06755-220002,
Keonjhar 06766-255437
Cuttak 0671-2507842.
Andhra Pradesh Emergency Nos. for Cyclone as on 11 October 2013:
Srikakulam – 0894-2240557/ 9652838191
Visakhapatnam- 1800425002 Toll Free: 1077/0892-2236947
East Godavari- 0884-2365506
West Godavari- 0881230617
Krishna- 086722525 Toll Free – 1077
Guntur- 08632345103/ 08632234990
Nellore- 08612331477
Odisha Central Control Room is 0674-2534177
|District Collectors of all nine Coastal Districts alerted regarding the IMD's Pre-cyclone watch bulletin and instructed to open control rooms in all vulnerable areas and to remain alert. All line departments such as Fire & Emergency services, Police, Irrigation, Transport, Electricity, R&B, RWS, MA &UD, Fisheries Departments alerted.
|NDMA Do's and Dont's : http://ndma.gov.in/ndma/cyclonesdosdont.html
Emergency Contact Numbers: https://twitter.com/malviyamit/status/388720572386906112/photo/1
Google PersonFinder: http://google.org/personfinder/2013-phailin
Google Crisis Team Spreadsheet: https://docs.google.com/spreadsheet/lv?key=0AjZz-QLzN_xOdDlyb3M1OWpTWGRmU2JtUGE0RFI3OVE&usp=sharing
Updated http://www.google.org/crisisresponse/ Easy to use google crisis map for those NGOs and Volunteers
|Traditional l Media and blogs
|Reuters India: http://reut.rs/185i9RG
Zee News: http://bit.ly/1gwREYW
Around 1.2 crore people likely 2 be affected, says NDMA http://t.co/8SydSWyw7d
IBN Live feed http://ibnlive.in.com/newstopics/cyclone.html
Associated Press http://hosted.ap.org/dynamic/stories/A/AS_INDIA_CYCLONE?SITE=AP&SECTION=HOME&TEMPLATE=DEFAULT&CTIME=2013-10-12-08-16-12
|#CyclonePhailin #Phailin #Odisha #Andhrapradesh
|NDMA: http://on.fb.me/17khecv IMD: http://on.fb.me/1ee80DG
|Maps and Situational awareness reports
|GDACS update: http://bit.ly/1ah3rEs
Hindustan Times: http://bit.ly/1afVFdZ
India's National Disaster Mgt. Authority: http://bit.ly/1afWkvX
India Meteorological: http://www.imd.gov.in/
PDC : http://dynamic.pdc.org/snc/prod/40489
Google CrisisMap: http://google.org/crisismap/2013-phailin?hl=en&llbox=27.17%2C11.24%2C97.79%2C72.98&t=TERRAIN&layers=2%2C5%2C1
CrisisMap: http://google.org/crisismap/a/gmail.com/2013-phailin-cyclone
ESRI: http://www.esri.com/services/disaster-response/hurricanes/latest-news-map
Google CrisisMap in Hindi: http://www.google.org/intl/hi/crisisresponse/2013-phailin.html
|More than 1,600 NDRF personnel have been dispatched to Odisha & AP as part of preparations to handle "very severe" cyclone Phailin http://on.fb.me/1ee80DG
|Odisha: http://22.214.171.124/hfw/Important_Map.asp?GL=1
Andhrapradesh: http://www.ap.gov.in/Pages/Govt-Hospitals.aspx
Hotels in India on Wikipedia: http://en.wikipedia.org/wiki/List_of_hospitals_in_India
|IAF assets have been kept on standby at various bases including — Raipur, Nagpur, agdalpur, Barrackpore, Ranchi and Gwalior. It has also kept two C130J aircraft, 18 helicopters, 2 AN-32s aircraft on a standby to move at a short notice besides asking Eastern Air Command to coordinate relief operation with the task force positioned at Barrackpore.
Twenty-eight teams of National Disaster Response Force (NDRF) have been deployed in Odisha as a preventive measure to handle emergency situations, once cyclone Phailin hits the coast.
Over 700 NDRF personnel have been deployed in four districts – Jagatsinghpur, Khurda, Puri and Ganjam. Fifteen NDRF teams of about 450 personnel will assist the Andhra Pradesh government at Srikakulam district, where the cyclone is expected to hit. NDMA will deploy more teams if needed, and take the assistance of the Navy and the Coast Guard http://on.fb.me/17khecv .
Defense Minister AK Antony on Friday asked the armed forces to be ready to move in to Odisha and Andhra Pradesh in view of the impending severe cyclonic storm Phailin.
|Odisha: http://www.osdma.org/multsecretary.aspx?vchglinkid=GL007&vchplinkid=PL040&vchslinkid=SL017
Andrapradesh: http://disastermanagement.ap.gov.in/website/shelters.htm
|The relatives of a missing person can telephone toll-free number 1949 and give the cell phone number of the missing person. Then BSNL will give the location of the last call made from that cell phone.
|Use Text messaging or update your social media status if voice lines are busy. Also, you can contact the Red Cross Society for assistance in locating missing family members.
Google PersonFinder: http://google.org/personfinder/2013-phailin
|Red Cross
|Followed by the meeting with Govt. the IAG Odisha has called a meeting at 12:30 pm at Red Cross's office on 11th october. agencies and possible Joint need assessment once Cyclone hits the coast.
Dr. Mangala Prasad Mohanty, Honorary Secretary,
Indian Red Cross Society,
Odisha State Branch:
Red Cross Bhawan, Bhubaneswar – 751022.
Code: 0674 (O) 2392389
Fax : 2402389, 2401242, 2407712, 2400205, 2402757 Branches: 31
E-mail : [email protected] [email protected], [email protected]
Website : www.odisharedcross.in
|other NGOs
|Animals in Disaster
|Society for the Prevention of Cruelty to Animals Orissa
Animal Welfare Organizations + Individuals Involved in Care, Rescue, Rehabilitation
People for Animals (India)
International Animal Rescue
List of veterinarians in India: http://vetsinindia.blogspot.com/
List of Veterinary Hospitals: http://www.indiasite.com/Hospital/Veterinary
|Blood Donations
|Donations (In kind or physical donation needs)
|IAG Odisha- 1. Dr. Mangala Prasad Mohanty, Convener IAG Odhisa, [email protected] +91- 9439193110
|Urgent Needs Information
|Transportation, Infrastructure and Road Conditions:
|Bhubaneswar airfield closed. All aircraft moved up, available on call. LIVE updates: http://ndtv.in/GKEvgX
Trains to Bhubaneswar and other parts of the state cancelled. Flights have also been cancelled. http://goo.gl/7ZXdvH
|Picture Links
|Volunteers active
|Paras, Alice, Cindy, Bettie, Christoph, Lloyd, Terri, Aline, Gerald, Judith, Magi, Scott, Nadia


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Proliferating cell nuclear antigen (PCNA), a processivity factor for DNA polymerases δ and , is involved in DNA replication as well as in diverse DNA repair pathways. In quiescent cells, UV light-induced bulky DNA damage triggers the transition of PCNA from a soluble to an insoluble chromatin-bound form, which is intimately associated with the repair synthesis by polymerases δ and . In this study, we investigated the efficiency of PCNA complex formation in response to ionizing radiation-induced DNA strand breaks in normal and radiation-sensitive Ataxia telangiectasia (AT) cells by immunofluorescence and western blot techniques. Exposure of normal cells to γ-rays rapidly triggered the formation of PCNA foci in a dose-dependent manner in the nuclei and the PCNA foci (40–45%) co-localized with sites of repair synthesis detected by bromodeoxyuridine labeling. The chromatin-bound PCNA gradually declined with increasing post-irradiation times and almost reached the level of unirradiated cells by 6 h. The PCNA foci formed after γ-irradiation was resistant to high salt extraction and the chromatin association of PCNA was lost after DNase I digestion. Interestingly, two radiosensitive primary fibroblast cell lines, derived from AT patients harboring homozygous mutations in the ATM gene, displayed an efficient PCNA redistribution after γ-irradiation. We also analyzed the PCNA complex induced by a radiomimetic agent, Bleomycin (BLM), which produces predominantly single- and double-strand DNA breaks. The efficiency and the time course of PCNA complex induced by BLM were identical in both normal and AT cells. Our study demonstrates for the first time that the ATM gene product is not required for PCNA complex assembly in response to DNA strand breaks. Additionally, we observed an increased interaction of PCNA with the Ku70 and Ku80 heterodimer after DNA damage, suggestive of a role for PCNA in the non-homologous end-joining repair pathway of DNA strand breaks.
Proliferating cell nuclear antigen (PCNA) is a processivity factor for DNA polymerases δ and (pol δ and ) (1,2) and is an essential protein for replication of chromosomal DNA (3,4). PCNA forms a toroidal trimer in S phase with replication factor-C (RF-C) and DNA in an ATP-dependent manner and enables the loading of DNA pol δ and onto the complex (3). The close association of PCNA with kinase complexes involved in cell cycle machinery (4) indicates that PCNA has a regulatory role in cell cycle progression (5–8). PCNA also participates in the processing of branched intermediates that arise during the lagging strand DNA synthesis (9,10). The amount of PCNA in eukaryotic cells is ~10-fold more than that of pol δ and RF-C, indicating that PCNA has additional roles other than being a processivity factor. In support, PCNA has been demonstrated to be an integral component of diverse DNA repair pathways such as nucleotide excision repair (NER; 11–13), base excision repair (BER; 14–18) and mismatch repair (19). Reconstitution of an NER reaction in vitro using purified factors indicates a critical role for PCNA in the repair resynthesis step (1).
Two forms of PCNA exist in cells: (i) a detergent-insoluble trimeric form stably associated with the replicating forks during S phase and (ii) a soluble form in quiescent cells in G1 and G2 phases (20). Treatment of quiescent cells with DNA damaging agents like UV-C irradiation (21–23), X-irradiation (24), alkylating agents (25) and hydrogen peroxide (25,26) triggers the redistribution of PCNA from a soluble to an insoluble chromatin-bound complex analogous to that found in S phase cells. The functional transition of PCNA in quiescent cells after DNA damage is an indicator for the repair activity mediated by DNA pol δ and . This approach has been successfully used to determine the efficiency of the NER pathway in cells derived from patients belonging to different genetic complementation groups (A–G) of the human photosensitive disorder, xeroderma pigmentosum (XP). NER deficient human XP-A, XP-F and XP-G cells fail to show any detectable PCNA complex after UV-irradiation (21,27). PCNA complex formation therefore correlates with NER activity in human cells. A good correlation also exists between PCNA complex formation and the BER activity in human cells following oxidative DNA damage induced by hydrogen peroxide (26). A direct link between PCNA and cellular DNA repair processes comes from the observation that PCNA mutations cause increased sensitivity to DNA damaging agents in budding and fission yeast (28–31).
In the present study, we examined the formation of PCNA complex in human cells in vivo after treatment with ionizing radiation (IR) and a radiomimetic drug, Bleomycin (BLM). Both these agents induce single-strand breaks (SSBs) and double-strand breaks (DSBs) in the genomic DNA. We compared the PCNA complex formation of normal cells with radiation sensitive cells derived from Ataxia telangiectasia (AT) patients. Our studies show that PCNA complex formation, indicative of DNA pol δ and activity, was efficiently induced by γ-radiation and BLM. The PCNA–chromatin association formed fairly rapidly after DNA damage and almost reached the level of untreated cells by 6 h, suggestive of the completion of repair events. AT cells showed an identical response to that seen in normal cells, indicating that the assembly of PCNA–chromatin complex in response to DNA strand breaks occurs independent of the ATM gene product. Efficient PCNA complex formation in AT cells despite their abnormal p53 induction further suggests that PCNA-dependent repair activity is independent of p53 associated pathway(s) in human cells.
Normal human dermal fibroblasts (NHDF) were obtained from Clonetics. Human normal (WI38 and MRC5) and AT (GM5823C and GM2052C) primary fibroblast cells were procured from Coriell Cell Repository (Camden, NJ). All the cells were routinely maintained in 2× Eagle's minimal essential medium supplemented with 15% fetal bovine serum, vitamins, essential amino acids, non-essential amino acids and antibiotics (Gibco BRL). The cultures were maintained at 37°C in a humidified 5% CO2 atmosphere.
Normal and AT cells were synchronized at G0/G1 phase by growth in low serum (0.5%) containing medium for 2–3 days prior to treatment with DNA damaging agents. Alternatively, cells were synchronized at G0/G1 by growing them to confluence and maintaining them in this state for a week. Flow cytometric analysis revealed that >95% of the cells were in G1 phase of the cell cycle. γ-Ray irradiation was done using a 137Cs source delivering a dose rate of 0.98 Gy/min (Gamma cell 40, Atomic Energy of Canada). Cells were irradiated with different doses (5–30 Gy) and incubated for various post-incubation times. For BLM treatment, cells were washed twice with phosphate-buffered saline (PBS) containing 1 mM CaCl2 and permeabilized with 40 µg/ml of l-α-lysophosphatidylcholine (Sigma) in PBS–CaCl2 for 2 min on ice. The solution was carefully removed and the cells were treated with BLM (Calbiochem; 10 mU/ml) in serum free medium for 30 min at 37°C. After treatment, the medium was removed and the cells were washed once with PBS. The cells were incubated in complete medium for different recovery times.
Cells were grown on glass chamber slides for immunofluorescence (Labtek). The cells were exposed to either γ-rays or BLM and incubated for the indicated times. The slides were treated with a hypotonic lysis solution (buffer I) containing 10 mM Tris–HCl pH 7.4, 2.5 mM MgCl2, 1 mM PMSF and 0.5% Nonidet P-40 for 8 min on ice. In some cases, the slides were treated with buffer II (25 mM sodium phosphate buffer pH 7.4, 0.5 M NaCl, 1 mM EDTA, 0.5% Triton X-100, 10% glycerol, 5 mM MgCl2 and 1 mM PMSF) for 10 min on ice. They were then washed once in PBS and fixed in ice-cold acetone–methanol (1:1). The slides were washed in TBST (20 mM Tris–HCl pH 7.4, 137 mM NaCl and 0.2% Tween-20) and incubated for 30 min in TBST containing 5% non-fat dried milk (NFDM). The slides were washed in TBST and incubated with mouse monoclonal PCNA antibody (1:10 dilution in TBST–5% NFDM; Santa Crutz Biotechnology) for 1 h at 37°C. The slides were washed three times (5 min each) in TBST buffer, incubated for 1 h with goat anti-mouse IgG2a antibody conjugated with fluorescein isothiocyanate (FITC; Boehringer Mannheim) diluted 1:20 in TBST–5% NFDM. The slides were washed in TBST, counterstained for DNA with DAPI (0.1 µg/ml prepared in Vectashield mounting medium; Vector laboratories) and covered with a cover glass. The variously fluorochromed images were captured using a Cytovision imaging system equipped with a charged coupled device camera and the PCNA fluorescence intensity per cell was measured using Adobe Photoshop.
MRC5 cells in G1 phase were irradiated with 20 Gy of γ-rays and incubated in the presence of 100 µM BrdU for 3 h at 37°C. The slides were immunostained for PCNA and fixed in 2% formaldehyde in PBS containing 50 mM MgCl2 for 10 min at room temperature followed by denaturation in 0.07 M NaOH in 70% ethanol for 2 min and dehydrated through cold ethanol series. The slides were immunostained using mouse primary BrdU antibody (1:50 dilution, 1 h at 37°C; Boehringer Mannheim) followed by biotinylated anti-mouse IgG (1:100 dilution, 1 h at 37°C; Vector Laboratories) and Texas red-conjugated Avidin (1:100 dilution, 1 h at 37°C; Vector Laboratories). The slides were washed thoroughly in TBST buffer, counterstained for DNA with DAPI (0.1 µg/ml prepared in Vectashield mounting medium; Vector laboratories) and covered with a cover glass. The images were captured using an Axioplan2 Imaging microscope (Zeiss) using the Isis software programme of MetaSystems.
Soluble and insoluble proteins were isolated following the protocol of Prosperi et al. (32). At different post-incubation times, 6–8 × 106 treated and mock treated cells were lysed for 8 min on ice in 500 µl of buffer I. The cell lysates were centrifuged at 735 g (Eppendorf Centrifuge 5415C) for 5 min and the soluble proteins (supernatant) were transferred to a fresh tube. The pellet fraction containing the detergent insoluble proteins was lysed for 20 min with 200 µl of buffer II. The proteins were recovered by centrifugation at 11 750 g for 5 min. Protein concentration was determined by Pierce protein assay kit. Aliquots of 20 µg of soluble and insoluble proteins were fractionated by 4–20% polyacrylamide gradient gel electrophoresis and blotted onto PVDF membrane following the standard protocol (Novex). The membrane was incubated with TBST buffer containing 5% NFDM for 60 min at room temperature. It was then incubated with mouse monoclonal antibody to PCNA (PC10, 1:1000 dilution in TBST–5% NFDM; Santa Cruz Biotechnology) for 1 h and washed three times (5 min each) in TBST buffer. The membrane was incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody (1:2000 dilution in TBST–5% NFDM; Vector Laboratories) for 1 h followed by repeated washing in TBST buffer. The signal was detected using the enhanced chemiluminescence technique (ECL) following the manufacturer's protocol (Amersham). Immunodetection of actin (Mouse IgM, 1:2000 dilution; Oncogene) and p53 (D0-1, Mouse IgG, 1:1000 dilution; Santa Cruz Biotechnology) was carried out as described above. The PCNA band intensity was measured using the Scion Image Programme (National Institutes of Health).
The trimeric structure of PCNA was preserved for SDS–PAGE by the addition of glutaraldehyde to a final concentration of 0.1% to the insoluble protein extracts. The samples were kept on ice for 20 min and the reaction was terminated by addition of lysine to a final concentration of 0.1 M (33). The samples were briefly heated at 80°C for 5 min prior to electrophoresis.
Total cellular proteins were extracted using RIPA buffer (10 mM sodium phosphate buffer pH 7.2, 2 mM EDTA, 50 mM sodium fluoride, 150 mM NaCl, 0.2 mM sodium vanadate, 0.1% SDS, 1% sodium deoxycholate, 1% Nonidet P-40 and 10 U/ml Aprotinin) from control and γ-irradiated (20 Gy) WI38 cells. Proteins (500 µg) were pre-cleared with normal mouse serum and 25 µl of protein G agarose beads (Boehringer Mannheim) for 1 h at 4°C with constant mixing. The samples were spun at 8160 g for 20 s and the supernatant was carefully transferred to new Eppendorf tubes. PCNA antibody–agarose conjugate (25 µl; Santa Cruz Biotechnology) was added to the samples and mixed constantly for 4 h at 4°C. The immunoprecipitated complex was pelleted down by centrifugation and washed four times in RIPA buffer under stringent conditions. The pellet was resuspended in SDS–PAGE loading buffer (100 µl), boiled for 5 min, vortexed and centrifuged. Supernatant (25 µl) was loaded in a 4–20% gradient gel and transferred to PVDF membrane using the manufacturer's specifications (Novex). The membranes were probed with either Ku70 or Ku80 antibody (1:1000 dilution; Neomarkers) followed by HRP-conjugated secondary antibody (1:2000 dilution; Vector Laboratories). The signal was detected using the ECL method (Amersham).
The treatment of NHDF cells with BLM (10 mU/ml) was done essentially as described earlier and the cells were collected by trypsinization at 0 min and 3 h after treatment. Alkaline single gel electrophoresis was done following the method described by Angelis et al. (34).
In order to determine the involvement of PCNA with IR-induced DNA damage, normal human primary fibroblast cells synchronized at G1 phase were irradiated with γ-rays and the chromatin-bound PCNA complex was analyzed by both immunofluorescence and western blot techniques. Fixation of cells with methanol revealed both soluble and insoluble forms of PCNA. In the initial experiments, cells were fixed and stained for PCNA without hypotonic buffer I treatment. The PCNA intensity detected by immunofluorescence and western blot was comparable between control and γ-irradiated (5 Gy) cells (Table (Table1,1, Fig. Fig.1A).1A). To determine the chromatin-bound PCNA after DNA damage, cells were treated with buffer I containing 0.5% Nonidet P-40 to release the unbound PCNA. In the untreated cells, a major proportion of PCNA was in the soluble form and the chromatin-bound PCNA was barely detectable. In cells irradiated with 5 Gy of γ-rays, intense PCNA staining was observed in the nuclei of all the cells (Fig. 1B). The PCNA complex appeared to form rapidly during irradiation, as the cells fixed immediately after 5 Gy of γ-irradiation (irradiation time, 5 min; no recovery time) also showed intense chromatin-bound PCNA foci (data not shown). Quantitation of PCNA fluorescence intensity in control and irradiated cells revealed that the PCNA complex formed after IR was resistant to high salt extraction (buffer II), indicating a very tight binding of PCNA to DNA (Table (Table1).1). The chromatin association of PCNA was lost by DNase I treatment evidenced by greatly diminished staining of PCNA (data not shown). The time course kinetics of PCNA foci formation was next determined in normal WI38 cells exposed to 10 Gy of γ-rays (Fig. 2A–F). For this purpose, 50 randomly chosen cells were analyzed for each time point. Microscopic examination of cells under high magnification (1000×) revealed a nice speckled pattern of 567 ± 10.3 (mean ± SE) focal sites of PCNA per cell immediately after irradiation (0 min recovery). The number of foci then declined to 166 ± 1.6/cell and 53 ± 0.7/cell after 3 and 6 h, respectively. A representative nucleus from each time point showing the time course distribution of PCNA foci is shown in Figure Figure22 (Fig. (Fig.2A2A and B, 0 min recovery; Fig. Fig.2C2C and D, 3 h recovery; Fig. Fig.2E2E and F, 6 h recovery).
In order to determine whether the PCNA foci associate with repair sites, co-localization of PCNA- and BrdU-labeled repair sites was carried out by immunofluorescence microscopy. With the exception of S phase cells, BrdU repair incorporation was not detected in the unirradiated cells (Fig. (Fig.2G).2G). Cells exposed to 20 Gy of γ-rays displayed repair foci detected with BrdU antibody after continuous labeling of cells with BrdU for 3 h after irradiation (Fig. (Fig.2H 2H and J). As BrdU labeling shorter than 3 h resulted in less consistent labeling patterns, co-localization of PCNA and BrdU sites could not be done earlier than 3 h after irradiation. In order to co-localize PCNA- and BrdU-labeled repair sites, PCNA (Fig. (Fig.2I) 2I) and BrdU sites (Fig. (Fig.2J)2J) were detected by FITC- and Texas red-conjugated antibodies, respectively. Analysis of 50 randomly chosen cells revealed that each interphase nucleus exhibited an average of 223 ± 2.6 (mean ± SE) PCNA foci and 183 ±1.5 BrdU-labeled repair sites. The merging of PCNA foci and BrdU sites showed the regions of overlap as yellow color (Fig. (Fig.2K2K and L). Microscopic analysis indicates that 40–45% of PCNA foci co-localized with repair sites labeled with BrdU.
AT cells are extremely radiosensitive and exhibit defects in the regulation of different cell cycle phases after damage. The gene mutated in AT patients (ATM) is considered to play an important role in radiation-induced damage-signaling pathways in mammalian cells. We therefore wished to determine whether or not ATM plays a role in regulating the efficiency of PCNA complex formation in response to DNA damage. The PCNA complex formation after IR was studied in two AT cell lines that carry homozygous mutations (indexed in the Coriell Cell Repository Catalogue). Immunofluorescence analysis of PCNA complex in buffer I extracted control and irradiated (5 Gy) cells revealed identical staining patterns in the two normal (NHDF and WI38) and the two AT (GM5823C and GM2052C) cell lines (Fig. (Fig.3).3). In order to determine whether the PCNA complex formation exhibits a dose-dependent response, normal (NHDF) cells were irradiated with 5, 10, 20 and 30 Gy of γ-rays and the proteins isolated 30 min after irradiation were analyzed by western blot. The enrichment of PCNA in the insoluble pool of proteins showed a 3-, 7.8-, 10- and 11.4-fold increase over control for 5, 10, 20 and 30 Gy of γ-irradiation, respectively (Fig. (Fig.4A4A and B) illustrating a dose-dependent PCNA complex formation.
The time course kinetics of PCNA complex formation were then analyzed by western blot in all four cell lines. The PCNA complex was readily detectable soon after irradiation (irradiation time, 10 min; no recovery time; Fig. Fig.5A5A and B) in the insoluble proteins and gradually declined to the control level (≥1) by 6 h in WI38 and two AT cell lines (Fig. (Fig.5B).5B). The NHDF cell line, however, showed a 2.7-fold increase in the induction of PCNA over control at 6 h, which declined to 1.9-fold at 24 h. Although there was variation among the cell lines, the kinetics of PCNA complex formation (peaking at ≥0 min recovery and declining at 6 h post-irradiation) was essentially similar in all the four cell lines. The time-dependent association of PCNA with chromatin appears to coincide with the fast kinetics of DSB repair reported during the first 30 min after IR (35,36). In order to verify the equal loading of proteins, the same membranes were stripped and immunoreacted with actin antibody (Fig. (Fig.55A).
Irradiation with γ-rays induces a wide spectrum of lesions including SSBs, DSBs, oxidative base damage and DNA–protein crosslinks. To evaluate a direct involvement of PCNA in the repair of DNA strand breaks, PCNA complex was analyzed in both normal and AT cells following treatment with an anti-tumor drug, BLM, which induces DNA strand breaks. As the BLM uptake may be variable among cells, BLM treatment was carried out in lysolecithin permeabilized cells. NHDF cells were treated for 30 min with different concentrations of BLM (0, 5, 10, 20, 30 and 40 mU/ml) and the soluble and insoluble proteins were analyzed for PCNA. The extent of DSBs induced by the highest concentration of BLM (40 mU/ml) is expected to approximately equal that generated by 15 Gy of X-rays (37). BLM effectively triggered PCNA redistribution at all concentrations but, unlike IR, no dose-dependent increase in PCNA complex was observed (Fig. (Fig.6).6). It is likely that the PCNA complex formation reached a saturation level even at the lowest BLM concentration. As there was no dose response, a concentration of 10 mU/ml was chosen for subsequent experiments. Western blots of insoluble proteins extracted from normal and AT cells showed a 4–5-fold increase in PCNA induction as compared to control cells immediately after 30 min BLM treatment (0 min; no recovery) and this PCNA level persisted up to 1 h after treatment (Fig. (Fig.7A 7A and B). The PCNA level was reduced to 1.7-fold by 3 h after treatment. In order to verify equal loading of proteins, the same membranes were stripped and probed with actin antibody. Alkaline single gel electrophoresis of BLM-treated (10 mU/ml) NHDF cells was carried out to determine the initial induction and repair of DNA strand breaks. The initial induction of strand breaks expressed as Olive tail movement was found to be 6.8 at 0 min after BLM treatment, which then declined to 1.8 after 3 h of repair, demonstrating the repair of 74% of the DNA strand breaks. Thus, there is a good correlation between the time course of PCNA complex and the repair kinetics of DNA strand breaks. The efficient PCNA complex formation induced by both IR and BLM in AT cells suggest that the ATM gene product is not required for PCNA complex assembly in response to DNA strand breaks.
A recent study has indicated that p53 regulates the expression of PCNA in rat embryo fibroblast cells upon exposure to IR (38). In order to verify this possibility in the human cells, time course induction of p53 was compared with that of PCNA in normal and AT cells after γ-rays and BLM. In γ-irradiated normal cells, maximal induction of p53 was observed between 2 and 6 h and the p53 level declined at 8 and 24 h (Fig. (Fig.8). 8). In addition to p53, a low molecular weight protein was detected with D0-1 antibody in NHDF cells. This band was observed in NHDF cells but not in MRC5 and WI38 cells. In contrast to p53 induction, the PCNA level peaks immediately after irradiation and drops to the level of unirradiated cells by 6 h in both normal and AT cells (Fig. (Fig.5A5A and B). In spite of a greatly attenuated p53 induction (Figs (Figs7A7A and and8),8), AT cells showed the PCNA complex formation immediately after irradiation like that observed in normal cells. Likewise, p53 accumulation did not correlate with PCNA complex formation after BLM treatment (Fig. (Fig.7A),7A), indicating that the p53-dependent pathway is not critical for DNA damage-dependent PCNA response.
The functional form of PCNA is often phosphorylated and exists as a trimeric structure, which is loaded onto DNA template in the presence of RF-C (20). In order to determine whether the chromatin-bound PCNA formed after DNA damage also occurs in a functional trimeric form, the insoluble proteins isolated from NHDF cells were crosslinked with glutaraldehyde and the PCNA trimeric structure was analyzed by western blot using PCNA antibody. The PCNA trimeric structures were rapidly triggered after treatment (0 min; no recovery) and the induction was 4- and 6-fold more than that of control cells in γ-rays and BLM-treated cells, respectively (Fig. 9). The PCNA trimeric structures constituted 22 and 37% of the total insoluble PCNA detected immediately after exposure to γ-rays and BLM, respectively, and the remaining fraction was in the monomeric form. The specific enrichment of PCNA trimer in a time- and damage-dependent manner in normal cells indicates the involvement of PCNA in strand break repair activity.
The damage-dependent redistribution of PCNA in normal and AT cells clearly reflects its role in the repair of DNA strand breaks. In order to determine whether the repair function of PCNA is mediated through its interaction with known DSB repair proteins such as Ku70 and Ku80, immunoprecipitation studies were carried out using PCNA antibody. Western analysis indicates that Ku70 protein was much more abundant than Ku80 and the levels of Ku70 and Ku80 remained grossly similar in both control and irradiated samples (Fig. (Fig.10A).10A). Western blot analyses of immunoprecipitated complexes using antibodies to Ku70 and Ku80 indicated a tight physical interaction of PCNA with the Ku70/80 heterodimer and this interaction was increased at 3 h after irradiation (Fig. (Fig.10A).10A). The immunoprecipitation was highly specific as omission of either primary or secondary antibody did not result in the precipitation of Ku70/80 proteins. Immunoprecipitation done using p53 antibody also did not precipitate the Ku proteins (data not shown). The observed interaction of PCNA with Ku70/80 heterodimer raises the possibility that PCNA may be involved in non-homologous end joining (NHEJ) pathway. Immunoprecipitation results were confirmed by immunological co-localization of PCNA and Ku70/80 heterodimer in WI38 cells 3 h after irradiation (Figure (Figure1010B).
The chromatin association of PCNA was observed in earlier studies following oxidative damage (25,26), and bulky DNA adducts (21,25,39,40) induced by hydrogen peroxide, UV and alkylating agents. The functional transition of PCNA from a soluble to an insoluble form observed after DNA damage is considered to reflect the repair activities mediated by pol δ and (20). We have studied the efficiency and time course kinetics of PCNA complex assembly in response to DNA strand breaks induced by γ-rays and BLM. Both agents triggered rapid PCNA complex formation in a time-dependent manner in human primary fibroblast cells. The maximal level of chromatin-bound PCNA observed in cells sampled immediately after exposure indicates that the process occurs even during the treatment time. Similar rapid formation of PCNA complex in radiosensitive ATM deficient cells indicates that ATM gene product is not required for this phenomenon. PCNA relocation was observed in the nuclei of damaged cells but not in mock treated cells, suggesting that the process is highly specific in relation to DNA repair activities.
The spatial distribution of PCNA foci in γ-rays and BLM-treated cells was homogenous throughout the nucleoplasm and this pattern was very similar to that obtained after UV damage (22,39). In both NER and BER pathways, PCNA is implicated in the repair resynthesis step and the repair patches vary from 5–7 to 25–30 bp for BER and NER, respectively (41,42). Unlike UV, which induces bulky DNA adducts, γ-rays and BLM predominantly produce SSBs and DSBs and the repair of these lesions is not expected to involve the resynthesis step. If PCNA is required only for the resynthesis step, it would be difficult to explain the formation of PCNA complex in response to DNA strand breaks. Miura et al. (24) have documented the formation of PCNA foci in X-irradiated cells of normal and NER-deficient XP-A cells. It is likely that the lesions generated by X-rays are repaired by PCNA-dependent long patch BER pathway. Although compelling evidence is lacking, the correlation observed in this study between the time course kinetics of PCNA complex and the repair of BLM-induced strand breaks (detected by single gel electrophoresis) suggests a role for PCNA in strand break repair activity in human cells.
We observed a rapid assembly of chromatin-bound PCNA foci soon after γ-irradiation, followed by the disappearance of majority of foci 6 h after treatment. Although the functional significance of this complex remains largely unclear, the time course of PCNA foci formation correlates with well-established kinetics with half-lives of 15–30 min and 1–3 h for SSB and DSB repair, respectively (35,36). The present study raises two possibilities for the potential participation of PCNA in the repair of DNA strand breaks. First, PCNA may scan the damage in the genomic DNA and effectively mediate strand break repair by recruiting the factors involved in both NHEJ and homologous recombination (HR) repair pathways. Secondly, PCNA may help in loading the DNA pol δ and onto the damaged DNA template and enhancing their processivity. Experimental evidence supports both of these possibilities. The first possibility is supported by the rapid functional transition of PCNA that may participate in a damage-signaling pathway. Recently, PCNA has been demonstrated to recruit chromatin assembly factor to SSBs and gaps (43). This suggests a potential role for PCNA not only during the repair synthesis but also in nucleosome assembly following DNA repair. Alternatively, PCNA may also serve as a loading dock for the catalytic turnover of repair proteins, thereby effectively mediating strand break repair activity in cells. Such a role for PCNA has been proposed for the NER pathway (11). The increased interaction of PCNA with Ku70/80 heterodimer after damage may be supportive of such a role for PCNA in mediating the NHEJ pathway.
The second possibility is strengthened by the time- and dose-dependent formation of PCNA trimeric structures observed in IR- and BLM-treated cells, which reflect DNA polymerizing activity, as PCNA trimeric structures are often associated with active DNA synthesis (44). The association of PCNA foci with the synthetic sites of repair as well as the formation of PCNA trimer observed after DNA damage strongly suggests the involvement of PCNA-dependent polymerization activity during the repair of strand breaks. In corroboration with our results, very long repair patches ranging from 3 to 150 nt were found after DSB repair in IR- and BLM-treated human cells at G1/S border (45). These long repair patches were abolished upon inhibition of pol δ and by aphidicolin and cytosine arabinoside (45). This study suggests that some of the strand breaks may be processed by DNA pol δ and , possibly requiring PCNA as an accessory factor for processivity. Whisnant-Hurst and Leadon (45) showed that the long repair patches constituted 20% of the repair label, which is in agreement with the fraction of PCNA trimeric structures observed after IR and BLM treatments in this study. Furthermore, we found that 40–45% of the PCNA foci co-localize with BrdU-labeled repair sites indicating the PCNA-dependent polymerizing activity in γ-treated cells. Also, treatment of cells with the DNA polymerase inhibitors aphidicolin and hydroxyurea after γ-irradiation resulted in the persistence of PCNA foci up to 24 h (data not shown). Budd and Campbell (46) have shown that aphidicolin-sensitive polymerases are involved in the repair of plasmid DNA damaged by γ-rays and BLM. DNA pol δ and have been found to be involved in the repair of IR-induced DNA damage in yeast (47,48). These studies provide supporting evidence for the role of pol δ and in the repair of strand breaks.
One of the novel findings of the present study is the demonstration of an efficient PCNA complex in radiation-sensitive AT cells. ATM, the gene mutated in the AT patients, encodes a 370 kDa protein with a phosphatidylinositol kinase (PI-3) domain, a feature shared by several other proteins involved in DNA damage response, cell cycle control and telomere maintenance (49,50). ATM phosphorylates p53 on serine15 and leads to the stabilization of p53 following DNA damage (51,52). Recently, the requirement of ATM and c-Abl has been demonstrated for the IR-induced assembly of a recombination complex comprising Rad51 and 52 proteins (53). However, the DNA damage response of PCNA to IR and BLM occurs independent of both ATM and p53 induction in AT cells. Foray et al. (54) have demonstrated that the repair of strand breaks is faster in AT cells than normal cells during the first 6 h after irradiation. Furthermore, the repair profile was grossly similar in both normal and AT primary fibroblast cells treated either at 4 or at 37°C with 30 Gy of γ-irradiation (35,36). Thus, the rapid PCNA complex formation observed corroborates the efficient strand break repair reported in AT cells. In IR- and BL-treated cells, a punctuated pattern of PCNA distribution is found homogenously throughout the nucleoplasm and the PCNA foci are tightly bound to nuclear substructures. In contrast to the kinetics of PCNA complex formation, human Mre11 and Rad50 form foci 8 h after irradiation in exponentially growing cells (55). It is likely that a small fraction of DSBs are repaired in mammalian cells by the HR pathway involving the Mre11 and Rad50 complex, and this pathway may be much slower than that of the PCNA-dependent repair pathway.
In this study, we have demonstrated efficient PCNA complex formation in a time-dependent manner following DNA damage in normal and AT cells. The rapid formation of a PCNA–chromatin complex, comprising monomer and trimer forms, in response to strand breaks suggests a role for PCNA both in damage recognition and in the rapid repair of SSBs and DSBs. Future studies are required to understand the precise mechanism of PCNA complex formation and its functional significance in the repair of IR- and BLM-induced DNA damage.
The authors wish to thank Dr M.P.Hande for helpful comments on the manuscript. The authors are grateful to Dr T.S.Kumaravel and Mr Althaf Lohani (Gerontology Research Center, NIA, NIH, Baltimore, MD) for help with alkaline single gel electrophoresis. This study was supported by research grants from NIH/NCI (CA75061 and CA49062) and DOE (DEFG02-98ER62667) awarded to C.R.G.
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Personalised Diary - Don't Publish
Great purchase
Worth the money, great product and good quality
By Khanom from London
Personalised Diary - Don't Publish
brilliant job
Lovely custom made diaries.
By happy from UK
Personalised Diary - Don't Publish
Good purchase
Quick delivery as ever, and I'm sure the wife will be pleased with it as a gift from our sons!
By Wozzy Chops from Ashford
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Benq GW2470HM. Display diagonal: 60.5 cm (23.8"), Display brightness: 250 cd/m², Display resolution: 1920 x 1080 pixels. RMS rated power: 2 W. Colour of product: Black, Heavy metals free: Hg (mercury). Mean time between failures (MTBF): 60000 h, Windows operating systems supported: Windows 10 Education,Windows 10 Education x64,Windows 10 Enterprise,Windows 10 Enterprise x64,Window. Power consumption (typical): 30 W, AC input voltage: 90-264 V, Power consumption (off): 0.5 W
Everyday Enjoyment for Leisure and BusinessThe GW2470HM monitor with Eye-Care design brings viewing pleasure to work and play with exquisite details on striking visual performance.Key features:- 3000:1 High Contrast Ratio- HDMI input- Built-in speakers- Flicker-free Technology- Low Blue Light Mode