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Time is running out to save on tickets for the 4th Annual Hearts of Love Gala
Buy your tickets now and save!
Time is running out to save on tickets for our 4th Annual Hearts of Love Gala: Our Hearts are Soaring. Tickets purchased by January 31, are only $150 per person. Starting Feb. 1, tickets are $175 per person. Buy your tickets today! A few sponsorships and advertising opportunities are still available as well.
The gala takes place at 6 p.m. on Saturday, February 22nd at the Sanibel Harbour Marriott Resort and Spa, 17260 Point Drive, Fort Myers. The highlight of the evening will be Tino Wallenda of the talented stunt performers, The Flying Wallendas, performing a tight rope act. Krista Fogelsong from NBC-2 will be the Master of Ceremonies. The gala will include an hour of hors d'oeuvres and an open bar, a 3-course dinner with wine, and live auctions, followed by music and dancing. Our Mother's Home will also present their annual Graduate of the Year and Mother of the Year awards. The ticket prices also include valet parking. All proceeds benefit Our Mother's Home in their life-saving mission of keeping teen mothers and their children together in a safe and supportive environment.
The event will offer the Southwest Florida community an opportunity to enjoy a night of entertainment, while supporting Our Mother's Home as they work toward making a positive impact in the lives of teen mothers who are in the foster care system or victims of human trafficking. In addition to an unforgettable opportunity to see a performance by Tino Wallenda of the famous Flying Wallendas, attendees will enjoy dinner, drinks, music, and exciting live auctions. Our Mother's Home will also take this opportunity to recognize their Mother of the Year and Graduate of the Year for 2013.
The funds raised at this event will go toward supporting the teen mothers who seek refuge at Our Mother's Home. The organization provides these teen moms a safe and loving home where they can learn to love and respect themselves and their children, as well as the resources to become self-sufficient and productive members of society. The Florida Department of Children and Families provides assistance for the girls, but not their babies. Our Mother's Home receives 52% of the funding they need from DCF, and the other 48% of necessary funds must be raised in order to help support these teen mothers.
This year's sponsors include Astrid and Rene Photography, Digestive Specialists, South Fork Grille, Bob Forget, Ben Few and Company, Restorative Health and Human Center, and Vein Specialists at Royal Palm Square. Sponsorship opportunities are still available for this gala event.
"The community contributions raised at this gala help Our Mother's Home continue to provide vital services to foster teens and human trafficking victims that want to keep and raise their children in a supportive environment," says Karen Watson, Executive Director of Our Mother's Home of Southwest Florida, Inc. "This year, we are very excited to offer our supporters the unique opportunity to see a performance by Tino Wallenda."
Seating is limited. Get your tickets now to take advantage of the special rate of $150 per person. For tickets or information on sponsorship and advertising opportunities, visit www.ourmothershome.com or call 239-267-4663.
About Our Mother's Home of Southwest Florida, Inc.
Our Mother's Home of Southwest Florida is a non-profit organization that aims to break the cycle of foster care and generational poverty by providing teen mothers, ages 12-18, who are in the foster care system or victims of human trafficking and other traumas, with a safe and nurturing home where they can learn to love and care for themselves and their children, while becoming productive members of society. As a privatized 501(c)3 organization, Our Mother's Home raises 50 – 60% of its own funding to provide teen mothers with a supportive environment to stay with their children, learn life skills, and finish their education through its Mentored Living Program. There are no federal, state or city funding provided for the infants of the teen mothers. They depend heavily on dedicated volunteers, and contributions of funds, commodities and services. With over 200 mothers and children being helped over its 13 years of operation, Our Mother's Home continues to be a model for success in decreasing the rate of children in foster care. For more information, call (239) 267-4663, email email@example.com, or visit http://www.ourmothershome.com.


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Hire a 3D Modeller
Orçamento $30-250 USD
Procuro alguem para modelagem, texturizaçao e renderizaçao 3d para jogos, em especifico Second life. Se voce conhece o jogo, nos contacte.
Segue exemplos abaixo.
I'm looking for someone for modeling, texturizing and 3d rendering for games, in specific Second life. If you know the game, contact us.
Here are examples below.
13 freelancers estão ofertando em média $203 para esse trabalho
Hi, Visit our profile page and see some jobs https://www.freelancer.com/u/xplow.html Ask us if you have any question that we will respond as soon as possible. Thanks
Entregarei modelo 3d conforme previo desenho 2d ou instruções de design a ser desenvolvido. Posso também criar o UV layout e enviar versões Low/High ou normal map se necessários.
I am here to bring your product to life. I'm a mechanical engineer and I have 8 experience years. Work type: 3D, 2D models, Render, Sheet metals, Machine and Devices, industrial product, Plastic molds, plastic parts Mais
I am an architect specializing in 3d design. Please see my works on Freelancer Professional use of all 3d software Experience 3 years in the field of design
I'm consists of experts in every field related to 3D, video and animation. We can cover all your 3D from the initial design, script and storyboard to modeling, texturing. I have over 6 year experienced in designin Mais
I've been playing SL since before the merger of the teen and standard grids, I've been building for the game for just as long, I love the game and I'd greatly appreciate the opportunity to build for you.
I am an architect and urban planner and currently work as a professional 3d modeling and rendering [login to view URL] / tonalves

subset: commoncrawl


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Wow! We have been SO busy these last few weeks -- I'm sure you can all relate! It is the season of standardized testing (blech!!!) which for my second graders means an altered schedule and lots of silent walking down the halls. But amidst all of that, we have had time to seriously kick some fraction BOOTY! I will admit, our pre-test scores were bad y'all. Like, my highest math kid got a 0% ... ZERO! SO I know I was going to need something FUN and ENGAGING to help these kiddos really understand. I created this fraction pack that is circus themed just for the occasion!
Check out the preview to see what we've been up to in our room!
On top of all of that circus fun, I found some fabulous resources from other teachers! If you've got a fraction unit coming up, or you're just on the search for some great activities for next year... check these out!
This sort is Free from The Teacher Wife! Click the picture to download a copy.
These adorable fraction kites are courtesy of Christina Bainbridge! Click here to check her out!
And these *adorable* little fraction caterpillars are from Cara Carroll!
We also made some name fractions. They turned out really cute and they were a great little intro into parts of a whole! I whipped up a quick little printable to go with these that you can download by clicking below!
So there you have it! Everything you need to have a blast with fractions. OH! By the way -- I mentioned how my class didn't do so hot on the pre-tests? WELL! 100% of my friends scored an 80% or higher on their Post-Tests. HOLLA! :)
I'm sure you've heard by now about the absolutely FABULOUS sale going on at TPT for a little Teacher Appreciation. You can hear it from me, too! TEACHERS ARE AWESOME! I'm so blessed to know so very many incredible teachers -- with beautiful hearts, souls, and minds! Let's celebrate teachers and all the hard work we do with a little shopping.
My store will be 20% off on Tuesday and Wednesday! Use the Promo code TAD13 at check-out for a total savings of 28% Now is the perfect time for some last minute activities AND to stock up on fabulous finds for next year. I can't wait to go shopping! Here are some new(ish) items from my store and some of my oldies but goodies for you to check out this week! :)
Alright, Thank you TEACHER for visiting my blog. But thank you even more for everything you do daily in the lives of children. We do NOT have an easy job. We are met with challenges every day. There are times when we cry the whole way home, there are times when we laugh the whole way home. Know this -- You are appreciated. Your hard work does not go unnoticed and your dedication to the lives of our youth is nothing short of incredible! :)


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Dear friends,
I'm back from vacation abroad. It was a joyous time with a few surprises in the mix like my sister giving birth 3 weeks early, just in time for me to meet my latest niecy, Zahra. I saw the snow, wet as rain, kiss Brooklyn streets, watched city boys perform kicking dance moves in the tight spaces of subways. I gorged on yogurt, dried craisins and mixed nuts, snacks I think should be far more affordable in Jamaica and ate a bit of fast food in my attempt to put on a few pounds :).
Continue reading "Vacation's Over"


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I was riding on the bus the other day when I noticed a statue that I had never seen before. It was sitting in the shadow on a tree. A branch was near it's head, threatening to cover his face as if nature was slowly erasing the remembrance of this man.
No one wants to be forgotten, so here's another .15 seconds of fame for some of the statues on Princes Street in Edinburgh.
Wellington. Arthur Wellesley, 1st Duke of Wellington. 1769 – 1852. A statesman and one of the leading military and political figures of the nineteenth century. He is often simply referred to as "The Duke of Wellington".
Livingstone. 1813 – 1873. As in, "Dr, Livingstone I presume?". Born in Scotland, David Livingstone was an explorer and missionary.
Sir Walter Scott. 1771 – 1832. A prolific Scottish historical novelist and poet. Famous titles include Ivanhoe, Rob Roy, The Lady of The Lake, The Heart of Midlothian and The Bride of Lammermoor. He was born in Edinburgh and not only has a huge monument on Princes Street, but the Waverley train station which stands not far from this monument is the only train station to be named after a novel.
Adam Black. 1784 – 1874. Born in Edinburgh, he was a publisher, and founded the A & C Black publishing company. Twice Lord Provost of Edinburgh, and represented the city in parliament from 1856 to 1865.
John Wilson. 1785 - 1854. A Scottish writer who was for a time the principal writer of Blackwood's Magazine, a magazine printed from 1817 to 1980.
Allan Ramsay. 1686 -1758. An Edinburgh poet and publisher who was involved in the early 18th century revival of Scots vernacular poetry. Best remembered for his five-part work, "The Gentle Shepherd." The statue was unveiled in 1865.
Memorial of The Royal Scots Grey. Commemorates the Royal Scots Greys who left Edinburgh in 1899 to fight in the South African War.
Thomas Guthrie. 1803–1873. A Scottish philanthropist who was one of the most popular preachers of his day in Scotland.
His statue reads: An eloquent preacher of the gospel. Founder of the Edinburgh Original Ragged Industrial Schools, and by tongue and pen, the apostle of the movement elsewhere. One of the earliest temperance reformers. A friend of the poor and of the oppressed.
Sir James Young Simpson. 1811 – 1870. A Scottish doctor and an important figure in the history of medicine. Simpson discovered the anaesthetic properties of chloroform and successfully introduced it for general medical use.
Maybe I'll take pictures of the statues on George Street soon ...


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Home /
Damac Properties might uphold 25% dividend in 2016
Damac Properties might uphold 25% dividend in 2016
Damac Properties expects to maintain its 25 per cent cash dividend policy for 2016 despite profit declines in the first three quarters, its group chief financial officer said on Sunday.
The developer's board set a minimum 25 per cent cash dividend target in 2014 for the following two years. The board followed the recommendation in 2015, however, is yet to announce a dividend for 2016. "There is no reason to speculate we will not adhere to this," Adil Taqi told Reuters at Damac's headquarters in Dubai, adding that it would be unlikely to be more than the target and would require regulatory approval. Profit at Damac, which is building a US$6 billion golf complex with Donald Trump, fell 23 per cent to Dh2.84 billion (US$773.23 million) in the nine months to September, according to Reuters calculations. Dubai's property market, where Damac's business is concentrated, has softened over the past two years as buyers became more cautious amid weaker oil prices and cuts to regional government spending.
The market is likely to move "sideways" in 2017 compared with 2016, when the company expects to reach around Dh7 billion in sales, Taqi said."I don't envisage huge growth," he added.
Damac expects Dubai's market to rebound in the "second or third quarter of 2019" in the lead up to the Expo 2020 the emirate is set to host. He also said Damac was considering its options for refinancing debt maturing over the next two years, including a US$100 million sukuk in March, of which it has already paid off US$25 million, and US$650 million sukuk in 2019.
Other Facilities
Gulf Property
Pan Asian Media MFZ-LLC
P.O. Box: 39865, Dubai
United Arab Emirates
Office Tell: +971 4 228 1021
Office Fax: +971 4 228 1051


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Trust is an essential underpinning of life in the digital age. We trust our friends on Facebook not to share our private family photos. We trust our email clients and antivirus software to keep viruses and spam at bay. But for many people, the risks of using the internet are scary enough to curb their online activities.
Earlier this year, the U.S. Department of Commerce's National Telecommunications & Information Administration (NTIA) looked at the results of a survey by the U.S. Census Bureau in 2015. Out of 41,000 U.S. households, 19% reported security breaches, identity theft, or other malicious activity in the previous 12 months. Among households with mobile data plans, 22% had experienced an online security breach. The most pressing concern, cited by 63% of online households, was identity theft, followed by credit card or banking fraud, and various forms of data collection.
What's of greater concern is the chilling effect this has had on online activities. Nearly half of online households said that their worries had stopped them from engaging in financial transactions, buying goods or services, posting on social networks, or commenting on political issues online; 30% refrained from at least two of these activities. It's not surprising that if users were concerned about a particular risk, they would avoid a related activity. Thus, 35% of households worried about identity theft had decided not to conduct financial transactions in the 12 months prior to the survey.
"Privacy and security concerns deterred each of these important activities in millions of households," the NTIA's Rafi Goldberg wrote. "In addition to being a problem of great concern to many Americans, privacy and security issues may reduce economic activity and hamper the free exchange of ideas online."
This is a disturbing finding; imagine if larger groups of people or even entire business sectors curtailed their online activities so significantly. The importance of information and communication technologies (ICT) to economic prosperity cannot be overstated. ICT accounted for some two-thirds of U.S. total factor productivity growth between 1995 and 2004 and about one-third since then, according to the Information Technology and Innovation Foundation (ITIF), a nonpartisan think tank in Washington. Here in Japan, companies, universities and R&D centers have developed advanced technologies but many firms still use outmoded, inefficient means of communication such as fax machines. Japan hasn't been among the top 10 most developed ICT nations since 2012, according to an International Telecommunication Union ranking. It's no wonder that the country has lagged its G7 peers in productivity growth, and its service sector is only half as productive as that of the U.S. Japan should be looking at ICT and cybersecurity as a business enabler, a topic I took up earlier this month.
Source: http://www.forbes.com/sites/williamsaito/2016/08/03/the-big-chill-lets-not-let-cybersecurity-threats-slow-productivity-and-economic-growth/#592d52552860


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I hold my hands up! I haven't shared a tune of the week post in a long time. I have been really busy lately and haven't had the time to sit down and check out new music as much as I'd like to.
I had a full on listening spree this week and VANN Music totally blew me away with their new song "Tina" and it's accompanying video. These guys just get better!


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The best adult community for homemade porn photos and amateur videos.
Welcome to No.1 amateur homemade porn site on the net where sexy amateur couples from all over the world exchange and submit their homemade porn movies. Personal Clips is not merely a homemade porn video site, it is also a friendly club for true amateur home made porn lovers where every member can have a good rest, meet real housewife sluts and couples, participate in forum's life, read exciting homemadeporn porn stories of our submitters and win prize money. Join to Personal Clips, submit your naughty amateur video and find thousands other homemadeporn movies.
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|Jori Anne
|Date of Birth
|Race / Ethnicity
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|Body Type
|i'm a light/social smoker
|i'm a light/social smoker
|I am light/social drinker
|I am light/social drinker
|Turn Ons
|Tall, tattoo covered, bald, bow legged, Mexicans with a swagger.
|Her awesome oral skills and tittyfucking talents.
|Turn Offs
|Being rushed or pressured for sex
|Very thin ladies.
|Taking care of her husband, cooking, candle making, tomahawk and knife throwing, bra collecting.
|Keeping up with her, shooting, fishing, Harley Davidsons, woodcarving, sketching in pen and ink.
|Metallica, Marilyn Manson, Megadeth.
|Heavy Metal, Alice Cooper, Kiss, ACDC.
About myself/ourselves
I can separate love from sex but my wife still has a problem with doing so. I have no interest in having sex with another woman, my wife is all I need but for a long time I've wanted to watch her being tittyfucked and giving oral sex to another man or men. She seems to get an ego boost from the great pleasure she bestows upon me from her extreme oral talents and she has no problem swallowing, ( she feels it would be rude to spit or gag ). Nothing turns me on more than to see her vdrenched in cum. She has never been a slut but before vwe got together she had more than perfected the art of the blowjob and she took some satisfaction in being thecovst. She agreed when I asked her the first time but the man we chose was in car accident. She would do a bukkaki if she didn't have to fuck and blow everyone . Deep inside I know she enjoys being the object of so much sexual desire, of other men wanting to fuck her because she has always had low self esteem this would boost her ego just like posting all the photos of her covered in cum with my dick in her mouth.
People I'm/We are looking to meet
Good looking well mannered non-agressive, non-pushy, down to earth, patient men who would like to have the blowjob and tittyfuck of a lifetime while at the same time building a woman's self esteem. Being hung at least average would be good but the bigger the better. Most likely no actual intercourse unless she wanted it but she can make a man cum over and over.
JoriandBalzag's friends:
Please don't be rude, never post URLs, emails, IM IDs. Violation will be a strong reason to delete your account. Let's be nice and make this site the most friendly site ever.


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B-Ray's Knockout of the week: Strong Pimp Hand [Video]
Two guys are at a gas station and one guy doesn't like what the other guy is saying. So what happens? A strong dose of his pimp hand that's what! It may be hard to knock someone out with a closed fist but try slapping someone unconscious.... takes a lot of years in the pimp game to master...
I think this is hands down the best way to start a fight and how to finish it. When it comes down to it, It didn't matter what they where arguing about all that matters is that someone got slapped into a dark and beautiful dream.
Big ups for the guy putting his poor victim into the recovery position so he doesn't swallow his tongue lol.


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My girl Cyd of The Sweetest Occasion also featured more great shots of the dining tablescape, and Confetti Pop has two tutorials of the fab DIYs.
And yes, this popcorn and several mini salted caramel cupcakes made it into my mouth during the shoot. Don't judge me.
ps - I'm in LOVE with that ring.


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Kings, Evil Courtiers, and the Intended Audience of the Historia Langobardorum
Currie, Edward
MetadataShow full item record
The Historia Langobardorum (History of the Langobards) was written by Paul the Deacon in the 780s-790s. It is an important work of historiography, but little attention has been paid to the power of the text as political propaganda. This study sets out to analyze how Paul promotes harmony between Carolingians and Langobards, uplifts the image of the old Langobardic king Grimoald I, and encourages an agreeable view of the contemporary prince of Benevento, Grimoald III, who rebelled against Charlemagne in 791.


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Prior to its release, 'Goddess of War' was the most common term used to describe Heavenly Sword, and for good reason. The main character, Nariko, wields a sword that splits into dual chain-blades, and the game emphasizes large-scale combat against hordes of enemies. Ninja Theory, the UK-based development team behind this title, hasn't responded negatively to the moniker, and for good reason. Heavenly Sword was built around a strong narrative, and its combat system really is a sight to behold.
Heavenly Sword, which is named after the legendary sword used in the game, will have you playing as Nariko, a young woman who was thought to fulfill a great prophecy before she was born, but is now an outcast. The sword is a dangerous weapon that grants tremendous power to whomever wields it, but at the cost of that person's life. Though her people have protected the sword for many years, King Bohan wants the sword for himself and has sworn to kill anyone who stands in his way. Nariko is accompanied by her friend Kai, who follows Nariko on her journey to stop Bohan and save their clansmen from Bohan's wrath.
When it comes to the core gameplay, this is a hack-n-slasher through and through. There's a few twists to the combat system to inject some added depth, but it's not completely necessary to learn everything within Nariko's extensive move list, at least not until later in the game. The reason for this is that the game doesn't require learning the unique combos until enemy difficult ramps up several hours in, allowing those who are new to the genre to quickly become acquainted with the controls.
The heavenly sword can be broken down and used in three separate forms. Each form transfers to a specific attack that Nariko can use. Speed attacks are performed by default, since Nariko is automatically in speed stance. But in order to select her range stance and power stance, the L1 and R1 buttons need to be selected, respectively. Attack combos can be strung together by pressing square and triangle in specific patterns, or by switching from one stance to the next during an attack.
Using combos and different styles are key to defeating enemies quickly, as well as unlocking super moves and bonus content. Each consecutive attack you perform without getting hit fills a meter that's divided into thirds. Perform enough combos and you'll earn a new combo; reach the second and third tiers and you'll unlock making-of movies, artwork, and more. You'll also gain access to super-style attacks along the way. When you've earned enough style points, you can hit the circle button and trigger a short cut scene where Nariko finishes off a foe with creative flair. Again, not a requirement, but it does help out a lot during the more difficult encounters.
Now here's where the twist come in. The first thing you'll notice is that there is no block button. Now I know what you're saying. How can you have a combat system without a block button? Well, as it turns out, Nariko automatically blocks whenever she's not attacking. There's also no way to cancel a move and suddenly block an attack. Everything needs to be timed and thought out correctly, and this can be a big challenge as it goes against all conventions within the genre.
The block system also ties into enemy types and stances. Your enemy will flash a specific color right before they attack you, and that color depends on the type of attack they are about to unleash. Colorless attacks require no player input to successfully block. Range attacks are blue, and Power attacks are yellow, and require selection of the shoulder buttons to successfully block. Unblockable attacks glow red, and as you can guess these are impossible to block. Instead, use the right analog stick to roll Nariko out of the way. Unblockables are the slowest of the bunch, so don't worry about them too much.
Counter attacks are performed by quickly pressing the triangle as an enemy attack is about to hit you. These are the fastest way to kill an enemy, and also the most difficult. The timing is very tight, but very satisfying, especially during crowded battles. If you screw up the timing on a block or a counter, Nariko will go flying in the air. But if you shake the controller (or press X) while in mid-air, Nariko will toss chain blades at that same enemy and attack them. This is just one of the many attacks you'll discover as you progress through the game.
Along with all the hacking and slashing you will encounter some areas where you will require the use of a cannon, a handheld weapon, or a crossbow. The aftertouch system, which slows down time, is used to control these weapons/objects from a first-person perspective. It's a unique take on bullet time that requires use of the Sixaxis to aim, and does provide much needed relief from the heavy combat segments. Nariko's friend, Kai, is integral to this system as she carries one of the coolest crossbows ever seen. In fact, it's almost bigger than her.
Kai is an integral character, and an amazing character study on how psychological damage can both help and hurt individuals. In fact, every character within this story is both believable and unforgettable. The bosses all have over-the-top personalities, and the story is filled with action and suspense (with the occasional bits of humor). Andy Serkis did a stand up job directing the motion-captured performances, and the game stands far above other titles within its genre because of it.
What helps out the believability of these characters, as well as the fantastical world they inhabit, are the graphics. Though the shadows are a bit jagged, every other aspect is done very well. Huge vistas, detailed walkways, and an organic layout to each level definitely sells the tangibility of this world. Everything is living and breathing, and the game has definitely come a long ways from the GDC previews. The number of enemies onscreen during the final battle is just absolutely ridiculous, and is helped out by the robust physics engine. This is one of the best looking PS3 games of 2007, and a wonderful example of what the PS3 is capable of. But even with all this praise, I still had a few issues with the game.
First off, the quicktime events are annoying. They pop up without any warning whatsoever, and take several tries to pull off successfully (and only because you start memorizing them). Even if you press the right buttons at the right moments, the event will still fall apart and occasionally lead to your death. It also would have been nice to have a block button that could override your moves. Some combos take a long time to complete, and enemies will sometimes complete attacks before you have a chance to finish. The developers should have made stance-blocking optional, but have an added benefit, such as taking in less damage than using the block button, for instance.
It's an epic game, but comes in a little short at just under 8 hours to complete. The length only becomes noticeable because the story is so streamlined and compelling. There are no sidequests, and no optional characters to deal with. I wish it was longer, but the content that's there is great. The boss fights are insane, and the graphics immerse you into the action like few others can. Heavenly Sword is a great title that definitely warrants a purchase.
PlayStation LifeStyle's Final Score
Gameplay style is more a button masher.
Great use of SIXAXIS motion control.
Shockingly good facial animations in cut scenes.


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http://www.peffcorp.com/#home-1 About Power Efficiency Corporation provides comprehensive one-stop solutions to the grid stability problems and takes advantage of large and actively growing market opportunity for energy storage, which is projected to reach $45 billion by 2020. PEC offers solutions to grid operators, utilities, and Commercial and Industrial (C&I) customers. Our focus is on renewable Distributed Energy Resources (DER) and Battery Energy Storage Systems (BESS) for grid balancing services to the Independent System Operators (ISOs); load shifting for utilities; energy management through storage for C&I customers; and micro grids for energy security. We build, own, and operate these facilities to develop a portfolio of income producing projects.
Shares Outstanding as of 8/9/16: 92.3 million Shares Floating (est): 27 million Current Market Cap (8/18/16 $0.012 close): $1.1 million
Recent News: Power Efficiency Corporation Wins 2 Year 12 MW Bid in Con Edison BQDM Auction (8/8/16) http://www.otcmarkets.com/stock/PEFF/news 11 MW x $4.5 million = $49.5 million 1 MW x $3.7 million = $3.7 million $49.5 + $3.7 = $53.2 million for PEFF
10-12G Filing 8/12/16 (first filing 4 years!): http://ih.advfn.com/p.php?pid=nmona&article=72203223&symbol=PEFF PEC is voluntarily filing this Registration Statement in order to make information concerning itself more readily available to the public. PEC believes that being a reporting company under the Exchange Act could provide a prospective business partner or customer with additional information concerning PEC, possibly making PEC more attractive to do business with. As a result of filing this Registration Statement, the Company becomes obligated to file with the SEC certain interim and periodic reports, including an annual report containing audited financial statements. The Company anticipates that it will continue to file such reports, notwithstanding the fact that, in the future, it may not otherwise be required to file such reports based on the criteria set forth under Section 12(g) of the Exchange Act.
On April 17, 2012, PEC filed a Form 15 to terminate its registration under Section 12(g) of the Securities Exchange Act of 1934 (the "Exchange Act") and suspend its duty to file reports under Section 13 and 15(d) of the Exchange Act. From that date until the present, PEC has been a shell company, meaning that it has no or nominal business operations and either no or nominal assets, or assets consisting of a small amount of cash and cash equivalents and nominal other assets. PEC's Common Stock has continued to trade on the OTC Pink Sheets.


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- GENEVA — A senior official at the U.S. National Oceanic and Atmospheric Administration says solar storms pose a growing threat to criticial infrastructure such as satellite communications, navigation systems and electrical transmission equipment. Full story:
* Scientists' research warns humanity may be facing 'vortex of death'... Major Solar Flare Erupts, May Make Auroras Visible in Northern U.S... THERE'S A HOLE IN THE SUN!... Solar flares could paralyse Britain's power and communications, Liam Fox says...Discovery ( Aug 25, 2010): IS THE SUN EMITTING A MYSTERY PARTICLE?...LiveScience(29 July 2010): Antarctica Experiment Discovers Puzzling Space Ray Pattern... National Geographic (November 19, 2008): "High-energy electrons captured over Antarctica could reveal the presence of a nearby but MYSTERIOUS ASTROPHYSICAL OBJECT that's bombarding Earth with cosmic rays... Scientists baffled by unusual upper atmosphere shrinkage... Scientists: solar activity could hit London 2012 Olympic Games... Dr. Michio Kaku Warns of 2012 Solar Storm... NASA: Sun's Nemesis Pelted Earth with Comets, Study Suggests...'Blocked' jetstream to blame for freak weather in Russia and Pakistan, say scientists... Sun storm to hit with 'force of 100m bombs':


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Currency Displayed in
This sleek light-bearing holster makes it convenient to carry our BLACKHAWK!® weapon light on a concealed handgun. The light's slim profile creates no added bulk and fits securely in this compact Level 2 holster.
• SERPA® Auto Lock™ release
• Passive retention detent adjustment screw
• Speed-cut design for rapid draw, target acquisition and re-holster
• Includes Belt Loop Platform and Paddle Platform
• Accommodates Night-Ops® Xiphos™ NTX Weapon Mounted Light
• Fits Shoulder, S.T.R.I.K.E., Quick Disconnect and Tactical Holster Platforms
Featuring our highest level of security, the Level 3 Light Bearing Duty Holster makes it possible to wear a weapon-mounted light while on duty. (Compatible with the BLACKHAWK!™ Xiphos™ NT Light only)
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With the holidays almost upon us, it's time once again for small and midsized businesses (SMBs) to focus on protecting themselves and their customers. The shopping rush has the potential to swamp company websites, and cyber thieves will be waiting for an opportunity to exploit weaknesses in data-rich systems. But by taking a few simple steps up front, Identity Theft 911 says SMBs can keep their customers — and their own networks — safe from holiday-minded hackers.
Related Topics : Unisys, Stimulus Package, Security Breaches, Symantec, Electronic Surveillance
To protect the company from those insiders who abuse their privileged access and from hackers with stolen credentials, many companies are turning to a privileged access management (PAM) solution. ... More >>
Is fake news a security issue? Some say yes, as it can be used as a social engineering tool to spread disinformation and conceivably to get unsuspecting users to click on malicious links. ... More >>
Blockchain comes with many costs and is surrounded by confusion. Here, we examine realistic use cases, drawbacks and the potential of blockchain. ... More >>


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ISTANBUL, Aug 14 (Reuters) - Turkish Prime Minister Tayyip
Erdogan on Wednesday called on the U.N. Security Council and
Arab League to take immediate steps to stop a "massacre" in
Egypt, saying international silence had paved the way for the
Egyptian authorities' violent crackdown.
"The international community, especially the U.N. Security
Council and Arab League, must act immediately to stop this
massacre," Erdogan's office said in a statement.
(Reporting by Jonathon Burch; Writing by Nick Tattersall;
Editing by John Stonestreet)


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Q: A pregnant friend told me that she does kick counts every day. What are they, and should I be doing them?
A: Fetal kick counts are a simple, noninvasive way to monitor your baby's well-being. "Fetal movement is a reassuring sign," explains William M. Gilbert, M.D., an OB-GYN specializing in maternal-fetal medicine at Sutter Memorial Hospital in Sacramento, Calif. "When babies are in trouble, they don't move as much." Gilbert recommends that all pregnant women begin doing kick counts daily starting at week 28. Choose the time of day when your baby is typically most active—usually it's in the evening—then time how long it takes to detect 10 movements, or kicks, while you rest comfortably.
"If a mother does kick counts every day, writes down the length of time it takes to feel 10 movements and brings her records to her doctor for review, it reduces her risk of stillbirth by 70 percent," Gilbert says. On average, it should take about 12 minutes to count 10 movements. "If it takes two hours or more, call your doctor," Gilbert advises. "Also call if there's a significant change in movement, especially toward the middle or end of your third trimester."


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Section 1. The "extreme" pro-life view. [1]
I agree with Thomson that the view that abortion is impermissible even to save the mother's life is an extreme pro-life view. I believe that abortions are justified if the mother's life is in immediate jeopardy. [2] She does wonder how we are supposed to weigh the mother's life against the unborn child's when the mother's life is at stake, but at that point her right to self-defense should be asserted.


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There is only a stardard version of 'Romance' available on the net,
but I have the high quality 1080p version for you!
Enjoy Caroline Ducey in the Catherine Breillat film 'Romance' (1999).
1920x1080 / 2:30 / 100 mb:
**** Dead Link ****
Check out my site for the explicit scenes from 'Romance' in 1080p !


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Romans 5:8 …'But God commendeth his love toward us, in that, while we were yet sinners, Christ died for us.'
The love of God is a great and deep subject that will fill the minds of the redeemed for eternal ages to come. Oh, the mystery of redemption! The love of God for a world that did not love Him! A love so complete that God sent His dearly beloved Son, His only begotten Son, into this world of sin, to make an infinite sacrifice, so that we sinners may have life through Him.
Dear reader. Have you really contemplated the love of God? This is a love that cannot be matched. The sacrifice made for us is of so great a value, and yet the majority of the world either reject it, or just take it for granted. Satan has so misrepresented the character of God, that many have a false view of God. But do you realize what was done for you?
John 3:16 …'For God so loved the world, that he gave his only begotten Son, that whosoever believeth in him should not perish, but have everlasting life.'
We are told in the Bible that God gave His only begotten Son. Now for God the Father to be able to 'give His Son', what did He need to have to send? A SON! That's right friend, God didn't just send His 'co-God', He sent His very own beloved Son.
Just imagine having to send your child to a place where you knew he or she would be rejected, mocked and beaten. And you had to stand back and watch all of this take place without intervening. Then you had to lay all the sins of those unworthy people upon your child, turn away from your child and allow him to be killed in a most cruel way. That is what God did for you! The creator of the universe could have just simply destroyed everyone, but because of His great love for His creation, He allowed His beloved Son to be afflicted for our sins and take the punishment that we deserve. And Christ agreed to make this sacrifice for us. What love!
1 John 3:1 …'Behold, what manner of love the Father hath bestowed upon us, that we should be called the sons of God.'
God could have simply wiped out anyone who was disobedient to His law and government. But God does not want a creation that serves Him through fear. He is a God of love, and wants His creation to serve Him in love. This is why God did not destroy Lucifer when he originally rebelled. Instead, God chose to let sin manifest itself, so that all the universe could see the nature and result of sin, and also see the love and character of God in His dealing with sin, by giving His own beloved Son as a ransom, to redeem mankind.
Acts 4:12 …'Neither is there salvation in any other: for there is none other name under heaven given among men, whereby we must be saved.'
"In order to fully realize the value of redemption, it is necessary to understand what it cost … There are many whose hearts are no more deeply stirred by the humiliation and death of Christ than by the death of the martyrs of Jesus. Many have suffered death by slow tortures, and some by crucifixion. In what does the death of God's dear Son differ from these? … bodily pain was only a small part of the agony of the beloved Son of God as he hung upon the cross. The sins of the world were upon him, and also the sense of his Father's wrath against the sinner, as he suffered the penalty of the law. It was these that crushed his divine soul. It was the hiding of his Father's face, a feeling that his own dear Father had forsaken him as he drank the cup which the sinner so richly merited, that brought despair to his soul. The separation that sin makes between God and man was fully realized and keenly felt by the innocent, suffering Man of Calvary. He was oppressed by the powers of darkness, and had not one ray of light to brighten the future. His mental agony on this account was so great that man can have but a faint conception of it … It was in this terrible hour of darkness, the face of his Father hidden, legions of evil angels enshrouding him, the sins of the world upon him, that from his pale lips were wrenched the words, 'My God, my God, why hast thou forsaken me?" (E.White, Bible Echo, Jan.1 1887)
Oh! If we could appreciate what has been done for us! If we could love God as He loves us, our lives would be forever changed!
Dear reader! Have you contemplated God's love for you? He has not forsaken you, but has given His only begotten Son to pay for your sins. But Christ did not stay in the grave. No, His victorious life and sacrifice brought about His resurrection from the grave. And Christ now intercedes on your behalf with God the Father in heaven. Even though you may have been rejecting Christ all your life, He is still sending rays of hope, through His divine Spirit into your life, so that you may turn to Him and be saved.
Isaiah 49:15-16 …'Can a woman forget her sucking child, that she should not have compassion on the son of her womb? yea, they may forget, yet will I not forget thee. Behold, I have graven thee upon the palms of my hands; thy walls are continually before me.'
Romans 8:32-39 …'He that spared not his own Son, but delivered him up for us all, how shall he not with him also freely give us all things? … It is Christ that died, yea rather, that is risen again, who is even at the right hand of God, who also maketh intercession for us. Who shall separate us from the love of Christ? shall tribulation, or distress, or persecution, or famine, or nakedness, or peril, or sword? As it is written, For thy sake we are killed all the day long; we are accounted as sheep for the slaughter. Nay, in all these things we are more than conquerors through him that loved us. For I am persuaded, that neither death, nor life, nor angels, nor principalities, nor powers, nor things present, nor things to come, Nor height, nor depth, nor any other creature, shall be able to separate us from the love of God, which is in Christ Jesus our Lord.'
Dear reader. A great sacrifice has been made for you and all mankind. God has shown us His love through Jesus Christ His Son. And there awaits an eternal life of indescribable joy for all those who accept this great gift of love – a gift that is FREE! Will you accept God's love today? Will you surrender your life to the only one who can save you? Please don't delay. Give your life to Jesus Christ today!
1 Corinthians 2:9 …'But as it is written, Eye hath not seen, nor ear heard, neither have entered into the heart of man, the things which God hath prepared for them that love him.'
"By transgression man had separated himself from Him who alone is light and love. The sinner was 'alienated from the life of God,' 'dead in trespasses and sins.' The only hope for the fallen race was found in their becoming reconciled to God. Satan had so misrepresented God that man had no true conception of the divine character. But in carrying out the plan of salvation, Christ revealed that 'GOD IS LOVE'. (E.White, Bible Echo, Aug.1, 1892)


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Today is the final entry in my Bible journal online. I have shared this with you this week just to give you a feel of what you might do to make your Bible reading more meaningful. Really thinking about biblical truth and prayerfully walking in light of that truth is an essential aspect of spiritual formation. If we are to be disciples of Jesus, taking in biblical truth and being transformed by it will be central to the task. Here is our last reading from Psalm 127 . . .
127 Unless the Lord builds the house,
those who build it labor in vain.
Unless the Lord watches over the city,
the watchman stays awake in vain.
2 It is in vain that you rise up early
and go late to rest,
eating the bread of anxious toil;
for he gives to his beloved sleep.
3 Behold, children are a heritage from the Lord,
the fruit of the womb a reward.
4 Like arrows in the hand of a warrior
are the children of one's youth.
5 Blessed is the man
who fills his quiver with them!
He shall not be put to shame
when he speaks with his enemies in the gate.
Journal: The imagery of verse one is powerful. There is real laboring and real watching on the part of people, but unless God is in an under all of it, empowering it, it is all in vain. How often do I fail to consider God's work? How often to I fail to go to Him asking for strength to be faithful in the work? If anyone lacks wisdom, let him ask God (James).
Vanity in vv. 1&2 brings me back to Ecclesiastes (meaningless, meaningless).
It is not wrong to rise early of stay up late, Jesus did both. But it is wrong to do these things in anxious toil. When your work, your earning, becomes anxious toil, it is a bad sign for your soul. I have been having a hard time with this very thing lately because of the state of our church in a time of much suffering. In addition, several fine families have moved away recently. That is hard. I wonder about the future of the church. And lo and behold I have been experiencing something in the last few months I have never experienced . . . poor sleep. I have been waking up in the middle of the night. This verse hits me . . . "He gives his beloved sleep." Not to say if a person has sleep problems they don't trust God but for me it is important.
God is sovereign over my house, my work and my children. Good biblical picture of children as a gift. I am hesitant to endorse the idea of the "quiverfull" movement, that we should have as many children as we can. I can see a biblical argument against birth control but put it in the category of disputable matters from Romans 14. But the idea that I am not doing right if I don't have as many children as possible is something I can't see in Scripture. "Be fruitful and multiply, fill the earth" is a command to a healthy population rate but I do not believe it must be carried to an extreme by every family. On the other hand, many times in our culture we do not see the value of children or put economic stability and mobility over the value of children. There is a place for responsibility (be hesitant to have more children than you can care for) but there is also a time when we need to take a careful look at our priorities (are we chasing money and forgetting people?). These are issues Christian couples need to think through carefully and I believe they are also ideas with which we must be gracious with others.
Lord, you told me to come to you, learn of you and find rest for my soul. I am anxious about so many things but one thing is needful. Set me on the path of rest in my labor and building and in my family. In Jesus' Name, Amen.


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Tuesday, February 3, 2009
Santogold - Say A-Ha - Remixes (2008)
I really am easily pleased, I mean, transforming lyrics (on and off, on and off for those of you not down with the lingo) has been around for years but I'm still a sucker for them and the Tepr remix here just does it so well. I like Santogold, she's cool and there's no doubt she's on the edge of being massive. Lap it up folks before it appears on 'Now That's What I Call Music'.
1. Say A-Ha (Original)
2. Say A-Ha (Tepr Remix)
3. Say A-Ha (Phones Cosmic Mix)
4. Say A-Ha (Tom Neville Remix)
Download @ 320kbps
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Duquesne Sophomore Receives Honorable Mention for Goldwater Scholarship
Sara Katrancha, a sophomore biochemistry major at Duquesne University, received an honorable mention for the Goldwater Scholarship for 2011.
The impressive scholarship is given annually to sophomores and juniors in engineering, computer science, mathematics and science to foster and encourage outstanding students to pursue studies in these fields.
Katrancha is the first Duquesne student to achieve this designation, according to Dr. Lewis Irwin, coordinator of the Office for National Fellowships.
"It's a nationally prestigious award," Irwin said. "The Goldwater Scholarship is aimed at increasing math, science and engineering proficiency within American society. Its goal is to promote the study of those disciplines, so they're looking at the top students in their respective fields. The fact that Sara has been named as an honorable mention indicates that she has a very bright future within her field of biochemistry."
Besides holding an excellent quality point average and an impressive list of honors and awards, Katrancha is an Honors College student from Dunlo, Cambria County, who was valedictorian in her class at Forest Hills High School and "has a very detailed and very robust research agenda," Irwin said.
At Duquesne, Katrancha has been involved in research with Dr. Rita Mihailescu in the Bayer School, analyzing the RNA binding properties of peptides that play a role in Fragile X syndrome, the most prevalent genetic cause of mental retardation.
"I am not surprised that Sara received the honorable mention for the Goldwater Scholarship for 2011," Mihailescu said. "She has a very inquisitive mind, her genuine interest in her research comes across from the type of questions she asks, and more importantly, tries to answer herself by reading more on the particular subject.
Katrancha, who is studying abroad in Italy this semester, said in her application that her motivation to be involved in biomedical research stems from her brother having a genetic disability.
Duquesne University
Founded in 1878, Duquesne is consistently ranked among the nation's top Catholic universities for its award-winning faculty and tradition of academic excellence. Duquesne, a campus of nearly 9,500 graduate and undergraduate students, has been nationally recognized for its academic programs, community service and commitment to sustainability. Follow Duquesne University on Facebook, Twitter and Instagram.


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Wallace State softball captures second NJCAA Division I national championship
ST GEORGE, UTAH – Wallace State softball has won its second national championship.
The Lady Lions, seeded eighth, defeated Salt Lake City (Utah) CC 7-3 in Saturday's NJCAA Division I national title game, using another big offensive inning in order to capture it all at the Canyons Complex in St. George Utah.
Tournament MVP and Most Outstanding Pitcher Lindsey DeFee scattered two earned runs on five hits in seven innings, freshman Kori Benson added her school-record 23rd home run and the Lady Lions put together a five-run seventh inning to pull away and earn the college its sixth national title overall.
"I can't say enough positive things about this team. It seems like for the last month we've had a lot of clutch hits from people up and down the lineup. I can't even begin to name them all, but that's helped us achieve this ultimate goal, along with our two sophomore pitchers," said Wallace State coach Jayne Clem, whose team won its first national crown in 2008. "Nobody thinks Alabama can play softball in junior college, so we never get much recognition. We still may not get any after this, but we'll continue to prove it on the field."
A year after finishing third nationally, Wallace State's only hiccup in the tournament was an 8-0 loss in Saturday's opener, forcing the Lady Lions to win the title in the "if" game.
The Lady Lions (55-12) were up to the task. Their forte throughout the entire tournament was giving the pitching staff a comfortable lead by exploding for big innings. With the game tied at 2-2, Wallace State decided the seventh was the magic inning in the finale.
For the third time in the tournament Wallace State batted around, sending 10 to the plate. Victoria Frith drove in the first run with an RBI single, and Katie Gentle and Kori Benson each added two-run singles for a 7-2 advantage.
After allowing a one-out solo homer and single in the bottom of the seventh, DeFee induced a 4-6-3 double play to start the celebration.
"Words can't describe what this means to this team. It's the best feeling I've had in my life," said DeFee, a Eufaula native. "We proved ourselves the entire tournament. I knew our hitters were going to come through and take the pressure off the pitching staff."
DeFee was pressed into duty earlier than she expected during Saturday's opener, relieving BreeAnna Blevins, and readjusted to Salt Lake hitters in the championship game. DeFee (26-5) earned three victories in the tournament, including two against second-seeded Salt Lake.
"It took a while for me to get ready mentally, and after we lost that first one I told myself we weren't going to lose the second one," DeFee said. "I knew my teammates weren't going to let it happen either."
Wallace State reeled off a perfect march at the Alabama Community College Conference (ACCC) tournament earlier this month to punch its national tournament bid and was nearly as dominant at nationals. The Lady Lions felt they were slighted by earning a No. 8 seed in Utah and they made each opponent pay.
Along their route to the title, Wallace State defeated top-seeded Butler (Kan.) CC, No. 2 Salt Lake and fifth-seeded Pima (Ariz.) CC.
"We play as a team—that's all it is. From the first game at the state tournament to the last game here, we played as a team. Butler (CC) had tons of girls already signed with Division I programs, but that didn't matter or intimidate us," said Wallace State sophomore catcher Paige Rowland. "We knew what our mission was and we were going to get it. It also helped out we came so close last year (when we finished third.) We knew what to expect and some teams here didn't."
Benson, Wallace State's third baseman, was named the Most Outstanding Defensive Player of the tournament and she wasn't a slouch with her bat either. She hit three homers in the tournament, including a two-run shot in the championship game. She finished 2 for 4 with four RBIs.
"When I committed to play at Wallace State in high school, I knew I was going to be a part of a national championship team," said Benson, an Oak Grove grad. "We came closer as a team after we lost some games we shouldn't have in April. That's part of growing up, and we've grown up a lot in the last month."
Also named to the All-tournament team were Wallace State's Gentle, Madeline Lee and Brittany Gillison.
Clem was named the winner of the Karen Sykes Outstanding Coach of the tournament, earning the recognition for the second time in her eight national tournament appearances.
For more information about Wallace State athletics, visit wallacestate.edu.
Russell Moore
Staff Writer
Wallace State Community College
P.O. Box 2000, Hanceville, AL 35077
1-866-350-9722 Direct line: 256-352-8443
256-352-8314 fax
Visit us online at www.wallacestate.edu


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Welcome to CPtherapy.
CPtherapy is a highly specialised, private occupational therapy service for young children with cerebral palsy located in Melbourne, Australia. We specifically focus on providing early intervention services for children with unilateral cerebral palsy (hemiplegia), aged 0 to 5 years. Our two occupational therapists, Brian Hoare BOT; PhD and Susan Greaves B.App.Sc (OT); PhD have over 40 years combined clinical experience in the treatment of children with cerebral palsy.
CPtherapy provides therapy services that are based on high-quality research evidence. Effective therapy for children with cerebral palsy is hard work and there is no "one size fits all" approach. CPtherapy adopts a range of treatment approaches including modified constraint-induced movement therapy, bimanual upper limb therapy, goal-directed training and upper limb strength training. Outcomes of all our programs are evaluated using objective measurement tools, designed specifically for children with cerebral palsy. Under the direction of your treating therapist it is a requirement that all children and families participate in a parent/caregiver led home program during active treatment programs.
If you are interested in accessing our services, please complete the contact form and we will endeavour to get back to you as soon as possible. Please remember, CPtherapy is highly specialised and services are restricted to young children with a diagnosis of unilateral cerebral palsy (hemiplegia).


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Structural maintenance of chromosomes (SMC) proteins play central roles in regulating higher order chromosome dynamics from bacteria to humans. As judged by electron microscopy, the SMC homodimer from Bacillus subtilis (BsSMC) is composed of two antiparallel, coiled‐coil arms with a flexible hinge. Site‐directed cross‐linking experiments show here that dimerization of BsSMC is mediated by a hinge–hinge interaction between self‐folded monomers. This architecture is conserved in the eukaryotic SMC2–SMC4 heterodimer. Analysis of different deletion mutants of BsSMC unexpectedly reveals that the major DNA‐binding activity does not reside in the catalytic ATPase domains located at the ends of a dimer. Instead, point mutations in the hinge domain that disturb dimerization of BsSMC drastically reduce its ability to interact with DNA. Proper hinge function is essential for BsSMC to recognize distinct DNA topology, and mutant proteins with altered hinge angles cross‐link double‐stranded DNA in a nucleotide‐dependent manner. We propose that the hinge domain of SMC proteins is not a simple dimerization site, but rather it acts as an essential determinant of dynamic SMC–DNA interactions.
Structural maintenance of chromosomes (SMC) proteins are ubiquitous among three phyla of life and play fundamental roles in ensuring the faithful segregation of chromosomes during cell division (reviewed by Koshland and Strunnikov, 1996; Cobbe and Heck, 2000; Hirano, 2002). In eukaryotes, at least six members of the SMC protein family are found in individual organisms. SMC1 and SMC3 form a heterodimer that functions as the core of the cohesin complex involved in sister chromatid cohesion (Losada et al., 1998; Toth et al., 1999; Tomonaga et al., 2000), whereas SMC2 and SMC4 act as components of the condensin complex responsible for chromosome condensation (Hirano et al., 1997; Sutani et al., 1999; Freeman et al., 2000; Schmiesing et al., 2000). The third complex containing SMC5 and SMC6 is implicated in DNA repair and checkpoint responses, but its exact functions remain elusive (Fousteri and Lehmann, 2000; Taylor et al., 2001). Each of the three complexes has a unique set of non‐SMC subunits, which confer additional structural and functional diversity on the eukaryotic SMC complexes. Most, if not all, of the bacterial and archaeal genomes contain a single smc gene, and its gene product functions as a homodimer. Disruption of the smc gene in Bacillus subtilis causes decondensation and mis‐segregation of chromosomes, indicating that bacterial SMC proteins share related functions with their eukaryotic counterparts in vivo (Britton et al., 1998; Graumann et al., 1998; Moriya et al., 1998).
SMC proteins are large polypeptides (between 1000 and 1500 amino acids) with a unique structural organization. Two nucleotide‐binding motifs, the Walker A and Walker B motifs, are located in the conserved N‐ and C‐terminal domains, respectively. The central domain is composed of a moderately conserved 'hinge' sequence that is flanked by two long coiled‐coil motifs. An electron microscopy (EM) study showed that the B.subtilis SMC (BsSMC) homodimer has a two‐armed structure with a flexible hinge, and each arm is composed of an antiparallel coiled‐coil (Melby et al., 1998). This antiparallel configuration allows association of the N‐ and C‐terminal sequences to assemble an ATP‐binding 'catalytic' domain at the distal end of each arm. This domain is structurally related to the nucleotide‐binding domain (NBD) of the ATP‐binding cassette (ABC) transporter family of proteins (Lowe et al., 2001). What is not fully established is how the two subunits are folded in the BsSMC dimer. The original model by Melby et al. (1998) proposed that dimerization is mediated by coiled‐coil interactions between the two different subunits. However, point mutations in the hinge domain disturb dimerization, raising the alternative possibility that two self‐folded monomers may dimerize by a hinge–hinge interaction (Hirano et al., 2001).
In recent years, substantial progress has been made in our understanding of the biochemical activities associated with SMC proteins. Perhaps the best characterized example is the eukaryotic condensin complex, which has been shown to possess an ability to introduce positive supercoils or positive knots into DNA in an ATP‐dependent manner (Kimura and Hirano, 1997; Kimura et al., 1999, 2001; Hagstrom et al., 2002). The presence of the non‐SMC subunits unique to condensin, as well as their cell cycle‐dependent phosphorylation, are vital to these activities, suggesting that these are not universal activities shared by all SMC proteins (Sutani and Yanagida, 1997; Kimura et al., 1998; Kimura and Hirano, 2000). Consistent with this notion, the cohesin complex displays remarkably different DNA‐binding properties from those of condensin (Losada and Hirano, 2001). The distinct biochemical functions of condensin and cohesin could be attributed, at least in part, to their different arm conformations and hinge angles (Anderson et al., 2002). On the other hand, the BsSMC homodimer binds preferentially to single‐stranded DNA (ssDNA) and makes large protein–DNA aggregates in an ATP‐dependent manner (Hirano and Hirano, 1998). It has been proposed that closing or opening of the central hinge allows association of two catalytic domains within a dimer or between different dimers, respectively, thereby modulating the ATPase cycle of BsSMC (Hirano et al., 2001).
Despite the increasing amount of information, an integrated molecular picture of how SMC proteins might work at a mechanistic level is still missing. In particular, very little is known about how the two‐armed SMC proteins interact with DNA. In this study, we have used BsSMC as a model system to understand the common theme of SMC action. Site‐directed, protein–protein cross‐linking experiments show that a BsSMC dimer is formed by a hinge‐mediated interaction of self‐folded monomers. By characterizing a number of mutants with altered dimerization properties, we show that the two‐armed conformation of BsSMC, and not its catalytic domains, is essential for proper binding to DNA. Our results emphasize the importance of the hinge domain, which not only acts as the dimerization site but also plays an active role in determining the mode of SMC–DNA interactions.
Construction of BsSMC mutants
We had reported previously that mutations in the hinge domain synergistically perturb the global conformation of BsSMC and affect its ability to form a stable dimer (Hirano et al., 2001). In particular, when four conserved glycine residues (G657, G658, G662 and G663) were all mutated into alanines, the resulting mutant was found to form a single‐armed monomer in solution, as judged by both electron microscopy and hydrodynamic analyses. For simplicity, wild‐type BsSMC is referred to as GGGG (Figure 1A, left and B, lane 1) whereas the quadruple alanine mutant is called AAAA (Figure 1A, middle and B, lane 2). In the same study (Hirano et al., 2001), we also constructed a so‐called hinge‐less mutant, in which the N‐terminal (1–476) and C‐terminal (672–1186) fragments were co‐expressed to make a single‐armed BsSMC with no hinge sequence (Figure 1A, right). As shown below, we have now found that the hinge‐less mutant displays an extremely poor DNA‐binding activity, whereas the binding of AAAA to DNA is substantial. This observation was surprising because the only difference between the two mutant proteins was the presence or absence of the hinge domain. To understand better the DNA‐binding properties of SMC proteins, we constructed a series of BsSMC derivatives that contain different point mutations in the hinge domain or deletions in the non‐hinge regions. In the first set of mutants, some or all of the glycine residues conserved in the hinge domain were replaced by aspartic acids (D) or leucines (L) to test whether the acidic or hydrophobic residues might perturb hinge functions differently from alanines. The resulting mutants are referred to as GGDD, AADD, DDDD and LLLL (Figure 1B, lanes 3–6). As judged by sucrose gradient centrifugation (Figure 1C; data not shown), the four mutant proteins had a virtually identical sedimentation coefficient (4.2S), which was much smaller than that of GGGG (6.3S) but was indistinguishable from that of AAAA (4.1S). The results suggested that these hinge mutants are all single‐armed monomers in solution.
In the second set of mutants, the N‐ and C‐terminal regions were deleted systematically. The head‐less mutant (160–1037) lacked the catalytic domain (Figure 1B, lane 7), whereas the hinge–stalk mutant (262–861) further lacked the 'neck' region that connects the catalytic and coiled‐coil stalk domains (Figure 1B, lane 8). The sedimentation coefficients of the head‐less and hinge–stalk mutants were 5.5S and 4.4S, respectively (Figure 1C), values consistent with the prediction that they form dimers with elongated shapes. We also made variants of the hinge–stalk mutant, in which the conserved glycines in the hinge domain were replaced by aspartic acids (referred to as hinge–stalk DDDD; Figure 1B, lane 9) or leucines (hinge–stalk LLLL; Figure 1B, lane 10). The two mutants have a sedimentation coefficient of 3.0–3.1S (Figure 1C), again in agreement with the idea that the hinge mutations convert a dimer into monomers. Finally, the hinge domain with no coiled‐coil stalk (476–672) was expressed (Figure 1B, lane 11). The sedimentation coefficient of this hinge‐only mutant was 3.3S (Figure 1C), a value predicted from the formation of a globular dimer (note that elongated molecules display anomalously smaller sedimentation coefficients compared with globular molecules of the same molecular mass).
Architecture of BsSMC as revealed by site‐directed, protein–protein cross‐linking
The original model by Melby et al. (1998) proposed that dimerization of BsSMC is achieved by coiled‐coil interactions between the two different subunits (Figure 2A, model I). Our current and previous studies convincingly show that mutations introduced into the hinge domain of BsSMC (AAAA, DDDD and LLLL) disrupt its dimerization, resulting in the formation of self‐folded monomers (Hirano et al., 2001). These observations raise the possibility that wild‐type BsSMC may dimerize by a hinge–hinge interaction of two self‐folded monomers (Figure 2A, model II). To distinguish between the two models, we performed site‐directed, protein–protein cross‐linking experiments using bis‐maleimidohexane (BMH), a cross‐linker specific for free sulfhydryl groups. Wild‐type BsSMC has four cysteine residues (C119, C437, C826 and C1114) that are cross‐linkable with this reagent. We first replaced three or all of the cysteines by serines to make mutants that contain either a single cysteine at 1114 (C1114) or no cysteine. We further mutated these polypeptides by replacing one or two serines by cysteines, and constructed mutants that have two cysteine residues in single polypeptides. The locations of these cross‐linkable residues were selected on the basis of the crystal structure of an SMC catalytic domain from Thermotoga maritima (Lowe et al., 2001) (Figure 2B). The first mutant (S142C/C1114) was designed so that the N‐ and C‐terminal sequences within a single catalytic domain can be cross‐linked with each other. In the second mutant (S55C/S1070C), the two cysteines introduced into the N‐ and C‐terminal sequences are too far apart to be cross‐linked within a single catalytic domain. However, when the hinge is closed and two different catalytic domains interact with each other (Hopfner et al., 2000), the S55C residue in one domain would be close enough to be cross‐linked to the S1070C residue in the other. If BsSMC dimerizes by a coiled‐coil interaction between the two different subunits, cross‐linking of the first mutant (S142C/C1114) would yield a dimeric circular polypeptide (Figure 2C, GGGG, model I) whereas the second mutant (S55C/1070C) would form circular monomers (Figure 2D, GGGG, model I). If dimerization of BsSMC is mediated by a hinge–hinge interaction, then the cross‐linking patterns would be reversed (Figure 2C and D, GGGG, model II). To judge the electrophoretic mobilities of cross‐linked products without ambiguity, we introduced the corresponding cysteine mutations into the single‐armed DDDD mutant. In this case, cross‐linking between S142C and C1114 would occur readily within a monomer (Figure 2C, DDDD), whereas cross‐linking between S55C and S1070C would be greatly suppressed, because two catalytic domains are not in the same molecule and barely interact with each other (Figure 2D, DDDD).
The mutant proteins were purified and cross‐linked with BMH in the presence or absence of ATP or ATPγS. After quenching the cross‐linking reaction, protein samples were analyzed by SDS–PAGE followed by immunoblotting. When two cysteines were present at positions 142 and 1114 in the two‐armed GGGG form, a specific band appeared just above the linear monomer (Figure 2E, lanes 10–12). A similar product was obtained with the single‐armed DDDD form (Figure 2E, lanes 13–15), suggesting that this band corresponds to a circular monomer. In contrast, when cysteines were present at positions 55 and 1070, a subset of high molecular weight bands appeared (Figure 2F, lanes 10–12). These bands are likely to be the products in which two different subunits are cross‐linked together. Consistent with this interpretation, a similar set of products was observed in the single‐armed form, but the cross‐linking reaction was far less efficient (Figure 2F, lanes 13–15). The specificity of the cross‐linking reactions was confirmed by using mutants with none or a single cysteine residue (Figure 2E and F, lanes 1–9). The presence or absence of the nucleotides had little, if any, effect on the cross‐linking properties in any cases. Thus, the results from the two complementary sets of cross‐linkable mutants are consistent with model II (Figure 2A), in which two self‐folded monomers dimerize by a hinge‐mediated interaction.
DNA‐binding properties of BsSMC mutants
Having established the folding pattern of the BsSMC dimer, we used a gel shift assay to examine the role of the hinge domain in the DNA‐binding activity of BsSMC (Figure 3A). The wild‐type BsSMC protein (GGGG) displayed a strong affinity for ssDNA (Figure 3A, upper panel, lanes 2–5) and a weaker affinity for negatively supercoiled double‐stranded DNA (dsDNA; Figure 3A, lower panel, lanes 2–5). The binding to both substrates was independent of ATP, as described previously (Hirano and Hirano, 1998). The hinge‐less mutant failed to shift both forms of DNA under the same condition (Figure 3A, lanes 6–9). Interestingly, we found that point mutations in the hinge domain greatly affected the ability of BsSMC to bind to ssDNA. The affinity of the hinge mutants for ssDNA was reduced progressively in the order AAAA, GGDD, AADD and DDDD (Figure 3A, upper panel, lanes 10–25). Binding to dsDNA was even better in AAAA than in GGGG, but was extremely low or negligible in the other mutants (Figure 3A, lower panel, lanes 10–25). One possible interpretation of these data is that a specific DNA‐binding site resides in the hinge domain, and the introduction of acidic residues into this domain disturbs its DNA‐binding activity due to electrostatic hindrance. This apparently is not the case, however, because the LLLL mutant displayed a very weak binding activity comparable with that of DDDD (Figure 3A, lanes 26–29).
To determine how the dimeric form of BsSMC interacts with DNA, we tested the DNA‐binding properties of the deletion mutants described in Figure 1. Unexpectedly, we found that the head‐less and hinge–stalk mutants have DNA‐binding activities comparable with that of wild‐type BsSMC (Figure 3B, lanes 2–9). When dimerization of the hinge–stalk mutant was perturbed by combining with the DDDD mutations, however, the DNA‐binding activity was completely abolished (Figure 3B, lanes 10–13). A combination with LLLL also substantially reduced the binding activity (Figure 3B, lanes 14–17). Finally, the dimeric hinge‐only mutant failed to bind to DNA (Figure 3B, lanes 18–21). These data show that the catalytic domains may not play a major role in DNA binding of BsSMC. Instead, hinge‐mediated dimerization of coiled‐coil stalks appears to be essential. We suggest that, even though all of the hinge mutants are single‐armed monomers in solution (Figure 1C), the hinge of AAAA (and to a lesser extent that of GGDD and AADD) is partially active and is able to support dimerization only when DNA is present.
ATPase activities of hinge mutants
The ATPase activity of BsSMC has a very unique and complex character. It has DNA‐independent and DNA‐stimulated modes that respond differently to KCl or MgCl2 titration (Figure 4, panels 1–6, GGGG; Hirano and Hirano, 1998). Although the AAAA mutant largely loses its DNA‐independent ATPase activity (Figure 4, panels 1, 3 and 5, AAAA), it retains a normal level of ssDNA‐stimulated ATPase under a low salt condition (Figure 4, panel 4, AAAA; Hirano et al., 2001). This is consistent with the finding that AAAA can bind to ssDNA. Thus, stimulation of ATP hydrolysis by ssDNA offers a highly sensitive assay for BsSMC–DNA interactions. To understand better the role of the hinge domain in the action of BsSMC, we performed KCl and MgCl2 titration experiments with the new series of hinge mutants. In the absence of DNA, the titration curves of the hinge mutants were almost indistinguishable from one another, although they were strikingly different from those of wild‐type BsSMC (Figure 4, panels 1, 3 and 5). In the presence of ssDNA, however, the different hinge mutants displayed different titration curves. In a KCl titration experiment, the rate of ATP hydrolysis decreased in the order AAAA, GGDD, AADD, DDDD (Figure 4, panel 2). This is in excellent agreement with their decreasing affinities for ssDNA as judged by the gel shift assay (Figure 3A). Similarly, MgCl2 titration curves of the hinge mutants at 5 mM KCl corresponded well to their DNA‐binding activities (Figure 4, panel 4). At 50 mM KCl, virtually no ssDNA‐dependent stimulation was observed in GGDD, AADD and DDDD, although AAAA retained a substantial activity that was comparable with that of GGGG (Figure 4, panel 6). The behavior of the hinge‐less mutant was similar, if not identical, to that of DDDD (Figure 4, panels 1–6, hinge‐less). These results provide additional evidence that the hinge of the AAAA mutant is partially active in the presence of DNA. In contrast, replacement of the conserved glycine residues by aspartic acids disrupts the dimerization interface more completely, thereby impairing the DNA‐binding and DNA‐stimulated ATPase activities of BsSMC.
Hinge mutations alter the mode of DNA recognition by BsSMC
The finding that AAAA has an altered, but not completely defective, hinge function prompted us to test whether such mutations might affect the mode of DNA recognition by BsSMC. A gel shift assay using different forms of dsDNA revealed that the wild‐type BsSMC protein binds to negatively supercoiled DNA but not to positively supercoiled DNA (Figure 5A, lanes 2–7). The addition of ATP or ATPγS (a slowly hydrolyzable ATP analog) had little effect on the dsDNA‐binding properties of wild‐type BsSMC. In contrast, the hinge mutant AAAA bound equally well to negative and positive supercoils, and its binding to both substrates was slightly enhanced in the presence of ATPγS (Figure 5A, lanes 8–13). Interestingly, two intermediate mutants, AAGG and GGAA (Hirano et al., 2001), displayed an affinity for positive supercoils only in the presence of ATPγS (Figure 5A, lanes 14–25).
To test further the differential responses to nucleotides between wild‐type BsSMC and the hinge mutants, we performed a spin‐down assay. In this assay, BsSMC was first incubated with different forms of DNA in the presence or absence of nucleotides, and then the mixture was spun by low‐speed centrifugation (Hirano and Hirano, 1998). When incubated with ssDNA, wild‐type BsSMC formed precipitable complexes in the presence of ATP or ATPγS (Figure 5B, GGGG, lanes 4–6). AAAA supported a similar reaction in the presence of ATPγS (Figure 5B, AAAA, lanes 4–6). Remarkable differences were found between the two proteins when dsDNAs were used as binding substrates. Although wild‐type BsSMC formed very few precipitable complexes with dsDNA (Figure 5B, GGGG, lanes 7–12), AAAA readily assembled aggregates with both negative and positive supercoils in the presence of ATPγS (Figure 5B, AAAA, lanes 9 and 12). The AAGG and GGAA mutants exhibited intermediate phenotypes (Figure 5B, AAGG and GGAA), as expected. These results show that the hinge mutations synergistically alter the DNA‐binding property of BsSMC. We suggest that two factors, an open hinge structure and binding of ATPγS to the catalytic domains, stabilize a protein–protein interaction that promotes cross‐linking of dsDNA (see Discussion).
Folding of eukaryotic SMC2–SMC4 heterodimers
In eukaryotic organisms, CAP‐C/SMC4 and CAP‐E/SMC2 form an SMC heterodimer that acts as the core of the condensin complex (Hirano et al., 1997; Schmiesing et al., 2000). We wished to test whether the conclusion regarding the architecture of BsSMC is also applicable to that of the eukaryotic dimer. Taking advantage of the heterodimeric nature of the eukaryotic SMC proteins, we set up a coiled‐coil interaction assay. If dimerization is mediated by coiled‐coil interactions between the two different subunits, the N‐terminal half of the CAP‐C would associate with the C‐terminal half of CAP‐E to form an intermolecular antiparallel coiled‐coil (Figure 6A, model I). Likewise, the C‐terminal half of CAP‐C would interact with the N‐terminal half of CAP‐E. Alternatively, both CAP‐C and CAP‐E may self‐fold by an intramolecular coiled‐coil interaction (Figure 6A, model II). In this case, the N‐terminal half of CAP‐C (or CAP‐E) would associate with its own C‐terminal half. To distinguish between the two models, C‐terminal fragments of human CAP‐C (hCAP‐C) of different lengths were co‐translated in vitro with an N‐terminal fragment of either hCAP‐C or hCAP‐E (Figure 6B, left). A specific antibody was then used to immunoprecipitate the C‐terminal fragments of hCAP‐C from the reaction mixtures. We found that the N‐terminal fragment of hCAP‐C, but not of hCAP‐E, co‐precipitated with a subset of the C‐terminal fragments of hCAP‐C (Figure 6C, left panels). We also set up a reciprocal experiment (Figure 6B, right) and obtained consistent results: a subset of the C‐terminal fragments of hCAP‐E associated with the N‐terminal fragment of hCAP‐E, but not with that of hCAP‐C (Figure 6C, right panels). These results show that, like BsSMC, the eukaryotic SMC2–SMC4 heterodimer is composed of two antiparallel coiled‐coils that are folded intramolecularly. No hinge sequences were included in the current assays, suggesting that the coiled‐coil sequences contain information that determines their binding specificity.
Architecture of SMC and related proteins
The current study is aimed at understanding the structure–function relationship of SMC proteins. First of all, we wanted to determine unambiguously how SMC subunits fold to make a dimer. On the basis of their primary sequence, Saitoh et al. (1994) predicted that SMC proteins have the potential to make antiparallel coiled‐coils by either intermolecular or intramolecular interactions. A subsequent EM study by Melby et al. (1998) demonstrated that BsSMC has a two‐armed structure in which two antiparallel coiled‐coils are connected by a flexible hinge. Although the original images were interpreted so that dimerization is mediated by an intermolecular coiled‐coiled interaction, our recent mutational analysis of the hinge domain argued in favor of an alternative model in which self‐folded subunits dimerize by a hinge–hinge interaction (Hirano et al., 2001). Site‐directed, protein–protein cross‐linking experiments presented in the current study provide compelling evidence that the second model is correct. We have also extended this conclusion to the eukaryotic heterodimer of CAP‐C/SMC4 and CAP‐E/SMC2 using a coiled‐coil interaction assay. Our results are in good agreement with those of Haering et al. (2002), who have very recently reported the architecture of the yeast cohesin complex (containing SMC1 and SMC3) based on a pull‐down assay using epitope‐tagged recombinant fragments. Thus, hinge‐mediated dimerization of self‐folded coiled‐coils is applicable to all SMC proteins, from bacterial SMC homodimers to eukaryotic SMC1–SMC3 and SMC2–SMC4 heterodimers.
Rad50 is an SMC‐like protein that is implicated in double‐strand DNA break repair (reviewed by Connelly and Leach, 2002; D'Amours and Jackson, 2002). A study using scanning force EM reported that, like SMC, the Rad50 polypeptide folds intramolecularly to form an antiparallel coiled‐coil (de Jager et al., 2001). Very recently, the hinge domain of Rad50 has been shown to form a zinc‐binding hook that can flexibly support either dimerization or tetramerization of the Mre11–Rad50 complex and thereby bridge two broken DNA ends (Hopfner et al., 2002). The hinge domain of SMC has no zinc‐binding motif, and is much bigger (∼150 amino acids) than that of Rad50 (∼30 amino acids). Thus, the hinge domains of SMC and Rad50 appear to be different both structurally and functionally.
The two‐armed structure of SMC is essential for its DNA binding
A previous study using small fragments of yeast SMC proteins fused with GST reported that the major DNA‐binding site of SMC proteins may reside in the C‐terminal globular domain (Akhmedov et al., 1998). A subsequent and similar approach showed that some internal coiled‐coil fragments could also bind to DNA (Akhmedov et al., 1999). Given our current knowledge of the SMC architecture, however, it seems that the constructs used in those early studies were too artificial and inappropriate to test the DNA‐binding properties of SMC proteins. The current study therefore represents the first systematic analysis of DNA‐binding properties of SMC proteins using physiologically relevant constructs. Our results show that neither the catalytic domain nor the hinge domain alone is sufficient to bind to DNA, as judged by gel shift assays. Instead, two coiled‐coil stalks constitute the major DNA‐binding surface only when they are linked together by a flexible hinge. Deletion of the hinge domain, which creates single‐armed monomers, results in complete loss of the ability of BsSMC to interact with DNA. Another remarkable observation is that point mutations in the hinge domain differentially and synergistically affect the DNA‐binding properties of BsSMC. The crystal structure of an SMC hinge domain from the bacterium T.maritima (Haering et al., 2002) has demonstrated recently that the conserved glycine residues mutated in our previous and current studies are clustered at the dimerization interface. Replacement of these glycines by alanines perturbs the interface, thereby altering the global conformation of BsSMC and its ability to interact with DNA. Replacement by aspartic acids has a more drastic effect as it results in the formation of single‐armed monomers whose functional properties are comparable with those of the hinge‐less mutant. Taking all these results together, we suggest that there may be no local high affinity site for DNA in BsSMC molecules. It is most likely that the two arms connected by a flexible hinge act like a 'hook' to trap DNA strands. The catalytic domains at the ends of a dimer would certainly play an important regulatory role in the subsequent manipulation of DNA, but may not be essential for the direct interaction with DNA. This DNA‐binding property of SMC is clearly different from that of Rad50, which requires ATP‐dependent dimerization of the two catalytic domains (Hopfner et al. 2000).
Does the hinge angle modulate the DNA‐binding mode of SMC proteins?
Our results suggest that the hinge of BsSMC is not a simple dimerization site but has an important regulatory function in its DNA‐binding activity. Comparison between the wild‐type protein (GGGG) and the partially active hinge mutants (GGAA, AAGG and AAAA) is particularly informative. Previous hydrodynamic analysis showed that these mutations synergistically decrease the sedimentation coefficient of BsSMC, being indicative of arm opening (Hirano et al., 2001). Having established the folding problem in the current study, we can now speculate that these mutations make the hinge angle wider, promoting increasingly open conformations (and eventually loosening the hinge‐mediated dimerization). Conceivably, a closed conformation of the wild‐type hinge or its structural flexibility contributes to the specific recognition of negative supercoils of DNA, although the underlying mechanism remains elusive. Such flexibility would allow BsSMC to take a variety of conformations and support dynamic and plastic interactions with DNA (Figure 7, left). On the other hand, the constitutively open conformation of AAAA leads to deregulated DNA binding, and places the two catalytic domains far apart from each other. This fixed conformation would then allow the catalytic domains of one dimer to interact stably with those of another dimer in the presence of ATPγS, resulting in robust cross‐linking of dsDNA (Figure 7, right). Thus, we propose that the hinge angle per se (or its flexibility) may have the potential to modulate the DNA‐binding mode of BsSMC.
Does this idea have any implications for the action of the eukaryotic SMC proteins? A recent EM study has revealed that condensin and cohesin display different arm conformations with characteristic hinge angles (Anderson et al., 2002). The hinge of condensin is largely closed whereas the hinge of cohesin is wide open with an average angle of ∼90°. The current mutational analysis of BsSMC supports the hypothesis that the different hinge angles of condensin and cohesin may be one of the key factors that determines their drastically different DNA‐binding properties (Losada and Hirano, 2001). If this is indeed the case, the AAAA mutant would be expected to display 'cohesin‐like' rather than 'condensin‐like' functions. The ATPγS‐dependent dsDNA cross‐linking activity supported by AAAA could reflect one such activity. Clearly, future work is required to understand exactly how two‐armed structures with specific hinge angles might be able to recognize different forms of DNA.
Toward a unified understanding of SMC action
Although the current study provides important insights into the role of the hinge domain in SMC functions, it remains to be established how this class of two‐armed ATPases works at a mechanistic level. A variety of different approaches are now beginning to catch a glimpse of their modes of action. By electron spectroscopic imaging, Bazett‐Jones et al. (2002) have demonstrated that a single condensin complex is able to trap two positive supercoils of DNA in an ATP hydrolysis‐dependent manner. Yoshimura et al. (2002) have used atomic force microscopy to show that condensin has an ability to form DNA loops. On the other hand, Haering et al. (2002) have proposed, on the basis of biochemical data, that cohesin might hold two sister chromatids together by 'embracing' duplicated DNA strands within its arms. Whatever the mode of SMC–DNA interactions might be, it is most likely that the two catalytic ATPase domains modulate the opening and closing of the two coiled‐coil arms (Hirano et al., 2001). In the case of condensin, there is evidence that the non‐SMC subunits that bind to the catalytic domains directly modulate the ATP binding and hydrolysis cycle of the SMC heterodimer (Kimura and Hirano, 2000; Anderson et al., 2002; Yoshimura et al., 2002). Until very recently, BsSMC has been thought to function as a simple homodimer in B.subtilis. A recent genetic study, however, suggests that two non‐SMC components (termed ScpA and ScpB) may work together with the SMC protein in vivo (Mascarenhas et al., 2002). It is of great interest to determine whether these proteins interact with the BsSMC dimer in vitro and modulate its ATPase and ATP‐dependent activities. Such efforts will further clarify the relationship between the eukaryotic and prokaryotic SMC protein machines, and will enhance our understanding of the action of this class of chromosomal ATPases that undoubtedly holds the secrets of higher order chromosome dynamics.
Materials and methods
Construction of BsSMC mutants
A plasmid (pSO133) that expresses wild‐type BsSMC with a His6 tag at its C‐terminal end was described previously (Hirano et al., 2001). Point mutations were introduced into this construct by a using QuikChange site‐directed mutagenesis kit (Stratagene) and confirmed by sequencing.
Deletion mutants were constructed as follows. For the head‐less mutant, a DNA sequence encoding amino acids 160–1037 was amplified by PCR and inserted into the BamHI site of pET23b(+) (Novagen). For the hinge–stalk and hinge‐only mutants, DNA sequences encoding amino acids 262–861 and 476–672, respectively, were amplified and inserted into the NdeI–BamHI site of the same vector. All deletion constructs have a His6 tag at their C‐termini.
Purification of BsSMC mutants
BsSMC mutant proteins were expressed in the Escherichia coli strain BL21(DE3)pLysS and purified as described previously (Hirano et al., 2001), with the following modifications. Typically, 10 ml of lysate were obtained from a 250 ml culture. After a spin, the supernatant was loaded onto a 2 ml Ni‐NTA metal affinity column (Qiagen; column size, 0.8 × 4 cm). The column was washed, and bound proteins were eluted with 8 ml of buffer containing 50 mM Na‐phosphate pH 7.5, 300 mM NaCl, 10% glycerol, 5 mM 2‐mercaptoethanol and 500 mM imidazole. The peak fractions were pooled (∼4 ml) and dialyzed against buffer M [20 mM K‐HEPES pH 7.7, 1 mM EDTA, 10% glycerol and 0.1 mM phenylmethylsulfonyl fluoride (PMSF)] containing 50 mM KCl and 5 mM 2‐mercaptoethanol. The dialysate was applied to a 1 ml HiTrapQ column (Amersham Pharmacia Biotech), and fractionated with a 5 ml KCl gradient (100–600 mM) in buffer M containing 5 mM 2‐mercaptoethanol. The peak fractions were pooled (∼0.5 ml), dialyzed against buffer M containing 50 mM KCl and 1 mM 2‐mercaptoethanol, aliquoted and stored at −70°C. The typical yield of BsSMC from a 250 ml culture was ∼0.4 mg. Purification of the hinge‐only mutant was performed in the same way except that HiTrapQ was replaced with a 1.5 ml SP‐Sepharose FF column (Amersham Pharmacia Biotech; 1 × 1.9 cm). The concentrations of purified BsSMC were determined by SDS–PAGE followed by Coomassie Blue stain using bovine serum albumin (BSA) as a standard. To compare the activities of the two‐armed and single‐armed proteins directly, all protein concentrations are expressed in moles of BsSMC arms rather than moles of dimers or monomers.
Site‐directed, protein–protein cross‐linking
Protein–protein cross‐linking was performed as described previously (Hirano et al., 2001) except that BMH (Pierce) was used at a final concentration of 0.08 mM.
In vitro transcription/translation of hCAP‐C and hCAP‐E fragments
DNA fragments encoding different regions of hCAP‐C or hCAP‐E were amplified by PCR and inserted into pRSETA (Invitrogen). In vitro transcription/translation reactions were performed using TNT Quick Coupled Transcription/Translation Systems (Promega) according to the manufacturer's instructions. On the assumption that a coiled‐coil fragment can fold properly only when a folding partner is present, we expressed two coiled‐coil fragments simultaneously by adding two different plasmid constructs into a single reaction tube. Reaction mixtures (50 μl) contained two plasmid DNAs (10–20 μg/ml each) and [35S]methionine (1000 Ci/mmol) at a final concentration of 0.4 mCi/ml. After incubation at 30°C for 60 min, 250 μl of dilution buffer (20 mM HEPES pH 7.7, 100 mM KCl, 2.5 mM MgCl2, 0.1% Tween‐20 and 0.5 mg/ml BSA) was added and the mixtures were spun at 10∼000 g at 4°C for 15 min. The supernatants were used for immunoprecipitation with anti‐hCAP‐C or anti‐hCAP‐E. Affinity‐purified rabbit antibodies that recognize the C‐terminal peptide sequences of hCAP‐C and hCAP‐E were described previously (Kimura et al., 2001). The precipitated polypeptides were resolved by SDS–PAGE and analyzed by autoradiography.
Other assays
Gel shift and ATPase assays were performed as described previously (Hirano and Hirano, 1998; Hirano et al., 2001). Positively supercoiled DNA was prepared by using a recombinant form of the archaeal histone HMfB as described previously (Starich et al., 1996; LaMarr et al., 1997). Sucrose gradient centrifugation was carried out as described previously (Hirano et al., 2001) with minor modifications. A 5 ml sucrose gradient (5–20%) was made in buffer containing 20 mM Tris–HCl pH 7.5, 5 mM KCl and 2 mM MgCl2, and spun at 189 000 g for 18 h in an SW50.1 rotor (Beckman).
We are grateful to Kyoko Yokomori for hCAP‐C and hCAP‐E cDNAs, and Kathleen Sandman for an rHMfB expression plasmid. We thank members of the Hirano lab for critically reading the manuscript. This work was supported by grants from the National Institutes of Health (to T.H.).
- Copyright © 2002 European Molecular Biology Organization


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A native of Arkansas, Brandy Donaldson came to the Quad Cities looking for something different. "I moved to the Quad-Cities almost 8 years ago with no expectation of making this my home," says Donaldson. "To my pleasant surprise, I found a vibrant area with much to offer. This community immediately embraced me and infused me with a spirit of growth and prosperity."
She continues, "It was September when I got here. Quickly after I arrived, it started snowing—which I was unfamiliar with. So that was a big shock to me."
Donaldson, an honoree for 2015 YP of the Year at the May 28 Community Impact Awards, credits the Quad Cities with helping her unlock her desire to serve. "You know the saying, 'It takes a village to raise a child,' Donaldson asks. "Well, it takes a village to raise a young professional. When I dove into the question, 'What can I do to help,' I really gained an appreciation for the Quad Cities." Donaldson continues. "It is a give and take. The Quad Cities gives me a lot and I take that and turn it into energy to give back in whatever way I feel I can add to it."
Her involvement with local organizations like the NAACP, Relay for Life, Big Brothers Big Sisters, the QC Empowerment Network, and YPQC have helped Donaldson develop a regional picture of the Quad Cities. She explains, "I was 25 when I moved here. [The Quad Cities] helped me come into the person I wanted to be: career-wise, socially, on a civic level. I developed all of that in the Quad Cities." Donaldson adds, "I don't target one city or one area to benefit. I think of what can I do for this region. Honestly I do think of the Quad Cities as one large city. When I immersed myself in this city that's what I developed. I don't think of it as Rock Island, Bettendorf, Davenport, Moline - I think of it as one large city."
Join Brandy Donaldson and our other honorees on May 28 for the Community Impact Awards at the Stern Center in downtown Rock Island.


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Hey guys! I know it's been a while, but I've been working on getting a surprise up and running! I recently decided that I was going to purchase my own domain (mostly so I could customize my page more and get more pictures on my blog, but yeah) so I have a new website! It's still Runs for Pancakes, but now it's runsforpancakes.com instead of a .wordpress link! I'm super excited to play with this new site and I'd love if you'd check it out!
Have a great weekend filled with lots of fun adventures!