text:
ONGC win Savio Cup
Mumbai: Oil & Natural Gas Corporation (ONGC), Uttarakhand underlined their supremacy over Indian Overseas Bank (IOB), Chennai by posting a convincing 70-52 win in the men's final of the UPL 9th Savio Cup All-India basketball tournament, sponsored by Tridhaatu Realty and Indian Oil and organised by the Don Bosco Recreation Centre.
Trideep Rai top scored for ONGC with 22 points, while Yadwinder Singh scored 19 and Vishesh Bhriguvanshi netted 13 points. ONGC were richer by Rs 1.25 lakh, while IOB received a cash prize of Rs 65,000. In the women's final, Southern Railway beat Maharashtra 67-62.
ONGC's Yadwinder Singh shoots to score in the Savio Cup final


text:
Add to Calendar
Enjoy the sights, sounds and smells of this amazing woodland in spring. Throughout this portion of The Greenbelt a gorgeous palate of wildflowers and the colorful fanfare of migrating spring birds will highlight an afternoon of forest bathing.
Participants will meet in the Nevada Avenue Parking Lot

subset: commoncrawl


text:
Re: New to the group
Expand Messages
- Hi Mary,
We are a fairly small group. We have 5 regulars, and many more who
attend sporadically. We meet every Wednesday from 7 p.m. until they
kick us out. Our get-togethers are at Caribou Coffee on Harlem Ave in
Tinley Park.
We range in age from 20's to 50's, but have had teenagers and seniors
attend, they just don't seem to come as often.
The staff at Caribou seem to like us. They mentioned that to me the
other day when I posted another sign on the bulletin board.
We are a very friendly group. Please come knit with us.
I look forward to seeing you tomorrow.
Becca (Tzurriz)
- Hi Mary Bridget!Hope you can join us some Wednesday!--
Jen Donnelly
-------------- Original message --------------
From: "Mary Bridget" <elefink2@...>
Hi, I was a member of SNBWB, but got a new job in Homewood a few
months ago, and can't make the drive to Wheaton after work anymore.
Anyway, I just wanted to introduce myself to the group and find out
about where the meeting are held, and a bit about who goes to them.
Mary Bridget
Your message has been successfully submitted and would be delivered to recipients shortly.


text:
Founded in 1987, MacNaughton Associates is a national, full-service retained executive search firm located in Southern California. By design, the firm has remained a compact, boutique firm that concentrates primarily on education and not-for-profit organizations.
We consider ourselves generalists and work across disciplines at the president down through the director levels. As a result of more than twenty years of refining executive search expertise.
MacNaughton Associates is able to offer our clients the effectiveness of a nationwide network of contacts and its substantial experience matching the specific leadership needs of an organization with the talents and judgment of seasoned professionals from compatible institutions around the country.


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Maximizing Space Utilization through Electronic Event Scheduling: A Case Study of Best Practices. Keith Stiles Blue Ridge Community College jk\[email protected] Information About BRCC. Location: Flat Rock, NC 128 acres, 13 buildings, 358,000 sq. ft.
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Blue Ridge Community College
Michael S. Rudden. (2008). "Ten Reasons Why Colleges and Institutions Undertake Campus Master Planning (And How to Align Your Campus Planning Effort to Best Address Them). Planning for Higher Education 36 (4), 33-41.
"We believe that improved accountability is vital to ensuring the success of all the other reforms we propose." (Final Report of the Commission on the Future of U.S. Higher Education)
"Institutional researchers can assist administrators in overcoming technical and institutional barriers to most effectively use space information and assist them in managing their complex enterprises" (Watt, 2007).
Analysis of Current Space Issues
Modeling of Future Space Needs
Modeling future building configurations using space and scheduling data to arrive at more efficient configurations of academic space.
Creating multi-year projection models for decision-makers concerning likely future academic space needs especially concerning classrooms and labs coupled with analyses of current scheduling practices.


text:
|31 Jul 2013
cat 5 or cat 6 cables - differences in service providers ?
In normal residential internet service wiring....is there any difference it the coaxial cables run by Cox Cable (or others) vs Direct TV ? Does either or both use a different type of cabling than normal cat 5 or whatever (cat) is commonly used today ?
|My System Specs
|31 Jul 2013
Windows 7 Professional SP1 - x64 [Non-UEFI Boot]
I think coaxial cables are most used for connecting devices between them...while Patch cables should be used to internet, network...Cat5 or Cat6.
I'm using at home for network V7 CAT6 Patch Cables just more reliable than the Cat5e for gigabit networks.
PS: Looks like for TV broadcasting, the Cat6 should be better for handling high MHz.
|My System Specs
|02 Aug 2013
What are the types of cabling that Internet Service Provider use
Is there a difference between Cox (my current provider} and Dish Network and/or Direct TV as far as types of installation cables they use ?
Can/will Direct TV use existing Cox coaxial cabling...visa-versa...or does each service have to have their own proprietary cabling inside and outside the home
|My System Specs
|02 Aug 2013
Windows 7 Pro. 64/SP-1
I have had DirecTV use Comcast cabling with no problems. It has been about 5 years ago. The cabling might be different now days or better.
|My System Specs
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Hello, I would like to remove these game providers(WildTangent, mostly) but whenever I go to uninstall it, it just displays this: Capture.PNG What can I do? I've waited for a while and It doesn't do anything. Thanks for helping. (Btw, I'm new, so if this isn't in the right place please tell me)
Add Search Providers to Internet Explorer
I have Windows Premium on an HP Laptop G60-535DX. Whenever I use either IE or Firefox; a tab keeps opening up "Add Search Providers to Internet Explorer". I've added all that I want. I've closed the tab, and as soon as I open a new tab, the same "Add Search Providers tab opens again. How do I stop...
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An excerpt from www.HouseOfNames.com archives copyright © 2000 - 2017
Bavaria, Germany is the original homeland of the Reininger family. Reininger is a patronymic name, a type of surname that derived from the given name of the father of the original bearer. Reininger was derived from the Old Germanic personal name Raginhari, which is composed of the Old German elements ragin, which means counsel, and hard, which means hardy.
Reininger Early Origins
Bavaria, where the name became noted for its many branches within the region, each house acquiring a status and influence which was envied and enrolled by the princes of the region. Chronicles first mention Reinerus Reynberti of Rostolk around 1280. The Latin "Renerus" also became "Reinher" from which "Reinhard" was formed. In their later history the Reiners became a power unto themselves and were elevated to the ranks of nobility as they grew into this most influential family.
Reininger Spelling Variations
Reininger Spelling Variations
One can encounter great variation in the spelling of surnames: in early times, spelling in general, and thus the spelling of names was not yet standardized; and later, spellings would change with branching and movement of families. Variations of the name Reininger include Reiner, Reine, Reinere, Rhiner, Reiners, Rainer, Rainers, Rainert, Rainere, Renerus (Latin) and many more.
Reininger Early History
Reininger Early History
This web page shows only a small excerpt of our Reininger research. Another 177 words (13 lines of text) covering the years 1728, 1606, 1647 and 1728 are included under the topic Early Reininger History in all our PDF Extended History products and printed products wherever possible.
Reininger Early Notables (pre 1700)
Reininger Early Notables (pre 1700)
Prominent among members of the name Reininger in this period include Vincentio (Vincenzio, Vincenzo) Reinieri (Renieri, Reiner) (1606-1647), an Italian mathematician and astronomer, close friend and disciple of...
Another 28 words (2 lines of text) are included under the topic Early Reininger Notables in all our PDF Extended History products and printed products wherever possible.
The Great Migration
The Great Migration
The great European flow of migration to North America, which began in the middle of the 17th century and continued into the 20th century, was particularly attractive to those from Bavaria who wished to escape either poverty or religious persecution. For many Bavarian tenant farmers, the chance to own their own land was a major incentive. So the widespread colonization of the United States began in 1650, when many immigrants from Germany settled in pockets in Pennsylvania, Texas, New York, Illinois, and California. In Canada, German settlement centered in Ontario and the prairie provinces. Among those of this surname listed in various historical records were:
Reininger Settlers in United States in the 18th Century
Reininger Settlers in United States in the 19th Century
Reininger Family Crest Products
Reininger Family Crest Products
See Also
See Also
Other References
The Reininger Family Crest was acquired from the Houseofnames.com archives. The Reininger Family Crest was drawn according to heraldic standards based on published blazons. We generally include the oldest published family crest once associated with each surname.
This page was last modified on 9 September 2015 at 08:13.
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Voltec's fluorescent retractable reels are constructed of tough ABS and mount easily for use in any work environment. The durable compact casing is easy to maneuver into the tightest of spots and the ergonomic handle makes it quite comfortable to lengthy hold times. Out E-zee Lamp replacement also makes bulb replacement simple. No more splitting the case or removing the handle base, simply remove the lens and easily replace the bulb. 26-Watt fluorescent angle retractable reel with magnet and outlet in handle, length: 50 ft. wire type: SJTW, Gauge: 16/3, plug: 5-15P, receptacle: 5-15R, 10-Amp 1250-Watt.
California residents: see Proposition 65 information
- 26-Watt fluorescent work light
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- Dual rotating hooks
- Ergonomic handle design
- 5 locking positions
- Convenient carry handle
- Mounting hardware included
- UL/CUL approved
- Click here for more information on Electronic Recycling Programs


text:
simple-fax.de FaxPrinter revolutionizes fax-sending for all Mac users.
FaxPrinter by simple-fax.de allows direct faxing from any program making faxing easy, quick and convenient. With its new fax software for Mac OSX simple-fax.de makes sending a fax as easy as printing. After installing FaxPrinter you can choose "send to simple-fax.de" from the print dialog. After selection a preview window where the recipients fax number(s) can be entered opens. Alternatively, you may choose the simple-fax.de app from the programs menu and select a PDF document from your hard drive. After the document is sent we will confirm its delivery by email, as usual.
You require a customer account to use the services of simple-fax.de. If you have not registered yet you can do so here.
FaxPrinter is available for free in Apple's AppStore. Click here..
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text:
Quiet neighborhood away from the city but only 10 mins to shops and restaurants in Monument and 15 min to North Colorado Springs. 20 min to USAFA Football Stadium. 15 min to hiking trails in Pike National Forest.
Queen bed with shared bath
The space
Great mountain views in a quiet neighborhood.
Guest access
Close to USAFA and shops. Castlerock outlet mall is 30 min drive
Other things to note
Hot tub available on the deck with views of the mountains.
No pets allowed.
No Pets
No smoking
Quiet time 10PM-6AM
7 Reviews
After spending over 20 years traveling with the army (my husband Otis is a retired Special Forces officer) we have settled in Monument, Colorado. We own a Seniors Helping Seniors (R) franchise. We provide non-medical home care for seniors by seniors. Otis is also the head coach of a nationally ranked high school club rugby team.
When not working, we try to visit our three children. Our oldest is getting her Doctorate in Physical Therapy at Texas Woman's University in Houston. We have a senior at Texas A&M who is in the corps and will go in the army after graduation. Our youngest is a freshman at the University of Arizona where he plays rugby.


text:
Cosmology has quite a few math symbols. Some are familiar using standard physics notation, others are easy to confuse (such as how "T" can mean Temperature or Frequency) or "misremember." Adding to that confusion, cosmology math symbols often have different meanings in other closely related fields.
So here's a cheat sheet of common symbols used in Cosmological math. It could be helpful to Cosmology students and for those of us who have a lot of other things to remember and would like to keep our brain free for more important details. (At least that's the excuse I'm going to use.)
If I've missed any vital ones, or Hubble Forbid . . . made any errors, send me a quick note and I'll fix it as soon as I can.
PS (Here's a list of common physics notations for background.)


text:
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Join Date: Jun 2007
Location: GA
Age: 30
Posts: 11,920
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Photobucket Alternative for Your Images
With Photobucket now charging to host Images many of you maybe wondering where to host your images. Below are 6 alternatives*
1. http://imgur.com/
No account required
2. http://imgtc.com/
No account required, though having a free account allows you to upload 20mb at once vs 10
3. http://postimage.org/
No account required
4. http://www.imagetitan.com/
No account required, can upload a bunch at one time, just have to do Captcha
5. http://imgup.net/
No account required but can connect facebook ro google+ account
6. http://cubeupload.com/
No account required, more of a drag and drop image uploader
While of course you can use any web hosting you would like these are a few alternatives to keep those builds coming.
If you see someone having an issue with photobucket please direct them to this announcement.
* Zilvia.net is not affiliated with any of these websites. These views and opinions of each of these sites do not necessarily represent those of Zilvia.net, Staff and/or any/all contributors to this site.
|08-21-2011 until 08-21-2021
Custom administrator user text
Join Date: Sep 2002
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Before paying via PayPal as "Gift"...
Remember, that paying as "gift" removes your protection as a buyer, because paypal sees it as nothing disputable if it was a gift.
This leaves the door WIDE open for someone to screw you on a transaction with you left no available options to dispute and get your money back.
If we catch anyone openly requesting to be paid as "gift" then it will be treated as if they're trying to take someone for their money and the thread will be closed, deleted or edited accordingly. I know that many are doing it because paypal fees, despite being in terms and conditions that all who use it agreed to, are ball-bustingly high these days (<--sarcasm). As a staff, we are tired of the PM that goes:
At the end of the day, if you trust a stranger enough with your money to give it to them in a manner that you are not protected, then you are partially in control of your own peril.


text:
This product is no longer available.
30-Day LED Glow-in-the-Dark Lantern
Going on a camping trip somewhere where it's too dry to start a fire? Then bring along this lantern. They'll use it when the sun goes down but they're not ready to get into their tent and curl up in a sleeping bag.


text:
Results 1 to 1 of 1
02-20-06, 03:42 #1Registered User
- Join Date
- Apr 2005
- Posts
- 203
Unanswered: problem reciving check box values using post method
Hi all i got a form with many checkboxes and i want to pass checkbox varaibles to a new page using post method but the problem is that each time the first variable get pasted .Beleow u see part of php code that recives the passed variables. When i run this script by selecting 3 check boxes which has values :1 first check box, 2 second checkbox and 3 3th checbox. i get this
select filename,title from wimpy where id=1
instead of
select filename,title from wimpy where id=1 OR id=2 OR id=3
I be happy if an expert help me fix this problem and be able to pass all values of check box to second page and be able to buil query such :
select filename,title from wimpy where id=1 OR id=2 OR id=3
part of check box form code:
<form method="POST" action="./playlist.php" name="mp3Play"> ... ... ...
<?php //echo $_POST['id']; $user = "root"; $pw = ""; $db = "mp3sversion5"; $mysql_access = mysql_connect("localhost", $user, $pw); mysql_select_db($db, $mysql_access); if (isset($_POST['Id'])) { $fragments = explode(',',$_POST['Id']); $loopCount = sizeof($fragments); for ($loop = 0; $loop < $loopCount; $loop++) { if ($where != '') $where .= ' OR'; $where .= ' id='.$fragments[$loop].' '; }} $query = "select filename,title from wimpy where $where"; echo $query; $result = mysql_query($query);


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The B3 case is designed to carry 3 Blunts (also fits Black & Milds, Cigarillos and most Electronic Cigarettes) in your back pocket. They are crush-proof and handmade in the USA of fine American walnut with a hand-rubbed tung oil finish. They are guaranteed unbreakable, they are water resistant and they Float.


text:
[Hearts] [160527] ナツイロココロログ 初回版Original Soundtrack + Bonus DVD + Drama CD + Manual
Release Year: 2016/05/27
Original name: ナツイロココロログ 初回版
Studio Name:Hearts
File size: 4.4 GB
getchu880903 + Original Soundtrack + Bonus DVD + Drama CD + Manual.rar
Highlight the problem fragment mouse and press CTRL + ENTER.
In the window that appears, describe the problem and send a notice of the Administration site.
For more details, please refer to [ How can I install the Japanese language pack? ] on the Frequently Asked Questions page.


text:
Separate names with a comma.
Posted By save$,
Jul 27, 2012 at 12:29 PM
A few pics of the July blooms
Beautiful. I love summer!
Lovely! And no fungus or insect damage. Last time I tried to grow sunflowers the critters ate the heads right off just as they were starting to unfurl.
I really like the purple coneflower and phlox pic.
We're in synch. Our echinacea and phlox have started blooming too.
You have Ents.
Very nice.
Love the petunias & the cone flowers, and I adore the dwarf red maple in the back ground I have one, and I know it's at least 50 years old !
I too like the cornflowers. Here is a couple more of them
You should send those to Better Homes and Gardens or something.
Very nice.


text:
I want to have only the necessary licenses because now I have about 140 ! In licenses folder and loading of avic takes time!
I have Greece, TomTom and here maps,poi
I have skin pongo also.
which licences do I need only? thank you
Sent from Zopo speed plus 7 via tapatalk


text:
After meeting the Discipline requirement and before applying for the Examination, the following requirement must be met:
Minimum of 15 clock hours of continuing education activities applicable to diabetes within the two (2) years prior to applying for certification.
Learn more about the details for this requirement in the current Exam handbook, Appendix II.
Click here for the details on the discipline requirement.
Click here for the details on the professional practice experience requirements.
Click here to review the continuing education recognized providers.
Click here to review the fee information.
Unique Qualifications Info/Application


text: Hi there, Slightly odd one this but would be very grateful for somebodies advice on this. The current deal in the UK means that if two couples book a two week stay in a two bed villa for 4 people, along with the 14 day (7 free) ultimate ticket they will get free dining for everybody. If one couple leaves after a week, but the other stay on in the same accommodation for the remaining week, providing nobody tells Disney, is it possible for the remaining couple to use the dining credits left on the departed couples cards? The only barrier I can think of is if the cards need to be used at the park turnstiles before using them at a restaurant. Thanks for your help


text:
|01 May 2011
Task Scheduler not running??
Anyone getting this message or had it and corrected it? see attachment. I''ve been on Google for a loong time and not really finding a fix. HP system 3 weeks old and this seems to be a problem for a ton of people.
I check the services and its running and grayed out so I can't touch it.
|My System Specs
|02 May 2011
wow 83 views and not one person has seen this? I Google it and it get tons of hits but no fixes. No problem - if anyone does see it and have a suggestion let me know.
Thanks folks!
|My System Specs
|02 May 2011
Windows 10 Pro X64
Did you see this one: Task Scheduler service is not available
|My System Specs
|02 May 2011
Win 7 Ultimate 64-bit. SP1.
Hello Tactics....
Read throught the link supplied to see if any of the steps help you.
Task Scheduler not running
I note you say your computer is 3 weeks old.
A new computer should not be presenting with the problem you have listed.
I strongly suggest that you should seek further advice from HP asap.
Under Warranty terms a duty of care applies.
|My System Specs
|02 May 2011
Yep seen it. Its a brand new W7 box so I don't want to create a new acct. I haven't changed the name either.
I don't have this in W7
"I came across this error after syspreping a [COLOR=#000096 !important][COLOR=#000096 !important]Windows[/COLOR][/COLOR] 2008 server (basically changing the computer name), Fix will also apply to Vista
• Open windows explorer
• Browse to C:\Windows\System32\Tasks\Microsoft\WindowsCalenda r\
• Select Reminders - <username>
• Right click Reminders - <username> and select Delete
• Click Yes to delete the task
• Close windows Explorer
• Open Task Scheduler
• Error should not appear
NOTE: There might be more than one Reminders - <username> task that needs deleting."
Can't do this:
Go to C:\Windows\system32\Tasks and make a backup of all the tasks
there somewhere else on your drive. I copied the whole folder content to
my Documents folder.
I try to add to .rar because every time I try and copy to C:\ or desktop I get hit with tasks folder already exist and asks me if I want to overwrite /copy files and folders etc etc..?? and I know for a fact its not there.
If I click any task in the scheduler I get the other error. oh well its not a big issue. I knows Windows 7 and because a ton of people have it - hopefully there will be fix in SP2 because I can find a fix anywhere.
PS its running according the my services.msc
|My System Specs
|02 May 2011
Hello Tactics....
Read throught the link supplied to see if any of the steps help you.
Task Scheduler not running
I note you say your computer is 3 weeks old.
A new computer should not be presenting with the problem you have listed.
I strongly suggest that you should seek further advice from HP asap.
Under Warranty terms a duty of care applies.
|My System Specs
|02 May 2011
Click on Windows event log service and see if it gives you any errors. What I am wanting to know will the event log service run.
|My System Specs
|02 May 2011
Plan "B"
Well you can rule out what I was thinking.
I found this:
The Task Scheduler engine manages a queue of tasks that are scheduled to run. A task is put in the queue when the service determines the task should run. The queue has a quota that defines the number of tasks allowed in the queue. The quota is defined in a registry key value.
I haven't found the key yet. I assume if the queue was full no further tasks could run. I'll keep looking.
|My System Specs
|02 May 2011
I found this in Windows Seven Forums (imagine that)
Attachment 152503
Attachment 152504
I also attached what my reg looks like.
|My System Specs
|Thread Tools
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Task scheduler not running??????
My daughter has an HP laptop that she uses in nursing school. It started acting funny and I found a bunch of junk on the computer that she says she doesn't know where it came from. But me problem is that automatic updates has stopped working along with the disk defag. When I try to run the...
|General Discussion
Task Scheduler Fails when Running VB Compile EXE
Hi Everyone, I am puzzled. I created an exe with Visual Studio 2010 to automate the execution of a 2nd program. This exe requires 2 command line arguments. When I run this exe from a Short cut with the arguments defined everything works as desired.
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Task Scheduler not Running Tasks
I created two tasks (Utorrent and Flashget) to run at 12:15 am every night but the tasks refuse to startup. I usually hibernate my computer at night so i have the two tasks "wake computer up to run this task" (the computer does wake itself up) The only way i can get it to run is if i manually run...
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Task Scheduler not running
I can't install anything on my computer or run disk defragger because I keep getting error messages. Here is one: "Disk Deragmenter cannot start because the task scheduler service is not running. Start the task scheduler service and try again. I tried to mess around with the task scheduler...
|General Discussion
task scheduler not running missed task
When i set task scheduler to run a program it works. But if the pc is not on at the time it will not run unless task scheduler is opened. It is set to run as soon as poss after scheduled time and task scheduler itself starts and runs properly
|General Discussion
Task Scheduler & Running application in foreground....
Hi, My requirement is to start an application (UI based) in the foreground as soon as the PC boots (whether user is logged in or not). I added a task to the task scheduler & configured it to run under the same account as my foreground logon account. I am seeing the task is scheduled but it...
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text:
Frankie Frain is an independent filmmaker, animator, and podcaster, best known for feature films such as A-Bo the Humonkey and Sexually Frank, and viral cartoons that lampoon Hollywood directors. In 2013, he directed his fourth feature film titled Having Fun Up There (written by Geoff Tarulli), a story about an artist in crisis. Here, Frankie describes the entire story behind Having Fun Up There, including the films that preceded, the writing and casting, the nine grueling production days, the edit and color correction, and the film festival circuit and distribution woes. Learn about the technology, techniques, and philosophies behind Frankie and crew's production in this entertaining tool for any beginning filmmaker.
©2014 Frankie Frain (P)2014 Frankie Frain
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text:
Marijuana politics is popping up in some US Senate races, pot arrests are still going up in some states, the Guam medical marijuana initiative faces a legal challenge, Stanton Peele takes on the disease model of addiction and liberals who love it, Jerry Brown signs the California Fair Sentencing Act, and more. Let's get to it:
Addiction psychologist Stanton Peele rips into liberals who embrace the disease model of addiction. (YouTube.com)
Marijuana Policy
Marijuana Policy in the Kentucky US Senate Race. Pot politics is playing a role in the Kentucky Senate race. Both incumbent Republican Sen. Mitch McConnell and Democratic challenger Alison Grimes agree that industrial hemp is a good thing, but Grimes said last Thursday she wants to have a discussion on legalizing marijuana, especially for medical purposes. McConnell retorted last Friday that he opposes legalizing marijuana for any reason because the state has a heroin problem and it would send the wrong message.
Nebraska Democratic US Senate Candidate Open to Legalization. The Democratic nominee for the US Senate from Nebraska is open to marijuana legalization. "I think we should give very serious consideration to legalization of marijuana," Dave Domina said Thursday. "If I were a philosopher king, we wouldn't have it, but I'm not and we need to spend our law enforcement dollars as intelligently as we can. That's not on marijuana enforcement. "What we should do is to make safe and dispense responsibly the drugs that are the most common, the least dangerous," he said. "I'm not saying they are desirable. I don't think cigarettes are desirable, frankly. But nonetheless, I think that's what we need to do. We need to methodically make marijuana available to people on a rational basis. It needs to be safely distributed and it needs to be taken away from crime as a profit center." The Republican nominee, Ben Sasse, didn't take a firm stand on the issue.
Marijuana Arrests Up in 17 States. Marijuana legalization may be inevitable, but some states haven't gotten the message. According to a new report from marijuana researcher Dr. Jon Gettman, even though national marijuana arrests peaked in 2007, marijuana arrests increased in 17 states between 2008 and 2012. Those states and their rates of annual increase are: South Carolina (11.6%), Washington, DC (7.7%) South Dakota (7.7%), North Dakota (5.5%), Utah (4.5%), Illinois (4.3%), Montana (3.5%), Idaho (3.2%), Virginia (2.6%), New York (2.4%), New Jersey (2.4%), Oregon (2.1%), Tennessee (1.7%), Wisconsin (1.2%), Vermont (1%), Michigan (1%), and West Virginia (1%). The report is Marijuana in the States 2012.
Overblown Analogy Department. The Polk County, Florida, Sheriff's Office is sounding the alarm over high-THC cannabis oil, popularly known as "wax" or "dabs," but its spokesperson is relying on an old bogeyman to get her message across. "This particular drug is to marijuana the way crack is to cocaine," Donna Wood said.
Medical Marijuana
Guam Attorney Seeks to Block Medical Marijuana Initiative. Voters in Guam are set to vote on a medical marijuana initiative submitted by the territorial legislature next month, but a Guam attorney has asked the US District Court there to block the vote. Howard Trapp argues that the legislature can't legally "pass the buck" to voters, even though the island's Supreme Court said it could in an August ruling. The election commission has until October 7 to respond to the filing.
First Illinois Patients Get Their Cards. Jim Champion, an Army vet who suffers from multiple sclerosis, is apparently the first Illinois patient to get his medical marijuana card. His came last week. He is the first of more than 2,000 Illinois residents who have applied under the state's new law.
New York Governor Asks Justice Department to Allow State to Acquire Medical Marijuana from Other States. Last Friday, Gov. Andrew Cuomo (D) sent a letter to Deputy Attorney General David Cole asking the Justice Department to extend a narrow, time-limited exception to federal law to allow the importation of certain strains of medical marijuana from other states for use by children in New York with severe forms of epilepsy. The letter follows a similar letter sent last month by the Cuomo administration to Attorney General Eric Holder.
Drug Policy
Dr. Stanton Peele on the Disease Theory of Addiction. Drug war critic and iconoclastic addiction psychologist Dr. Stanton Peele has penned a provocative screed critical of the disease theory of addiction and political liberals who embrace it. The article is "Why Liberals Love the Disease Theory of Addiction, by a Liberal Who Hates It."
California Fair Sentencing Act Signed Into Law. California Gov. Jerry Brown Sunday signed into law the Fair Sentencing Act, which will undo racial disparities in the sentencing of cocaine offenders under laws passed during the crack cocaine hysteria of the 1980s. Brown did not issue a signing statement. The law, Senate Bill 1010, eliminates sentencing disparities between crack and powder cocaine offenses. It also eliminates related disparities in probation and asset forfeiture guidelines for those offenses.
Uruguay Presidential Candidate Says Pot Smoker Registry Could Be Used to Identify People Needing Drug Treatment. Former President Tabare Vazquez, who is running to return to the office, has proposed using the state's registry of marijuana users to expose them to drug treatment. He was attempting to frame the law as a rehabilitative measure, but critics say it could dissuade users from registering with the state. Vazquez's remarks also run afoul of provisions in the law that limit revealing such data "without the individual's express written consent."
(This article was published by StoptheDrugWar.org's lobbying arm, the Drug Reform Coordination Network, which also shares the cost of maintaining this web site. DRCNet Foundation takes no positions on candidates for public office, in compliance with section 501(c)(3) of the Internal Revenue Code, and does not pay for reporting that could be interpreted or misinterpreted as doing so.)
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Junior college offensive lineman Scott Haverkamp will be visiting the University of Georgia officially this weekend. Haverkamp is an offensive guard out of Butler Community College and he could be...
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Relations between Great Britain, its North American colonies, and the Native Americans were often difficult. The British wanted to solve these problems so they could reduce the number of British troops needed and thereby reduce expenses. One attempt to bring peace was the Proclamation of 1763. This edict created a line of settlement beyond which colonists were forbidden to settle. A transcription is included below in Support Materials.
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text: Integrity, compassion and accountability are hallmarks of this organization. First responders during both domestic and international disasters, Samaritans Purse strives to be first to offer assistance as a tangible expression of God's love. Projects under its umbrella include Operation Christmas Child, Children's Heart Project, World Medical Mission and many others.


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Mathematical Software. Mathematical Research. Mathematical Education. Tvalx Products.
Let's start with Sine y = sin(x) :
Note that near origin graph of y=sin(x) is close to graph of y=x. We see that Sine is a periodic function. Sine has a (single) period 2π, that is sin(x)=sin(x+2π) for any x. The domain of Sine is entire real line and the range is interval [-1, 1] . By convention brackets are used for denoting interval when endpoints are included into interval.
Next is Cosine y = cos(x) :
At x=0 the graph of y=cos(x) is close to graph y=1. Cosine is also a periodic functions with period 2π. Note that graph of cos(x) coincides with graph of sin(x) if we shift it by -π/2+2πn :
Note that for shifting graph to the left by s units we add s to the argument x: f(x+s). For shifting graph to the right we subtract s units from argument: f(x-s) .
Tangent by definition is sin(x)/cos(x). When cos(x)=0 (at x=π/2+2πn) the expression sin(x)/cos(x) is not defined. Thus tan(x) is not defined at x=π/2+2πn for each integer n.
Let's look at the graph of y = tan(x) :
From the left to each "critical point" x=π/2+2πn y goes to plus infinity (that is goes up) and from the right y goes to minus infinity (that is goes down) .
Cotangent by definition is 1/tan(x) = cos(x) / sin(x) . Thus where Tangent has zero Cotangent has infinity and where Tangent has infinity Cotangent has zero:
Both Tangent and Cotangent have period 2π.
In situations when graph of function goes to infinity at some critical point, a vertical line passing through the critical point is called a vertical asymptote. In general, an asymptote of function is a (straight) line which graph of function approaches "infinitely close". Pressing button "asympt" we draw asymptotes for Tangent and Cotangent:
By clicking on intersection of dark blue line and dark gold dashed line in right window we get coordinates of the point where Cotangent is zero and tangent is infinity. that is x=π/2. In general x=π/2+2πn. There different notations used for Tangent: tan, tng, tg - and for Cotangent: cot, ctg, ctn .
Secant and Cosecant are less known trigonometric functions. They are present in Math Center Level 2 . There are different notations used for Secant: sec, sct - and for Cosecant: csc, cst, cosec.
By definition sec(x)=1/cos(x) and csc=1/sin(x). Thus Secant has vertical asymptotes where Cosine has zeroes and Cosecant has vertical asymptotes where Sine has zeroes.
Let's look at y = sec(x):
Now look at y = csc(x) :
Secant and Cosecant have period 2π.
Let's recall trigonometric equalities and explore them with help Graphing Calculator 2D from Math Center Level 1 and Math Center Level 2 .
Recall sin2(x) + cos2(x) = 1 . On the picture below red graph is for sin, green for cos, blue for sin2, purple for cos2, and yellow is for sin2 + cos2 :
Recall sin(2x)=2sin(x)cos(x) :
More rear identity sin(3x)=3sin(x)-4sin3(x) :
© 2008 Tvalx


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76 Macfarlan Avenue Blairgowrie 3942 VIC
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text: English fringe theatre director and academic Albert Hunt was a guest director for the fringe component of the 1974 Queensland Festival of the Arts. During his time in Australia he also worked with the Popular Theatre Troupe (1975-1976), providing several of his own plays and co-writing others with Richard Fotheringham. He later worked at the Victorian College of the Arts developing community theatre courses.


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Around Rim Country
Arizona Rim Country is an area dominated by the tourism, retirement, retail and vacation industries. Cooler weather, terrific air quality and outdoor activities cause many Arizona residents and visitors to migrate to Arizona Rim Country. Countless thousands of migratory birds, nature trails rarely hiked, and refreshing afternoon rains beckon everyone to relax and enjoy themselves.
The Rim Country Regional Chamber of Commerce serves as the "front door" to Payson. Our office offers visitors and newcomers with information about the area in addition to local business. Rim Country Regional Chamber of Commerce serves the Town of Payson, Town of Star Valley, Christopher Creek, Pine, Strawberry, Rye, Gisela, and all the small communities in between.
Maps of Rim Country Trails and Places of Interest
PATS (Payson Area Trails System) Map
A Lesson in History Map


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Trying to decide between getting a degree in education, agronomy or psychology. I'm aware none of them have any relation but they all really interest me. Just trying to find something likeable with a demanding field?
Psychology majors are a dime a dozen. Go to any college and they make up the bulk of the student body. Interesting subject, little to no math, perfect for the typical person who has no idea what to do with their life! So you can imagine what the field is like. Unless you're dead set … Read more


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Pre-Notched Trims and Flashing Components Save Time at the Jobsite
Common problems that occur in metal panel trim and flashing installation include maintaining the correct overlap distances. As well, heavier gauges and multiple bends or complex shapes may make field notching difficult. The quality of workmanship in creating trims and flashings in the field are not as easily controlled compared to factory fabrication, and the time to perform this operation in the field is typically longer.
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Petersen Aluminum expands its Precision Series line of metal wall panels with the addition of seven new Highline products. The Precision Series Highline siding panels feature varying rib patterns and widths for creative architectural effects in residential, commercial, institutional or many other applications. The Highline panels are 1 3/8 inches deep and showcase a range of rib patterns for visual interest and architectural creativity. Options include either 12-inch or 16-inch widths, a no-clip panel, or a clip-fastened panel to accommodate thermal expansion and contraction.
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Restore Domes with 22-karat Gold Restoration Film
Used to beautify church domes throughout the U.S., Europe and Asia, the 22-karat gold film features a 20-plus-year outdoor warranty. Guaranteed non-fading and non-delaminating for 20 years, the maintenance-free exterior grade of the gold film is durable and features a non-stick surface resistant to airborne contaminants and fowl waste.
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Spanish clubs investigated over possible match fixing - .PDF file.
By Ceyhun Pehlivan and Jorge Monclús
The Spanish Professional Football League (LFP) has opened an investigation into the recent match of the second division (so-called 'Liga Adelante') between the Racing Santander and Hercules for possible case of match-fixing.
Alarm bells of the UEFA started to ring after detecting that allegedly unusual levels of online betting, up to 60 times higher than the ordinary level for this category of games, were oddly placed on Racing winning with several goals. Since the LFP's jurisdiction extends only to the clubs and officials, it has requested the Spanish Football Federation to open its own proceeding into the players involved in the game…
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Multiple cyclin-dependent kinases (CDKs) control eukaryotic cell division, but assigning specific functions to individual CDKs remains a challenge. During the mammalian cell cycle, Cdk2 forms active complexes before Cdk1, but lack of Cdk2 protein does not block cell-cycle progression. To detect requirements and define functions for Cdk2 activity in human cells when normal expression levels are preserved, and non-physiologic compensation by other CDKs is prevented, we replaced the wild-type kinase with a version sensitized to specific inhibition by bulky adenine analogs. The sensitizing mutation also impaired a non-catalytic function of Cdk2 in restricting assembly of cyclin A with Cdk1, but this defect could be corrected by both inhibitory and non-inhibitory analogs. This allowed either chemical rescue or selective antagonism of Cdk2 activity in vivo, to uncover a requirement in cell proliferation, and non-redundant, rate-limiting roles in restriction point passage and S-phase entry.
During G1 phase of the division cycle, a eukaryotic cell commits to a round of duplication or enters a quiescent state (G0) (Morgan, 2007). When conditions are appropriate for proliferation, growth factor signaling promotes passage through the restriction (R) point, after which further cell-cycle progression becomes growth factor-independent (Pardee, 1974). This transition coincides with initiation of a transcription program directed by the E2F family of DNA-binding factors, which regulate genes critical for DNA synthesis (S) phase and mitosis (Bracken et al., 2004). Analysis of gene expression in single cells suggests that the switch governing E2F activation is bistable in response to increasing growth factor concentration, attaining an ON or OFF steady state with no intermediate states of partial activation (Yao et al., 2008).
G1/S transcription is controlled by both activator and repressor E2Fs that bind to a family of pocket proteins comprising the retinoblastoma tumor suppressor protein pRb, p107 and p130 (Cobrinik, 2005). In G0 and early G1, gene expression is inhibited by repressor E2Fs, in complexes with p107 or p130 that bind E2F-responsive promoters and recruit histone-modifying enzymes to impose a repressive chromatin state (Frolov and Dyson, 2004). Later in G1 and S phase, repression is relieved and free activator E2Fs, dissociated from pocket proteins, promote transcription. Cyclin-dependent kinases (CDKs) play a decisive role in this switch by phosphorylating pRb, p107 and p130. In vitro, cyclin D-, E- and A-dependent kinases phosphorylate pRb with overlapping but distinct site-specificity (Zarkowska and Mittnacht, 1997), and multiple CDKs contribute to complete pRb inactivation in vivo (Lundberg and Weinberg, 1998). Phosphorylations on distinct sets of residues—targets in vitro of different CDKs—decrease affinity between discrete regions of pRb and E2F (Rubin et al., 2005; Burke et al., 2010). The deregulation of pocket protein function and its control by CDKs is a hallmark of many cancers (Classon and Harlow, 2002).
Cdk2 was originally implicated in this regulatory circuit because its period of cyclin-binding and activity extended from late G1 until just prior to mitosis (Pagano et al., 1992; Rosenblatt et al., 1992; Pagano et al., 1993), and expression of dominant-negative Cdk2 arrested cells in G1, S and G2 phases (van den Heuvel and Harlow, 1993; Hu et al., 2001). This model was challenged by the discovery that Cdk2 is not essential for mouse viability (Berthet et al., 2003; Ortega et al., 2003). In cells lacking a full complement of catalytic subunits, Cdk1 can form complexes with cyclins D and E (Aleem et al., 2005; Santamaria et al., 2007); compensation by Cdk1 may explain how cells lacking Cdk2, Cdk4 and Cdk6 can proliferate in culture (Santamaria et al., 2007). It remained unclear whether events early in the cell cycle depend exclusively on Cdk2 in wild-type cells, or if Cdk1 can perform these functions normally.
To begin to address this question, we previously determined the relative amounts of Cdk1 and Cdk2 bound to various cyclins during the course of a human cell cycle (Merrick et al., 2008). Cyclins E and B bound almost exclusively to Cdk2 and Cdk1, respectively, whereas cyclin A formed complexes with both CDKs in strict temporal order—predominantly with Cdk2 until mid-S phase, and only thereafter with Cdk1. Cdk2 has priority despite being ~10-fold less abundant than Cdk1, possibly as a consequence of different activation mechanisms for the two kinases. In vivo, Cdk1 and Cdk2 follow distinct paths to full activity even though they are ~65% identical in sequence, have overlapping cyclin-binding profiles, and are targets of the same CDK-activating kinase (CAK)—the Cdk7 complex. The primary pathway for Cdk2 comprises two steps: first, phosphorylation of the activation (T-) loop by Cdk7 and then, binding to cyclin (Merrick et al., 2008). Cdk1, conversely, cannot be phosphorylated by Cdk7 in the absence of a cyclin, but cannot form a stable complex without that phosphorylation, meaning that the two steps must occur in concert (Larochelle et al., 2007). In vitro, a yeast CAK that phosphorylates monomeric Cdk1 can force it into the Cdk2 pathway, switching cyclin A-binding preference from Cdk2 to Cdk1 in extracts (Merrick et al., 2008). This suggested that pairing rules for CDK-cyclin assembly derive from kinetic insulation of activation pathways.
Therefore an important function of Cdk2 may be to exclude Cdk1 from cyclin complexes until DNA replication has commenced, restricting Cdk1/cyclin A activity to later in S phase and potentially preventing precocious firing of late replication origins (Katsuno et al., 2009). When that scaffolding function is preserved, moreover, Cdk2 activity might be required for early cell-cycle events, even though there is not a strict requirement for Cdk2 protein. Removing Cdk2 by gene disruption abolishes both catalytic activity and a non-catalytic function in restraining Cdk1 activation. To distinguish and define those roles, we needed an alternative strategy that would allow: 1) specific inhibition of Cdk2 catalytic activity; and 2) control over Cdk2-cyclin pairing and, consequently, Cdk1 complex assembly.
Here we replace wild-type Cdk2 in human epithelial cells with an analog-sensitive (AS) version modified to bind bulky adenine analogs. A Cdk2as allele is hypomorphic; the mutant protein has lost its competitive advantage over Cdk1 in binding cyclin A. That priority can be restored by different analogs, which either promote activation or inhibit activity of Cdk2as. We thus created a chemical "allelic series" that allows both rescue and selective antagonism of Cdk2 functions. Specific inhibition impedes cell proliferation, demonstrating an essential requirement for the catalytic activity of Cdk2. Both catalytic and scaffold functions are required for normal responses to growth factors and timing of R-point passage. Therefore, chemical genetics reveals Cdk2 to be a non-redundant, rate-limiting regulator of G1/S progression in human cells.
Gene disruption did not reveal a requirement for Cdk2 in mouse cell proliferation. The removal of Cdk2 protein, however, allows atypical CDK/cyclin complexes to compensate for, and thereby obscure, any exclusive functions it might perform (Aleem et al., 2005; Santamaria et al., 2007). Furthermore, available small molecules effective against Cdk2 also inhibit other CDKs. Therefore, to inactivate Cdk2 specifically while maintaining normal expression levels, we combined chemical genetics (Bishop et al., 1998) with homologous gene replacement in human somatic cells (Papi et al., 2005; Larochelle et al., 2007).
Most protein kinases contain a bulky "gatekeeper" residue in the ATP binding pocket that can be mutated to a smaller Ala or Gly residue, sensitizing them to bulky adenine analogs that do not inhibit wild-type kinases. We targeted the endogenous Cdk2 locus, in human telomerase-expressing retinal pigment epithelial (RPE-hTERT) and HCT116 colon carcinoma cells, with recombinant adeno-associated virus (rAAV) vectors containing a Cdk2F80G AS mutation (Kraybill et al., 2002) (Figures 1A, S1A, S1B). In this way we created an allelic series: Cdk2as/+, which expresses normal levels of Cdk2, half of which is AS; Cdk2as/neo, in which only Cdk2as is expressed but at half the wild-type level due to silencing of one allele by a retained neoR gene; and Cdk2as/as, in which Cdk2as is expressed exclusively and at wild-type levels (Figures 1B, S1C). AS kinases can use bulky ATP analogs as substrates (Kraybill et al., 2002), so to test if Cdk2as expressed in human cells was active, we added radiolabeled N6-(benzyl)-ATP to extracts from the various lines (Figure 1C). As expected, there was no labeling above background in extracts of wild-type cells. In mutant cell extracts, however, there was phosphorylation of discrete proteins, which increased with the number of expressed Cdk2as alleles. The labeling patterns were grossly similar in Cdk2as/as HCT116 and RPE-hTERT extracts, but with differences in intensity of some substrates. We conclude that Cdk2as encodes an active, AS kinase in both human cell types.
We next asked if specific inhibition of Cdk2as affected cell proliferation (Figures 1D, E, S1D, E). In the absence of drugs, all four RPE-hTERT lines grew at similar rates, although Cdk2as/as HCT116 cells grew more slowly than did wild-type cells. Growth of Cdk2as/as cells—both RPE-hTERT and HCT116—was inhibited by the pyrazolopyrimidine-based, allele-specific inhibitor 3-MB-PP1 in a dose-dependent manner. Wild-type cells were impervious to 3-MB-PP1 concentrations up to 10 μM, establishing that effects of the drug were strictly dependent on the Cdk2as mutation. In both drug titrations (Figure 1D) and time courses at 10 μM 3-MB-PP1 (Figure 1E), Cdk2as/+ and Cdk2as/neo RPE-hTERT cells had sensitivities intermediate between those of Cdk2+/+ and Cdk2as/as cells—a dependency on gene dosage that validates the chemical-genetic strategy. The indistinguishable sensitivities of Cdk2as/+ and Cdk2as/neo cells imply that either wild-type Cdk2 itself (in Cdk2as/+), or other CDKs such as Cdk1 (in Cdk2as/neo), can compensate for inhibition of Cdk2as expressed from a single gene copy. Also consistent with this interpretation, mouse embryonic fibrobalasts (MEFs), derived from Cdk2−/− cells but expressing Cdk2as at ~ten times the physiologic level (data not shown), were more sensitive to 3-MB-PP1 than were human cells expressing Cdk2as at 50–100% of normal levels (Figure S1E). [Construction and characterization of Cdk2as MEFs will be described elsewhere (D.H., N.E.H. and A.G., unpublished observations).] Although overexpression of Cdk2as could interfere with Cdk1 functions, specific inhibition of Cdk2as expressed at exactly wild-type levels slowed or blocked proliferation of both non-transformed RPE-hTERT and HCT116 cancer cells. The latter were resistant to reductions in Cdk2 protein or activity levels by dominant-negative or antisense approaches (Tetsu and McCormick, 2003). This difference illustrates the power of chemical genetics to uncover essential functions missed by other methods.
Mammalian cells can tolerate loss of Cdk2 protein, but not acute, selective inhibition of Cdk2as expressed at physiologic levels, perhaps because normal CDK-cyclin pairing was preserved in the latter scenario. We tested this with an assay we developed previously to compare the relative amounts of Cdk1 and Cdk2 bound to each cyclin (Merrick et al., 2008). The nearly exclusive binding of cyclin E to Cdk2 was maintained in Cdk2as/as HCT116 cells, but Cdk2as had a defect in binding cyclin A (Figure 2A, B). In asynchronously growing wild-type cells, cyclin A complexes contained ~2-fold more Cdk2 than Cdk1, but in the mutant cells the ratio was inverted (Figure 2A, compare lanes 1 and 2 with lanes 5 and 6). We obtained similar results in RPE-hTERT cells; introduction of one or two expressed copies of Cdk2as increased cyclin A-binding to Cdk1, at the expense of Cdk2 (Figure S2A). The reduction in Cdk2as/cyclin A correlated with decreased Cdk2as T-loop phosphorylation (Figures 1B, ,2C).2C). In vitro, Cdk2as readily formed complexes with cyclin A (data not shown), suggesting that the mutation specifically impaired the ability of Cdk2as to compete with Cdk1 in vivo. These results seemed to indicate that normal CDK-cyclin pairing had not been maintained in the mutant cells as we supposed.
An active-site mutation that reduced ATP affinity of Cdk2 (Kraybill et al., 2002) disrupted its scaffold function and prevented proper assembly with cyclin A in vivo. We reasoned that a tight-binding ATP-analog inhibitor might correct the conformational defects of the mutant protein (Papa et al., 2003). Indeed, 3-MB-PP1 stimulated cyclin A-Cdk2 binding in Cdk2as/as HCT116 cells in dose-dependent manner—restoring the normal ~2:1 ratio of Cdk2 to Cdk1 in cyclin A complexes (Figure 2A, lanes 11 and 12)—but had no effect on distribution of cyclin A in wild-type cells, or of cyclin E in either cell line (Figure 2B). We next asked if the increase in cyclin A-binding coincided with rescue of T-loop phosphorylation (Figure 2C). To test this, we released G0-arrested RPE-hTERT cells into fresh media containing DMSO or 3-MB-PP1; Cdk2-Thr160 phosphorylation occurred upon cell-cycle reentry in wild-type cells, but this response was diminished in DMSO-treated Cdk2as/as cells (compare lanes 2 and 5). Treatment of Cdk2as/as but not wild-type cells with the analog increased Thr160 phosphorylation in a concentration-dependent manner. The T-loop phosphorylation defect could be complemented by multiple inhibitors of AS kinases (Figure S2B), built on two distinct scaffolds; degree of rescue in vivo correlated with inhibitory potency in vitro (Zhang and Shokat, 2007). The defect in binding cyclin A was accompanied by reduced association with the CDK inhibitor (CKI) p21Cip1, which binds preferentially to CDK/cyclin complexes (Harper et al., 1995); p21Cip1-binding was also restored by 3-MB-PP1 treatment (Figure S2C). Therefore, defects in cyclin A- and CKI-binding, and in phosphorylation by CAK, were rescued by allele-specific inhibitors. In the absence of drugs, impaired complex formation might cause Cdk2as/as cells to resemble Cdk2−/− cells, and could account for their cell-cycle defects (see below). Addition of 3-MB-PP1 restored the normal distribution of cyclin A between its CDK partners, however, thus fulfilling the condition we sought to establish by the AS kinase strategy.
Although AS-kinase inhibitors could rescue conformational defects of Cdk2as, they would inhibit any active kinase that might form in vivo, and are therefore unlikely to complement Cdk2as phenotypes. We hypothesized that a different small molecule might restore priority in cyclin A-binding to Cdk2as without blocking its activity. Such a compound would occupy the expanded ATP-binding pocket preferentially, but could be competed off by ATP at physiologic concentrations.
A priori, one class of compounds likely to fulfill both criteria was the N6-modified adenines. These are nucleobase components of ATP analogs used by AS kinases, designed to bind with the N6-substituent group occupying space normally taken up by the gatekeeper side chain (Kraybill et al., 2002), but apt to bind less tightly than the corresponding nucleotides due to their lack of a ribose sugar and phosphates. We screened several adenines by monitoring phosphorylation of the Cdk2as T-loop upon release from contact inhibition (Figure 3A). One compound, 6-benzylaminopurine (6-BAP), rescued phosphorylation in a dose-dependent manner (Figure 3B). Treating cells with 6-BAP also corrected cyclin A- and p21Cip1-binding defects (Figures 3C, S2C), and increased labeling by Cdk2as with [32P]N6-(benzyl)-ATP in extracts (Figure 3D), suggesting a positive effect on Cdk2as activation in vivo. Compared to 3-MB-PP1, 6-BAP was a weak inhibitor of Cdk2as in vitro (IC50 of ~3 nM and ~1 μM, respectively), and neither compound inhibited wild-type Cdk2 (Figure 3E, F). Importantly, 6-BAP, unlike 3-MB-PP1, did not slow Cdk2as/as cell proliferation at the highest dose we tested (80 μM, Figure S3), suggesting that it might be capable of chemical complementation. Cdk2as could then be turned ON or OFF with different small molecules in vivo (see below).
We developed a mathematical model of CDK activation (Merrick and Fisher, 2010), which recapitulates the normal temporal order of Cdk2/ and Cdk1/cyclin A complex formation in vivo (Figures 4A, left, and S4A). To predict how changing kinetic parameters of Cdk2 activation would affect partner selection by cyclin A, we plotted the ratio of Cdk2 to Cdk1 bound to cyclin A at the point (i.e., the cyclin A concentration) when the two complexes are equally abundant under physiologic conditions (Figure 4A, right), which corresponds to late S phase. Deviating from those conditions, either by decreasing the rate of Cdk2 monomer phosphorylation by CAK or reducing affinity of unphosphorylated Cdk2 for cyclin A, did not significantly impair the ability of Cdk2 to compete with Cdk1. Only when both parameters were changed in the same simulation was there a switch to predominant Cdk1-binding, suggesting that reinforcing mechanisms ensure the normal, preferential pairing of cyclin A with Cdk2.
Because the Cdk2as mutation abrogated normal pairing rules, it presented an opportunity to test predictions of the model: that, in the absence of analogs, both affinity of Cdk2as for cyclin A and phosphorylation of monomeric Cdk2as should be reduced, relative to wild-type Cdk2. To test the first prediction, we incubated wild-type and Cdk2as/as HCT116 cell extracts with ATP and a limiting amount of recombinant, His-tagged cyclin A, and isolated exogenous cyclin A on Ni2+ beads to determine its relative binding to endogenous Cdk2 and Cdk1. This assay recapitulated the loss of Cdk2- and gain of Cdk1-binding in the mutant cells (Figure 4B, compare lanes 2 and 5). When 3-MB-PP1 was added to Cdk2as/as extracts prior to cyclin A, Cdk2-cyclin A assembly increased whereas the amount of bound Cdk1 decreased nearly to wild-type levels (lane 6). The Cdk2as we recovered was mostly unphosphorylated on the T-loop, indicating that 3-MB-PP1 restored cyclin-binding independent of activating phosphorylation, consistent with the predicted decrease in Cdk2as-cyclin A affinity. The results suggest, moreover, that 3-MB-PP1 directly altered Cdk2as conformation to restore a competitive advantage over Cdk1. Compared to 3-MB-PP1, 6-BAP was less effective at promoting Cdk2as-cyclin A pairing unless it was also included at pull-down and wash steps (Figure 4C, compare lanes 6 and 7), indicating a lower affinity of this compound for the AS kinase. When we omitted 6-BAP from post-incubation steps, we preferentially recovered phosphorylated Cdk2, suggesting that continued presence of the drug was needed to stabilize binding of cyclin A to unphosphorylated Cdk2as, but dispensable for stability of phosphorylated complexes, once they had formed.
To test the second prediction of the model, we asked if Cdk2as was able to follow the primary pathway of activation and become phosphorylated as a monomer in vivo (Figure 4A). Size exclusion chromatography of wild-type and Cdk2as/as HCT116 cell extracts revealed similar profiles of total Cdk2 (Figure S4B, C), but relatively less Thr160 phosphorylation in monomer fractions from Cdk2as/as extracts (Figure 4D). This could arise from inability of CAK to phosphorylate Cdk2as or increased susceptibility of Cdk2as to phosphatases. In support of the first explanation, Cdk2 monomer from wild-type cells could be phosphorylated in vitro by human Cdk7 or by the fission yeast CAK Csk1, whereas Cdk2as was not efficiently phosphorylated by either CAK (Figure 4E). Therefore, as predicted by a mathematical model, Cdk2as is impaired in two distinctive features of its activation pathway that play reinforcing roles in ensuring selectivity of cyclin A for wild-type Cdk2 over Cdk1.
During exit from G0, E2F activation is bistable in response to serum concentration (Yao et al., 2008), suggesting that early cell-cycle events might be more dependent on CDK activity when mitogens are limiting (Lee et al., 2010). Treatment of cells grown in 10% serum with 10 μM 3-MB-PP1 increased doubling time from ~26 to ~40 hr, whereas 1 μM 3-MB-PP1 was less effective at slowing proliferation (doubling time ~33 hr). In 1% serum, however, 1 or 10 μM 3-MB-PP1 was equally effective at prolonging doubling time, to 96 hr, compared to ~28 hr for mock-treated cells (Figure 5A and data not shown). Therefore, growth factor limitation sensitized RPE-hTERT cells to specific inhibition of Cdk2.
We next asked if the Cdk2 activity requirement for S-phase entry was sensitive to changing serum concentration. Cdk2as/as HCT116 cells were synchronized by serum starvation and released into media containing varying concentrations of serum with or without 10 μM 3-MB-PP1. Nocodazole was added, to trap any cells that progressed through S and G2 phases in mitosis, and cells were collected for flow cytometry 24 hr after release. In 10% serum, 3-MB-PP1 caused a delay in accumulation of cells with 4N DNA content, which became more pronounced as serum concentration was lowered (Figure S5). Upon release into a medium containing 1% serum, selective inhibition of Cdk2 caused a fraction of cells to arrest with 2N DNA content (Figure 5B).
In parallel, we asked if specific inhibition of Cdk2 affected phosphorylation of pRb, p107, and p130 (Figure 5C). Treatment with 3-MB-PP1 reduced phosphorylation of pRb on residues recognized preferentially by Cdk2 (Thr821) or by both Cdk2 and Cdk4 (Ser795), but not on those targeted exclusively by Cdk4 (Ser807/811) in vitro (Zarkowska and Mittnacht, 1997). In addition, Cdk2 inhibition limited accumulation of hyperphosphorylated isoforms of p107 and p130, indicating that Cdk2 activity is required for phosphorylation of all three pocket proteins in vivo. In 10% serum, the effects of Cdk2 inhibition on pocket protein phosphorylation were less severe (Figure 5D and data not shown), consistent with its diminished impact on G1/S progression (Figure S5). Therefore, requirements for Cdk2 activity to phosphorylate pocket proteins and drive S-phase entry become more stringent when growth factors are limiting.
Activation of E2F-dependent transcription correlates with passage through the R point, so we tested whether inhibition of Cdk2 affected timing of this irreversible transition. We first asked how RPE-hTERT cells respond to stimulation with different concentrations of serum, by measuring the fraction that passed into S phase after release from contact inhibition (Figure 6A). Even in the absence of drugs, Cdk2as/as cells were more sensitive than wild-type cells to decreased serum concentration. In the presence of 3-MB-PP1, Cdk2as/as cells became more sensitive still, and a large fraction remained arrested in G0/G1 even at saturating serum concentrations. In contrast, releasing Cdk2as/as cells into media containing 6-BAP shifted their serum response closer to that of wild-type cells, suggesting that this compound could indeed complement Cdk2as in vivo. Neither drug affected serum-responsiveness of wild-type RPE-hTERT cells (Figure S6A). These results, together with the increased sensitivity of HCT116 cells to Cdk2 inhibition in low serum (Figures 5A, B), suggest a general requirement for Cdk2 activity in the response to mitogens.
We next asked if modulating Cdk2 activity with small molecules affected the timing of R-point passage in response to saturating levels of growth factors. Cells were released from contact inhibition into media containing 10% serum and either DMSO, 6-BAP or 3-MB-PP1. The medium was removed at various times, and replaced by a serum-free medium containing the same compound present during the first incubation. Finally, we scored R-point passage by measuring BrdU incorporation 40 hr after release. Compared to wild-type cells, Cdk2as/as cells were delayed in passing the R point, suggesting that the mutation per se hindered the ability to respond to mitogens. Treatment with 3-MB-PP1 caused a further delay in passage of the R point, whereas 6-BAP advanced the timing of R-point passage to near that of wild-type cells (Figure 6B). Neither drug had any effect on R-point timing in wild-type cells (Figure S6B). Therefore, Cdk2 activity is rate-limiting for cell-cycle commitment.
We sought to determine when Cdk2 activity is needed: during growth factor stimulation, to pass the R point; after serum removal, to execute a post-R-point function required for S-phase entry; or both. We repeated the R-point experiment with a variation: when 6-BAP or 3-MB-PP1-treated cells were transferred to serum-free media, they were either maintained in the same compound or switched into a medium containing the other drug (Figure 6C). There was no difference in the BrdU-positive fraction between cells kept in 3-MB-PP1 for the entire experiment and those switched from 3-MB-PP1 to 6-BAP. This indicates that Cdk2 activity is required to drive cells past the R point, and that establishment of permissive conditions only after serum withdrawal is not sufficient for continued progression. The reciprocal treatment—6-BAP during stimulation and 3-MB-PP1 upon withdrawal—revealed time-dependent effects: at 8 and 12 hr, the number of BrdU-positive cells was reduced compared to the population maintained in 6-BAP; but this effect was lost at 16 and 20 hr, possibly due to breakdown of cell synchrony. This suggests a window of time, after the R point, during which Cdk2 activity is still required to negotiate the G1/S transition. Therefore, Cdk2 is a non-redundant regulator of both R-point passage and S-phase entry in human cells.
Gene disruptions revealed that Cdk2 protein is dispensable for viability (Berthet et al., 2003; Ortega et al., 2003), and that Cdk1 can perform all essential functions of interphase CDKs when it is able to form complexes with the relevant cyclins, i.e., when their usual partners are missing (Santamaria et al., 2007). These studies sidestepped whether Cdk2 performs essential functions, exclusively, in wild-type cells. This question is important for understanding how different CDK/cyclin complexes collaborate to make metazoan cell cycles run smoothly, and for efforts to target CDKs therapeutically. The viability of Cdk2−/− animals has been invoked both to discourage and to justify attempts to inhibit Cdk2 in cancer cells (Malumbres and Barbacid, 2009). One line of reasoning holds, simply, that drugs selective for Cdk2 are unlikely to be effective because absence of Cdk2 protein fails to block cell proliferation. A counter-argument—that certain tumors might be more dependent on Cdk2 and therefore susceptible to its inhibition, whereas normal cells might be relatively resistant—gained support from studies implicating Cdk2 in suppression of oncogene-dependent cellular senescence (Campaner et al., 2010; Hydbring et al., 2010). That role was inferred, however, from comparisons of Cdk2−/− and wild-type cells, and from responses to drugs that target other CDKs besides Cdk2.
Our previous work, showing that Cdk2 excludes Cdk1 from active complexes throughout G1 and early S phase (Merrick et al., 2008), suggested a unique function for Cdk2 during the interval when cells commit to a round of division. That role is likely to be general, i.e., not restricted to particular cell types; and to depend critically on Cdk2 levels, because lowering the amount of Cdk2 would advance onset of Cdk1-cyclin assembly and reduce dependency on Cdk2 (as it might, for example, in Cdk2as/neo cells), whereas increasing it would interfere with normal activation of Cdk1 (as it might in MEFs overexpressing Cdk2as).
Here, by a chemical genetic strategy that maintained physiologic expression levels, we showed that Cdk2 activity is required for proliferation even though Cdk2 protein is not essential. Drug sensitivity was dependent on mutant gene dosage (Cdk2as/as > Cdk2as/neo ≈ Cdk2as/+ > Cdk2+/+), implying that ~50% inhibition is sufficient to slow, but complete blockade is needed to arrest, cell division. We probably never achieved 100% inhibition of Cdk2as with 3-MB-PP1 (or complete rescue of function with 6-BAP), so cannot rule out a more stringent dependence on Cdk2 in vivo. We can be certain, however, that conventional CDK inhibitors could not have uncovered this requirement. In vitro, CVT-313 and -2584 are ~8–10-fold more potent towards Cdk2 than towards Cdk1, but ~100-fold less potent than is 3-MB-PP1 towards Cdk2as (Brooks et al., 1997; Campaner et al., 2010). Thus, concentrations required for equivalent inhibition of Cdk2 in vivo would also inhibit other kinases. Consistent with this interpretation, 10 μM CVT-313 blocked S phase more effectively in wild-type cells than did 10 μM 3-MB-PP1 in Cdk2as/as cells (Figure S6C). Therefore, even the most Cdk2-selective drugs available probably have off-target effects that contribute to their phenotypes—a problem obviated by the AS kinase strategy.
A mathematical model indicated that cyclin A pairing rules are enforced by: 1) ability of Cdk7 to phosphorylate Cdk2 monomer and 2) high affinity of unphosphorylated Cdk2 for cyclin A. Introduction of Cdk2as into human cells allowed us to confirm the model's prediction that both properties would need to be disrupted for Cdk1 to become the preferred partner in vivo.
The order of Cdk2 and Cdk1 activation may coordinate the temporal program of replication origin-firing (Katsuno et al., 2009). In addition, Cdk1/cyclin A has been specifically implicated in attenuating expression of histone genes at the end of S phase (Koseoglu et al., 2008); premature onset of Cdk1/cyclin A assembly could therefore lead to histone depletion and genomic instability. Cdk1 activity is normally kept low until mid-S phase, both by preferential formation of Cdk2/cyclin A complexes (Merrick et al., 2008) and by inhibitory phosphorylation of Cdk1 (Katsuno et al., 2009). Therefore, reinforcing mechanisms evolved to ensure that activation of Cdk2 precedes that of Cdk1 in mammalian cells. Consistent with strong selection to maintain that order, Cdk2as/as cells, in which the barrier to Cdk1/cyclin A activation is lowered, have cell-cycle delays and defective responses to mitogens in the absence of analogs.
The biochemical defects caused by a gatekeeper mutation, and their rescue by small molecules, suggest an important role of active-site structure in normal Cdk2 activation. Similar observations were made for the Ser/Thr kinases Akt, B- and C-Raf, and PKC isoforms α and ε. For each kinase, an ATP-competitive inhibitor induced T-loop phosphorylation in vivo, but by a different mechanism in each case. Drug-binding had two distinct effects on Akt—enhanced membrane localization and a conformational change—both of which favored phosphorylation (Okuzumi et al., 2009). In contrast, Raf inhibitors promoted activating phosphorylation through kinase dimerization (Poulikakos et al., 2010). Cdk2as appears most similar to a PKC gatekeeper mutant with weakened nucleotide binding and reduced T-loop phosphorylation, which was complemented by ATP analogs (Cameron et al., 2009), but may be unique in its impaired binding to an allosteric regulator, cyclin A. This might be analogous to effects of a gatekeeper mutation on interactions between the plant kinase Pto and its bacterial effector proteins, which were also corrected by PP1-based analogs (Salomon et al., 2009).
Here we recapitulated the Cdk2as-activation defect and its rescue by small molecules in vitro, to yield insights into how active-site conformation influences the steps of Cdk2 activation. Cdk2as is a poor substrate for both Cdk7 and Csk1, which was surprising because Csk1, unlike Cdk7, readily phosphorylates monomeric Cdk1 (Merrick et al., 2008). This suggests the T-loop is generally unavailable for protein-protein interactions in the Cdk2as monomer. Because the T-loop contributes to cyclin-binding (Jeffrey et al., 1995; Russo et al., 1996), its mal-orientation could also explain a lower affinity for cyclin A.
We were able to correct these defects in vivo with 3-MB-PP1, a strong inhibitor, or 6-BAP, which is weakly inhibitory in vitro and had no negative effect on cell proliferation. Cdk2as is therefore a three-position switch (Figure 7): defective for activation in the absence of drugs; bound normally to cyclins, but inhibited, in the presence of 3-MB-PP1; and closer to wild-type Cdk2 in both cyclin-binding and activity in the presence of 6-BAP. Chemical rescue of a kinase hypomorph has been described previously; active-site mutations in the Tyr kinases Src and Csk impaired their activity, but imidazole could act as a prosthetic group to restore catalytic function (Williams et al., 2000; Qiao et al., 2006). 6-BAP is likely to rescue Cdk2as by a different mechanism—transient binding to allow complex formation and phosphorylation, and subsequent displacement by ATP to permit catalytic activity. It will be interesting to see if 6-BAP can rescue other AS kinases in which gatekeeper mutations impair function.
In response to growth-factor signaling, Myc drives expression of cyclin D, which activates Cdk4 and Cdk6, leading to partial phosphorylation of pocket proteins and derepression of E2F targets including cyclin E, which promotes further phosphorylation of Rb family members and full E2F activation (Yao et al., 2008). Inhibition of Cdk2 disrupted normal R-point control, possibly by interrupting this positive feedback loop. Even in the absence of drugs, S-phase entry was more sensitive to lowered serum concentration in Cdk2as/as than in wild-type cells; that sensitivity could be enhanced by 3-MB-PP1 or suppressed by 6-BAP. Similarly, E2F expression in rat fibroblasts was more sensitive to CDK inhibition by CVT-313 at low serum concentrations (Lee et al., 2010). Manipulating Cdk2as activity with 3-MB-PP1 or 6-BAP delayed or accelerated R-point passage, respectively, revealing a rate-limiting function of Cdk2 and, possibly, of positive feedback. When mitogens are limiting, blocking that function arrests cells in G1, but in 10% serum, signaling through other pathways can override inhibition of Cdk2 to allow progression, albeit delayed, through the cell cycle. Indeed, this result might have been predicted by a mathematical model, in which increased amplitude of Myc expression converted the R-point switch from a bistable to a monostable one (Yao et al., 2008).
Once budding yeast cells pass Start—analogous to the mammalian R point—they proceed through division independent of environmental cues (Johnston et al., 1977). Also like the R point, Start depends on CDK/cyclin complexes that activate a transcription program important for later cell-cycle events (Spellman et al., 1998; Skotheim et al., 2008). The cyclin Cln3 initiates the transition by promoting transcription dependent on the activators SBF and MBF, targets of which include CLN1 and CLN2, which also encode cyclins that act, through Cdk1 and SBF/MBF, to promote their own expression (Dirick et al., 1995). Removal of this positive feedback by deletion of CLN1 and CLN2 had two main effects on G1/S transcription. First, activation of the program took longer and occurred with greater cell-to-cell variability. Second, there was a variable delay between onset and maximal expression of target genes. The aberrant timing and incoherence of gene expression in cln1Δ cln2Δ cells led to increased frequency of cell-cycle arrest, demonstrating the importance of positive feedback for normal cell division (Skotheim et al., 2008).
We hypothesize that Cdk2 activity is specifically required for timely and coherent expression of E2F-dependent genes—functions analogous to those of G1 cyclin/CDK complexes in yeast. Selective inhibition of Cdk2as in human cells dampened the response to growth factor signaling in G1, impeded passage of the R point and impaired proliferation. This is consistent with the previous finding that S-phase entry was defective in MEFs exiting G0 without cyclin E (Geng et al., 2003), which binds nearly exclusively to Cdk2 in human cells (Merrick et al., 2008). The ability to escape quiescence and proliferate, often under suboptimal growth conditions, is a feature of oncogenic transformation. Therefore, specific functions of Cdk2 uncovered by chemical genetics could yield new insights into how CDKs in general, and R-point control in particular, might be effectively targeted by anticancer therapy.
To replace wild-type Cdk2 with Cdk2as, we followed the strategy used to create Cdk7as/as cells (Larochelle et al., 2007). We constructed an rAAV vector containing two contiguous regions of the Cdk2 locus amplified from BAC clone RP11-973D8 (Children's Hospital of Oakland Research Institute) flanking a loxP-neoR-loxP cassette, mutated exon 3 to change Phe80 to Gly and create an EcoRI restriction site for screening purposes, and performed two rounds of targeting as follows: 1) rAAV infection and selection for G418-resistance, PCR screening for correct integration and verification of mutations by sequencing; 2) Cre recombinase-mediated excision of neoR to yield Cdk2as/+ clones, verified by PCR and reversion to G418-sensitivity; and 3) repetition of steps 1 and 2 to yield homozygous Cdk2as/as clones.
Extract preparation, analyses of CDK assembly and activation, Superdex 200 chromatography, immunoblotting, and immunoprecipitation were carried out as described (Merrick et al., 2008).
Labeling with [γ-32P]N6-(benzyl)-ATP was performed as previously described (Larochelle et al., 2007; Merrick et al., 2008). 3-MB-PP1, 6-BAP and other N6-modified adenines were dissolved in DMSO and used at indicated concentrations. IC50 was measured by incubating purified Cdk2/cyclin A with varying concentrations of 3-MB-PP1 or 6-BAP, prior to kinase reactions containing 25 mM HEPES pH 7.4, 150 mM NaCl, 10 mM MgCl2, 200 μM ATP, 5 μCi [γ-32P]ATP, 5 μg histone H1, ~3 nM Cdk2/cyclin A. Incorporation was visualized by autoradiography and quantified by phosphorimager.
To test a requirement for Cdk2 activity we seeded 100 cells/well in 96-well dishes with varying amounts of 3-MB-PP1. After 7 d, medium was removed and cells were stained with crystal violet. For proliferation curves, asynchronously growing cells pre-treated for 48 hr with DMSO or 3-MB-PP1 (to allow correction of the cyclin A-binding defect) were plated at 105 cells/well in a 6-well dish and cell number determined in triplicate every 24 hr with a hemacytometer. To analyze Cdk2 functions in G1/S, we synchronized cells by serum starvation for 72 hr (HCT116) or by contact inhibition (RPE-hTERT). HCT116 cell-cycle progression was monitored by analyzing DNA content with propidium iodide-staining and flow cytometry. To quantify cells that entered S phase, we released RPE-hTERT cells from G0 in chamber slides containing medium + 10 μg/mL BrdU. Cells were fixed in 70% ethanol, DNA was denatured with 1 M HCl for 10 min, and the rest of the staining protocol was performed as described (Terret et al., 2009). Incorporation was detected with anti-BrdU antibody (1:100) and visualized with secondary antibody conjugated to DyLight488 or Cy5. Cells were counterstained with DAPI to determine total numbers. R-point assays were performed in the same way except that, at various times after release, serum-containing medium was removed, cells were washed twice, and serum-free medium containing BrdU was added.
Figure S1, Related to Figure 1: This figure contains details of the screening and genotyping we performed in the course of generating Cdk2as/as HCT116 and RPE-hTERT cells used in this study (Figure S1A, B). Additionally, it shows that Cdk2as/as HCT116 cells have the same amount of Cdk2 protein as wild-type cells (Figure S1C), that Cdk2as/as HCT116 cells are sensitive to 3-MB-PP1 (S1D), and that Cdk2−/− MEFs ectopically expressing Cdk2as are sensitive to 3-MB-PP1.
Figure S2, Related to Figure 2: Here we show that substitution of Cdk1 for Cdk2 in cyclin A complexes occurs in cells expressing one or two copies of Cdk2as ((S1A), that AS inhibitors built on a two distinct scaffolds can restore Cdk2as T-loop phosphorylation to wild-type levels, and chemical rescue correlates with inhibitor potency (S1B), and that the gatekeeper mutation impairs, and ATP analogs restore, binding of Cdk2 to the CDK inhibitor p21 (S2C).
Figure S3, Related to Figure 3: This figure demonstrates that a high dose of 6-BAP (80 μM) does not inhibit proliferation of wild-type or Cdk2as/as RPE-hTERT cells.
Figure S4, Related to Figure 4: Figure S4A shows how the temporal dynamics of CDK-cyclin complex formation is affected by altering parameters of Cdk2's activation pathways, thus providing a more complete view of the modeling results presented in Figure 4A. Here we also show the complete sizing column profiles from wild-type (Figure S4B) and Cdk2as/as (Figure S4C) HCT116 extracts and indicate the fractions we pooled for our experiments with monomeric Cdk2 (Figure 4D and E).
Figure S5, Related to Figure 5: In this figure we release Cdk2as/as HCT116 from serum starvation into medium with varying serum concentration. We find that decreasing the serum concentration sensitizes cells to Cdk2 inhibition justifying the experiments in Figure 5C-D, performed with cells grown in 1% serum.
Figure S6, Related to Figure 6: Here we show that 6-BAP and 3-MB-PP1 do not affect the serum-response curve of RPE-hTERT wild-type cells (Figure S6A), and these compounds do not affect R point timing in wild-type cells (Figure S6B). Figure S6C provides evidence that the phenotype produced by CVT-313 treatment depends on inhibition of other kinases besides Cdk2.
We thank K. George and P. Jallepalli for advice and assistance in gene targeting, D. Morgan for baculovirus encoding Cdk2as, and members of the Fisher lab for helpful discussions and critical review of the manuscript. The work was supported by fellowship support of the Deutsche Forschungsgemeinschaft to L.W. (WO1456) and the California Breast Cancer Research Program to D. H. and N.E.H., and by NIH grants GM056985 to R.P.F., EB001987 to K.M.S. and CA136717 to A.G.
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- "I have now finished your finely written, provocative and fascinating book. Your epilogue was simply FUN stuff. Very well done!"Steve Wells, Tacoma, WA
About Jeremiah Brown
Rowing & Training
Here is where you can find my thoughts on rowing and training in general. The way we train the body is very similar to how we cultivate our minds for high performance.
In The Community
A collection of images, updates, and ongoing community initiatives that my teammates and I are taking on as ambassadors of sport in Canada.
The 4 Year Olympian
I began rowing in 2009. I won a silver medal at the 2012 Olympics with my teammates three and a half years later. Here's what I learned…
Latest articles
Fear of Failure
I listened to a friend talk about her fears very openly today. Athletes and musicians–anyone...
Remember Your Own Narrative
Since the Olympics, I've been studying music with the kind of discipline that was present...
Here's how to go from desiring an outcome to making that outcome a product of...
The Ego Battle
The biggest problem is the ego telling us what we can't do. It's the little...


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This 3-star hotel is ideally located opposite New Street Rail Link.
You and your group will have easy access to the surrounding areas and attractions. It's right in the thick of it all – within walking distance of the Bullring Shopping Centre and the seriously vibrant China Town. The rooms are traditional with modern en-suite bathrooms, and tea and coffee making facilities for that all-important and very strong coffee the morning after. But, the best is the relaxing bar and restaurant, renowned for its carvery menu - perfect food for sorting out your hangover. It also serves up a massive fry-up or a delicious buffet breakfast, guaranteed to set you up for the rest of the weekend.


text: Ben Collins wrote: > > The only way to manage this, is to set up a group wheel, use the > > "auth required pam_wheel.so" line, add user "you" to group > > wheel and do it the above way leaving out the "apply=you" option. > > It shouldn't according to the docs (yes I read that particular caveat, but > the logic is still there for it to work). For passwordless access, you could > make the pam_wheel.so module "sufficient" which means that belonging to the > group "root" gives them access to su without a password. I'm sorry. It doesn't work. I'll stay with the method described in my last posting which is sufficient for my needs. Thanks again for your interest. -- Andreas Kurth Mannheim, Germany


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Re: Mobley's in Maryland
In reply to:
Re: Mobleyu2019s in Maryland
John Mobley
John Mobley was my 9th Great GrandFather.He married Rachel, I have her name as Pindall Or Pindell.
He had sons Edward Mobley Born Oct 8, 1716,Married Mary ?
Edward Died June 1777 in Wake co., North Carolina
Mordicai Mobley born 1714, Married Dorcas Reed.
Mordicai Died 1792, Wake Co., North Carolina
Hammond Mobley born 1718, Married Avorilla(no further info
James Mobley born May 2, 1720, Married Chloe (nif)
If there were more children I have no information on them
Mordicai Mobley had ten children, nine daughters and one son.His daughter Susannah (Sukey) Married her cousin Ezekial Mobley, son of Edward Mobley,II born 1740
Edward Mobley & Rachel Pindellhadfour children that I have listed. If you have further info I would sure like to knowEvalee Russell


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Over Mistakes I Have Madei really beat myself up over the past.i find myself appologizing over and over sometimes.i understand when pepole get mad,but they don`t understand my point of view.and i need to remember this as well.and try to over look what they say.
ghostofmyself 36-40 1 Response 1 Mar 20, 2009
Obsession On My PicturesI got bored last saturday so i got my phone and took pictures of myself. until now, i'm staring at those two great shots of mine
dazzlingeyes 18-21, F 2 Responses 0 Apr 29, 2010
Fast and EasyIt always starts with passion, then it becomes obsession! That's how I do things. I should keep the balance. Obsessions is lethal.
LordVoldemort 36-40, F 2 Responses 0 Mar 17, 2009
On What? Well It Depends.I tend to obsess about things. When I decide I like something or am interested in something I obsess about it constantly, every day, all day long until I have exhausted every aspect of it at least 3 times. lol This month, it's dreads. In the past it has been Witchcraft and...
FellidayaFiresong 36-40, F 0 Sep 10, 2013