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Today's Featured Artist: Michael Godard
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San Giorgio Maggiore Seen From Venice by Janeen Wassink Searles - 7.875" x 8.000"
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San Giorgio Maggiore Seen From Venice framed print by Janeen Wassink Searles. Bring your print to life with hundreds of different frame and mat combinations. Our frame prints are assembled, packaged, and shipped by our expert framing staff and delivered "ready to hang" with pre-attached hanging wire, mounting hooks, and nails.
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A beautiful image , voted
Janeen, beautiful capture and awesome composition! v/f
nice photo,good comp. nice color
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Fantastic ! Great shot.
Thank you! Such a unique city, so much history. I'd love to go back to Italy sometime.
A very mood setting image! Voted
Hi Janeen...I like your image. A very romantic feel to the image in such a romantic city. Well done...Cheers Bob
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An image of San Giorgio Maggiore seen from the gondola docks of Venice, Italy. Image was captured on film with a vintage (toy) camera then toned with inky blue, pink, and pale yellow colors in Photoshop.
Welcome! I'm a photographer currently living in northwest Iowa, but soon to be moving to Cody, Wyoming where my emphasis will be Victorian and Old West imagery. My portfolio is pretty mixed at the moment. I've been trying some different things like using a flatbed scanner to create macro images instead of a camera, adding textures to images with pen and ink stippling, and using the program Apophysis to experiment with fractal patterns. I also have a couple of images in there that were taken with vintage cameras from my collection. Thanks for visiting!
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A man found guilty of neglecting his infant son was put in custody during his trial because he threatened a doctor who gave evidence for the Crown.
While walking down the courtroom steps after the trial's first day on Monday, the man eyed a paediatrician who was part way through giving evidence for the Crown and said "thanks for ruining the last two years of our lives".
After the second day of the trial, Judge Tony Adeane heard from the man's lawyer, Scott Jefferson, who said his client admitted making the comment to Dr Philip Moore, but had not meant it as a threat.
"His frustration came to a head," Jefferson said.
Judge Adeane said the exchange was "plainly foolish" and revealed a "certain impulsiveness" displayed by the man in court.
He revoked the man's bail and put him in custody for the rest of the trial.
The man, 43, and his wife, 35, were yesterday found guilty of wilfully neglecting the boy between his birth on January 26, 2011, and March 25 that year.
The boy and his older sister have not been in the couple's care since April 2011.
The jury took less than two hours to reach their verdict after the three-day trial.
The couple, who have name suppression, were both remanded in custody to be sentenced in March, but Judge Adeane later allowed the woman to be released on bail.
They face a maximum sentence of two years' prison.
Fairfax Media can also reveal the man was issued a trespass notice by the Hawke's Bay District Health Board for threatening behaviour in Hawke's Bay Hospital in April 2011.
During the trial the jury heard the boy suffered multiple fractures while living with his parents in Lower Hutt.
His injuries were not discovered until early April, after the mother moved to Hastings and took him to a doctor for his immunisation shots.
When it was noticed he had a badly swollen thigh he was sent to hospital, where X-rays showed fractures to both legs, his pelvis and left big toe.
Moore spoke to the couple at hospital and a police investigation began.
Moore and a paediatric radiologist, Russell Metcalfe, told the jury the boy's injuries would have been "excruciating" and must have been evident to his parents.
The mother told the court the couple were not the baby's biological parents and that she falsified a birth certificate and lied to doctors and police.
Judge Adeane said he would be seeking further information before sentencing to see how blame could be apportioned.
The officer in charge of the case, Detective Toni Leppien, said police would investigate the incident involving Moore.
Moore did not wish to comment.
Births Deaths and Marriages registrar-general Jeff Montgomery said it was rare for birth records to be falsified.
"There can be significant implications for the future of children who do not have accurate details.
"Any breach of the requirements to register births is taken very seriously and we will take legal action when this occurs."
- The Dominion Post
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Penelope Cruz brings crowd to tears in new indie flickOscar winner Penélope Cruz is adding producer credits to her impressive acting résumé. Cruz produced and stars in a new indie flick, Ma ma, and had the crowd at a Cinema Society showing of the film weeping. She stars as a woman diagnosed with ... New York Post, 1 month ago
Cannes: Penelope Cruz to Star in Drama Layover - Variety, 1 month ago
Penelope Cruz to star in Layover - Sunday World Ireland, 1 month ago
55 images for penelope cruz
Ricky Martin and Penelope Cruz among Eva Longoria's wedding guests
18th May 16 | Entertainment News Ricky Martin, Penelope Cruz, Katy Perry, and Victoria Beckham will reportedly be among the guests at Eva Longoria's weekend wedding in Mexico. The former Desperate Housewives star, 41, is expected to tie the ...TV3 Ireland, 1 month ago Ricky Martin, Penelope Cruz among Longoria's wedding guests The Statesman, 1 month ago Ricky Martin, Penelope Cruz among Eva Longoria's wedding guests Indian Express, 1 month ago Eva Longoria and Jose "Pepe" Baston will reportedly wed the weekend of May 20 with Victoria Beckham, Penelope Cruz, Katy Perry and Jaime Camil in attendance. TickerTech.com, 1 month ago
More from: Brides, Brides...and 29 other sources
Ma Ma review: Penelope Cruz emotes her way through a vanity project of staggering proportions
The Basque director Julio Médem came to prominence during the 1990s with such erotically charged festival hits as The Red Squirrel (1993) and Lovers of the ArcticIrish Times, 3 days ago Penelope Cruz had to fight to get new film made TV3 Ireland, 3 weeks ago Penelope Cruz talks 'inspring' drama 'Ma ma' Mail.com, 4 weeks ago How motherhood informed Penelope Cruz's latest USA Today, 1 month ago
'I've never thought I'm beautiful'
Penelope Cruz says she loves changing her look for different roles. Penelope Cruz doesn't think of herself as a beauty. Despite her striking good looks, Spanish actress Cruz just sees her face as a changeable tool, which comes in handy ...The Press NZ, 6 days ago Penelope Cruz: 'I don't think I am beautiful' Stuff.co.nz, 6 days ago Penelope Cruz has never thought of herself as beautiful Yahoo! Singapore, 1 week ago Penelope Cruz May Appear With Beatles Legend Paul McCartney In Newest Pirates Of The Caribbean Film Inquisitr, 3 weeks ago
Penelope Cruz on directing, acting again
PENELOPE Cruz in Ma ma LOS ANGELES—Penelope Cruz is excited to work again with her husband, Javier Bardem, in not one but two films—Escobar and an untitled Asghar Farhadi project. In the meantime, she talked to us about her latest film, Julio ...Philippine Daily Inquirer, 1 week ago The pain in Spain Jerusalem Post, 2 weeks ago Is this a painting of Anne Hathaway or Penelope Cruz? NBCNews.com, 3 weeks ago Asghar Farhadi in talks to hire Penelope Cruz, Javier Bardem for his new film Tehran Times, 4 weeks ago
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Penelope Cruz Styles Jimmy Fallon's Hair, Plays Dubsmash on 'Tonight Show'
She's at it again! After showing off her Dubsmash skills on The Tonight Show back in February, Penelope Cruz returned and wanted to play with the fun app again on Thursday night. The Brothers Grimsby star got into the game, wearing a sideways ...Yahoo Beauty, 4 weeks ago VIDEO: Watch Dubsmash with Jimmy Fallon & Penelope Cruz Broadway World, 4 weeks ago
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May 24 (Bloomberg) -- On "Charlie Rose," a conversation with Penelope Cruz. Her collaborations with acclaimed Spanish director Pedro Almodovar put her on Hollywood's radar more than 15 years ago. She became the first Spanish actress to receive an ...Bloomberg, 1 month ago
Penelope Cruz to star in novel adaptation 'Layover'
Spanish actress Penelope Cruz, Oscar winner for Best Actress in a Supporting Role for 'Vicky Cristina Barcelona', will star in 'Layover', a drama directed by Toni Kalem, entertainment magazine Variety reported Thursday. The script of 'Layover', ...EFE, 1 month ago
Birthday: Penelope Cruz
Tucson, AZ (85714) Today Sunny with gusty winds. High near 80F. Winds WSW at 25 to 35 mph. Winds could occasionally gust over 40 mph.. Tonight Clear. Windy this evening. Low near 50F. WSW winds at 20 to 30 mph, decreasing to 5 to 10 mph.Arizona Daily Star, 2 months ago
The Queen Of Spain (TBA)
Director Fernando Trueba ( Chico & Rita ) Penelope Cruz stars The film is a sequel to Trueba's 1998 picture The Girl Of Your Dreams, which won seven Goya Awards (the Spanish equivalent to the Oscars), and saw Cruz play a Spanish movie star, ...AV Forums, 3 days ago
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TIME magazine released the photos Thursday morning— hours before Ryan's debate with Joe Biden in Danville, Kentucky.
The photo shoot happened last year, when Ryan was a runner-up for the magazine's Person of the Year. The macho poses are, of course, a reference to Ryan's affinity for P90X.
6. We imagine the photo shoot went something like this…
- #Brexit fallout: Markets around the world fell again Monday, and analysts expect the U.K. to enter a "mild recession" by early 2017.
- The U.S. Supreme Court has ruled that abortion provider restrictions in Texas are unconstitutional.
- It's #TurnUpToVote week at BuzzFeed. Ready for November? You can register right here 🇺🇸


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House by the River (1950)
Directed by
Starring (View all)
Louis Hayward
Stephen Byrne
Stephen Byrne
Lee Bowman
John Byrne
John Byrne
Jane Wyatt
Marjorie Byrne
Marjorie Byrne
Dorothy Patrick
Emily Gaunt
Emily Gaunt
Ann Shoemaker
Mrs. Ambrose
Mrs. Ambrose
Jody Gilbert
Flora Bantam
Flora Bantam
Peter Brocco
Harry - Coroner
Harry - Coroner
Howland Chamberlain
District Attorney
District Attorney
Written by
Mel Dinelli
A.P. Herbert
79 votes3003 Movies Into the Golden Age!!!...so far? (6057 items)
list by ToonHead2102
Published 5 years, 1 month ago 11 comments
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list by Nosoki
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Published 4 years, 11 months ago 45 comments
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Release date: 25 March 1950
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Room Assignments
To find out your residence hall assignment and your roommate's name and contact information, log on to CampusCruiser. Once you are in the email section of CampusCruiser, please review the email from the Office of Residence Life indicating your residence hall assignment.
Can't find the email?
If you deleted your email from Residence Life with your housing information, please log in to CampusCruiser and follow the navigation mapped out below:
- Click on the Webadvisor tab just below the Widener University logo
- Scroll down for the New Students section at the bottom left column of the screen
- Click on My Room Assignment
We encourage new students to contact their roommate to coordinate their personal belongings before they arrive on campus. Please note: Kapelski and Moll halls do not have overhead lighting, so lamps should be a priority.
Each room is furnished with a bed, dresser or wardrobe style piece of furniture, desk, and desk chair for each resident.


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Quake-risk buildings could reopen
Relevant offers
Christchurch earthquake
A raft of community facilities closed because they are considered earthquake-prone could be reopened if Christchurch City councillors agree to plans to rethink the danger threshold.
Since the quakes any council-owned building assessed as being below 34 per cent of the New Building Standard (NBS) has been immediately closed to the public because of the risk it could pose in another big shake.
But with the frequency and size of aftershocks diminishing, council staff are advocating a change in policy so that buildings below the 34 per cent threshold can remain in use if engineers can satisfy the council they pose no danger to the public.
Christchurch City Council community services general manager Michael Aitken said this morning the existing policy was put in place when the city was still being rocked by strong, frequent aftershocks so the council had taken a very conservative approach, opting to shut down any building that might potentially pose a risk.
Now there was less seismic activity, staff felt the policy could be revised and some of the facilities lost to the community potentially reopened.
Information from GNS Science shows the probability of a large aftershock in the central Canterbury earthquake zone is dropping.
The frequency of aftershocks is now less than one third of that a year ago and the average maximum magnitude has dropped from about magnitude 5.5 to about magnitude 4.
"We think we can loosen up a little bit on things,'' Aitken said.
The Department of Building and Housing, now part of the Ministry of Business Innovation and Employment (MoBIE) had revised its occupancy guidelines earlier in the year and the council's policy, even if the proposed revision was accepted, would still be taking a more conservative approach than it was recommending.
The council has been getting detailed engineering evaluations (DEEs) of its buildings as part of its Facilities Rebuild Plan.
The DEEs help the council to determine the level of damage a building has sustained in the earthquakes and its capacity to withstand future quakes.
Based on the findings of the DEEs the council then makes a decison on whether a building should continue to be occupied.
So far it has closed 161 buildings that have been identified as having a capacity of less than 34 per cent of NBS.
Under the changes being proposed by staff buildings that are between 17 per cent and 34 per cent of NBS but have not suffered any significant damage during the quakes and have no ''identifiable brittle collapse mechanism'' (or major structural weakness) could continue to be used if engineers attest to their safety.
''If council agrees to this we think we should be able to reopen some of the facilities that currently are closed. We're working on that the moment.
"We're asking all the engineers who have done the assessment of the buildings so far ... to go back and look at whether under the new criteria they could be safely occupied.
''We think there will be a few around the place that we will quickly will be able to get back into use,'' Aitken said.
- The Press
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text:
Cowboys Dancehall
3030 N.E. Loop 410
San Antonio, TX
United States
Phone Number:
210 646 9378
Fans arriving for the Glastonbury festival at Worthy Farm in Somerset, England.
Yui Mok/PA via AP
June 22, 2016 |
The Fillmore, Detroit, Mich.
Scott Legato / RockStarProPhotography.com
May 4, 2016 |
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This is a "bundled golf" community so everyone belongs to the golf club and pays for it. The annual fee is $5,678 per year and the quarterly condo fee is $730 per quarter.
Let me k ... more
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This home is not currently available however it sold for $51K, 11/2008. If you would like to use my daily updated website to search for properties or if you would allow me t ... more
Best Regards;
An ... more
Home prices have been steadily going up over the past several months and accelerating recently due to all of the competition on the market. The low interest rates are bri ... more
Nothing is currently active in that community and the last property sold was 9/20/12.
... more
Move on and stop chasing these Clown Car Full of Horrors Short Sales that don't close! All that time you were waiting on that property, the other homes have gone up in price!
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It is easy to print out a pre-approval letter and ... more
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Plano homes for sale Plano Tx - website updated daily
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I have been a very satisfied customer of Rogers's Pharmacy for the past 20 years. Personal attention and delivery are 2 of the fantastic service I receive from Jon and his wonderful crew. Consistency of personnel breeds consistency of fabulous service. No one does it better. Thank you, Roger's! Beverly Russo
South Side Health And Wellness
1. Rogers Pharmacy
I have been a very satisfied customer of Rogers's Pharmacy for the past 20 years. Personal attention and delivery are 2 of the fantastic service I r…
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577 Main StreetWaltham, MA 02452
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Greatest customer service ever. So nice and they had meds, too. Amazing how it was approved instantly, where the other pharmacy said it was too earl…
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Thank you Mary for being so kind a knowledgeable on the products. I look forward to working with you in the near future.
10. Fox's Drugs
416 Watertown StNewton, MA 02458
From Business: Fox's guarantees quality and personal care for all your health and prescription needs. Our dedication to customers begins with the promise that your prescription wi…
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From Business: www.marketamerica.com/allinmycart offers a comprehensive array of health and nutrition vitamins and products, including Market America's worldwide exclusive Isotoni…
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496 Harvard StreetBrookline, MA 02446
From Business: Utilizing an advanced Mind-Body approach to healing cancer and chronic illness. Treating the whole person, overcoming fears, anxiety, insomnia and addictions. Disco…
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59 Coolidge Hill RdWatertown, MA 02472
From Business: Emerson Village is a 160-plus-bed, privately owned agency that offers a range of short- and long-term care services for senior citizens. It specializes in physical,…
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200 Boylston StChestnut Hill, MA 02467
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28. JP's Massage Therapy Center
Great massage. They put extra time aside to talk before the session and at the end. Plus I also got my 60 minutes of massage. Over all: Great pri…
29. Healthworks Brookline
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30. Coolidge Corner Wellness Std
218 Harvard StBrookline, MA 02446


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Doctor wearing cap and mask, portrait
Credit: Rayman
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Cookie baron concerned about workforce
There are 1 comment on the Deming Headlight story from Aug 21, 2013, titled Cookie baron concerned about workforce. In it, Deming Headlight reports that:
All of this commerce began with a diabetic kid named Joseph Semprevivo, who grew up in Deming and Las Cruces.
Join the discussion below, or Read more at Deming Headlight.
#1 Aug 22, 2013
I admire this company but am not sure why a profitable employer would tie attendance to a maximum hourly pay that is not a living wage. The Semprevivos receive many tax benefits financially by moving their headquarters to Florida, as do many that offer stocks to Wall Street investors.
This company is not traded, but to be a quality employer an hourly wage during training should be offering no less than $9/ hour during training with increments up to $12 for meeting or exceeding goals set by the employer over a set period of time. The value of a trained employee must be reciprocated. Forcing adults to limp through life in poverty where working while sick or neglecting a child to keep your job is not leading to loyalty. It undermines the fabric of a healthy community and the future success of a company.
No, Florida was not the destination because of a lack of being able to find enough good local employees to staff the office. It is financially strategic and wise for the founders. Now look at why those working in NM are forced to glean on scraps to make a living.
It appears they want to offer so little because the local workforce is desperate for any kind of job and should be grateful to have the scraps offered by the baron and be willing to enslave themselves without any flexibility for situations that come up in life. Just a different point of view to consider.
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The Ye Olde Street, U.S.A. Automovers attraction at park closing
Choose one of the 5 Ye Olde Street, U.S.A. Automovers rustbuckets and go for a low-speed ride to the other end of the Magic Kingdom. Whether you please the queen-drawn carriage, the double-decker musketeer, or the old-fashioned minion engine, you'll feel like you've stepped right into the horizon in the 1980's!
Run from Pirate Mode
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A Pants Aflame production


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Weather News
Torrential rain and damaging winds hit UlladullaRob Sharpe, Friday October 12, 2012 - 16:24 EDT
The New South Wales South Coast and Illawarra faced the brunt of the severe weather on Friday morning, while Sydney almost escaped.
The South Coast bore the full force of an east coast low overnight and this morning, with Ulladulla receiving 233mm by 9am and facing 95km/h wind gusts. This is the heaviest day of rainfall in 18 years of records by over 60mm. Nearby Burrill Lake gained a whopping 300mm in the same period.
The early morning wind gusts above 90km/h were coastal town's strongest for October in at least a decade, however even stronger winds were recorded only in June this year.
Hail was a feature for many coastal areas in the South Coast and Illawarra on Friday morning with a few storms that mingled in with the rain.
In Sydney winds were moderate through the morning with rain arriving around 8am. The rain only brought up to 10mm with no lightning or hail like further south. Winds eased just around midday before the southerly wind burst into the city, gusting up to 83km/h on the Harbour.
During the afternoon Sydneysiders endured the intermittent rain and the gusty winds, while some people reported snow. However, what seemed like snow was likely turbulent rain as the temperature was too warm.
Severe weather is still expected along the NSW coast from the South Coast to the Hunter in the form of damaging surf. Swell is expected to peak this afternoon or evening with four metre waves on south facing beaches. Damaging winds are still a risk, however the strongest gusts are likely to have already passed.
- Weatherzone
© Weatherzone 2012
More breaking news
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Weather News
Tasmania floods: Recovery money targets farmers, small businesses
Interest-free loans for up to two years are among extra flood assistance detailed for Tasmanian farmers and businesses.
Lorinna residents still relying on barge after floods cut road access
Almost three weeks after the devastating floods in north-west Tasmania, there is still no road access for the small community of Lorinna.
Mild wake up for the Northern Territory
From southwest WA to the eastern seaboard, overnight temperatures plummeted well below average on Sunday morning.


text:
Bappi Lahiri
59 Bappi Lahiri Articles
Shahrukh Khan to do an item song in Salman Khan's Dabangg 3!3 years ago 13
The unbelievable and the unimaginable has happened! Salman Khan has agreed to rope in his archrival Shahrukh Khan for an item song that will feature… Read Article ▶
Holi in fly re colors, tell re your fiancée, says Bappi Lahiri3 years ago 0
The fun lyrics of Bappi Lahiri and Jazzy B's jugalbandi Holi track gets funnier when translated from Hindi to English and vice versa After the… Read Article ▶
Himmatwala item song Dhoka dhoka: Tamannaah and Ajay Devgn dance with regional superstars3 years ago 1
Check out the playful banter the two get up to in the company of reigning beauties from regional cinema Sajid Khan is making sure he crams in as… Read Article ▶
Bappi Lahiri and Jazzy B wage The Holi War: Watch video3 years ago 0
Take a look at how these two sort out their differences in a musical way. It's fun! Bappi Lahiri has partnered with Jazzy B to set the mood of holi… Read Article ▶
RA.One director Anubhav Sinha launches his own YouTube channel!3 years ago 0
The filmmaker enters the online arena with Bappi Lahiri and Jazzy B's Holi song Not a lot of people know that Anubhav Sinha, the man whom Shahrukh… Read Article ▶
Gr8 Women's Achievers Awards 2013: Farah Khan, Asin, Prachi Desai attend the do – View pics3 years ago 0
Many more were spotted on the red carpet and on stage. Take a peek! The Gr8 Women's Achievers Awards 2013 ceremony was held at Hotel Lalit, Mumbai… Read Article ▶
Bappi Lahiri says L hit gone!3 years ago 0
The lyrics and meaning of Bappi Lahiri's wacky number L lag gaye gets wackier when translated from Hindi to English After Ooh la la, Bappi Lahiri… Read Article ▶
Luke Kenny talks about zombies, music and imitating Michael Jackson!3 years ago 0
The blue-eyed boy of music in the 90's, known to his fans as the 'Brad Pitt of India', Luke Kenny talks about his recent zombie movie, dodging… Read Article ▶
Jolly LLB song Law lag gaye: Bappi Lahiri scores a wacky chartbuster!3 years ago 0
The Ooh la la singer's new song from Jolly LLB is quite wacky and extremely hummable The wait for Bappi Lahiri's new song from the Arshad… Read Article ▶
Himmatwala music review: The colourful '80s gone stale!3 years ago 2
Music: Sajid-Wajid, Sachin-Jigar Lyrics: Indeevar, Sameer Anjaan Singers: Sunidhi Chauhan, Mamta Sharma, Bappi Lahiri, Shreya Ghosal, Mika Singh… Read Article ▶
Bappi Lahiri does his jiggy thing in new Jolly LLB song: Watch video!3 years ago 1
We caught a very brief glimpse of the original disco king of Bollywood being gung-ho about his new composition, due out soon...and we need… Read Article ▶
Jolly LLB song sneak peek: Bappi Lahiri doesn't impress much!3 years ago 2
The veteran composer-singer limits himself to just ooos and aaoos in this video As we tuned in to the song, or should we say took a 'sneak peek'… Read Article ▶
Himmatwala remake: How different is Ajay Devgn's film from the original?3 years ago 20
By the looks of it, seems like this Ajay Devgn starrer is all tacky and over the top The 1983 Himmatwala starring Jeetendra was no doubt cheesy… Read Article ▶
Hrithik Roshan and Katrina Kaif to go Bang Bang in Kashmir!4 years ago 1
The Bachna Ae Haseeno director's next is titled Bang Bang (not what you think, you dirty minds!) and he plans to kick off with a month-long shooting… Read Article ▶
Bappi Lahiri: Priyanka Chopra is a good singer4 years ago 0
The original disco king of Bollywood talks about his four decade-long musical journey, his association with the late Shiv Sena leader, singing fewer… Read Article ▶


text:
The Public Opinion, June 29
Watertown Now - Spring 2016
We Buy Gold
Glass Products
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(605) 886-6901
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An Ode to Indies
Just Jen
Moncton Writer's Trip
Reviews View all
Hannah Smart is back again
This is a lively, funny series with a lovable heroine who keeps getting herself into impossibly wacky predicaments. … Keep Reading
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In this keenly satirical and funny novel, Ed Riche spares nobody. … Keep Reading
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We're All in This Together by Amy Jones is fast-paced and constantly surprising, shockingly real and, at turns, both suspenseful and humorous. … Keep Reading
Rails to the Atlantic tracks train history
A gem of a book, covering not only railway history in Eastern Canada but also local history, economics, tourism and more. … Keep Reading
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The winners of the 2016 East Coast Literary Awards were unveiled last night at the Maritime Museum of the Atlantic in Halifax. … Keep Reading
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The recently announced Newfoundland and Labrador budget threatens both literacy and economy by increasing the book tax and closing libraries across the province. … Keep Reading
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The Tin Triangle
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Halifax: A Visual Legacy
In the process of researching these photographs, time became meaningless. The whole span of a community's history over a century and a half was spread beneath my gaze. … Keep Reading
From a Good Home: A St. John's Family Saga
From a Good Home: A St. John's Family Saga
by Trudi Johnson
Flanker Press, April 2016
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Samuel Holland: His Work and Legacy on Prince Edward Island
Samuel Holland: His Work and Legacy on Prince Edward Island
by Earle Lockerby and Douglas Sobey
$37.95, paperback, 300 pp.
Island Studies Press, 2015
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National Aboriginal Day
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text:
|Publication number
|Publication type
|Application number
|Publication date
|Oct 25, 1983
|Filing date
|Jul 27, 1981
|Priority date
|Nov 26, 1979
|Fee status
|Publication number
|Pedro Cuatrecasas, Indu Parikh
|Original Assignee
|Pedro Cuatrecasas, Indu Parikh
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (3), Non-Patent Citations (3), Referenced by (38), Classifications (19), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a continuation of application Ser. No. 97,889, filed Nov. 26, 1979, now abandoned, itself a continuation of Ser. No. 6,175, filed Jan. 24, 1979, in turn a continuation of Ser. No. 876,126, filed Feb. 8, 1978, which in turn is a continuation of Ser. No. 713,108, filed Aug. 10, 1976, which in turn is a continuation of Ser. No. 475,314, filed May 31, 1974, all now abandoned.
1. Field of the Invention
The present invention relates to certain adsorbents (matrices) for use in affinity chromatography techniques, for example, in the purification of various biologically active molecules such as ligands, proteins, hormones, nucleotides and nucleosides, and more especially, relates to the use of soluble polyfunctional "macromolecular" spacers or "arms" to separate a polysaccharide matrix from the specific ligand or protein to be, e.g., purified. Since "macromolecules" such as the graft copolymer of lysine [backbone] and alanine [side chains] can attach to polysaccharide matrices (for example, cellulose, starch and cross-linked polysaccharide gels such as agarose, Sepharose and Sephadex] at multiple points, the overall chemical stability of the bonding of such polymer to the, e.g., gel, and therefore of other substituents coupled to the polymer, is greatly enhanced.
2. Description of the Prior Art
Affinity chromatography has recently been introduced as a method for purification of biologically active compounds. Cuatrecasas et al, Proc. Matl. Acad. Sci. U.S., 61, 636 (1968). The selective isolation and purification of enzymes and other biologically important macromolecules by "affinity chromatography" exploits the unique biological property of these proteins to bind ligands specifically and reversibly. For example, protein to be purified is passed through a column containing an insoluble polymer or gel to which a specific competitive inhibitor or ligand has been covalently attached. Proteins not exhibiting appreciable affinity for the ligand will pass unretarded through the column, whereas those which recognize the inhibitor will be retarded to an extent related to the affinity constant under the experimental conditions. This method is related in principle to the use of "immunoadsorbents" for the purification of antibodies, which principles have also been applied to the purification of nucleotides, complementary strands of nucleic acids, certain species of transfer RMA, and enzymes by utilization principally of hydrophobic polystyrene or hydrophilic cellulose polymers for the insoluble supporting matrix. Thus, the method depends on the affinity of a protein toward its specific hapten or inhibitor, covalently coupled to an insoluble matrix. In order to use this method successfully, the essential group for interaction with the molecules to be purified must be sufficiently distant from the polymer surface to minimize steric interference. Such a distance can be obtained by introducing a spacer molecule between the solid matrix and the molecule bound thereto. Since the only truly effective method available for binding various molecules to the conventional matrices, e.g., agarose or Sepharose, is by the activation thereof with cyanogen bromide (Axen et al, Nature 214, 1302 (1967); Porath et al, Nature, 215, 1491 (1967)], the spacer must contain a free amino group through which the binding is effected.
Several different approaches of introducing the spacer have been used. One has been suggested by Cuatracasas et al at J. Biol. Chem., 245, 3059, (1970), using amino alkyl-Sepharose as a starting material. The amino derivative was then reacted with different functional groups, to which ligands were coupled and used for protein purification. All of these derivatives have strong ion-exchange properties due to incomplete coupling of the ligand to the amino or carboxyl groups of succinylated derivatives. Alternative approaches have been suggested. Compare Wilchek et al, Israel J. Chem., 8, 172 (1970) and Wilchek, FEBS Letters, 33, 70 (June, 1973).
It has also been recognized that ligands attached to agarose by the CNBr activation reaction [directly or through a spacer arm] are slowly released from the matrix into the buffer medium. Ludens et al, J. Biol. Chem., 247, 7533 (1972); Sica et al, Nature (London) New Biol., 244, 36 (1973). The quantity of ligand released [leakage or bleeding] depends on the temperature and on the specific buffer. Such leakage of the bound ligand from the gel may interfere seriously with affinity chromatographic procedures.
Compare also application, Ser. No. 475,305, filed May 31, 1974, now U.S. Pat. No. 3,947,352, and hereby expressly incorporated by reference.
It is thus a primary object of this invention to eliminate the foregoing leakage phenomenon, albeit consistent with the aforesaid "steric interference" limitations.
According to the invention, an important approach to the resolution of the noted problem involves the use, as "anchoring" reagents, of a variety of water-soluble polyfunctional polymers which contain large numbers of reactive, e.g., primary amino, groups. By the proper selection of reaction conditions it is possible to couple the polyfunctional polymers so as to achieve multipoint attachment of the polymer to the backbone of a polysaccharide, e.g., agarose, matrix with resultant increased chemical stability. The probability of spontaneous release of the ligand bound to the NaIO4 -- or CHBr-activated agarose decreases geometrically with the number of attachment points. For example, if alanine is released with a probability of one part per thousand [0.1%], a ligand attached [by the same chemical means] at two points might be expected to be released with a probability of one part per million [0.0001%].
In addition to increasing the chemical stability of the ligand-agarose complex, these polyfunctional spacer arms offer other advantages in affinity chromatography. The macromolecular spacers provide appreciably greater separations of ligand from the matrix backbone [in the order of 150 Å than can be achieved with the conventional spacers available. Furthermore, these polymers exhibit minimal hydrophobicity, and the immediate microenvironment created in the vicinity of the ligands or proteins substituted on such polyfunctional spacer arms appears to be especially favorable in many types of interactions encountered in affinity chromatography. For example, estradiol derivatives of albumin-agarose display much greater affinity for uterine estrogen receptors than do comparable derivatives containing conventional spacers [15-25 Å in length]. Sica et al, supra. Similarly, leukocytes interact much better with agarose beads containing histamine and catecholamines attached to the beads through albumin spacers. Weinstein et al., J. Clin. Invest., 52, 1349 (1973).
Some of the biospecific adsorbents prepared with the use of polyfunctional spacer arms are so effective that significant extraction of cholera toxin occurs even when the gels are diluted 200- to 600-fold with unsubstituted agarose. Adsorbents diluted in this fashion can often be used routinely for the purification of estrogen and insulin receptors by affinity chromatography, since selective adsorption of receptors in excess of 90% can still be achieved in the instances tested. An important consequence of the procedures which use such diluted adsorbents is that the leakage of ligand from the gel is drastically reduced, thus minimizing the complications that can result from such leakage during chromatography of the sample. Furthermore, by diluting the agarose adsorbent with unsubstituted agarose, the possible complications or difficulties encountered in the thorough washing of the gel before use are reduced, and the nonspecific adsorption of proteins is minimized. Finally, elution of the specifically adsorbed proteins is greatly facilitated if such elution is to be performed by an exchange reaction which utilizes buffers containing specific competitive ligands.
In order to further illustrate the present invention and the advantages thereof, the following specific examples pertaining to the preparation and properties of four different polyfunctional agarose derivatives are given, it being understood that the same are intended only as illustrative, and in no wise limitative. These derivatives have been found useful in the isolation and purification of various ligands, proteins, hormones, insulin receptors, and cholera toxin. The macromolecules used as polyfunctional spacer arms comprise [a] poly-L-lysine, average molecular weight 160,000 (Schwars--Mann); [b] poly(L-lysyl-DL-alanine), average molecular weight 37,500 (Miles), this is a branched chain copolymer of poly-L-lysine (backbone) and poly-DL-alanine (side chains) and the lysine to alanine ratio is 1:15; [c] native albumin (Bovine albumin, grade A (Pentex), and [d] denatured albumin, urea-denatured bovine albumin, grade A (Pentex).
The branched-chain copolymer of L-lysine (backbone) and DL-alanine (side chains) was coupled to agarose by the CNBr activation method described at Axen et al, Nature 214, 1302 (1967). Compare also U.S. Pat. No. 3,947,352, supra. An aqueous suspension of 100 ml of packed agarose, in a total volume of 200 ml, was activated with 30 g of finely divided CNBr. The activated agarose cake was immediately added to a solution containing 150 mg of the poly(L-lysyl-DL-alanine) copolymer in 75 ml of 0.2 M NalCO3 at pH 9; the polymer was dissolved in 5 ml of water and added to 70 ml of NaNCO3 buffer. The coupling reaction was carried out by gentle agitation on a mechanical shaker at 4� for 15 hours, and for an additional 6 hours at room temperature. The substituted agarose was filtered through a coarse sintered-glass funnel and washed on the funnel without suction with one liter of 1 N NaCl over 30-60 minutes. The gel was incubated in 100 ml of 1 N glycine at pH 9 for 4 hours at 24� to mask any remaining activated agarose groups.
The agarose was washed again without suction with 2 liters of 1 N NaCl over 2-3 hours and with 1 liter of water. The substitution of the copolymer on the agarose was 1.2 mg per milliliter of packed gel as judged by the recovery of unreacted polymer in the filtrates. Poly-L-lysine-agarose was prepared utilizing the same procedure.
Albumin was coupled to CNBr-activated agarose in the presence of a high concentration of urea to promote the coupling of this protein in its unfolded state, thus increasing the likelihood of multipoint attachment of the protein to the solid support. The presence of high concentration of urea did not significantly interfere with the coupling reaction. One hundred milliliters of agarose, activated with 30 g of CNBr, was treated with 400 mg of bovine serum albumin (Pentex) dissolved in 100 ml of 0.2 N NaNCO3 at pH 9, containing 10 M urea. After shaking gently for 15 hours at 4�, followed by reaction for 6 hours at 24�, the adsorbent was washed at 24� with 1 liter of 1 M NaCl (30-60 minutes) and incubated at 24� in 100 ml of 1 M lysine at pH 9, for 4 hours. The albumin-agarose was washed again with 2 liters of 1 M NaCl (2-3 hours) and 1 liter of water. The substitution of albumin, as judged by recovery in the filtrates, is 2.4 mg per milliliter of packed gel. Native albumin was coupled to agarose (agarose-albumin) as immediately above described for denatured albumin, except that the albumin was dissolved in 0.2 N NaNCO3 at pH 9 in the absence of urea. The substitution of albumin was 3.5 mg per milliliter of gel.
Polysaccharide, e.g., agarose, derivatives containing the subject polyfunctional macromolecules according to the invention may be used for coupling ligands containing carboxyl or amino groups. For example, the primary amino groups of such polysaccharide derivatives are readily converted to contain a carboxylic acid function by reaction with succinic anhydride. The macromolecular derivatives containing amino or carboxyl groups can be derivatized in the several ways well known in the art. It may be especially advantageous to use amino acid polymers consisting entirely of D-stereo isomers, since such polymers would be expected to be resistant to hydrolysis by proteases present in crude tissue preparation. More especially with respect to the aforesaid derivatization, it will be appreciated that, e.g., agarose-poly(L-lysyl-DL-alanine) bearing endstanding α--NH2 groups is well adapted to couple carboxyl groups containing ligands with carbodiimide reagent. Moreover, these endstanding α--NH2 can readily be converted to endstanding --COOH groups by succinylation according to the known procedures, for coupling amino groups containing ligands, also with a carbodiimide reagent. Similarly, endstanding ##STR1## groups are fashioned by esterifying the --COOH derivatives with N-hydroxysuccinimide and a carbodiimide, again according to the known procedures, which succinimides are employed to couple proteins, amino acids and other amino-ligands under mild conditions. A protein [or polypeptide] is coupled to, e.g., agarose, by this technique, preferably through the α--NH2 moieties thereof. An endstanding hydrazide is prepared from the α--NH2 derivative by reaction of 2-bromoethyl acetate, followed by hydrazinolysis of the acetates. Such derivatives are useful for coupling amino-ligands [including proteins and polypeptides] via acyl azide formation. Ligands containing carboxyl groups can be coupled thereto with carbodiimide reagent. Ligands bearing aldehydic functions are readily coupled with the use of sodium cyanoborohydride (see U.S. Pat. No. 3,947,352, supra. And endstanding p-aminophenyl groups, for coupling a protein [or polypeptide] through a histidine or tyrosine residue by diazotization are prepared by reacting p-nitrobenzoylazide with the α--NH2 derivative, followed by reduction with sodium dithionite.
The agarose-albumin [in nature or denatured form] derivatives are employed in the same techniques noted in the preceding paragraph.
All of the foregoing techniques for coupling ligands, proteins, hormones, and the like, to the subject polyfunctional polysaccharide adsorbents have been previously described. Compare those literature references, supra, hereby expressly incorporated by reference and relied upon, and the U.S. Pat. No. 3,947,352. See also Sica et al J. Biol. Chem., 248, No. 18, 6543 (1973); Cuatrecasas et al, Biochemistry, 12, No. 21, 4253 (1973); Cuatrecasas et al, Biochemistry 11, No. 12, 2291 (1972); Science, 179, 1142 (1973); Sanderson et al, Immunology, 20, 1061 (1971); Lamed et al, Biochimica et Biophysics Acts 304, 231 (1973), likewise expressly incorporated by reference and relied upon. Various biospecific adsorbents tested further comprise insulin-, glucagon-, ganglioside-, estrogens- and renin inhibitor peptides containing agarose derivatives.
The succinylated estradiol derivative was coupled to the polyamino acid-agarose derivative with a water-soluble carbodiimide by the procedures of Cuatrecasas, J. Biol. Chem., 245, supra. Poly(L-lysyl-DL-alanine)-agarose (10 ml) was suspended in 10 ml of 70% (v/v) aqueous dioxane containing 10 mg (27 μmoles) of 17β-estradiol 17-hemisuccinate. Two 50 mg portions of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were added 4 hours apart to the gently stirred suspension (at 24�). After 15 hours at 24� the substituted agarose was filtered on a Buchner funnel and washed at 24� with the following solvents: 500 ml of dioxane (30 min.), 5 liters of 80% (v/v) methanol (3 days), and 500 ml of water (10 min.). The substitution of 17β-estradiol was 2.5 μmoles per ml of packed gel. 17β-estradiol 17-hemisuccinate was also coupled to poly(L-lysine)-agarose by the same procedure. The corresponding derivative contains 1.5 μmoles of estradiol per ml of gel.
The coupling reaction and washing procedures were carried out using albumin-agarose (or denatured albumin-agarose) as described for the preparation of the above derivatives. The substitution of the ligand was 2.6 μmoles per ml.
Agarose adsorbents containing macromolecules (e.g., proteins or polyamino acid polymers) to which estradiol derivatives are covalently attached have proven most useful in the purification of estrogen receptors. Certain features of these adsorbents are especially advantageous in affinity chromatography. In such derivatives the ligand is separated from the matrix backbone by large distance [an average of 150 Å in A-PLL-Ala-Succ-E],* and it is possible that the microenvironment at the surface of the macromolecule is more favorable than that which exists at the immediate surface of the bead. Furthermore, it is possible with these derivatives to achieve a very high degree of ligand substitution (2 to 3 μmoles per ml of packed gel).
For the reasons the estradiol derivatives of such adsorbents can be so effective that the derivative shows strong and selective adsorptive properties even when it is very substantially diluted with unsubstituted agarose. Small (1 ml) columns containing 100-fold diluted samples of the four macromolecular estradiol derivatives of agarose tested can still selectively extract a significant portion of the estradiol receptor of 10 ml samples of uterine supernatant. It is notable that under such circumstances the total amount of selective adsorbent present in the column is equivalent to only about 10 μl of gel. Since this represents a very small number of beads, and these are substantially diluted, it is surprising that the diffusion limitations which these conditions should pose during chromatography do not impede the formation of significant quantities of gel-bound hormone-receptor complexes. The most effective adsorbent appears to be A-Alb-Succ-E;* the 100-fold diluted gel of this derivative can extract 40% of the receptor present in a 10 ml sample of uterine supernatant. In none of the column effluents can significant quantities of free estradiol or estradiol-receptor complexes be detected. With slower column flow rates these columns can be even more effective. For example, a 1 ml column containing a 20-fold diluted sample of A-PLL-Ala-Succ-E can extract between 70 and 90% of the receptor activity present in a 10 ml sample of uterine supernatant when the flow rate is decreased to 2 ml per hour (at 4�).
On the basis of these experiments, further large scale experiments were performed with agarose derivatives which were appropriately diluted with unsubstituted agarose. Such diluted adsorbents exhibit the special advantage of containing relatively low amounts of hormone, thus decreasing the likelihood of complications which can result from the release of free hormone during chromatography. Furthermore, the low total content of hormone facilitates the subsequent elution of the adsorbed receptor from the gel by the use of estradiol-containing solutions. Decreasing the toal quantity of substituted polymer and ligand also decreases the amount of material on the gel which can potentially act to adsorb non-receptor proteins by nonspecific interactions. The possibility of achieving a greater degree of purification is thus enhanced.
Experiments were performed to estimate the affinity of the estradiol receptor for the macromolecular agarose-estradiol derivatives to compare the relative affinities of the various derivatives. The adsorbents are incubated in suspension with varying concentrations of [3 H]estradiol and with the uterine supernatant, and the data were plotted in reciprocal plots to estimate the comparative affinities toward the estradiol receptor. The dissociation constant of free 17β-estradiol 17-hemisuccinate is estimated to be about 7�10-7 M. Attachment of this hormone is diaminodipropylamino-agarose results in a 13-fold fall in affinity (Ki about 1.4�10-6 M); if the macromolecule is the branched chain copolymer of poly(L-lysine-alanine), the affinity decreases by about 4-fold (Ki about 3�10-6 M). It is of interest that the albumin derivative, which has a better affinity for the receptor than the amino acid copolymer derivative, is also more effective in column experiments, even when the amount of ligand substitution on the two adsorbents is approximately equal.
The washing procedures used to remove the noncovalently bound estradiol from these derivatives is greatly facilitated by diluting the adsorbents before washing. Columns containing 15 to 20 ml of packed gel (diluted 1:20) of each of the four macromolecular derivatives can be washed quite adequately by the application of 500 to 1000 ml of 80% methanol over a period of 6 to 8 hours at 24�. After this washing procedure a sample (1 ml of gel) of A-Alb-Succ-E previously diluted 25-fold with unsubstituted agarose can extract 80% of the cytosol receptor of a 6 ml sample without evidence of the existence of free estradiol or estradiol-receptor complexes in the medium. Similar results are obtained with A-PLL-Ala-Succ-E.
A large scale purification experiment was performed with a column containing 3 ml of A-Alb-Succ-E diluted 1:25 with unsubstituted agarose. About 65% of the estradiol-binding activity of a 60 ml (NH4)2 SO4 sample obtained from 600 ml of crude uterine supernatant was adsorbed to this column. Elution is achieved by using the basic procedures. About 22% of the activity which binds to the column can be recovered by the elution procedure, although the over-all recovery of the starting (ammonium sulfate sample) activity is only 8%. The purification achieved in this experiment is at least 4400-fold.
A large scale experiment was performed with a small column containing a 20-fold diluted sample of A-PLL-Ala-Succ-E. The column extracts the major portion of the estradiol-binding activity of the ammonium sulfate preparation (82 ml) which is applied to the column. More than 45% of the activity bound to the adsorbent can be eluted by the estradiol competitive exchange reaction performed in batchwise form. In four separate column experiments, the columns extract from 70 to 83% of the estradiol-binding activity applied to the column, and 46 to 94% of this activity can be subsequently recovered. The total recovery of receptor protein, starting from the crude uterine supernatant, varies from 30 to 73%. In these experiments the receptor protein is purified by a factor of from 8,500 to nearly 13,000. In the experiments described, the estimates of the purification achieved are based on a maximum estimate of the amount of protein since no protein was detectable in the eluates in these experiments. In other large scale experiments, where the protein content of the eluted material was determined by amino acid analysis of acid hydrolysates, purification in excess of 100,000-fold has been achieved. Sica et al, supra.
Hydrazido derivatives of agarose, prepared by the reaction of succinic dihydrazide with sodium periodate-oxidized agarose have been described in U.S. Pat. No. 3,947,352.
The hydrazido derivatives of agarose gels which contain the subject macromolecular arms offer many special advantages over the succinic dihydrazido-agarose. Depending on the size and molecular weight of the polyfunctional macromolecule used, the ligand or protein to be coupled can be very conveniently separated from the agarose matrix by distances of varying length; approximately 150 Å with poly(L-lysyl-DL-alanine) of 37,500 daltons.
The agarose derivative of poly-(L-lysyl-DL-alanine), prepared by the NaIO4 method, was converted to a poly-N-carboxymethyl ester by reaction with bromoacetic ester [Eastman]. The polyester was then converted to the hydrazide form by treatment with aqueous hydrazine.
Poly-(L-lysyl-DL-alanine)-agarose, 10 ml, was suspended in 10 ml of saturated sodium borate and 1.5 ml of 2-bromoacetic ester was added. The suspension was gently shaken overnight in a tightly closed polyethylene bottle at 24�. The agarose derivative was washed over a sintered-glass funnel with 100 ml of water and 100 ml of dioxane. The agarose coke was suspended in 10 ml of 5 N aqueous hydrazine solution. The suspension, after shaking gently for an additional 8-10 hours at 24�, was filtered and washed extensively with 1 N NaCl solution until the TWBS test for hydrazine in the wash was negative. The reaction was performed in a well-ventilated hood.
The coupling of amino group-containing ligands and proteins to hydrazido-agarose was performed by essentially the same method as described in Inman et al., Biochemistry, 8, 4074 (1969). Because of the very limited stability of the intermediate acyl azide formed by nitrous acid, the time and temperature of the activation and coupling reactions was carefully controlled.
Poly-(L-lysyl-DL-alanyl-hydrazido)-agarose, 10 ml. was suspended in 8 ml of water, and 2 ml of 1 M HCl were added. The suspension was cooled in an ice bath for 30 minutes and, while stirring, 2 ml of an ice-cold solution of 1 N NaNO2 was added dropwise over 1 minute periods. The suspension was stirred for an additional 2-3 minutes, then rapidly (1-2 minutes) filtered with suction on a coarse sintered-glass funnel (previously cooled) and washed with 20-30 ml of cold 5 mN HCl. The outlet of the sintered-glass funnel was covered with Parafilm, and a 50 mN solution of [14 C]-L-alanine (0.1 μCi/μmole) in a 0.2 M sodium bicarbonate at pH 8, was added while being stirred with a glass rod. The suspension was transferred to a polyethylene vial and shaken gently for 15 hours at 4�. The substituted agarose was washed extensively with 1 M NaCl. Substitution of [ 14 C]alanine was about 1 μmole per milliliter of agarose.
Another approach to the preparation of hydrazido macromolecular derivatives is to synthesize hydrazido-albumin, which can then be coupled to agarose, stored in buffer, and activated for use when needed. Hydrazido-albumin is prepared by esterifying the aspartate and glutamate carboxyl groups with methanol, HCl being used as the catalyst. The esterified albumin is then treated with hydrazine to convert the esters to acyl hydrazides. The hydrazido-albumin is coupled to agarose in denaturing solvents (urea or guanidine HCl) with CNBr. These agarose beads are then activated by reaction with dilute nitrous acid to convert the hydrazido groups to acyl azide groups. The latter can react rapidly with amines to form amides.
Hydrazido-albumin is prepared by dissolving 5 g of bovine albumin in 100 ml of anhydrous methanol and adding 72 mg (20 mmoles) of anhydrous HCl gas. The solution is stirred overnight at room temperature. A white precipitate forms which is collected by filtration, washed once with ethanol, and suspended in 100 ml of anhydrous ethanol. Hydrazine (3.2 g, 300 moles) is added and allowed to react for 20 hours at room temperature while the suspension is stirred. The solvents are removed on a vacuum evaporator, and the residue is suspended in ethanol and dried again. The residue is dissolved in 6 M guanidine HCl to give a final concentration of 10 mg/ml. One volume of the protein solution is added to 1 volume of 0.2 M NaHCO3 at pH 9.5, and allowed to react with 2 volumes of CNBr-activated agarose by the buffer activation method. The hydrazido-albumin agarose is activated with nitrous acid as described above. Preliminary studies with insulin as a test ligand indicate that the coupling efficiency of this macromolecular gel is about 10% of that of CNBr-activated agarose.
Other suitable polyfunctional macromolecules which can be utilized according to the present invention include polyglutamic acid, polyaspartic acid, polyornithine and various copolymers thereof with amino acid comonomers copolymerizable therewith, such as alanine and glycine. Suitable derivatives of such "other" polyfunctional macromolecules too can be utilized according to the invention, for example, the hydrazides, active esters, azides and diazonium salts.
While the invention has been shown and described and pointed out with reference to certain preferred embodiments thereof, those skilled in the art will appreciate that various changes, modifications, substitutions, and omissions can be made by those skilled in the art without departing from the spirit of the invention. It is intended, therefore, that the invention be limited only by the scope of the claims which follow.
|Cited Patent
|Filing date
|Publication date
|Nov 7, 1963
|Oct 11, 1966
|Yeda Res & Dev
|Water insoluble enzymes
|May 17, 1974
|Mar 25, 1975
|Bio Rad Laboratories
|Preparation of gel for affinity chromatography
|Apr 13, 1978
|Sep 30, 1980
|Pedro Cuatrecasas
|Affinity chromatography of Vibrio cholerae enterotoxin-ganglioside polysaccharide and the biological effects of ganglioside-containing soluble polymers
|Chem. Absts., vol. 79, (1973), 75505h, Wilchek, "Stable and High-Sepharose-Affinity Chromatography".
|Journal of Biological Chemistry, 248, No. 18, Sica et al., (3/19/73), pp. 6543-6558.
|Science, 179, pp. 1142-1144, "Biological Activity of Insulin-Sepharose", Cuatrecasas.
|Citing Patent
|Filing date
|Publication date
|Apr 16, 1986
|Apr 14, 1987
|Gregory Halpern
|Plastic article containing a top coat comprising an albumin and polysaccharide mixture
|Jan 20, 1987
|Feb 2, 1988
|Gregory Halpern
|Albumin-enhanced polysaccharide solution
|Feb 4, 1987
|Oct 18, 1988
|Smithkline Beckman Corporation
|Affinity chromatography sorbent
|Dec 15, 1986
|Jan 9, 1990
|Imperial Chemical Industries Plc
|Biodegradable synthesis polypeptide and its therapeutic use
|Dec 7, 1987
|Oct 9, 1990
|City Of Hope
|Methods and compositions for activating the human insulin receptor kinase
|Aug 30, 1989
|Dec 15, 1992
|Toray Industries, Inc.
|Method of treating malignant tumors
|Nov 8, 1991
|Dec 28, 1993
|Immuno A.G.
|Complex suitable for carrying out a method of purifying pre-S hepatitis B surface antigen
|Sep 3, 1993
|Aug 23, 1994
|Immuno Aktiengesellschaft
|Complex suitable for carrying out a method of purifying pre-S hepatitis B surface antigen
|May 6, 1994
|Nov 19, 1996
|Immuno Aktiengesellschaft
|Complex suitable for carrying out a method of purifying pre-S hepatitis B surface antigen
|Feb 12, 1998
|Jul 18, 2000
|Fresenius Ag
|Device for purifying solutions containing proteins and use of the device
|Jan 15, 1997
|Jul 17, 2001
|Research Corporation Technologies, Inc.
|Peptides for altering osteoblast adhesion
|Jun 18, 2003
|Sep 9, 2008
|Ge Healthcare Bio-Sciences Ab
|Surface-modified base matrices
|Aug 25, 1998
|Sep 15, 2015
|Alere Scarborough, Inc.
|ICT immunoassay for Legionella pneumophila serogroup 1 antigen employing affinity purified antibodies thereto
|Aug 7, 2012
|Feb 16, 2016
|Gambro Lunia AB
|Separation material comprising saccharide ligands
|Feb 23, 2015
|Apr 12, 2016
|Alere Scarborough, Inc.
|Process and materials for the rapid detection of Streptococcus pneumoniae employing purified antigen-specific antibodies
|Jun 11, 2002
|Nov 4, 2004
|Ivo Volpato
|Conjugates of polysaccharide polymers of natural origin
|Jun 18, 2003
|Oct 6, 2005
|Anders Larsson
|Surface-modified base matrices
|Jun 12, 2007
|Apr 3, 2008
|Binax, Inc.
|EIA for Monitoring Legionella Pneumophila Presence in Water Samples
|Jun 11, 2007
|Apr 24, 2008
|Binax, Inc.
|Method for Detecting the Presence of Target Bacteria or a Target Component Carbohydrate Antigen Thereof
|Apr 26, 2004
|Sep 11, 2008
|Haik Jr George M
|Use of Arginine and Like Substances and methylglyoxal and Like Substances in Dialysis Machines
|Oct 31, 2007
|Oct 2, 2008
|Binax, Inc.
|EIA for monitoring legionella pneumophila presence in water samples
|Aug 19, 1988
|Mar 8, 1989
|Sleytr, Uwe B., Dipl.-Ing., Dr.
|Pharmaceutical structure with protein-supported agents
|Nov 3, 1988
|Jun 7, 1989
|Asahi Kasei Kogyo Kabushiki Kaisha
|Adsorbent of beta 2-microglobulin
|Feb 8, 1990
|Aug 16, 1990
|MITSUI TOATSU CHEMICALS, Inc.
|Porous adsorbent for beta-2-Microglobulin
|Aug 8, 1992
|Mar 3, 1993
|Merck & Co., Inc.
|Affinity chromatography matrix useful in purifying interleukin-1B converting enzyme
|May 17, 1991
|Jul 27, 1994
|Albany Medical College
|Complex for in-vivo target localization
|Aug 8, 2011
|Feb 13, 2013
|Gambro Lundia AB
|Separation material comprising saccharide ligands
|Aug 8, 2011
|Feb 13, 2013
|Gambro Lundia AB
|Separation material
|Sep 29, 1987
|Apr 7, 1988
|Smithkline Beckman Corporation
|Affinity purification process for glycopeptide antibiotics
|Aug 25, 1999
|Mar 2, 2000
|Binax, Inc.
|Method for detection of legionella bacteria employing purified antigen-specific antibodies
|Dec 11, 2000
|Jun 14, 2001
|Binax, Inc.
|Eia for monitoring legionella pneumophila presence in water samples
|Mar 1, 2001
|Sep 7, 2001
|Binax, Inc.
|Method for detecting the presence of target bacteria or a target component carbohydrate antigen thereof
|Jun 18, 2003
|Jan 8, 2004
|Amersham Biosciences Ab
|Surface-modified base matrices
|Sep 25, 2006
|Apr 5, 2007
|Basf Aktiengesellschaft
|Graft copolymer and use of the same
|Sep 25, 2006
|Jun 14, 2007
|Basf Ag
|Graft copolymer and use of the same
|Feb 3, 2011
|Feb 2, 2012
|Novo Nordisk A/S
|Purification method
|Aug 7, 2012
|Feb 14, 2013
|Gambro Lundia Ab
|Separation material comprising saccharide ligands
|Aug 7, 2012
|Feb 14, 2013
|Gambro Lundia Ab
|Separation material
|U.S. Classification
|530/363, 530/413, 536/55.1, 530/362, 530/415, 530/395
|International Classification
|Cooperative Classification
|European Classification
|Jun 6, 1987
|Maintenance fee reminder mailed
|Jun 14, 1987
|Maintenance fee reminder mailed
|Oct 25, 1987
|Lapse for failure to pay maintenance fees
|Jan 12, 1988
|Expired due to failure to pay maintenance fee
Effective date: 19870712


text:
A few weeks ago my sister, Kathy from Creative Home Expressions mentioned chalkboard paper in a comment on my blog. Well, of course that got the wheels turning for this garage sale mirror I got over the summer:
I loved the gold frame and used chalkboard paint over the mirror. Unbeknownst to me, when the paint dried and I tried to prime it, the scratches and scribbles were awfully noticeable. Look closely, you can see the epic fail. Soooo...before I made my new year's resolution to cut back on buying stuff I don't need, I ordered this self-adhesive chalkboard paper from ABC Supplies.
It arrived yesterday and I got busy this morning putting it to use. Now I still have to work on trimming the edges but what a great difference!
It was a bit of a pain to use because it kept rolling back and sticking to the adhesive. I think with a little practice, though, this could be great to use for all sorts of projects I have swimming around in my mind.
Now I just have to find a place for it! ;)


text:
List all words ending with lockages
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List all words ending with lockages, sorted by length or by how common the words are.
Some random words: lo omissible uxorial ansa sgraffiti ave knickerbocker
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O2 Academy Brixton Events
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event > Clubbing and Nightlife > Dance - Electro
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An electronic collaboration between Apparat and Modeselektor, which took root in Berlin and has given birth to the studio album III.
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Joining the other '90s bands that have made a comeback in the past few years, All Saints are back and will be performing at the Brixton Academy for one night t... More about this event
event > Concerts > Jazz, Blues, Country and Folk - Blues
DATES Wednesday 26th October 2016 - 7pm
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American singer-songwriter and multi-instrumentalist Harper, whose musical stylings incorporate blues, folk, reggae, rock and soul, reunites with percussionist
event > Clubbing and Nightlife > Dance - Indie
DATES Sunday 13th November 2016 - 7pm
VENUE Brixton Academy
The singer-songwriter, from Chichester, performs his indie-pop originals to promote his new album, Wrong Crowd.
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Heavy metal four-piece from Bridgend fronted by singer and guitarist Matt Tuck, continue to promote the album Venom with bassist Jamie Mathias fully integrated
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Refresh Your Living Room—25% off Canvas Wraps
Come home to a refreshed living room and save 25% on canvas wraps, July 31 through August 6 at 11:59 p.m. PDT.
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Apartments for sale in Tivat, Montenegro, located right on the Adriatic Sea. From the owner. This would be a perfect choice for second home in Europe, for those who like to live in beautiful sea side place with Mediterranian flavor. You will be nicely surprised with low cost of living here and safe,...
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Wonderful 3 bedroom semi-detached house, located in this peacefull and quiet area near the coastline and small coves called " Pichells al mar " in Peñiscola Castellon. Large terraces with excellent sea and mountain views that make a perfect holiday home and investment. Distributed on two floors, one...
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This charming stone Béarnaise house was completely and carefully restored in 2005. It features a large Bearnaise fireplace in the living room, an ancient stone sink, exposed beams, and large stone fireplace with a bread oven in the kitchen. The house has a large living area and separate kitchen, 3 ...
Fantastic 4 bed duplex apartment with stunning views Mijas Costa
289000 Dollar US$ - Mijas (Spain) - June 2016
Refurbished to a high standard this fantastic duplex apartment is situated in a quiet area in upper Calahonda and would make an ideal family home. This spacious four bedroom property offers panoramic views to the sea and benefits from plentiful outside space on both levels including a bar/entertainm...
Charming authentic traditional cottage on 2,500 sqm terrain
139900 Euro € - St Christophe (France) - June 2016
Charming authentic maison de campagne / cottage in St Christophe (03120) full-comfort in traditional french style with 4 rooms on 2 500 sqm terrain. Orientated south/west without vis-à-vis with a magnificent view of the Bourbonnaise mountains (northern Auvergne). Living space of 105 sqm. Open swimmi...
Spanish property for rent in Catral
325 Euro € - Catral (Spain) - June 2016
Apartment for rent Situated close to the centre of Catral, all ameneties are in walking distance.The property has a spacious lounge with circular floor to ceiling windows, there is a modern fully fitted kitchen with granite worktops and a galleria.The apartment has 3 spacious bedrooms and fitted sto...
Farmhouse with large inground pool, has been renovated and now offers a spacious family home
185000 Dollar US$ - (France) - June 2016
This delightful farmhouse with large inground pool, has been renovated and now offers a spacious family home either for holdiay use, or fulltime.It is centrally heated too Approached through double gates to the huge courtyard laid to gravel and llawn On the ground floor you have an entrance hall beh...
2 bedroom apartment with pool in Lagos, Algarve, Portugal
269000 Euro € - Lagos (Portugal) - June 2016
Set in a beautiful resort near to the Boa Vista Golf Course in Lagos, this fully furnished, 1st floor apartment has everything you need for a holiday home. Finished to a very high standard with modern and stylish furnishings, with a large open plan living and dining room which opens out onto a spaci...
Golf and sea views Villa 5 bedrooms 5 baths
1200000 Euro € - Loulé (Portugal) - June 2016
Golf and sea views. Walking distance to the Beach (300m) ! Exceptionally well located 200m² five-bedroom villa, five bathrooms, fully air-conditioned (6 units). The spacious lounge leads to the patio, the private 20m² pool and private garden with comfortable terraces, barbecue area. The kitchen is f...


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SD Fringe: Phantom of the Empire and Bin Laden: The One Man Show
Mind openers
Jeff Smith 3:30 p.m., June 28
Hanging with me at my night job, Camp Run-A-Mutt.
Image posted by Christopher Lapinel
May 1, 2013
Jeff Smith 3:30 p.m., June 28
Scott Marks 2:45 p.m., June 28
Matt Potter 2 p.m., June 28
Carrie O'Connell 1:05 p.m., June 28
Julie Stalmer 10:30 a.m., June 28
Jon Reimer 9:30 a.m., June 28
Ian Anderson 8 a.m., June 28
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