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Montreal Impact 0-2 Seattle Sounders: Visitors prevail without Dempsey and Evans
Mar 23, 2014, 6:26 PM EDT
In the absence of some key players, Seattle's Lamar Neagle was the pivotal man as Seattle picked up its second win of the season while keeping Montreal winless in a 2-0 victory in Quebec.
Brad Evans and Clint Dempsey were unavailable for the Sounders, who fended off a shot-ready attack from Montreal. The Impact were credited with 25 shots in the match to Seattle's nine, though just six were on frame.
Troy Perkins made some big saves as usual, but it was his misfortune that got Seattle on the board. Gonzalo Pineda's free kick was headed off the frame by Neagle, but the ball hit Perkins and entered the goal to give Seattle a 1-0 lead in the 8th minute.
Perkins got his body on the second goal as well, this time a hand, as Obafemi Martins headed home a Kenny Cooper cutback cross to double Seattle's lead. And yes, he went flip city in celebration.
Frank Klopas is now staring at an 0-3 start to his time in Montreal. The only other team without a point this season is DC United, which has only played twice.
Seattle improves to 2-1-0, their lone loss coming against Toronto FC. Stefan Frei was strong between the sticks for the visitors, who also got several impressive moments from DeAndre Yedlin.
- talgrath - Mar 23, 2014 at 6:40 PM
There were a lot of nervy moments in the game for the Sounders, but overall I thought they looked very good even without Dempsey and Evans. That game very well could have been 4-0 if not for some excellent saves by Perkins.
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Missouri calls out its Guard; Holder to visit
Governor removes curfew
Posted: August 19, 2014 at 3:43 a.m.
FERGUSON, Mo. -- Gov. Jay Nixon lifted a curfew in this embattled city Monday, hours after deploying the Missouri National Guard, as officials struggle to control unrest that has paralyzed the community since an unarmed black teenager was killed by a white police officer.
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Most police officers don't fire their guns a single time over the course of their careers. But those rare moments when they do pull the trigger are fast, furious and frenetic.
And, as several New York policemen discovered early Saturday morning, sometimes things go tragically awry in the adrenaline-pumping, split-second heat of the moment.
When four officers fired 50 shots at bridegroom Sean Bell, a 23-year-old unarmed man leaving a bachelor party in the Jamaica neighborhood of Queens, the incident marked the latest in a rare but recurring problem for police departments around the country: When cops engage in "contagious shooting" against an unarmed suspect.
"Contagious shooting" refers to the phenomenon in which multiple officers will start shooting once one officer has opened fire -- even if no real threat is present. Experts say the reaction is a response to sudden fear.
"When a fellow officer starts shooting it sends off an alarm, warning the others that they must be in lethal danger," says Dr. Carole Lieberman, a Beverly Hills forensic psychiatrist and assistant clinical professor at UCLA's Neuropsychiatric Institute in Los Angeles, Calif. "Not only is there ostensibly a 'killer on the loose,' but they fear they can no longer count on their fellow officers to protect them."
Lieberman says the phenomenon is more common than many may realize. Thus, she says, it is important that officers be held accountable for their actions. "The officer who starts the shooting is responsible for where he aims and how many times he shoots at the alleged 'criminal,'" she says. "The other officers who become affected by the 'contagion' are responsible for panicking, becoming blinded by the outbursts of gunfire, and not restraining themselves enough to assess the situation."
Although the tragedy immediately reminded many of the infamous 1999 killing of Amadou Diallo, when NYPD officers fired 41 shots at the unarmed West African immigrant, that type of high-round shooting is actually quite rare in New York. The number of shots fired per officer in each of the 112 shooting incidents this year was 3.2. Last year, it was 3.7 shots fired per officer, down from a high of 5.0 in 1995.
"It's amazing that this doesn't happen more often when you think about the thousands of contacts that the police have with the public," says Edward Mamet, a law enforcement consultant and retired NYPD captain. "The New York police is very restrained. This type of contagious shooting happens out West -- it's a cowboy mentality. They like to shoot out there."
Indeed, high-round shooting has been a perennial problem in Los Angeles, Denver and other cities. Last June, six Los Angeles County sheriff's deputies fired more than 50 shots into the car in which drunken driving suspect Carl Williams was driving, after his car rammed a police vehicle following a chase. One deputy fired so many shots that he had to reload his weapon during the incident.
That shooting occurred a year after the Los Angeles County Sheriff's Department instituted several rules that restricted when deputies could fire at moving cars or fire multiple times.


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WebMD Health News
Joanna Broder
Reviewed by
Laura J. Martin, MD
March 16, 2010 -- When people think about skin cancer, often it's melanoma, the deadliest form, that comes to mind. But a new study suggests that nonmelanoma skin cancers, already the most common form of cancer in the United States, appear to be on the rise. From 1992 to 2006, cases of nonmelanoma skin cancer in the Medicare population rose an average of 4.2% a year, according to the study, which appears in this week's Archives of Dermatology. In 2006, there were an estimated 3.5 million nonmelanoma skin cancers overall in the U.S., and about 2.1 million people were treated for the disease. "These data give the most complete evaluation to date of the underrecognized epidemic of skin cancer in the United States," the authors say. The purpose of the study was to gauge the incidence of nonmelanoma skin cancer in the United States. Such an estimate is important because although nonmelanoma skin cancer has substantial associated costs and morbidity (deaths are fewer than for other cancers, but it is still significant), it is not reported in most cancer registries, the authors say. Hence, the actual incidence of nonmelanoma skin cancer is not known. "Understanding skin cancer incidence and treatment is important for planning prevention strategies and allocating resources for treatment," the authors say. To estimate the incidence and treatment of cases of nonmelanoma skin cancer in 2006 in the overall U.S. population, Howard Rogers, MD, PhD, a dermatologist in Norwich, Conn., and colleagues, evaluated Medicare databases and a national survey based on visits to doctor offices. "The data presented herein from national databases indicate that the incidence of skin cancer in the United States has substantially increased from 1992 to 2006 and is now about double the last published estimate from 1994," the authors write. The study has significant limitations in how it estimated the incidence of nonmelanoma skin cancer, including the assumption that one treatment procedure equates to one incident of nonmelanoma skin cancer. Still, "it provides a much stronger NMSC [nonmelanoma skin cancer] estimate than has previously been published," they write.
SOURCES:Howard W. Rogers, MD, PhD, Advanced Dermatology, Norwich, Conn.
Rogers, H. Archives of Dermatology, March 2010; vol 146: pp
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PAWTUCKET — Despite some ongoing protests from residents, city officials are moving forward with plans to privatize the city's waste transfer station on Grotto Avenue.
At a special meeting last week, the City Council voted unanimously to approve a five-year contract with Waste Haulers (WHM Holdings, LLC) to operate the Blackstone Valley Regional Transfer Station. Under the terms of the contract, the company will lease the city-owned facility for $10,000 a month and also make payments to the city based on tonnage.
The contract awaits signing by Mayor Donald Grebien, but it is expected to be finalized shortly, with Waste Haulers taking over operations by early next month. Grebien has been lobbying for the privatization, saying that it will mean an additional $750,000 to the city annually between the money from the lease and the savings of about $210,000 in operating costs being spent now.
Under the terms of the agreement with Waste Haulers, the company would be limited to accept 450 tons of waste per day, but could expand beyond that as long as it builds an access road off Concord Street. Once the access road is in place, the city could expand the license to allow for up to 2,000 tons of waste a day. However, any such expansion would require an additional public hearing and approval by the City Council.
About a dozen residents turned out to last Thursday's public hearing on the proposed plan, and the council voted later that night to authorize Grebien to go ahead with the contract. Thursday's meeting was a far cry from an informational session held on April 9 at the Nathanael Greene Elementary School, where about 30 neighbors showed up. Many expressed concerns about truck traffic, noise, odors and the effect on property values from the privatization and potential expansion, and some voiced outright opposition.
City Council President David Moran, who had attended the first meeting on the proposal, said there were far less people at the second meeting. He said he felt that the issues the neighbors had raised had primarily been addressed, including concerns about the devaluation of property in the immediate area. To that end, he said the council had promised to have a private company evaluate the property values should an expansion occur, and then voted to support this action at Wednesday's council meeting.
Moran said he liked the way the process was handled and was satisfied that anyone who wanted to speak on the issue had been heard. He pointed out that for any expansion to 2,000 tons per day to occur, the whole public hearing and approval process would have to start over. He said he supported the privatization plan primarily because of its financial benefit to the cash-strapped city, and also because it should help improve the unsightly industrial area as a whole. "That $750,000 a year will help with taxes and to reduce the deficit," Moran said.
Councilor James Chadwick, who represents District 6, said that while there are still some concerns on the part of residents, he thinks the privatization plan will greatly benefit the city as a whole. He said he felt "comfortable" with the contract as it has been presented.
Chadwick said that in response to concerns about property values, he and the mayor drafted a resolution requiring that an independent study be done of the effect that a cap of 2,000 tons of waste per day at the transfer station would have on the value of surrounding properties.
"We're working to stay on top of the issues. I fully expect we can keep the promises we made to residents," Chadwick stated.
Grebien said that the outsourcing of city transfer operations "will unlock the potential of a long-underutilized city asset and turn it into a significant revenue enhancer for the city while greatly improving operations there." He added that city resident permit holders will retain their disposal rights at the transfer station.
Grebien noted that the agreement with the vendor, which was chosen in an open competitive process, will ensure that the operation is not expanded beyond current capacity with proper safeguards and approvals, including state environmental and city reviews.
The mayor said that in two public meetings, he and his administrative team have "listened closely to the concerns of residents, including those who live nearby. We will continue to respond to those concerns." He added that truck traffic will not be allowed on any local roads that are not already used now, and that a proposed access road, if built, would further divert truck traffic from local roads.
The overall result of the privatization, Grebien said, will be "a cleaner, safer and much more modern facility and a significant financial benefit to the city."


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93 and 94 are AWESOME, 95 is a sweet free card.....96 may be a dip after like 5 badass cards in a row, it happens sometimes.
i'm excited to see Demian Maia beat Chael Sonnen's ass
Note to Willpunisher: You have lost signature privileges until November 1, 2010. You may remove this message, but may not add anything to your signature until November 1.
Note to Asshole Mods: Stop being assholes and teasing me. If you going to ban me because you guys are fucking dicks than just do it already. Fucking bitches.
this should be a fight night not a ppv event, lame card
With all the great MMA this month and the past 2 why is everybody bitching about a solid card without a marquee headliner that we get to watch for free? Counting the fight night thats 2 UFC cards that we get for free in the same month.
I guess you get demoted to the prelims when you dont bend over for Danas veiny cock & bow to his wishes about signing your lifetime image rights over to the UFC,FOREVER! Silly,silly Koscheck,how dare he want to retain the rights to his own image after hes done fighting for the UFC.
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Morris Sun Tribune
AnglingBuzz: The Walleye - Mayfly Connection
Morris Fire celebrates 125 years of service
Prairie Pioneer Days bigger and better
Area Student News: July 4, 2015
Bookends: July 2015
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Greg Schwartz Memorial Camp
Tigers take advantage of opportunities in 13-win season
JV Tiger baseball finishes season 7-6
Morris/C-A C squad
Carrington leads VFW to victory
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Gregory Arthur Durkee
Raymond Lembcke
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Greg Durkee
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Talking it over: A good kind of exhaustion
Sue's Views: Making an effort makes it worth it to shop local
Sen. Al Franken: Making rail safety a top priority in Minnesota
John Crabtree: What's value added mean anyway?
Talking it over: Physical activity
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July 2, 2015
Talking it over: A good kind of exhaustion
I am exhausted but that is a good thing. Over the weekend, my husband and I had our son's three daughters stay with us and on one of the nights, our other two granddaughters joined us. It was a fun and memorable three days. I had been thinking about trying to have the girls stay...
July 2, 2015 - 10:01am
June 27, 2015
Sue's Views: Making an effort makes it worth it to shop local
The last few weeks have been wildly busy in Morris and it is the stuff that makes small town living worthwhile. Helping Eul's Hardware Hank celebrate 85 years in business with a picnic lunch and cake is just about the best way to spend part of a Saturday afternoon that I know of. A...
opinion / local commentary
June 27, 2015 - 5:55am
Sen. Al Franken: Making rail safety a top priority in Minnesota
Minnesotans have always known how important safe, reliable rail service is to our state's economy.
opinion / editorials
June 27, 2015 - 5:53am
John Crabtree: What's value added mean anyway?
Whether enabling dairy farmers to expand their line of product offerings or helping farm families start businesses for unique products or providing capital for the creation of farm-identity preserved regional supply chains, USDA's Value-Added Producer Grant (VAPG) program has been...
opinion / editorials
June 27, 2015 - 5:51am
June 25, 2015
Talking it over: Physical activity
Being physically active and physically educated are both very important. Knowing what is good physical activity for yourself and those around you is critical. This could mean what is safe for you, what you are capable of doing and what your limitations should be. Last week my niece's...
June 25, 2015 - 9:42am
June 20, 2015
Talking it Over: Pictures can speak volumes
A life can be defined through pictures. Quite often for special events we use pictures to chronicle a life. We start with pictures of a person or couple when they are very young and move through the years with pictures that show some of the highlights of their life. Those pictures...
opinion / local commentary
June 20, 2015 - 5:03am
Rachael Meyer: Conservation, crop insurance and tax dollars
The federal crop insurance program provides an agricultural safety net, and crop insurance premium subsidies were created to increase usage of these risk management tools. The federal government subsidizes, on average, 62 percent of crop insurance premiums annually. Crop insurance...
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June 20, 2015 - 5:03am
Christopher Shoff: Fixing our transportation system should transcend election cycles
As the 2015 session finally came to a close last week, the Legislature was once again unable to come to agreement on a comprehensive transportation bill. This has become an annual disappointment for the members of the Association of Minnesota Counties (AMC). Freeborn County Engineer...
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June 20, 2015 - 5:01am
June 19, 2015
Letter: DFL legislature helped turn Minnesota around
Rep. Peppin, in response to your letter, I am proud to have been a member of the DFL led legislature that turned Minnesota around. It's because of our good work balancing Minnesota's budget fairly for the first time in a decade that Rep. Backer was able to vote for all of the increased...
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June 19, 2015 - 4:00pm
June 13, 2015
Literature in a Hurry: Weekends at home and weekends away
This weekend, I'm getting out of town. By 5:30 on Friday afternoon, I'll be on the road with friends, heading to my parent's cabin in Wisconsin for a long weekend. It's my first weekend at the lake this year and I couldn't be more excited. I told a friend in the audience at...
opinion / local commentary
June 13, 2015 - 5:15am
Blaine Gomer: EPA puts oil industry agenda ahead of American farmers
It hasn't always been easy for the average American farmer. Farmers have had to work tirelessly to stay afloat amid fluctuating crop demand and a tumultuous environmental and political climate. Despite the chaos, hope springs from domestic biofuel production, which has bolstered...
opinion / editorials
June 13, 2015 - 5:14am
June 12, 2015
Letter: Keep Morris beautiful
I know that some people in Morris like the sounds of the cemetery "chimes." I am not one of them. Neither was my mother-in-law, Kathleen Owen, who used to live in this house where my wife and I are now, only three doors up Fifth Street from the cemetery itself. Kathleen was a proper...
opinion / letters
June 12, 2015 - 3:13pm
Letter: There are many versions of Christianity
In a June 6 letter to the editor, Reverend Main suggested that I made "slurs against Christianity" in an editorial from May 30. This cannot be true, because I don't believe in the existence of Christianity. I believe in the existence of many Christianities. As a college...
opinion / letters
June 12, 2015 - 3:12pm
June 11, 2015
Talking it over: Remembering Issy
When my twin granddaughters were born, Issy Foss came up to me and said "I can't imagine why anyone would name their child Isabell." I thought afterward that I only hoped my Isabelle would be half as great of a person as this Isabell. This community, our church, her friends...
June 11, 2015 - 10:40am
June 5, 2015
Letter: Keep the stop signs on main street
I am wondering if anybody noticed how much better the traffic flow is on the main street of Morris since they removed the old stop and go lights. The four-way stop and go it is your turn is so much quicker and faster than waiting for the light to change. I know it depends a lot on...
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June 5, 2015 - 4:34pm
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Mount & Blade
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Mount & Blade Latest News
Paradox "handing over the reins" of Mount & Blade back to developer TaleWorlds
31 January 2014 | By /staff
As of today the Mount & Blade franchise is squarely back in its developer's hands, TaleWorlds, as Paradox Interactive give back the reins of the IP after five and a half years.
Paradox "feel honoured" to have been part of the journey and as a parting gift to fans, they'll be giving Early Access to Magicka: Wizard Wars in their library.
The Best in free PC games and mods: October 2012
02 November 2012 | By Simonkey75
Simon Williams presents us with the best PC games and mods from October 2012.
Paradox "close to 90%" digital sales, retailers 'bad' for creativity
07 July 2011 | By /staff
Fredrik Wester, Paradox Interactive CEO, has claimed it a "release" to not be reliant on retailer sales for their games, which are now "like a bonus".
He notes we "don't really need" retail chains any more, which have "not been good" for the industry - stifling creativity. It's "really relieving" to speak about it.


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I've made similar posts (focused more on total opportunities and home/road splits rather than ranking though) in the past in response to the "bettman favours the hawks" crap so I thought I'd respond. Generally ignored like this thread may be too by many.
I do have somewhat of an explanation for the Hawks ranking. They've been one of the better puck possession teams of the past few years and without going any deeper you'd expect them to have more PPOs than they do. The refs aren't robots though, so they have no choice but to let things go. If they did they'd be calling 4 and 5 penalties a period. The same idea as teams that interfere a lot getting away with more interference than teams that regularly don't for example. The "interfering team" would have more total interference penalties but they'd get away with a ton more. And the percentage of interferences called would probably be lower for them too. It's simplistic, but somewhat true. Just not sure I'm expressing it properly.
Play style is important here too obviously and explains more of it, but that's a longer post. In summary there are logical arguments for why the Hawks get so few opportunities relatively. There are few for the "refs favour the Hawks" however. I guess it doesn't really explain some of the teams high on the list though.
At the same time the Hawks do generally have one of the better PP/SH differentials. That actually might be a better indicator here. I'd suspect it would better correlate with success than the current list.
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Geforce 8200 users, I need your help with finding usb hanging issue
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Join Date: Sep 2002
Posts: 623
[PATCH] Re: Geforce 8200 users, I need your help with finding usb hanging issue
My official bug report:
Except my patch there is no any workaround. I have exactly the same bug as you.
I reported the bug to Nvidia and we had some information exchange about this bug.
I do not know if I'm allowed to talk about it so better ask someone from Nvidia Linux team about usb ohci hanging bug in MCP78S chipset. Maybe someone from Nvidia will say something here because this is growing problem as people upgrade their PCs to nForce 7xx family.
You will find my patch attached below.
It fixes:
+hang on usb plug in chance to avoid the bug: 99%
+hang during medium speed transfers (adsl usb modems, usb storages) 90% chance to avoid the bug
+hang during low speed transfers (usb keyboard and mouse) 98% chance to avoid the bug
It does
-usb hang after few minutes of heavy and continuous usb traffic devices: usb 1.1 audio, movie cameras, usb radio.
This patch is experimental. Test it before use to make sure it does not break anything else.
Linux by default uses fasteoi interrupt handler. This patch switches to level interrupt handler to recreate windows behavior for interrupts. Slower but less hangs.
I do not recommend using this patch.
I use it since few months and except occasional hangs it works for me.
How to use the patch:
Patch kernel sources you have. I designed this patch for kernel 188.8.131.52 but it can be applied on 126.96.36.199 or any other 2.6 kernel with less or more modifications required. After patching, building and installing kernel add this option to kernel boot parameters:
and reboot Linux.
How to see if the patch is active:
cat /proc/interrupts
If you see:
patch is active.
If you see:
it is not.
Recommended solution which workarounds the bug in 100%:
Buy and install external PCI/PCI-E card with usb ports. You can safely use mainboard usb connectors only for usb 2.0 devices. If you have usb 1.1 only devices - use connectors on PCI/PCI-E external usb card.
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Glee Season 4: Brody and Rachel's Cute Day Together at the Park (PHOTOS)
Rachel Berry's (Lea Michele) big move from Ohio to New York City hasn't been easy. One day, as Rachel sits in the park thinking about just how much she misses Finn (Cory Monteith), she is surprised by a charming visitor — her handsome new NYADA friend Brody Weston. We think it's very sweet how much Brody (Dean Geyer) care and wants to help cheer Rachel up, but does she have to look at him like that? We definitely get the feeling that these two might end up as more than just friends.
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Europe is being wowed by the 2013/14 UEFA Champions League Trophy Tour, presented by Unicredit, as UEFA.com found out in Berlin with UEFA ambassador Karl-Heinz Riedle and UniCredit ambassador Fabio Capello.


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CBNNews.com - EREZ CROSSING, Israel - A suicide bomber blew up himself and his pickup truck early Thursday on the Palestinian side of the Erez crossing between Israel and the Gaza Strip.
Two Palestinian terror groups claimed joint responsibility for the 6:35 a.m. bombing: Islamic Jihad said they planned the attack with the help of Fatah-affiliated al-Aksa Martyrs' Brigades.
A spokesman for Islamic Jihad described the attack as "a successful martyrdom operation," though Israel Defense Forces (IDF) soldiers fired on the truck, preventing it from reaching the crossing.
"The soldiers' deployment, readiness and conduct prevented a more serious attack," an IDF officer said. The pickup truck was filled with "hundreds of pounds of explosives," he said.
Following the massive explosion, IDF soldiers searched the area for more terrorists.
Several houses in the Israeli community of Netiv Ha'asara were damaged by the blast and the IDF instructed residents to remain inside their homes until they finished assessing the incident.
Like last month's well-planned terror attack at the Kerem Shalom crossing, in which 13 IDF soldiers were wounded, Palestinians used a combined assault with explosive-laden vehicles and gunfire.
The IDF believes Palestinians are trying to kidnap more Israelis to use as bargaining chips for the release of Palestinian prisoners serving time for terror attacks.
Sources: Haaretz, YNet news
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TV news crews robbed in S.F.; cameraman pistol-whipped
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Jennifer Garner and Ben Affleck expecting third child
Jennifer Garner and Ben Affleck are expanding their brood – the celebrity couple is expecting a third child.
They already have two daughters – five-year-old Violet and Serephina, two.
The happy news comes after the Garner's publicist denied the actress was expecting again earlier this year when she was spotted sporting what looked like a baby bump.
Garner and Affleck wed in 2005.
Ben Affleck tours Turkey with Istanbul mayor
Ben Affleck got a first-class tour of Turkey on Tuesday after the mayor of Istanbul volunteered to show him the sights.
The actor is shooting his upcoming political drama "Argo," about the Iran hostage crisis, in Turkey.
He jumped at the chance to get better acquainted with Istanbul after Mayor Mustafa Demir offered to accompany him around the city.
The pair walked around Sultan Ahmet Square, a plaza Affleck scouted as a potential shooting location for "Argo," before making a stop at Bilgi University.
He finished the day browsing the Grand Bazaar – the same market where he snapped up an expensive antique rug earlier this year.
Ben Affleck gets out of 'Gatsby' film
Ben Affleck has pulled out of director Baz Luhrmann's adaptation of F. Scott Fitzgerald's classic "The Great Gatsby."
Just hours after news broke about Isla Fisher joining the cast as the mistress of Affleck's character Tom Buchanan, "The Town" star has reportedly quit the project to concentrate on his next directorial role on Iranian hostage crisis drama "Argo."
Leonardo DiCaprio, Tobey Maguire and Carey Mulligan are still a part of the all-star cast.
Matt Damon applauds Ben Affleck for resurrecting career
Matt Damon has praised his best friend Ben Affleck for turning his career around after falling "out of favor" in Hollywood.
The pals wrote and starred in 1997's critically-acclaimed "Good Will Hunting," which won two Academy Awards.
Affleck's private life quickly became headline-making news because of his glamorous girlfriends, including Gwyneth Paltrow and Jennifer Lopez.
Affleck and Lopez went on to co-star in 2003 flop "Gigli," and Affleck saw his popularity wane further after starring in badly-received movies "Surviving Christmas" and "Jersey Girl."
The star subsequently turned his attention to movie-making and directed 2007 hit "Gone Baby Gone," which scooped the Hollywood Film Festival Award for Breakthrough Director of the Year.
And Damon has applauded his pal for his Hollywood comeback, which was cemented with last year's crime drama "The Town."
He tells CNN's Piers Morgan Tonight, "Ben kind of fell out of favor, he got just a crushing amount of publicity in his personal life and that made people, I think, loath to work for him a little while there, he was kind of in the penalty box.
"He did what we did originally did… 'Gone Baby Gone,' his directorial debut… he wrote that, directed it and then with 'The Town,' he couldn't get a job as an actor that he wanted so he wrote it for himself and directed it."
Ben Affleck: 'Recent success won't last forever'
Ben Affleck is refusing to allow the success of his film "The Town" to go to his head – because he is bound to be a box office failure again in the future.
The actor turned director on the heist movie, which was a hit with cinema-goers and earned star Jeremy Renner a Best Supporting Actor nod at this year's Academy Awards.
But despite the acclaim his third moviemaking venture has received, Affleck is determined not to allow his ego to be boosted – insisting he's learned a Hollywood career is full of ups and downs.
He tells Britain's Live magazine, "Right now I'm a success again but I'll be a failure again too, and that's ok. Failure is just a function of risk. I look at my great heroes as actors and directors and none of them have perfect records. I imagine I will have an even more imperfect record.
"I think you have to still be brave enough to take risks. Today I am enjoying the success. Being number one at the box office is pretty cool. But I've been round the block enough times to know it won't last forever."
Ben Affleck not looking to work with Jennifer Garner
Ben Affleck doubts he will ever work with his actress wife Jennifer Garner on screen, because he's still haunted by the box office failure that was "Gigli."
The actor filmed the romantic comedy with Jennifer Lopez, who he began dating after meeting her on set in 2002.
The movie was released a year later, but the media circus that surrounded their whirlwind romance and subsequent engagement did nothing to boost "Gigli's" profile, and critics savaged the film.
Affleck and Lopez split in 2004, shortly before they were due to wed, but the movie experience was so bad, it has put the actor/director off the idea of teaming up on the big screen with Garner, his wife of five years.
He tells Us Weekly magazine, "Jen is a great actress. I would be profoundly lucky to work with her. But something tells me that people don't want to see real-life couples together at the movies."
Jennifer Lopez opens up about doomed Ben Affleck romance
AP Photo/Evan Agostini
Jennifer Lopez is convinced her high-profile engagement to Ben Affleck was destined to fail, insisting no couple could have survived the intense media pressure the pair endured.
Lopez began dating Affleck in 2002, sparking a media circus that turned crazy for the couple when the actor proposed, but the glare of the spotlight became too much for the stars and they called off plans to wed the following year.
And Lopez admits the romance was doomed from the start. She says, "I love Ben. He's a great guy but it was a lot for both of us to be under that type of siege for two years straight. We were on the cover of every magazine every week, and it was just a weird thing that the media catches on to, and they just go crazy with it. Our relationship, I think, did suffer from it."
And the actress credits her husband, singer Marc Anthony, with teaching her how to live her life away from the glare of the paparazzi.
She tells U.K. TV host Graham Norton, "I think he has a totally different approach to fame, to celebrity — our work, our business. (He believes) you can have your career, you can promote, you can do all that stuff — but you can also have a personal life, and it can be quiet.
"I think, when you're in the throes of (fame), you don't realize that you have control over it. You kind of feel like, 'Oh, it's just happening, I can't stop it.' But he taught me that you can."
Lopez married Anthony in 2004, while Affleck is now happily married to actress Jennifer Garner.
Damon and Affleck gamble for charity
Best pals Matt Damon and Ben Affleck faced off at the poker table on Thursday night as part of a charity event in Las Vegas.
The Oscar-winners joined event host Don Cheadle and other celebrities including actors Brad Garrett and Danny Masterson and retired boxing champ Mike Tyson to help raise money for Ante Up for Africa.
The stars joined members of the public and dug deep into their pockets to join in the fun, handing over $5,000 as a buy-in for the gambling tournament, with all proceeds benefiting the Enough project and Refugees International — organizations that aim to raise awareness and provide help to the survivors of war-torn Darfur in Sudan.
Affleck/Garner School Fights Back In Paparazzi War
The preschool Jennifer Garner and Ben Affleck's daughter attends is set to be made anti-paparazzi if concerned parents and local councilors get their way.
The First Presbyterian Nursery School in Santa Monica, Calif., has become a popular target for photographers ever since little Violet, three, became a student there and parents and teachers claim the paparazzi are robbing kids of a peaceful arrival.
Some worried parents claim they've been physically and verbally abused by the snappers, and one alleges her 4-year-old was almost run over by an over-eager photographer.
Now, Santa Monica City Council member Richard Bloom is leading a charge to create school buffer zones, where the paparazzi cannot stand.
Bloom, an attorney, tells the Los Angeles Times, "If this were an isolated thing that had happened once or twice, I wouldn't be spending my time on it."
The councilor admits he has spoken with Garner and Affleck about their concerns: "They expressed to me that they understand this is a part of their lives that they have to accept, but it's not something that other people should have to experience."
Bloom joins Los Angeles City Council member Dennis Zine among those leaders trying to restrict paparazzi access in Tinseltown.
Zine has spent the past 18 months pushing measures to create buffer zones throughout Hollywood.
Affleck Taught His Daughter To Swear – In German
Ben Affleck was left red-faced during a recent restaurant outing with his 3-year-old daughter Violet, when he accidentally taught her how to swear in German.
While Affleck's wife Jennifer Garner took care of their 3-month-old baby daughter Seraphina, the actor treated Violet to a trip to a Mexican restaurant.
But their family bliss turned to chaos when the girl decided to spill both their meals on her dad's lap, prompting him to swear in German.
The actor thought he'd avoided teaching her a bad word by cursing in a foreign language, only to watch Violet recite the phrase loudly to the horror of other diners.
Affleck explains, "All the food is coming, spilling towards me. And I go, 'Shhhhh … and I turn it around and say 'sheisse.'
"Why I came up with that word, I don't know, I have not even been to Germany, but I guess that's a German swear word in my subconscious…
"I thought, 'I've averted disaster.' I look over at her and she says, 'What's sheisse? Why did you say sheisse?'
"I said, 'It's just a bad word, and we don't say that.' As soon as I told her it was a bad word, I was like, 'You idiot.'
"So she just goes, sheisse, sheisse, sheisse, sheisse, over and over again. It turned into a total nightmare for me. My daughter's swearing in German."


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jerking it again
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text:
Our weather report if calling for a possibility of snow/sleet on Saturday
Enjoy your snow and cold weather!!
My tolerance for stupid shit is getting less and less.
We have the AC on
We sure do. It's 80 here.
We are supposed to have 8C here tomorrow and the day after, dropping to 0C on Friday. Yeah, that's Canada.
[This message edited by lynnm1947 at 3:12 PM, December 3rd (Tuesday)]
"I could have missed the pain, but I would have had to miss the dance." Garth Brooks
The people you do your life with shape the life you live
dreamlife - Post pics of the snow!
Fear is the cheapest room in the house. I would like to see you living in better conditions.
― Hafiz
You can laugh at us on Saturday. We don't know how to handle the cold. We bundle up like we are headed to the North Pole when it gets close to 40.
Isn't that the truth honey!! MH and I were already contemplating on calling our landscaper to help us 'winterize' our sprinkler system because we have no clue how to
Dress warm for your travels up here for the Elf Party!!


text:
View Record Details
Pub. Type:
An Experimental Evaluation of the Phoenix Repeat Offender Program, Final Report
Subtitle/Series Name:
Pub. Date:
The ROU experiment involved the assignment of offenders randomly to two groups, experimentals and controls. The experimentals consisted of offenders assigned as targets of the ROU. The ROU worked closely with the Maricopa County Attorney's Office to document prior criminal records, to conduct followup investigations, and to devote extra effort to obtaining and maintaining victim and witness cooperation. The controls were handled through normal departmental procedures. The assignments to the experimental and control groups lasted 1 year; the last assignment was made on December 7, 1988 yielding 480 assignments: 257 to the experimental group and 223 to the control group. Every case was given at least 6 months prior to the determination of the effects of either the ROU program or normal processing. In June 1989 a data-collection team determined the followup outcomes. The analysis of case disposition patterns showed no significant increase in conviction rate for the experimental ROU cases, but there was a significant increase in the likelihood of imprisonment and the length of the term imposed. These increases were attributable to the ROU officers' success in the development of additional charges against the defendants and documentation of their prior records. 4 tables, 4 figures, and 21 references source
National Institute of Justice
Place of Production:
Washington, DC
Related Studies
This publication is related to the following dataset(s):


text:
A roundup of what The Globe and Mail's market strategist Scott Barlow is reading this morning on the World Wide Web.
China released economic growth data showing accelerating activity but some economists and pundits are viewing the report as a sign of weakness rather than strength. The economy was rescued by yet another surge in credit growth despite the government's previous admission that this strategy risked the longer term health of the Chinese financial system.


text:
Methods for modifying polyhydroxylated materials by the direct covalent bonding of nucleophilic ligands to the former sites of hydroxyl groups on the material are disclosed. More specifically, methods for activating the surface of polyhydroxylated materials such as silica, which can serve as stationary phases in various chromatographic methods, are disclosed. The silica is first contacted with a reagent, e.g., a phosphorylating agent, effective to cleave the O--H bond of at least one of said hydroxyl groups and introduce through an --O-- linkage a moiety amenable to nucleophilic displacement; and the product of step (a) is then contacted with a suitable nucleophilic ligand.
1. A method for preparing a modified polyhydroxylated material comprising:
(a) providing a polyhydroxylated material comprising an organic or inorganic backbone having a plurality of hydroxyl groups at its surface;
(b) contacting said polyhydroxylated material with an agent selected from the group consisting of phosphorylating agents, and compounds of the formulae Cl3 CC(O) NHCOCl, O(CF3)2, ##STR14## (c) contacting the product of step (b) with a nucleophilic ligand.
2. The method of claim 1 in which said polyhydroxylated material comprises an inorganic backbone.
3. The method of claim 2 in which said polyhydroxylated material is selected from the group consisting of silica and glass.
4. The method of claim 3 in which said polyhydroxylated material is silica.
5. The method of claim 1 in which said polyhydroxylated material comprises an organic backbone.
6. The method of claim 5 in which said polyhydroxylated material is selected from the group consisting of acrylic resins, cellulose, agarose, sepharose, polysaccharides, and oligosaccharides.
7. The method of claim 6 in which said polyhydroxylated material is selected from the group consisting of hydroxylated polystyrene/polyvinyl benzene, dextran, and cyclodextrin.
8. The method of claim 1 in which said nucleophilic ligand is selected from the group consisting of dyes, antibodies, antigens, drugs, antibiotics, lectins, proteins, peptides, amino acids, DNA and RNA.
9. The method of claim 1 in which said agent is a phosphorylating agent.
10. The method of claim 9 in which said agent is selected from the group consisting of phosphorous oxychloride and Hal-P(O)(OH)--SO2 OH were hal=a halogen atom.
11. A method for preparing a modified polyhydroxylated material comprising
(a) providing a polyhydroxylated material comprising an organic or inorganic backbone having a pluraliyto of hydroxyl groups at its surface;
(b) contacting said polyhydroxylated material with an agent selected from the group consisting of ClSO2 --Ph--CH3 and F3 CCH2 SO2 Cl; and
(c) contacting the product of step (b) with a nucleophilic ligand.
12. The method of claim 1 in which said agent is selected from the group consisting of compounds of the formulae Cl3 CC(O)NHCOCl, O(CF3)2, and ##STR15##
13. The method of claim 3 where said agent is a phosphorylating agent.
14. The method of claim 4 where said agent is a phosphorylating agent.
15. The method of claim 13 where said agent is selected from the group consisting of phosphorous oxychloride and Hal-P(O)(OH)--SO2 OH where Hal=a halogen atom.
16. The method of claim 14 where said agent is selected from the group consisting of phosphorous oxychloride and Hal-P(O)(OH)--SO2 OH where Hal=a halogen atom.
17. The method of claim 2 where said agent is a phosphorylating agent.
18. The method of claim 17 in which said agent is selected form the group consisting of phosphorous oxychloride and Hal-P(O)(OH)--SO2 OH were Hal is a halogen atom.
This invention relates to methods for modifying polyhydroxylated materials by covalently bonding nucleophilic ligands to said material. More specifically, this invention relates to improved support materials for use as stationary phases in various chromatographic methods, and to methods for preparing such support materials.
Chromatography is a separation technique whereby individual chemical compounds which were originally present in a mixture are resolved from each other by the selective process of distribution between two heterogeneous (immiscible) phases. The distribution of chemical species to be separated occurs in a dynamic process between a mobile phase and a stationary phase. The stationary phase is a dispersed medium, which usually has a relatively large surface area, through which the mobile phase is allowed to flow. The chemical nature of the stationary phase exercises the primary control over the separation process. The greater the affinity of a particular chemical compound (referred to as the solute) for the stationary medium, the longer it will be retained in the system. The mobile phase can be either gas or liquid; correspondingly, the methods are referred to as gas chromatography and liquid chromatography.
There are a wide variety of chromatographic methods, varying, for example, in the selection of mobile and stationary phases, techniques and solute measurement principles. As an example, ion exchange chromatography is a widely used form of liquid chromatography. It is based on selective ionic attractions between variously charged sample constituents and an ionized chromatographic matrix. The most commonly used ion exchangers consist of an organic polymeric backbone with either acidic or basic exchange sites on its porous surface. The charged resins are capable of exchanging their cations or anions with those ions in the liquid phase which have a greater affinity for the matrix. Exchange interactions that take place during the passage of various ions through the column cause separation into discrete ionic zones.
Thin layer chromatography is a technique in which the stationary phase is a suspension which forms a layer on a plastic or glass plate. It is most frequently an adsorbent (with a particle size of several microns) suspended in a suitable solvent, uniformly spread on a plate, and dried. The mobile phase is a liquid that ascends the plate by capillary action, and the components of the sample mixture are separated by the partition effect.
Reverse-phase chromatography is a type of chromatography in which hydrocarbons as well as polar samples are partitioned between a nonpolar stationary phase and a polar eluting phase. Under these conditions the most polar substances elute most rapidly. This is the reverse of the more common partition chromatography in which the stationary phase is polar and the least polar substances elute most rapidly with the nonpolar eluting phase. In reverse phase chromatography, the stationary phase often consists of a chain of atoms chemically bonded to an inert surface such as silica or glass, and the eluting phase is frequently aqueous methanol or aqueous acetonitrile.
Molecular sieve chromatography, often called gel chromatography, has resulted in tremendous progress in the chemistry of biomacromolecules. Separation in molecular sieve chromatography is based on a selective process of penetration of molecules of different sizes and shapes through a porous gel medium. The largest molecules in the mixture do not penetrate the porous structure at all; the medium-size molecules can penetrate only some pores; and the small molecules can diffuse rather freely inside the medium and can spend a considerably longer time there. Consequently, if the porous material is contained in a column, mixtures of components with differing molecular weights can be effectively resolved.
In any chromatographic process, some components of a given mixture will be retained on the stationary phase longer than others. This allows for extremely selective chromatographic separations. For example, in the method called affinity chromatography, molecules to be purified interact with immobilized ligands on the surface of the stationary phase and are strongly retained by the stationary phase material Passage of a multicomponent extract through a column of immobilized ligand results in selective adsorption of the recognized material to the column. Non-interacting material can be washed away, and bound components can be eluted biospecifically with competetive or affinity modifying reagents, or under denaturing conditions. It is therefore always necessary to seek a stationary phase material with a selectivity appropriate to a given separation problem.
Common to all of the above-described chromatography methods is the use of a stationary phase having at its surface a phase which will interact with the desired components of the mobile phase in the desired manner, e.g., the highly specific ligands attached to the stationary phase in affinity chromatography, or the acidic or basic exchange sites on the stationary phase in ion exchange chromatography. Development of stationary phases for various types of chromatography in general has focused on the attachment of various bonded phases to dextran (Sephadex), agarose, glass, silica and polymeric materials such as polyacrylamide, polymethacrylates or latex.
More specifically, for use in affinity chromatography, the chemistry of ligand immobilization using activation of agarose with cyanogen bromide has been the most popular methodology. The generation of carbonates and caoamates by reaction with 1,1'-carbonyldiimidazole or chloroformate with agarose, polyacrylamide, cellulose, glass beads or hydroxylated polystyrene or other polymers has also been employed. A major disadvantage of such chemistries is the production, on reaction with amines, of a relatively unstable amide bond resulting in continuous ligand leakage at a slow but measureabIe rate. Moreover, ionic contributions to non-specific protein adsorption are also observed, probably due to the formation of isourea groups. The use of carbonyl diimidizole activated supports which on reaction with amines form a urethane linxage, as well as use of bifunctional oxiranes, has reduced but not eliminated non-specific protein binding. Despite these improvements, agarose remains susceptible to microbial attack, is of limited usefulness in the presence of organic solvents, and is not amenable to easy scale-up and high flow rates. Other supports such as glass, while performing well in organic solvents, suffer from residual charged functions and non-specific binding. Polyacrylamide, while more resistant to microbial attack an agarose, does not form high flow capacity columns. Other polymeric material suffer from higher levels of non-specific interactions than agarose.
Ideally, stationary phases, or chromatography supports, should have good mechanical strength and flow properties, be available in a range of particle sizes, pore sizes and shapes, be chemically stable, possess a high level of hydrophilicity, be amenable to a number of modifications and possess little or no non-specific interaction with the components to be resolved. Silica has been shown to satisfy most of these criteria. Optical activation and performance of silica columns for use in affinity chromatography has been achieved with spherical 10 micron particles. No significant advantage was obtained with smaller particles, and substantial decreases in performance were observed with 20 micron materials. Important advantages accruing from the use of silica supports in affinity chromatography have been found, including high accessible capacity, complete resistance to microbial attack, ease and versatility of immobilization chemistry, high purification efficiency and excellent flow properties. See Hollis et al., J. Liq. Chromat., 10, 2349 (1987). Use of silica results in affinity chromatography systems where elution volumes are minimized and procedures are rapid and easily automated.
Previously, silica has been modified for use in chromatography via a series of reactions using various organosilane analogues and methodologies. These chemistries result in bonded phase attachment via an Si--O--Si linkage which is sensitive and labile to acid, base and other treatments. A generalized scheme representing silane activation of silica is a follows: ##STR1## As shown in Scheme 1, the reactive organo silane is directed towards available hydroxyl functions on the silica surface. The half-life of such supports are variable and unpredictable due to slow decomposition of the Si--O--Si bond. In particular, silane activated silicas are unstable above a pH of about 7.2. While this has not hindered the use of silica for applications requiring a pH in the range of 2-7.2, different supports such as polymeric beads must be used for applications requiring a higher pH. Many polymeric beads are stable at elevated pH but show poor flow characteristics and higher non-specific binding when compared to silica. In some cases, zirconium impregnated or polymer coated silicas have been prepared and are claimed to possess higher pH stability.
Thus, although silica provides a chromatographic support material which is advantageous in comparison to other materials in many respects, chemical instability stemming from the chemical approach of attaching a ligand is a severe drawback. There are many separation applications which would benefit from the ability to use a silica support having greater pH stability, e.g., biochromatographic methods involving the separation of proteins, DNA, RNA, cells or cellular particles in a format designed to maintain any associated biological activity. Examples of such biochromatographic techniques are ion exchange, hydrophobic interaction, size exclusion separations, as well as the above-described affinity chromatography. Because of the chemical instability of surface-modified silica, and because optimum bio-separations are usually observed at an alkaline pH, many such biochromatographic separations employ polymer packings possessing less efficient flow properties and substantially greater non-specific interactions than silica.
There is thus a need for methods for preparing silica supports in the absence of impregnated or polymer coating materials which would display little or no non-specific interaction with components to be resolved and which would maintain the excellent flow properties of silica. In addition, there is a need for methods for preparing silica supports which would have greater pH stability than the standard organo silane-activated silicas in use today.
Towards this end, we hypothesized that a silica particle, with its numerous hydroxyl functions, could be viewed as a "polyol" wherein some, if not all, hydroxyls are assumed to be in close proximity and in cis-configuratoin, i.e., as one encounters in a sugar molecule. It is known that a nucleophile may be introduced to a 1,2-cis-diol, such as ribose, by application of carbohydrate phosphate chemistry as illustrated in Scheme 2. ##STR2## Thus, we hypothesized that a nucleophile could be introduced to a silica surface using the same general chemistry, as illustrated in Scheme 3. ##STR3##
Tests have indicated that such chemistry can indeed be utilized to introduce nucleophiles suitable for chromatography on the surface of silica. Advantageously, in the materials prepared in this manner, the nucleophiles are covalently bound directly to a silicon atom of the backbone, yielding a Si-Nu linkage (Scheme 3) which is more stable than the acid-base-sensitive Si--O--Si--Nu linkage (Scheme 1) arising in standard organo silane-activated silicas, thus avoiding one of the major problems inherent in the use of silica chromatography supports to date. This work has implications far broader, however, than only the modification of silica materials. If it can be assumed that at least a portion of the hydroxyl functions on the surface of a silica support are in close proximity and in cis-configuration, and the work described herein bears out this assumption, then it can also be assumed that at least a portion of the hydroxyl functions on the surface of other polyhydroxylated support materials would also be so arranged. Thus, the activation/nucleophilic displacement chemistry described above can be extended to include the modification of a wide variety of polyhydroxylated materials, exemplified by those described in this patent, in a variety of ways.
This invention therefore relates to a polyhydroxylated material comprising an organic or inorganic backbone having a plurality of hydroxyl groups at its surface, which material has been modified by the direct covalent bonding to said backbone, at the former site of at least one of said hydroxyl groups, of a nucleophilic ligand. This invention further relates to methods for preparing such materials comprising (a) reacting a polyhydroxylated polymeric material having a plurality of hydroxyl groups at its surface with a reagent effective to cleave the O--H bond of at least one of said hydroxyl groups and introduce through an --O-- linkage a moiety amenable to nucleophilic displacement; an (b) reacting the product of step (a) with a nucleophilic ligand.
FIG. 1 is a graph of phospholipase A2 and phospholipase c activities of the various fractions collected from a column having a silica support, prepared according to this invention, with mellitin immobilized thereon. The curve with open circles represents endogenous activity and the curve with closed circles represents stimulatory activity.
FIG. 2 shows affinity chromatography of phospholipase c on silica prepared according to this invention.
FIG. 3 is the elution profile of cytochrome C tryptic peptides, insulin, cytochrome C, and Bacillus cereus from a column of silica prepared according to this invention, using reversed phase HPLC.
FIG. 4 is the elution profile of cytochrome c, trypsin inhibitor and phospholipase c from a column of silica prepared according to this invention, using reversed phase HPLC and after washing the column with ammonium hydroxide pH 9.5 for 16 hours.
FIG. 5 is the elution profile of small cytochrome c peptides, cytochrome scheme peptide, insulin, cytochrome c and PLC from a column of silica, prepared according to this invention, using reversed phase HPLC.
FIG. 6 is the elution profile of a mixture of aromatic amines from a column of silica, prepared according to this invention, using reversed phase HPLC.
FIG. 7 is the elution profile of the synthetic peptide Gly-Asn-Glu-Phe-Trp-Thr-Ser-Ile-Asp-Val from column using reversed phase HPLC. This peptide was synthesized from components attached to a fractogel prepared according to this invention.
Tests indicate that the methods described herein can be used to modify, i.e., directly and covalently bond a nucleophile to, the surface of any polyhydroxylated material. By polyhydroxylated, it is meant a material having a plurality of hydroxyl groups on its surface; however the preferred materials for use herein with various surface areas will have far in excess of only two hydroxyl groups. At least a portion of the hydroxyl groups must be at the surface of the support material, e.g., must be accessible to the components of the mobile phase used in the selected chromatographic method. The term "backbone is used herein to refer to the major structure of the polyhydroxylated material to which the hydroxyl groups are bonded. Examples of polyhydroxylated materials which are included within the scope of this invention include those with inorganic backbones such as silica and glass, and those with organic backbones including acrylic resins such as hydroxylated polystyrene/polyvinyl benzene, sepharose, cellulose, agarose, polysaccharides such as dextran and oligosaccharides such as cyclodextrin. Silica, which is the preferred support material, can be used in many forms, including but not limited to particles, beads of any size or shape, blocks, or impregnated into papers of any sort. Similarly, glass may be used in the form of particles, beads, tubes, plates, wool, fibers, capillaries, papers or impregnated glass fiber papers.
The polyhydroxylated material is modified, or activated, by the direct covalent bonding to its surface of a nucleophilic ligand with the specific characteristics required for the intended use of the polyhydroxylated material. By the term "direct" bonding, it is meant that the covalent bond is between the "backbone" of the polyhydroxylated material and an atom of the nucleophile itself, rather than, for example, through the hydroxyl oxygen atom yielding an ether linkage. This invention is not limited by the nature of the ligand, except that it must be nucleophilic in nature (i.e., an electron pair donor). For example, if the polymeric material is intended for use as a stationary phase in affinity chromatography, the ligand may be a material capable of interacting selectively with a component in a mixture to be chromatographed to thereby retain that component on the surface of the stationary phase for later elution. There are myriad other examples of the usefulness of polyhydroxylated polymeric materials modified according to this invention and, thus, of the types of nucleophilic ligands which might be used. These examples include:
Covalent attachment of any chemical, reagent group or material useful for amino acid sequencing by liquid, gas or gas flow techniques, such as polybrene or protein dyes, or functions for protein-peptide immobilization and solid phase sequencing;
Covalent attachment of any chemical, reagent group or material useful for electrophoretic or other transfer; Such groups may contain cleavable acid, base labite, disulfide or other functions. Examples are diethylaminoethylamine, carboxymethylamine, aliphatic or aromatic compounds normally associated with chromatography, polybrene or protein dyes;
Covalent attachment of any material, reagent, protein, DNA, RNA or chemical to a polyhydroxylated material useful for diagnostic or clinical purposes. Examples are attachment to small particles of materials for immunoassay or visualization in vitro or in vivo;
Covalent attachment of any chemical, reagent, natural product, protein, DNA, RNA, for oligonucleotide or peptide and protein synthesis, detection or purification;
Immobilization of enzymes, e.g., proteolytic or synthetic enzymes, for any purpose;
Immobilization of DNA, RNA or nucleotides for any purpose;
Immobilization of antigens, antibodies, antibody binding proteins or any protein or peptide for any purpose;
Immobilization of dyes (fluorescent, colored, radioactive electron dense or other) on sufficiently small particles for any purpose, e.g., for analytical purposes, in vitro or in vivo diagnosis, therapeutics, or detection of any substance separated by any technique or by electron microscopy;
Formation of co- or multi-conjugated particles containing any mixture of materials, chemicals, reagents, dyes or drugs for any purpose such as drug delivery or delivery of signal enhancing markers to specific sites for in vitro or in vivo diagnostics. Further illustrative examples are electron microscopy, fluorescence microscopy, antigen-hapten coupling, delivery to antibody producing cells for immunostimulation or suppression, and covalent attachment to magnetic particles of any substance for any purpose including diagnosis, drug delivery, purification or detection methods;
Immobilization of hydrophobic materials to make them behave in a hydrophilic fashion. Examples include immobilization of radiochromophores for inclusion in electrophoresis gels for replacement of external intensifying screens or for radio-flow detection.
Attachment of materials to mono, di or polysaccharides for any purpose. These include cyclodextrins or polyols of any molecular weight and composition. Materials to be attached, e.g., for drug delivery and diagnostic purposes, include but are not limited to: ligands, drugs, antibodies, antigens, dyes of any sort, specific markers of any sort, antibiotics, and growth factors. Using this approach (or alternate attachment chemistry if experimentally required) cyclodextrins, mono, di or polysaccharides or polyols can be used as bridges between site directed molecules and the functional molecule(s) to be delivered for any purpose. These molecules may be complexed individually or in any combination and proportion for any purpose. The approach is substantially different from the art where liposomes are used to package materials for delivery an in a limited number of cases site-directed molecules used to coat the surface by covalent or non-covalent interactions.
Detection of glycoproteins by contacting said glycoproteins (or a sample containing said glycoproteins) with an reagent (e.g., a phosphorylating agent) effective to cleave the O--H bond of at least one of hydroxyl groups on the glycoproteins and introduce through an --O-- linkage a moiety amenable to nucleophilic displacement, followed by reaction with a nucleophilic, fluorescent or radioactive such as fluorescein, dansylamine, rhodamine and their derivatives. In this way, the detectable dye is attached to the glycoprotein, allowing detection of the latter.
The aforementioned applications are exemplary only, and their recitation is not intended to limit the applicability of the chemistry described herein but, rather, to illustrate the broad applicability and versatility of that chemistry.
As further examples of the broad applicability and versatility of the modified polyhydroxylated polymer supports of this invention, the following are examples of targets which might be immobilized on the modified supports in any of the various applications listed above: glycine, gelatin, fucose, N-acetyl glucosamine, D-Ala-D-Ala, adenosine-3',5'-cyclic monophosphate, adenosine-5'-monophosphate, alanine, ε-aminocaproyl glucosamine, benzamidine, ω-aminooctyl, p-aminophenyl-2-acetamido-2-deoxy-β-thioglucopyranoside, m-aminophenyl boronic acid, p-aminophenyl-α galacto-pyranoside, ω-aminopropyl epoxy, avidin, biton, blue dextran, butyl, chloramphenicol caproate, cholic acid, cholesteryl hemisuccinate, coenzyme-A, concanavalin A (lectins), cysteamine, cytidine 5'-monophosphate, hemoglobin, heparin, S-hexylglutathione, wheat germ lectin, uridine 2', 5'& 3', 5,-diphosphate, uridine 5'-monophosphate, trityl, tryptophan, tyrosine, thyroxine, serine, spermine, oligo dT, protein A, protamine, polyriboinosinic acid, polylysine, O-phosphorylethanolamine, phosphodiesterase 3',5'-cyclid nucleotide activator, octyl, α-methylmannoside, insulin, histidine, lactalbumin, β-nicotinamide adenine dinucleotide, β-nicotinamide adenine dinucleotide phosphate, α-lactose, (+) melibiose, trypsin inhibitor, N-hydroxysuccinimide, imidazolyl carbamate or carbonyl imidiazol moiety, arachidonic acid, polymyxin (endotoxin-removal), 7,7-dimethyleicosadienoic acid, acuvicin (AT-125) (irreversible inhibitor of γ-glutanyl transpeptidase, furegrelate (U-63557A) (thromboxane A2 synthase inhibitor), dopamine antagonists, chiral ligands for resolution of optically active isomers, iminodiacetic acid (metal chelate), polyoxins, p-aminobenzamidine, octadecyl, Cibracon Blue F3Ga, aminoaryl, 8-hydroxyquinoline, Procion Red HE3B, (Nα-CBZ)-D-phenylalanine, poly-(L-lysine), histamine, methotrexate, pepstatin, ketanserin, L-(+)-tartaric acid, serontonin, fetuin, β-estradiol 17-hemisuccinate, glycyl-L-tyrosyl-azo-benzyl succinic acid, deoxycholic acid, diaminodipropylamine, dextran sulfate, p-aminobenzyl phosphonic acid, 2-aminoethyl dihydrogen phosphate, p-aminophenyl phosphoryl choline, boronic acid, p-chloromercuribenzoate, N-acetyl-D,L-homocysteine, L-alanyl-L-alanyl-L-alanine, p-aminobenzamidine, Tris(carboxy methyl)ethylenediamine, 3'-linked deoxyribose with protected pyrimidine or purine base (oligodeoxynucleotide synthesis support), protein G, polybrene, melittin, diethylaminoethylamine, and Coomassie Blue.
Many nucleophilic ligands which could be introduced on the surface of the polyhydroxylated support material according to this invention in order to immobilize targets such as those listed above are generally known in the art. One may use group selective or multi-specific ligands for biomolecule purification with affinity chromatography. Examples of group specific ligands are lectins and dyes. Lectins such as concanavalin A or wheat germ agglutinin are proteins which bind to characteristic sugar residues of the carbohydrate portions of glycoproteins. Many lectins from a variety of sources have been identified with differing sugar binding specificities. Glycoproteins bound to immobilized lectins can be eluted with an appropriate free competing sugar. Several dyes have been found to interact selectively with nucleotide requiring enzymes such as dehydrogenase, kinase, peptidase and phosphatase or growth factors. The nature of these interactions is not understood. More selective forms of affinity chromatography use immobilized protein A, protein G, various antigens, antibodies or anti-antibodies. These methods are usually referred to as immunoaffinity chromatography. Immobilized materials such as oligonucletoide binding dyes, etc., can be used for separations of DNA and RNA. Highly specific ligands such as pharmacological ligands and toxins have been used for the affinity chromatography of neurotransmitter and hormone receptors.
For successful affinity chromatography, ligand biomolecule interactions should be on the order of Kd=1-100 nM to allow retention and elution. Appropriate groups for ligand immobilization should be available without compromise of specificity or affinity. Since resin interactions follow mass action kinetics, increasing the concentration of the biomolecule to be purified will drive the reaction forward. The absolute dissociation constant (Kd), however, is characteristic of the biomolecule-ligand system. Adsorbed species are typically in an equilibrium binding state with the immobilized ligand dissociating and reassociating according to ligand-component concentration and system Kd. The concentration of ligand is usually in vast excess over the material to be purified. Immobilization of the ligand may enhance or decrease observed affinities either by steric hinderance or through coupling chemistry.
Biospecific elution can be achieved by using a ligand of higher or similar affinity for the bound component (at a higher or similar concentration) by preventing reinteraction, as the sample dissociates from the resin. Under these conditions purified components are collected in column or batch eluates. In some cases the affinity of a sample for an immobilized ligand can be increased or decreased by an allosteric regulator, modifying agent or changes in mobile phase. Advantage of these properties can be taken in both binding and elution from affinity resins. Eluting ligands are removed by dialysis, gel filtration or another chromatography step. In some cases sample-ligand affinities are such that biospecific elution yields only trace amounts of material. Denaturing agents such as sodium dodecyl sulfate (SDS), urea or changes in ionic strength or pH may be used to recover the sample. Care must be taken that this procedure does not alter the properties of the resin, particularly immobilized lectins or antibodies, protein A or protein G.
The first step involved in the direct covalent bonding of a nucleophilic ligand to the surface of the polyhydroxylated support material involves "activation" of at least a portion of the hydroxyl groups on the surface of the material by cleavage of the 0-H bond and introduction of a moiety amenable to nucleophilic displacement. This is accomplished by contacting the polyhydroxylated material with an agent selected from the group consisting of phosphorylating agents, such as phosphoric acid derivatives or phosphorous oxychloride, sulfonating agents, such as sulfonyl chloride derivatives, and other O-derivatizing agents as set forth below. In this way, the hydroxyl hydrogen is replaced with a moiety amenable to nucleophilic displacement such as a phosphate ester, cyclic phosphate, cyclic anhydride, etc. Examples of reagents which might be used, and the moieties which would thereby be introduced onto the surface of the polyhydroxylated materials through a linkage with the hydroxyl oxygen are as follows:
______________________________________Reagent Moiety______________________________________phosphoryl chloride P(O)3 ##STR4## P(O)(OH)SO2 HClSO2PhCH 3 SO2PhCH 3 ##STR5## OCNHC(O)CCl3F3 CCH2 SO2 Cl SO2 CH2 CF3 ##STR6## ##STR7## ##STR8## ##STR9## ##STR10## ##STR11##______________________________________
It should be appreciated that structural moieties derived from those specifically recited herein might be found which are amenable to nucleophilic displacement and which could cleave the O--H bonds on the surface of the support material as used herein. Since it is impossible to recite all such moieties, that list presented herein is not intended to be all-encompassing. This invention is deemed to be broad enough to include the use of other structurally related moieties which would function in the same way as the moieties recited herein.
In the preferred embodiment, the polyhydroxylated material is phosphorylated. This may be accomplished by phosphorylation methods generally known in the art. Preferably, the polyhydroxylated material is contacted with an organic base, such as pyridine, and a phosphorylating agent, such as phosphoryl chloride, under anhydrous conditions. The reaction tends to be exothermic. Following removal of the phosphorylating agent, and preferable washing with organic base and alcohol, the phosphorylated material is ready for introduction of the nucleophilic ligand.
Introduction of the nucleophilic ligand is accomplished by contacting the phosphorylated or otherwise "activated" polyhydroxylated support with the nucleophilic ligand under appropriate reaction conditions. Such conditions will vary, according to the selected nucleophile, but, in general, the nucleophilic displacement reaction occurs quite readily and no extraordinary reaction conditions are required.
The materials and methods of this invention, as well as their utility, are further illustrated by the following examples, which are not intended to limit the scope of this invention.
The reactions described in Examples 1 and 3 are illustrated in Scheme 4. ##STR12##
Silica (100 g; 200 micron, 200 Å pore size) was manually stirred at regular intervals with HCI (200 ml, 0.6N) and left standing overnight. After washing with distilled water (10,000 ml) by decantation the silica was collected by filtration in vacuo and over dried at 60� C. for 7 days (Yield: 80 g). 50 g of silica was treated with pyridine (300 ml) and the pyridine distilled off in vacuo until a thick sludge was left behind. After cooling at room temperature, an excess of phosphoryl chloride (50 ml) was added and the solution swirled at regular intervals. The flask was left at 60� C. for 7 days, the silica filtered in vacuo and washed with pyridine. After washing, the silica was vigorously swirled with a large excess of methanol (5000 ml) and allowed to stand overnight in methanol. Modified silica (I) was recovered by filtration. Analysis: Found: P 1.08%; FT-IR(KBr): 1105 cm-1 (P═O and P--O stretch).
Phosphorylated silica (I; 5 g) was mixed with cystamine (7 ml) and left at 60� C. overnight. Ethanol (50 ml) was added to the slurry and refluxed with slow magnetic stirring for 6 hours. Reacted silica was washed with a large excess of ethanol to yield cystaminylated silica (II; 4 g). Analysis: Found; C, 2.32; H, 0.62; N, 1.60%.
Silica (II; 2 g) was stirred overnight at room temperature with acetonitrile containing 4-pentene-1-0-tosylate. Filtration and washing with acetonitrile yielded a light yellow colored product (III). Analysis: Found; C, 2.08; H, 0.51; N, 0.67%.
Silica III (2 g) containing the 4-pentene moiety was stirred in chloroform (50 ml) containing m-chloroperbenzoic acid (2 g), filtered and washed extensively with chloroform to give the final support (IV) containing the desired epoxy ring. Analysis: Found; C, 4.24; H, 0.75; N, 0.15%.
A. Purification of Phospholipase Regulatory Protein
Phospholipase enzymes have been shown to play a key role in the proinflammatory production of various eicosanoids and to be subject to a variety of regulatory mechanisms. During the last decade advances have been made in the isolation and characterization of phospholipase and proteins which regulate enzyme activity. A steroid inducible mammalian protein "lipocortin" has been identified, isolated and suggested to exhibit phospholipase A2 (PLA2) activity. Melittin, a bee venom peptide of 2800 MW has been shown to stimulate PLA2 activity. The inventors have used anti-melittin antibodies to isolate from mammalian sources of PLA2 stimulatory protein termed phospholipase activating protein or PLAP. This work used silica affinity columns prepared via silane chemistry or CNBr activated agarose. Substantially better purification folds were obtained with silica than with agarose. The commercially available organo-silane activated silica showed lesser stability under the slightly alkaline pH condition used. The 10 micron particle size reportedly optimal for organo-silane activated silica was also poorly compatible with the biological extracts used. Column fouling therefore occurred after a minimum number of runs. Separation of PLAP using the novel chromatography supports disclosed herein is described below.
Rabbit polyclonal antibodies were prepared against glutaraldehyde crosslinked melittin and affinity purified using melittin immobilized to the silica IV support. The affinity purified antibodies were then immobilized to similar silica and used to isolate cross-reactive material from mammalian cells grown in culture. Stimulatory activity was defined as the difference between the activity observed in reactions containing cell free sonicates and any endogenous activity associated with fractions from the affinity column. One unit of activity was defined as that amount of purified protein required to produce a two-fold increase in observed PLA2 activity found for 1 mg/ml cell free sonicate. Elution and assay of fractions recovered from the antimelittin column revealed material capable of stimulatino PLA2 activity. Minimum column fouling occurred and in similar experiments extracts from 16 liters of cultured cells could be chromatographed with no apparent increase in column back pressure or loss of performance. This represents a substantial improvement over the art. A comparison of performance of silica IV at the 20 micron size to organo-silane activated 20 micron silica revealed substantially superior performance for silica IV.
Approximately 1 mg of synthetic melittin in phosphate buffer (200 mM; pH 7.5) was immobilized to a 4.6 mm�7.5 cm silica IV column (prepared by manual packing of column in acetonitrile) by recirculating at 25� C. overnight at 0.2 ml per minute. Spectral analysis of the recirculate showed 95% immobilization of the applied melittin. After washing with phosphate buffered saline, 2 ml of anti-melittin anti-sera was applied to the column at 0.2 ml/min. After washing, the bound antibody was eluted with 100 mM glycine at pH 3.0. Fractions were collected and the pH was adjusted to 7.5 with phosphate buffer. SDS polyacrylamide gel analysis showed essentially only the heavy and light chains of IgG in the purified preparation.
Affinity purified melittin antibodies in PBS were immobilized on a silica IV affinity column by recirculating (0.1 ml/min) 1 ml of affinity purified antibody solution (300 ug/ml) through the column overnight. PBS containing 0.05% Tween 20 was used to wash the column extensively. The column was then equilibrated with PBS containing the protease inhibitors phenylmethylsulfonylfluoride (10 uM), bacitracin, (100 ug/ml), benzamidine (lmM) and soybean trypsin inhibitor (5 ug/ml) and 0.05% Tween 20. Cell sonicate was then passed through the column at 0.1 ml/min. The cell sonicate was prepared as follows. Logarithmically growing cells were removed from five 150 cm2 Corning Tissue Culture Flasks (Corning, NY) and concentrated by centrifugation (500 g for 5 min). Cells were resuspended in 1 ml of Pucks Saline F (GIBCO, Grand Island, NY) containing 10 mM Hepes, protease inhibitors (10 ug/ml soybean trypsin inhibitor; 1 mM benzamidine; 100 ug/ml bacitracin and 10 uM phenylmethylsulfonyl fluoride) and detergents (0.05% Tween 20; 0.04% SDS and 1 mM deoxycholate) and quickly sonicated using a Branson sonicator. Sonicates were centrifuged in a microfuge for 20 min at room temperature and the resulting supernatant passed through a 0.2 um millipore filter (Millipore Corp., Bedford, MA).
The filtered sonicate was passed through the antimelittin antibody column at 0.1 ml/min. The column was washed for 30 min at 2 ml/min followed by elution using 50 mM sodium acetate pH 3.1. Fractions (0.5 ml) were collected and stored frozen at -70� until use.
Phospholipase A2 and phospholipase C activities were quantitated radiometrically using appropriate substrates. Reactions were buffered with 200 mM tris pH 9.0, according to conventional methods. In FIG. 1: the curve with open circle represents endogenous activity. The closed circles represent stimulatory activity.
Phospholipase C (PLC) is a term used to refer to a family of enzymes which cleave the polar head group of phospholipids, producing diacylglyceride. Peripheral blood monocytes and polymorphonuclear leukocytes from rheumatoid arthritic patients express elevated PLC activity levels compared to control cells. The elevated activity prefers phosphatidylcholine (PC) as substrate. The microorganism Bacillus cereus has been shown to prefer PC as substrate. The inventors have previously used anti-bacterial PLC antibodies and silane activated silica affinity columns to isolate a mammalian PC preferring PLC enzyme. A similar purification using the disclosed silica IV support is described below.
Rabbit polyclonal antibodies were prepared against Bacillus cereus PC preferring PLC and affinity purified using the antigen immobilized to silica IV. The affinity purified antibodies were immobilized and used to purify mammalian cross-reactive enzyme from cells grown in culture or drawn from human volunteers. Specifically, affinity purified antibodies, approximately 600 mg, were immobilized on a silica IV column. U937 cells, approximately 108 cells, were sonicated and applied to the column. The column was then eluted and the resulting fractions were assayed for phospholipase C activity using phosphatidylinositol and phosphatidylcholine as substrates. As shown in FIG. 2 this procedure resulted in the recovery of mammalian PC preferring PLC.
Phospholipase C isolated from B. cereus was obtained from Boehringer Mannheim (Indianapolis, IN) and crosslinked using gluteraldehyde (3% vol:vol) for 30 min at room temperature. The antigen (2000 units per injection) was mixed with a equal volume of Freunds adjuvant and injected intradermally into multiple sites of New Zealand White rabbits at 2 week intervals. The rabbits were bled 3-4 days following the second injections and the blood allowed to clot overnight at 4�. The next day the serum was removed and the immunoglobulin fraction enriched by precipitation using ammonium sulfate. Affinity purified antibodies were then prepared using silica IV as described.
Briefly, 300 ug of affinity purified antibody in 2 ml of phosphate buffered saline (PBS) pH 7.2, (GIBCO, Grand Island, NY) was circulated through a column of silica IV overnight at 0.2 ml/min. The next day the column was washed extensively using PBS containing 0.05% Tween-20 at 2 ml/min. Cells were concentrated by centrifugation (2000�g for 5 min), and resuspended in 2 ml of PBS containing 0.05% Tween-20 and protease inhibitors and sonicated. The protease inhibitors included: phenylmethylsulphonylfluoride (10 uM), bacitracin (100 ug/ml), benzamidine (1 mM) and soybean trypsin inhibitor (5 ug/ml). The cell-free sonicate was then centrifuged in a microfuge (13,000 �g for 20 min) and the supernatant filtered through a 0.2 uM filter (Millipore, Boston, MA). The filtrate was passed through the anti-PLC antibody affinity column (0.1 ml/min). The mobile phase consisted of PBS with 0.05% Tween-20. The column was then washed using the same mobile phase at 2 ml/min for 10 min and the bound material eluted using 50 mM sodium acetate pH 3.1 at a flow rate of 0.5 ml/min. Fractions were collected (2 ml) in tubes containing 200 ul of 10 x PBS and 400 ul of glycerol.
Reversed phase high performance liquid chromatography (HPLC) is a well accepted technique for the separation of many substances. Using the chemistry described herein we have prepared several typical bonded phases on 20 micron 200 A pore sized silica. While these particle and pore sizes are not optimal for reversed phase HPLC separations the materials were used to validate the chemical method.
Silica I (5 g) containing a trace amount of pyridine was treated with absolute ethanol (20 ml). The mixture was mechanically stirred and octadecyl amine (5 g) added and the reaction left at 60� C. for 6 days. Finally the solution was refluxed for 6 hours and filtered. The solid was washed with methanol, ethanol, and dried to yield the desired silica with C-18 unit (V; Scheme 4). Analysis: Found; C, 1.59; H, 0.55; N, 0.02%. To prepare silica with C-11 unit undecylamine was used in place of octadecyl amine to yield the desired silica C-11 product (VI; Scheme 4). Analysis: Found; C, 2.85; H, 0.54; N, 0.02%.
Silica V and VI was capped according to the art and 4.6 mm�7.5 cm columns packed by hand in 100% acetonitrile. Mobile phases were 0.1% trifluoroacetic acid (TFA) and 80% acetonitrile. Solvent gradients were produced according to the art using commercially available HPLC systems. Samples were cytochrome C, serum albumin, cytochrome C tryptic peptides, insulin and Bacillus cereus PLC. Peaks were detected by diode array ultraviolet absorbance or fixed wavelength detection at 214 nm. Shown in FIG. 3 are the results obtained using a 4.6 mm�7.5 cm column of silica VI. The applied components were cytochrome C tryptic peptides, insulin, cytochrome C, and Bacillus cereus PLC. Buffer A was 0.1% TFA. Buffer B was 80% acetonitrile. A linear gradient of 20 to 100% B over 5 minutes was used at a flow rate of 1.5 ml per minute. A similar sample was chromatographed after washing the column with 800 ml of ammonium hydroxide pH 9.5 for 16 hours (FIG. 4). A similar mixture was also chromatographed using silica V (FIG. 5). A separation of aromatic amines on silica V is shown in FIG. 6.
The reactions described in this example are illustrated in Scheme 5. ##STR13##
Fractogel (hydroxylated polystyrene/polyvinyl benzene, from Toyopearl, TSK HW-65; 32-63 micron; 500 ml) was filtered and the resulting cake swirled with dilute HCl and left standing overnight. After washing with distilled water (5000 ml) and methanol (2000 ml) by decantation the gel was collected by filtration and oven dried at 60� C. for 6 days. 50 g of gel was then slurried with pyridine (300 ml) and the pyridine removed in vacuo until a sludge was left behind. The flask was cooled in a dry ice bath and phosphoryl chloride (50 ml) added slowly to avoid any violent reaction. The flask was left at 60� C. for 2 days. Washing with pyridine, methanol, methanol-water, and methanol yielded the activated Fractogel (1; 40 g). Analysis: Found; H, 6.05; N, 1.14; P, 2.0%.
Activated Fractogel (1; 5 g) was mixed with cystamine (5 ml) and the resultant slurry left at 60� C. overnight. Absolute ethanol (50 ml) was added and the mixture refluxed overnight. The yellow colored product (2) was recovered by filtration after washing with methanol. Analysis: Found; C, 42.09; H, 5.78; N, 3.09%.
Activated Fractogel (1; 5 g) was reacted with diaminodiphenyldisulfide (5 g) as described for cystamine. The final product 3 showed on analysis: C, 51.42; H, 6.28; N, 2.47%.
Fractogel phosphate (1; 5 g) containing a trace amount of pyridine was slurried with octadecylamine (6 g) and left at 60� C. for 2 days. Toluene (100 ml) was added to the flask and the mixture refluxed for 24 hours. The mixture was then filtered and the gel washed with methanol to yield Fractogel C-18 (4). The resin was capped with methyltrimethoxy silane in toluene-pyridine at 60� C. for 24 hours. Analysis showed; C, 48.82; H, 6.26; N, 5 1.34%.
Fractogel phosphate (1; 5 g) and diphenylamine (5 g) were treated with toluene-pyridine (100:1) and refluxed for 24 hours. Subsequent steps were as described above. The final product Fractogel-diphenylamine (5) showed on analysis: C, 49.36; H, 6.30; N, 2.02%.
Fractogel with the cystamine chain obtained above (2 g) was stirred in toluene (50 ml) with a trace amount of pyridine. Trimethoxymethylsilane (0.1 ml) was added to the stirring solution and left for 24 hours. Filtration and washing as described above yielded capped Fractogel-cystamine. Analysis showed; C, 47.79; H, 6.60; N, 3.06%.
Cystamine-containing Fractogel (1; 1 g) was slowly stirred with 1 ml of 50% glutaraldehyde diluted to 4 ml with phosphate buffer pH 7. After 30 minutes at room temperature the reaction mixture at 40� C. was left for 4 days. After washing with methanol, filtration yielded the product (6). Analysis showed; C, 51.18; H; 6.65; N, 2.95%.
Fractogel derivativ (3, 1 g) in acetone (100 ml) was treated with carbonyldiimidazole at 60� C. for 6 days. The product (7) was obtained by filtration and washing with acetone. The imidazole containing product was treated with an equal weight of m-aminophenylboronic acid in 1 M sodium carbonate (20 ml, pH 10) for 4 days at 60� C. The solution was filtered and the final product (8) containing boronic acid (800 mg) obtained. Analysis showed: C, 53.04; H, 6.37; N, 2.80%. FT-IR(KBr): 3600-3100 cm-1 (--OH), and 1734 cm-1 (C=0).
Fractogel with cystamine chain (2; 1 g) was treated with the N-hydroxysuccinimide ester of biotin (150 mg) in dimethylformamide (DMF; 4 ml). The reaction mixture was stirred at room temperature for 6 days. The solution was filtered and washed with DMF and pyridine. The biotinylated resin (9) was capped with trimethoxymethylsilane as described above. Analysis showed; C, 48.93; H, 6.46; N, 3.14%.
Peptide synthesis is a technique where amino acids are sequentially linked to form peptide chains of various lengths. While the chemistry involved can be performed in solution, several systems have been developed for solid phase peptide synthesis. This technique uses a solid support (usually functionalized polystyrene) and frequently specialized equipment for automated delivery of reagents and solvents. The peptide chain grows on the resin and is cleaved from it after synthesis with hydroflouric (HF) or triflouroacetic acid (TFA). The preferred acid will depend on the nature of the amino acids used. BOC-amino acids require HF, FMOC-amino acids TFA. In addition to the synthesis of peptides for evaluation as therapeutic agents there is an increasing demand for peptides in studies of epitope mapping, purification of proteins, and use specific antibodies for cellular and physiological localization of biomolecules. Assays, diagnostics and a host of other applications also require peptides. These studies are frequently carried out in laboratories with little or no synthetic capabilities. We have sought to prepare reagents that would simplify peptide preparation. A particular advantage would be the elimination of the need for strong acids in cleavage from the resin and recovery of the peptide in a form compatible with HPLC purification as well as ready for attachment to a hapten for antibody production.
Herein we disclose the application of derivatized Fractogel in peptide synthesis. Our starting material was Fractogel containing cystamine which provided two features:
The NH2 group to attach any spacer by nucleophilic displacement.
An S-S bond cleavable under mild conditions.
For synthesis of peptides, an amino acid sequences were chosen to be synthesized and an automated peptide synthesizer used to deliver reagents and solvents.
After synthesis the resin was washed with methanol and the peptide cleaved from the resin by reduction of the S--S bond using dithiothreitol or mercaptoethanol. Alternatively the S--S bond was oxidized with performic acid. Organic modifiers such as acetonitrile or detergents necessary for peptide solubility were also added depending on the peptide sequence. (HF or TFA eluted peptides are not frequently recovered in a form compatible with HPLC purification and peptides may be insoluble after removal of the acid). Recovered peptides were directly subjected to HPLC purification and hapten conjugation for antibody production. Amino acid compositions of purified peptides wa confirmed by amino acid analysis. In FIG. 7 are shown the results obtained on synthesis of the peptide Gly-Asn-Phe-Trp-Cys-Glu-Phe-Trp-Thr-Ser-Ile-Asp-Val. This sequence was derived from the deduced sequence for the second extracellular loop of the hamster lung beta adrenergic receptor. The peptide was cleaved from the resin with 1 M dithiothreitol and purified by reversed phase HPLC according to the art. Composition of the recovered peptide was confirmed by amino acid analysis. The recovered peptide contained a free SH group at the site of cleavage and was coupled to the hapten Keyhole Limpet protein for antibody production using SH directed bifunctional crosslinking reagents according to the art.
An alternate approach to the preparation of peptides using readily cleavable S-S functions is to use cis-hydroxyl groups and periodic acid or otherwise acid or base labile bonds. For solution chemistry or as a coupler to already existing resins a BOC or FMOC reagent containing a cleavable function is appropriate. For example BOC or FMOC cystamine. This reagent would preempt the first amino acid in current protocols. All of the above described approaches differ from the art where cleavage occurs at the site of direct attachment to the resin.
We suggest that peptide synthesis may also be improved by the use of BOC or FMOC-dipeptides prepared according to the frequency by which such pairs occur in proteins. A similar concept is being explored for oligonucleotide synthesis. By reducing the number of coupling steps for a given synthesis, yield, purity, and achievable chain length should be increased.
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