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Saturday, April 10, 2010
© Copyright 2015 Albany Herald
ALBANY, Ga. -- The Dougherty County Board of Education will eliminate one of the last major hurdles still standing in the way of named Superintendent Joshua Murfree taking over the school system's reins in the near future.
Because Murfree does not possess state K-12 teaching certification, the Georgia Professional Standards Commission requires that Murfree apply for a permit and that "the system must verify ... that the applicant for the permit is the best-qualified applicant available."
"It's a hurdle we'll have to cross because we do want to follow the law," Board Chairman David Maschke said after a Feb. 24 board meeting.
Maschke provided Board member Milton Griffin an opportunity to add the "best-qualified applicant available" language into his motion when the Board named Murfree superintendent at its March 24 meeting, but Griffin didn't change his motion. As a result, the Board will likely vote Murfree as the "best-qualified applicant available" at Monday's 7 p.m. board meeting at the downtown DCSS Administration Building.
Because four of the seven Dougherty County Board of Education members were unable to attend Wednesday's called board meeting to discuss Murfree's proposed contract and the timeline of when he may start, the meeting was postponed. It will be rescheduled for the near future.
Per e-mail discussions with Maschke, Murfree was utilizing sample contracts provided by the Georgia School Boards Association, as well as the current contract of retiring Superintendent Sally Whatley, to write up his own contract proposal. Whatley's contract draft was written and developed by attorney Tommy Coleman, and the details were negotiated between Whatley and Board Chair C.W. Grant.
Whatley earned $173,143.32 during Fiscal Year 2009.
"I told him if he wanted to prepare and submit a proposed contract he could ... or that the school board attorney would prepare a proposed contract and he would send it to him," Maschke said. "(Murfree) opted to submit a proposed contract to us, and that's where we're starting. He submitted a proposed contract, which the board is now in the process of reviewing, and we're going to have a called board meeting to discuss the contract.
"The contract has to be a document that is satisfactory to both the board and Dr. Murfree and has to be approved by the majority of the board."
Maschke said Murfree wouldn't attend the first meeting on his contract, which will be discussed in a closed-door executive session.
"But there may be a subsequent meeting where Dr. Murfree or Dr. Murfree and his attorney could come and meet with the board to work out details in a more expedited manner, face-to-face," Maschke said.
Through a Georgia Open Records request of Murfree's Albany State University e-mail account, The Herald discovered that Murfree's correspondence with some board members began well before Whatley announced her retirement last August. Murfree had e-mailed board member Anita Williams-Brown May 6, 2008, along with 36 other people he referred to as friends in a subsequent May 12 e-mail, to pledge $1 to raise funds for his participation in the Muscular Dystrophy Association's lock-up fundraiser.
Williams-Brown voted to appoint Murfree as superintendent at the Board's Jan. 27 meeting, and The Herald later uncovered that she had a business relationship with Murfree dating back to 2003 and that Murfree wrote a letter of recommendation for her in 2001 when she was applying for a principal's position with the Dougherty school system.
Murfree's May 6, 2008, e-mail reads:
"Please click on the link belwo (sic) ... This sifor (sic) a great cause. I amasking (sic) all myfriends (sic) to participate. I have tried the link several times and it works. I am asking that easch (sic) of my friend (sic) send $1.00 in my name.
SUPPORT the Muscular Dystrophy Campaign and keep me out of JAIL!
Dr. Murfree"
Murfree received an e-mail from board member Velvet Riggins May 7, 2009, in regards to a Girl Scouts Day at Albany State University. The e-mail reads:
"Dr. Murfree,
Thank you for speaking with me today. The Program Manager has settled on Oct. 24, 2009. (sic) Girl Scout Day at ASU. Girls attending will be grades 6-12th. The Campus Girl Scouts will set up a campus tour with girls. We have schedule (sic) several Campus Tours all over the (sic) Georgia to give the girls an opportunity to visit colleges and hopeful (sic) expand their career opportunity (sic).
Velvet Riggins
Membership Manager"
In a Jan. 28 interview with The Herald, Riggins said she had met Murfree before through attending Albany State sporting events and because of her job at Girl Scouts of Historic Georgia. As membership manager, she has 56 ASU campus Girls Scouts.
Riggins has been one of Murfree's strongest supporters as she provided other prominent names of successful out-of-the-box superintendents from around the country at the Feb. 3 special called Board meeting. She also provided The Herald with a December 2008 Ebony magazine that briefly included Murfree in an article. Of the four board members to vote for Murfree to be named superintendent at the March 24 Board meeting, Riggins was the only one to comment on her decision -- via an e-mail after the meeting -- while James Bush, Williams-Brown and Griffin briskly left the meeting without talking to reporters.
Riggins sent Murfree an e-mail Nov. 11, 2009, that she forwarded from her mother and Dougherty County Commissioner Muarlean Edwards regarding a meeting with Murfree. It is believed to be in reference to a pilot program called Project SHIELD for which Edwards holds the volunteer director title. The program aims to combat truancy at Turner Elementary School. It is a partnership between the Albany State University Foundation, Turner Job Corps and the Dougherty County School System.
The meeting was set for Nov. 12, which interestingly is the same day Murfree's application for the Dougherty County superintendent position is dated. For a point of reference, the deadline for superintendent applications was Nov. 24. Since the Feb. 3 school board meeting, Edwards has become a fixture at board meetings.
Calls made by The Herald for clarification about the e-mails to Riggins and Edwards were not returned.
The e-mail, which included a PDF attachment titled "policy-attendance-law," reads:
"Dr. Murfree, I am looking forward to seeing you tomorrow, November 12. (sic) 2009 at 10:00a.m. (sic)
Attendance Law:
*Control of Child
*Early Pickup
*Unexcused Absent
*A.M. School Schedule
*Food Service
Saturday School:
Top Administration:
*Organization Structure
*Directors etc.
*Hands on
Thanks, Muarlean"
The Open Records request also revealed an e-mail exchange between Murfree and W. Frank Wilson, who writes columns for The Albany Southwest Georgian, a weekly paper circulated predominately in the African-American community. The Southwest Georgian has been the only media outlet Murfree has granted interviews with since he was appointed superintendent on Jan. 27.
Wilson is also an ASU employee and the director of the school's Center for the African-American Male (CAAM) and also is an academic adviser. Murfree previously was the CAAM director.
The Feb. 1 e-mail is in response to The Herald's coverage of board member Michael Windom asking board members to "reconsider" their choice of Murfree and requesting a special called board meeting. Wilson's e-mail reads:
"Added some stuff as a result Sunday newspaper (sic)"
Murfree responds:
"I don't read the Hell...rald [Devil's Paper]."
Wilson replies:
"Touche my brotha (sic)"
Murfree then asks:
"Explain (sic)"
Wilson writes:
"I was merely saying ok to the fact that you do not read the Herald (sic)
"I am so proud of your demeanor during this circus."
Murfree appreciated the compliment. He replies:
"Thank you. If it had not been for Martin, Malcolm, Medgar, Emmitt, Lucy Diggs-Slowe, W.E.B., Booker T., George Washington Carver, Sojourner Truth, Jesse, The Little Rock Nine, Wilson and Murfree would really be catching hell. It is our time ... If not us then the future of our children is [NOT]!!!"
Wilson ends the exchange:
"Success comes from confidence, (sic) Confidence comes from preparation (sic) we know what we know (sic) end of story (sic)"
On Feb. 2, Wilson told Murfree about recording a segment for WALB-TV. It reads:
"Just recorded Viewpoint at TV 10 (sic)
"FYI there are many( (sic) Black and White) who want you there so PLEASE don't let this deter you (sic)
"Channel 10 selected my comments from many that were submitted
-- Herald government reporter J.D. Sumner contributed to this story.
More like this story
- Board squabbles over request by ministers ( April 29, 2010 )
- Bush sought follower as leader ( February 25, 2010 )
- DCSS to vote on contract ( May 25, 2010 )
- Murfree lacks board support for contract renewal ( October 18, 2012 )
- Attorney general to mediate between press and school system ( April 21, 2010 )


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Favorite Office Time Wasters
Richard Marini finds the best and the brightest distractions.
Posts filtered on Category
Commercials we're sold on: Amstel Light
It's a little belated for Valentine's Day, but this ad asks the eternal question: Who says guys aren't romantic? Tip o' the tam to Laura L. for the link. [Read More]
Commercials we're sold on: The Wellington (NZ) Zoo Valentine's Day event
Way down under, the Wellington Zoo is promoting an adults-only Valentine's Evening event with this saucy little (and NSFW — audio only) ad starring some of their more startled denizens. [Read More]
Commercials we're sold on: An eyebrow-raising advert
From the folks who brought you the drum-playing gorilla. (Scroll down after clicking the link to find the embedded video). Tip o' the tam to Rene G. for the link. [Read More]
Commercials we're sold on: Virgin Atlantic celebrates 25 years in flight
Virgin Atlantic Airline is celebrating 25 years in business with this wayback ad that, from Asteroids to brick-sized cell phones to greed-is-good, suspender-wearing yuppies almost makes one pine for the long-ago Thatcher/Reagan years. As an aside, we used to write about the airline industry back in '84, and the swath Virgin Atlantic cut in that […] [Read More]
Commercials we're sold on: Clash of the philosophers
Who knew philosopher elections could get this down and dirty? [Read More]
Commercials we're old on: The Rise and Fall of Pete the Meat Puppet
Warning: This very strange video contains scenes depicting suckling, whoring, coke, self-cannibalism and urinal-cleaning. But you shouldn't miss it anyway. [Read More]
Commercials we're sold on: JC Penney
Who put you in the doghouse? (woof, woof-woof-woof-woof) [Read More]
Commercials we're sold on: Adecco. Better work, better life
In some cities, crosswalk signals make specific noises so the vision-impaired can tell whether or not it's safe to cross the street. And here's how it's done. [Read More]
Commercials we're sold on: Holiday Inn Express
Subtly subverting the "white guy acting goofy in comparison to cool black people" message. [Read More]
Show More

subset: commoncrawl


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Jennifer Aniston [1] and her husband-to-be Justin Theroux got engaged in August 2012 after meeting on the set of Wanderlust two years earlier. While prepping for their big day, the couple has shared tons of sweet moments, from red carpet cuteness to steamy beach PDA. In honor of Jennifer's 45th birthday today, we're taking a look back at her most adorable moments with Justin — keep reading to see them all now.
Getty [2], Fame Flynet [3], Universal Pictures [Array], Terry's Diary [4]
Justin was by Jennifer's side as she promoted Life of Crime at the Toronto Film Festival in September 2013.
Jennifer and Justin lounged on a boat during their vacation in Capri, Italy in June 2012.
Jennifer and Justin were beaming while out running errands around NYC in September 2011.
They tanned their toned bodies together in Cabo in January 2013.
Jen and Justin were the picture of perfect love on the Oscars red carpet in February 2013.
Jennifer and Justin took a picturesque stroll through NYC in September 2011.
They posed arm in arm at the LACMA's Art and Film Gala in October 2012.
The couple held hands as they arrived at NYC's Rockefeller Plaza in September 2011.
Jen wore a bikini next to a shirtless [5] Justin while in Cabo at the end of 2012.
Justin planted a smooch on Jen during her Walk of Fame ceremony in LA in February 2012.
The couple held hands during a day of shopping in NYC in May 2013.
Jennifer showed off her engagement ring during an October 2012 trip to Santa Fe, NM.
In September 2011, Justin put a loving arm around Jennifer during one of their many walks around NYC.
Jennifer and Justin shared a look of love in Paris in June 2012.
The cute couple toured the Vatican Museums in June 2012.
Jennifer and Justin layered up for a loved-up walk through NYC in September 2011.
The happy couple posed for photos at Elle's 18th Annual Women in Hollywood Tribute in October 2011.
Jennifer and Justin left the Shoreditch House in London while she was overseas promoting Horrible Bosses in July 2011.
They hung out with Drew Barrymore [6] and husband Will Kopelman in October at a LACMA 2012 Art + Film Gala in LA.
Jennifer was a nominee for her work on the Lifetime movie Five at the January 2012 Directors Guild Awards in LA. Justin was right by her side the entire night and the pair even snuggled up and happily posed for a few photos.
They snuck in some PDA during their December 2012 stint in Cabo.
Jennifer and Justin took a September 2011 stroll through the West Village.
They held hands in January 2012 after leaving a movie theater in LA.
In November 2012, Jennifer and Justin had a laugh on the red carpet together at the American Cinematheque Awards in LA.
Justin was full of laughs during Jen's July 2011 Hand and Footprint ceremony in LA.
Jennifer and Justin posed for photographer Terry Richardson following a NYC dinner date in June 2011.
Photo courtesy Terry's Diary [7]
Jennifer and Justin held hands and wore matching aviator sunglasses at the airport after attending Jen's London premiere of Horrible Bosses in July 2011.
They cuddled up for a romantic walk through NYC in September 2011.
Jennifer and Justin chatted with Reese Witherspoon [8] at Elle's 18th Annual Women in Hollywood Tribute in October 2011.
They arrived at NYC's Museum of Modern Art in November 2011 holding hands.
Justin held an umbrella over the couple as they ventured out during a rainy NYC day in September 2011.
The couple left their NYC building in October 2011 wearing matching hats.


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LANSING, Mich. (AP) -- High-level talks over fixing Michigan's deteriorating roads are at a standstill.
Republican and Democratic leaders can't agree much on how to even proceed.
Feeling burned by passage of a right-to-work law, Democrats won't consider tax increases without public assurances that Gov. Rick Snyder will veto other legislation. Democrats want a repeal of a law guaranteeing better wages on government construction projects taken off the table, along with talk of dividing the state's electoral votes proportionally.
Majority Republicans counter that Democrats must move beyond making demands and put forth their own road-funding proposals so everything is on the table.
Neither side appears to be budging. Snyder says progress is going slower than he'd like if the state budget is to be finalized by June.
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I read or hear one stupid coach or player says they made mistakes or its correctable I will flip out. The Dolphins were outcoached, outschemed and outhustled tonight in all aspects. QB situation needs to be cleared up, special teams coach needs to be fired and this team quit after the second half. Horrendous.
A truly disgraceful display of football and an embarrassment to NFL football in general.


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BRADFORD - On Friday morning, the University of Pittsburgh's Bradford campus received a bomb threat similar to those that have been plaguing the university's main campus for the past two months.
According to UPB Director of Communications and Marketing Pat Cercone, at 10:30 a.m. Friday the main campus received an email stating there was a bomb in Hanley Library on the Bradford campus.
Donald R. "Captain" Allegretto, 69, of 879 Rasselas Rd., Wilcox, died Friday morning, April 13, 2012 at his residence following a lengthy illness.
He was born on Aug. 25, 1942 in Buffalo, N.Y., son of the late Raymond and Betty Sample Allegretto. On Aug. 5, 1967 in St. Ann's Church, Wilcox, he married Katherine "Kaki" Gelsick, who survives.
He was a lifelong resident of Rasselas, having attended Wilcox and Johnsonburg schools. He was a Vietnam-era veteran of the U.S. Army, serving overseas as an SP4 with Co. B, 166th Eng. Bn.
The St. Marys Area Flying Dutch baseball team lost a 10-9, nine-inning contest to the Brookville Raiders Thursday afternoon at Berwind Park.
Brookville took the lead 7-5 after three innings of play. The Dutch scored four times in the fourth inning to take the 9-7 lead. Brookville would tie the game at nine with two runs in the sixth inning. Neither team would score until the top of the ninth when Brookville was able to score a run on one hit, a hit batsman and an error.
With trout season set to begin in the region on April 14, the Straub Brewery's annual Fishing Program was under way Thursday, with 75 tagged trout to be stocked in area waterways.
Don "Flip" Bobenrieth, sales representative for Straub's north central market, began the day by picking up the rainbow trout, ranging in size from 19 to 26 inches long, from the Lake Clear Creek Fish and Deer Farm in Emporium.
"I went down this morning and got them. Yesterday we stocked Emporium and today we'll be stocking the other creeks in DuBois, Johnsonburg, and Brockway," Bobenrieth said.
April has been designated Sexual Assault Awareness Month, a period in which outreach groups place extra emphasis on raising public awareness about sexual violence and educating people and the public at large about how to prevent it.
Beginning in the fall, the Elk County Catholic School System will incorporate iPads into their curriculum.
JAY TWP. - The Pennsylvania State Police have issued a warning to Jay Township residents urging them to lock their vehicles and take valuable items inside following a recent string of burglaries and thefts from motor vehicles in the area.
Trooper Craig A. Smith, the investigating officer in two recent thefts from a motor vehicle, said the criminal activity has peaked within the last month and the investigation has yet to yield any suspects.
Richard L. Carrara, 76, died unexpectedly on Monday, April 9, 2012, at the Cleveland Clinic.
He was born in Coal Hollow on March 31, 1936, a son of the late Joseph and Inez Contrini Carrara.
Residing in Fairview, Richard owned Carrara Steel & Carrara Steel Erectors, Amthor Steel, Serafini's Restaurant, the Inn at Presque Isle and Sparkling Ponds Winery. He was a member of St. Jude the Apostle Church.
RIDGWAY - Township supervisors from Elk County had concerns about the state's new oil and gas drilling law versus local ordinances at a recent meeting of the Elk County Gas Task Force.
Fox Township Supervisor and Roadmaster Randy Gradizzi and Jay Township Supervisor Murray Lilley questioned representatives from the State Department of Environmental Protection (DEP) about protections in the new law (Act 13 of 2012) and whether they were sufficient to adequately shield area residents and water sources from the impact of Marcellus Shale drilling.
RIDGWAY - Ridgway Mayor Guillermo Udarbe and Consul General of the Philippines Mario De Leon Jr. and his staff attended a meeting with Congressman Glenn Thompson [PA-5] Wednesday afternoon at his office in Bellefonte to encourage sponsorship of a bill aimed to allow the duty-free entry of Philippine garments into the U.S.
Currently the bill, H.R. 2387, Save Our Industries Act, has 20 bipartisan cosponsors hoping to grant the duty-free entry of Philippine garments into the United States if the textiles and fabrics are made in the U.S.


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Indoor-Outdoor Living Room
An industrial garage door brings the outdoors in.
This gorgeous live/work home used to be an old workshop in Montreal's Plateau area. Architect Natalie Dionne bought the bare-bones space and transformed it into her dream home with partner Martin Laneuville. The garage door with windows provides loads of natural light in the winter, and opens to create a patio-like space in the warmer months.
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Made in 1973, it should be fine. Pre WWI models should not be fed high velocity ammo
A buddy of mine lent me his 1973 Marlin 39A Mountie to take to the range. Can I use high velocity ammo with it? I plan to use CCI mini-mag, Velocitor, maybe some Stingers. Any idea where I can get info regarding older rifles and newer types of ammo? Thanks!
Made in 1973, it should be fine. Pre WWI models should not be fed high velocity ammo
I'm not as good as I once was but I'm better ONCE than I ever was.
Team 38-55 #29
Marlin League #80
Team Old Pharts #101
Team Shotgun #21
1973?? "Older rifle"?? Godamighty!! I got socks older than that.
If in good order and clean your 39A is safe in any 22RF ammo made today.Though I dought you'll see top accuracy out of the hyper velocity ammo you mentioned.I never could in any .22.
Not nearly old enough to have structural problems with HV ammo. Doesn't mean it'll shoot them accurately, though... Like the other poster said, it's the early guns prior to the 39A that will have problems. Try out some standard velocity ammo as well, though.
God, I feel antique when '73 is considered "old".
Brophy's book is the definitive work on Marlins, shows what the 39 bolts looked like before and after high-velocity manufacturing mods.
Have fun! You'll want one of your own when you finish.
Stan S.
Ain't that the truth!God, I feel antique when '73 is considered "old".
And I bought my first Marlin in 1964!!
I thought my '51 39A was just middle aged!
My Dad once said "1973? I've got hemorrhoids older than that."
Now Hypervelocity ammo should be avoided on principle. It's expensive and inaccurate. The worst of both worlds. Oh and it also is very hard on your recoil buffer (which can be upgraded), so make that all three worlds.
Costco should sell bullets in those bins, like walnuts. Or you could buy two five gallon buckets of bullets, shrink-wrapped together.
Coffee is hard to make when you haven't had your coffee yet. This is called the "pre-caffeinated paradox".
"The problem with internet quotes is it is very hard to determine their authenticity." --Abraham Lincoln
There is nothing to writing. All you do is sit down at a typewriter and bleed.
Ernest Hemingway


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New York Metropolitan Region
Goes to the Movies
October 26, 2013
Colin Firth as Mr. Darcy
Come join us as the New York Metro Region "Goes to the Movies: Celebrating Pride and Prejudice," a day long event featuring speakers Carrie Rickey, former film critic of the Philadelphia Inquirer; Professor Lisa Kasmer of Clark University; and Metro's Co-Regional Coordinator, Linda Dennery, all addressing how different films have approached different scenes and characters in Jane Austen's most loved novel. The 10:00 AM - 4:00 PM event will be held in Manhattan at the Fifth Avenue Presbyterian Church at 7 West 55th St, between Fifth and Sixth Avenues. Registration will open after Labor Day.
For more information email: firstname.lastname@example.com.
—Meg Levin


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Symbol Use and Symbolic Representation: Developmental and Comparative Perspectivesby Namy,Laura
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Symbol Use and Symbolic Representation: Developmental and Comparative Perspectivesis the proceedings of a workshop held at Emory University in 2002 to discuss the difficult and age-old issue of what makes a symbol symbolic. The issue shifts towards exploring the relation between apparent symbolic behavior and actual symbolic insight on the part of the user or recipient. The workshop discussed the pitfalls of inferring symbolic understanding from apparently symbolic behaviors and possible criteria that would enable us to ascertain when a symbol is being employed in an intentional, communicative, representational manner. Broken down into three parts, this volume: *focuses on the factors that influence the emergence of symbolic behavior in young, typically developing children; *turns to an examination of individual and population differences in symbolic development and the ways variability in symbol use can inform the cognitive mechanisms underlying symbolic insight; and *explores symbolic understanding in non-human animals. The text ends with a synthesis of recurring themes, questions, concerns, and conclusions, and offers a new perspective on the process of understanding the relation between symbol use and symbolic insight.


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All Posts Tagged With: "Apple"
Apple (AAPL) Sets Guinness Records
May 17th, 2011 • 0 comments • Filed Under • by Trifecta
The 2011 Guinness Book of World Records Gaming Edition has confirmed what most gamers already know- Apple is a serious contender in the world of gaming. As stated recently on Apple Insider, "Apple's success with the iPhone 4 and the App Store has earned the company several impressive records in the 2011 Gamer's Edition of […]
Apple (NASDAQ: AAPL) Sets World Records
May 16th, 2011 • 2 comments • Filed Under • by Trifecta
Apple (NASDAQ: AAPL) has made the news yet again. To the delight of Apple fans around the world the company is receiving lots of recognition for making it into the Guinness Book of World Records. Fans should be excited to know that they didn't receive just one recognition, but several. Rewards went out for the […]
Apple Involved in Yet Another Location-Information Legal Battle
May 13th, 2011 • 1 comment • Filed Under • by Trifecta
The United States District Court for the District of Puerto Rico is the latest to file a lawsuit against the technology company, Apple (NASDAQ: APPL). The lawsuit alleges that Apple transmitted phone users locations to third-party advertisers which constituted fraud and unfair trade practices. This lawsuit also names The Weather Channel and Pandora Media as […]


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Willa M. King, 75, of Columbia City, passed away Wednesday, Aug. 17, 2011 at 11:30 p.m. at Miller's Merry Manor, Columbia City.
Born Sept. 2, 1935 in Breathitt, Ky., she was the daughter of Willie and Clara (Little) Young.
Her formative years were spent in Kentucky where she completed her education.
On Oct. 6, 1956, she married Darrell King at Springfield, Ill.
In 1960, she moved to Whitley County from Illinois. She worked as a CNA at Whitley County Hospital for many years and retired from The Nursing Connection as a home health aide in 1999. She was a member of Thorncreek Bethel Church of God.
Survivors include her daughters, Susan A. Moehring and Judy A. (Jack) Smith both of Columbia City; brother, Chester (Ladean) Young of Columbia City; three grandchildren and one great-grandchild. She was preceded in death by her parents, husband, Darrell King on Jan. 30, 1999; sisters, Lucille Maloney, Alberta Clem and Olean Cassidy.
A graveside service will be held Saturday at 11 a.m. at Greenhill Cemetery, Columbia City.
In lieu of flowers, the family requests memorials be given in memory of Mrs. King to Thorncreek Bethel Church of God.
Arrangements have been entrusted to DeMoney-Grimes Countryside Park Funeral Home, Columbia City. Please visit www.demoneygrimes.com to send family condolences or sign the guest register book.


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text:
by Kit Williams (1979)
was the first of its kind, often imitated,
never surpassed,
this treasure hunt book inspired imaginations
around the world.
by David Blaine (2002)
features a $100,000 Challenge,
authored by "the son of John who
nearly stepped off a cliff."
by Edgar Allen Poe (1843) describes how
the carcass of an odd insect leads to
the discovery of Captain Kidd's
lost treasure.
by M.R. James (1904) tells the legend
of the treasure hunt for the seven-eyed
(1972) Mystery writer Laurence Olivier
invites loverboy Michael Caine to his
mansion of puzzles and games to exact
deadly revenge. Video - DVD
(1973) James Coburn is the sadistic host
of a continental party game to die for.
Written by Anthony Perkins & Stephen
Sondheim. Video - DVD
Blame these films for inspiring me to
do mystery and treasure hunt parties which
eventually led this Fool to his Errand.
featuring quotes from The
Devil's Dictionary by Ambrose Bierce.
by Nick Baxter
A tribute to antique wood and molded plastic,
recreated online.
by Robert Abbott
Don't miss his outrageous essay Video
Games Are Incredibly Stupid!


text:
This member hasn't added any personal information yet.
Leave Nikigreybird a message to find out more.
Posted 08/21/2013 10:04:53 EDT
Not true. Most who use the rear doors have Charlie cards.
Posted 12/19/2012 09:35:12 EST
A very helpful historical perspective on a complex issue. Scientists and fishermen are natural allies.
Posted 08/06/2012 02:14:45 EDT
Green Line ridership is up. The T could become more efficient with funding from congestion pricing and a gas tax.
Posted 07/29/2012 11:53:17 EDT
Romney is not a "Friend of Israel"; he is a friend of the Israeli Right Wing. Viser's reporting is accurate and appropriate.
Posted 07/15/2012 11:25:29 EDT
I thought that in recent years it had been concluded that the fire started due a gas leak in a fixture in the basement ceiling, sparked by a match struck by an employee.
Posted 06/05/2010 02:02:12 EDT
A minor correction to this article re: the Commonwealth Avenue Carriage Lane Promenade project. It's my understanding that abutters and other residents needing access to their homes will be able to dr more »
Posted 06/08/2009 04:30:27 EDT
The question is about a driver's ability, not age. Some drivers have excellent skill; they have always, and will continue, to drive safely without incident or accident, well into old age. Others are p more »
©2015 Boston Globe Media Partners, LLC
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text:
Program Number:
Program Type:
Nov. 1, 2012 | Thursday, 7:00pm
Multiple Times:
First Thursday of every month, 7:00pm-9:00pm
Location: Nature Research Center - Daily Planet Science Cafe, Raleigh
What's cooler than absolute zero? Find out at Science Trivia nights at the NC Museum of Natural Sciences, where questions feature a mix of science facts, current news, pop culture and scientific discoveries. Held in the new wing's Daily Planet Cafe on the first Thursday of every month, this is a great opportunity to mingle with friends, compete for prizes and bragging rights ... and perhaps even learn something cool.


text:
|Viewing 1-25 of 606 Record(s)
|Title: Keeping Austin green ( review of Environmental City: People, Place, Politics, and the Meaning of Modern Austin by William Scott Swearingen, Jr).
|Authors: Black,Lauren
|Source: Texas Books in Review, Winter 2010-2011, 30 :4 ,pg 12-
|Subjects: Swearingen William Scott/Austin- Books
|Additional Information: ID: 158
|Ref ID: 91497
|Title: Keeping Austin green ( review of Environmental City: People, Place, Politics, and the Meaning of Modern Austin by William Scott Swearingen, Jr).
|Authors: Black,Lauren
|Source: Texas Books in Review, Winter 2010-2011, 30 :4 ,pg 12-
|Subjects: Swearingen William Scott/Austin- Books
|Ref ID: 91573
|Title: Texas parenting news: How does your child talk and hear?
|Source: Texas Child Care, Winter 2010, 34 :3 ,pg 4 of insert-
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|Title: Different takes on an ugly institution (Review of "The Folly of Jim Crow: Rethinking the Segregated South edited by Stephanie Cole and Natalie J. Ring)
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|Source: Texas Books in Review, Summer 2012, 32 :2 ,pg 3+-
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|Source: Texas Child Care, Summer 2011, 35 :01 ,pg 24- 27
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|Ref ID: 91767
|Title: Early Childhood Intervention: How to refer children with suspected hearing loss
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|Title: The working life: Stories from the 9 to 5 - Michael Brewer, 80, clock repairman
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|Title: Behind the lines: Big D-vide - The John Wiley Price corruption investigation has only begun, but it has already reopened the age-old rift in Dallas between north and south and white and black. Will things be different this time?
|Authors: Sweany,Brian D.
|Source: Texas Monthly, Sep-11, 39 :9 ,pg 12+-
|Subjects: Dallas (Tex.) - Race relations Price John Wiley Political corruption Nealy Kathy Fain Dapheny Watkins Craig Lipscomb Al Collins Carr P. Sr. Williams Shawn Hill Don Farrington Sheila Lee D'Angelo Hodge Terri
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|Title: Coca-Cola Valued Youth Program high school winning essay: What will I remember the most?
|Authors: Rugg,Kayla
|Source: IDRA Newsletter, Sep-11, 38 :08
|Subjects: Peer group tutoring of students Tutors and tutoring Intercultural Development Research Association (IDRA)
|Ref ID: 91790
|Title: Postcards: Cuero bets on fracking
|Authors: Elbein,Saul
|Source: Texas Observer, Sep-11, 103 :14 ,pg 29-32-
|Subjects: Cuero (Tex.) Petrohawk Energy Corporation Eagle Ford Shale Petroleum industry and trade Meyer Sara Post Krause Charlotte Kleinecke David Reibschlager Elizabeth Hydraulic fracturing
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|Title: Employee privacy: getting the right balance - Technology makes it possible for employers to monitor workers
|Authors: Gladstone,Sheila
|Source: Texas Business Today, Second Quarter 2012, :
|Subjects: Employment - Law and legislation Business - Law and legislation Employees - Law and legislation Electronic monitoring in the workplace Privacy Right of
|Ref ID: 94661
|Title: Letter from Decatur: Here's the drill - An extraction technique known as fracking has unleashed not only a frenzied gas boom across the country but also crucial questions about contamination and regulation. For Barnett Shale residents Tim and Christine Ru
|Authors: Elbein,Saul
|Source: Texas Monthly, Oct-11, 39 :10 ,pg 74+-
|Subjects: Ruggiero Tim RUggiero Christine Hydraulic fracturing Wise County Aruba Petroleum Inc. Texas Commission on Environmental Quality Railroad Commission of Texas Petroleum industry and trade - Pollution
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|Title: Street smarts: A quickie guide to...Old Town Spring - Dozens of charming, century-old homes just north of Houston have been transformed into a historic shopping district, complete with wooden clogs and fried Oreos
|Authors: Breal,Jordan
|Source: Texas Monthly, Nov-11, 39 :11 ,pg 60+-
|Subjects: Spring (Tex.) - Restaurants Spring (Tex.) - Shopping
|Additional Information: 1-The Little Dutch Girl; 2-Loose Caboose BBQ and The Funnel Cake Express; 3-Crossroads Collection; 4-Wunsche Bros. Cafe; 5-Puffabellys Old Depot; 6-Thad's;7-Lana Williams Gallery; 8-Just for the Birds.
|Ref ID: 92780
|Title: The straight & narrow minded: El Paso's three-year feud over gay rights
|Authors: Del Bosque,Melissa
|Source: Texas Observer, May-12, 104 :5 ,pg 16- 21
|Subjects: Gay liberation Homosexuality and politics Homosexuality and religion Gays - Employee fringe benefits
|Ref ID: 94183
|Title: Coca-Cola Valued Youth Program alumni share their stories
|Authors: Cantu,Linda
|Source: IDRA Newsletter, May-11, 38 :05
|Subjects: Peer-group tutoring of students Tutors and tutoring
|Ref ID: 91050
|Title: Object lesson: What you'll find in...Dean Fearing's closet
|Authors: Ramirez,Kristie
|Source: Texas Monthly, Mar-11, 39 :3 ,pg 40-
|Subjects: Fearing Dean Chefs
|Ref ID: 91230
|Title: News from IDRA's Coca-Cola Valued Youth Program
|Source: IDRA Newsletter, Mar-11, 38 :3
|Subjects: Peer-group tutoring of students Tutors and tutoring
|Ref ID: 90519
|Title: Six teens win national essay contest award
|Source: IDRA Newsletter, June-July 2012, 39 :06
|Subjects: Peer-group tutoring of students Tutors and tutoring Intercultural Development Research Association (IDRA) - Programs
|Additional Information: Coca-Cola Valued Youth Program.
|Ref ID: 93374
|Title: Young tutors win national essay contest
|Source: IDRA Newsletter, June-July 2011, 38 :06
|Subjects: Peer-group tutoring of students Essays - Awards
|Additional Information: Coca-Cola Valued Youth Program.
|Ref ID: 91704
|Title: San Marcos springs eternal
|Authors: Patoski,Joe Nick
|Source: Texas Highways, Jul-12, 59 :7 ,pg 30- 37
|Subjects: San Marcos (Tex.) - Parks Aquarena Springs Springs
|Ref ID: 94769
|Title: Cy-Fair's 'Trick or Treat, So Kids Can Eat' drive a rousing success
|Source: Texas Lone Star, Jul-11, 29 :05
|Subjects: Food banks Caring in children
|Additional Information: Cypress-Fairbanks ISD.
|Ref ID: 91713
|Title: With love, from Lewisville: Four programs in North Texas district reach out to less fortunate
|Source: Texas Lone Star, Jul-11, 29 :05 ,pg 18- 20
|Subjects: Child volunteers Caring in children
|Ref ID: 91718
|Title: 'Fifteen Days of Caring': IB initiative has lasting effects in Longview ISD
|Source: Texas Lone Star, Jul-11, 29 :05 ,pg 36- 37
|Subjects: Child volunteers Caring in children
|Additional Information: International Baccalaureate (IB) program.
|Ref ID: 91727
|Title: Knit one, kindness, too: Students at Northside ISD's Scobee Elementary make dolls for disaster victims
|Source: Texas Lone Star, Jul-11, 29 :05 ,pg 38- 39
|Subjects: Disaster relief Caring in children
|Ref ID: 91728
|Title: Pflugerville teacher receives $25,000 Valentine surprise
|Source: Texas Education Today, January/February 2012, 25 :03 ,pg Online-
|Subjects: Teachers - Awards Mentoring in education
|Additional Information: 2011-2012 Milken Educator Award. TAP: The System for Teacher and Student Advancement.
|Ref ID: 93029
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Portfolio of Taiyab Raja, a freelance UI/UX designer in Birmingham, England.James VincentPosted in Web Design → Portfolio
We showcase beautiful offices from all around the world, ranging from new Startups, Co-Working Spaces to Freelancers Desks. Join us and get behind the scenes of your favourite startup.James VincentPosted in Web Design → Corporate
Lindsay Cornelissen is passionate when it comes to truly excellent wines from the less well known and smaller family producers. With her diploma from the Wine & Spirits Education Trust and years of experience within the financial services industry catering for demanding, time-pressed clients, she had everything she needed to build a business championing wines that punched above their weight.Si KeanePosted in Web Design → Food
Genio is a single page resume & personal portfolio template. It is suitable for designers, developers & freelancers. Genio has a super clean, minimal & professional looking that anyone seeking for simplicity needs.Mo AliPosted in Web Design → Portfolio
A simply stunning design aesthetic for a themed set of playing cards. You just desperately want to see the rest and hold them in your hands!Posted in Interface → Other
When eating is motivated by pleasure, rather than hunger. A combination of traditional craftsmanship and the quality of "made in italy". I love the atmospheric quality of this site.Posted in Web Design → Food
A simple and intuitive design for an admin dashboard.Posted in Interface → Web App
Website about responsive web design, a web design approach aimed at crafting sites to provide an optimal viewing experience across a wide range of devices.Lluís VilasecaPosted in Web Design → Design
An interesting and beautifully symmetrical design for a logo mark. I love the gradually development of the logo and the subtle sense of perspective and depth created by the gradients.Posted in Branding → Logo
Absolute is a clean, sophisticated, lightweight and adaptable business or portfolio Joomla 3+ fully responsive template. Built with LESS CSS compilation and CSS3 based on gantry framework 4+. It has a limitless color variations, k2 styling, bootstrap integration and much more.Ankit ParmarPosted in Web Design → Corporate
Recollection is a space for vintage fashion clothing and accessories in Florence, Italy. The website is instrumental in conveying the message of the site; making vintage fashion appear modern and cool.Alberto BPosted in Web Design → Misc
Pixate is the next generation of mobile interaction design, you can learn more about this amazing tool at www.pixate.com.Posted in Branding → Logo
App icon for software company focused on creating beautiful interfaces for every device. This heart shape made up of 4 devices such as iPhone, tablets, laptops, large screens.Posted in Interface → Element
We recently launched a fully redesigned version of this very successful e-commerce site. With a mobile browsing overtaking desktop this year the site had to be retina ready and responsive, and we took a lot of care developing a unique gallery system that works great on small touch devices and large desktop screens.THRIVE GOLD COASTPosted in Web Design → Design
Three examples of a pop-out notification interface. Similar to Notification Center in OS X, it has space for a description of the notification, a status icon, and a user profile image/photo.Posted in Interface → Web App
Magnetic is a free HTML5/CSS3 photography template. This template can be used as a portfolio by photographers or designer to show their best pieces of work, the magnetic template comes with three hand-coded and fully-responsive.Med IjjaPosted in Web Design → Portfolio
Kyochi is an elegantly crafted and fully responsive Ghost blogging theme built on the Backbone Responsive Framework. Boasting easy to customize styles, featured images/embedding, smooth animation effects, comment plugins, social sharing and support for all the latest features of Ghost 0.4.2 (Aton)!Posted in Web Design → Blog
Mr. Tailor is a design-driven shop theme for WooCommerce (2.1+) and WordPress. The theme includes everything you need to start an online store right away.Get BowtiedPosted in Web Design → Design
Captionit is a mobile app that allows people to play caption contest game. Since the app promotes fun and creativity, there is intensive use of hilarious illustrations, animations and humorous on-screen texts to create a funky, game-like experience.Alex ZhuPosted in Web Design → Software
Welcome to the brand new website of Fermette, your specialist distributor of fresh foods and cold meats. Feel free to wander through our assortment or check out the company. Using state-of-the-art HTML/CSS/jQuery techniques, this website truly represents the House Of Delicacies on all devices.Robin SteenPosted in Web Design → Corporate


text:
Pyruvate oxidase can be produced by culturing Pediococcus sp. B-0667, Streptococcus sp. B-0668, Aerococcus viridans IFO-12219 or Aerococcus viridans IFO-12317. It is useful for analysis for pyruvic acid, because it catalyzes the reaction of pyruvic acid, phosphate and oxygen to form acetylphosphate, carbon dioxide and hydrogen peroxide. A kit containing the various reagents for such analysis is also provided by this invention.
1. A process for the manufacture of pyruvate oxidase, which comprises culturing a pyruvate oxidase-producing microorganism belonging to a genus selected from Pediococcus, Streptococcus and Aerococcus, in a nutrient culture medium, and separating the pyruvate oxidase thus produced from the culture medium.
2. A process as claimed in claim 1, in which said microorganism is selected from the group consisting of Pediococcus sp. B-0667, Streptococcus sp. B-0668, Aerococcus viridans IFO-12219 and Aerococcus viridans IFO-12317.
3. A method for the analysis of pyruvic acid in a sample containing pyruvic acid or a pyruvate-liberating system, comprising contacting said sample with pyruvate oxidase and measuring a consumed component or a generated component, in which said reaction system contains at least pyruvate oxidase, FAD, thiamine pyrophosphate, phosphate and a salt which liberates ions selected from the group consisting of calcium cobalt, magnesium and manganese.
4. A method as claimed in claim 3, in which said system also contains an indicator for hydrogen peroxide.
5. A method as claimed in claim 4, in which said indicator for hydrogen peroxide is selected from the group consisting of peroxidase, 4-aminoanitipyrin, phenol, N,N-dimethylaniline and homovanillic acid.
6. A method as claimed in claim 3, in which a consumed component is measured and said consumed component is oxygen.
7. A method as claimed in claim 3, in which a generated component is measured and said generated component is hydrogen peroxide.
8. A kit for the analysis of pyruvic acid, comprising a plurality of separate compositions one of which contains at least pyruvate oxidase, FAD, thiamine pyrophosphate and phosphate and another of which contains at least a salt which liberates an ion selected from the group consisting of calcium, cobalt, magnesium and manganese.
9. A kit as claimed in claim 8, containing also an indicator for hydrogen peroxide.
10. A kit as claimed in claim 9, in which said indicator for hydrogen peroxide is a member selected from the group consisting of peroxidase, 4-aminoanitipyrin, phenol, N,N-dimethylaniline and homovanillic acid.
This invention relates to a process for the manufacture of pyruvate oxidase and its use in quantitative analysis and a kit for such analysis.
Pyruvate oxidase is a hitherto-known enzyme which catalyzes a reaction of pyruvic acid, phosphate and oxygen to form acetylphosphate, carbon dioxide and hydrogen peroxide and has heretofore been derived from a strain of Lactobacillus derbruckii.
It has now been found that an enzyme pyruvate oxidase can be produced by culturing bacterial strains B-0667 belonging to genus Pediococcus and B-0668 belonging to genus Streptococcus isolated from a soil sample collected in a radish field in Ohito-cho, Tagata-gun, Shizuoka-ken, Japan, and that the pyruvate oxidase produced therefrom can be used for pyruvic acid analysis in a sample containing pyruvic acid or various systems which liberate pyruvic acid. We have also found that this enzyme can be used for the quantitative analysis of pyruvic acid, the measurement of the enzyme activity of enzyme reaction systems which form pyruvates, and the quantitative determination of the enzymes and the substrates thereof. Pyruvic acid can be analyzed by reacting a sample containing pyruvic acid with a reaction system comprising at least pyruvate oxidase, flavin adenine dinucleotide (hereinafter called as FAD), thiamine pyrophosphate, oxygen and phosphate, and there has been discovered an excellent kit for pyruvate analysis and analytical methods for pyruvic acid.
Further, it has now been found that adding a salt which liberates calcium ions, cobalt ions, magnesium ions or manganese ions to the said reaction system results in an improvement of the analysis. Addition of chromogen or fluorescent indicators to the reaction system provides a convenient and excellent analytical tool. Also there has been discovered a process for the manufacture of an enzyme pyruvate oxidase.
An object of the present invention is accordingly to provide a kit for quantitative analysis, especially a reaction system containing pyruvate oxidase.
Another object of the present invention is to provide an analytical method for determining pyruvic acid in a sample containing pyruvic acid or a pyruvic acid liberating system, which method comprises treating a sample with a reaction system containing pyruvate oxidase and measuring the consumed component or generated component.
A further object of the present invention is to provide a process for the manufacture of pyruvate oxidase which comprises culturing a pyruvate oxidase-producing microorganism belonging to genus Pediococcus, Streptococcus or Aerococcus in a nutrient culture medium and isolating the pyruvate oxidase thus produced from the cultured medium.
Other objects, features and advantages of the present invention will become apparent from a consideration of the following description, taken in connection with the accompanying drawings, which are graphs illustrating the present invention, and in which more particularly:
FIG. 1 is a family of curves of optimum pH of pyruvate oxidase.
FIG. 2 shows the heat stability of pyruvate oxidase.
FIG. 3 shows the pH stability of pyruvate oxidase.
FIG. 4 shows the results of analysis of pyruvic acid by oxygen electrode using pyruvate oxidase.
FIG. 5 shows the results of analysis of serum by oxygen electrode using pyruvate oxidase.
FIG. 6 shows the results of analysis of pyruvic acid by colorimetric method using pyruvate oxidase.
FIG. 7 shows the results of quantitative analysis of serum pyruvic acid using pyruvate oxidase.
FIG. 8 shows the results of analysis of ADP using pyruvate oxidase.
FIG. 9 shows the results of analysis of glycerol, triglyceride and serum triglyceride using pyruvate oxidase.
FIG. 10 shows the results of analysis of GPT- and GOT-activity using pyruvate oxidase.
FIG. 11 is a correlation diagram of analysis of GPT-activity using pyruvate oxidase.
FIG. 12 is a correlation diagram of analysis of GOT-activity using pyruvate oxidase.
An enzyme pyruvate oxidase in this invention catalyzes an oxidative reaction of pyruvic acid, inorganic phosphate and oxygen to form acetylphosphate, carbon dioxide and hydrogen peroxide, and is preferably manufactured by culturing a pyruvate oxidase-producing microbial strain belonging to genus Pediococcus, Streptococcus or Aerococcus, for example Pediococcus sp. B-0667, Streptococcus sp. B-0668, Aerococcus viridans IFO 12219 or IFO 12317.
The isolated strains B-0667 and B-0668 hereinabove have the following taxonomical properties:
__________________________________________________________________________A. Observations on various media, cultured at 30� C. for 2 days: Strain B-0667 strain B-0668__________________________________________________________________________Tryptosoybroth: weak growth, homogeneously turbid, later wooly weak growth, homogeneously turbid, later wooly precipitation. precipitation.Tryptosoy agar slant: weak growth, pale yellowish gray, no lustre. weak growth, pale yellowish gray, no lustre. no production of soluble pigment. no production of soluble pigment.Tryptosoy agar plate: colony; small and flat. colony; small and flat.Gelatin slab: growth along stabbed line. growth along stabbed line. no gelatin liquefaction. no gelatin liquefaction.BCP milk (14 days): no change. no change.__________________________________________________________________________
______________________________________B. Microscopic observation: Strain B-0667 Strain B-0668______________________________________Shape: Spherical, ovoid, pairs, Spherical, ovoid, pairs, tetra-shaped or short tetra-shaped or short chain. chain.Size: 0.5-1.0 � 0.5-1.0 μ 0.8-1.0 � 1.0-1.2 μMotility: - -Spore: - -Gram's stain: + +Acid-faststrain: - -______________________________________
______________________________________C. Physiological properties: Strain B-0667 Strain B-0668______________________________________Growth temperature: 45� C. - - 37� C. + + 30� C. + + 26� C. + + 10� C. + + 5� C. � or (+) � or (+)Halotolerance: NaCL 10% + - 6.5% + - 5.0% + + 1.0% + + 0% + -OF-test: fermentative fermentativeBehavior in oxygen: facultative facultative anaerobic anaerobicNitrate reduction: - -Indole formation: - -Hydrogen sulfateformation: - -Gelatin hydrolysis: - -Starch hydrolysis: - -Esculin hydrolysis: + +Acetoin formation: - -MR-test - -Catalase: - -Oxidase: - -Urease (SSR): - -Urease (Christensen): - -Utilization of citricacid (Christensen): - -Acid formation from sugar:adonitol: - -L(+)-arabinose: - -cellobiose: + +dulcitol: - -meso-erythritol: - -fructose: + +fucose: - -galactose: + +glucose: + +glycerol: - -inositol: - -inulin: - -lactose: + +maltose: + +mannitol: + -mannose: + +melezitose: - -melibiose: + -raffinose: + -L(+)-rhamnose: - -salicin: (+) -L-sorbose: - -sorbitol: - -starch: - -sucrose: + +trehalose: + +xylose: - -Tolerance at 60� C. for30 min. - +______________________________________
Consulting "Bergey's Manual of Determinative Bacteriology", 8th Ed., 1974 and S. T. Cowan and K. J. Steel, "Manual for the Identification of Medical Bacteria", Cambridge Press, 1974, the strain B-0667 and B-0668 having the taxonomical properties hereinabove, especially Gram positive cocci, catalase and oxidase negative, fermentative acid formation from glucose, and no gas formation from sugar (glucose), is referred to as belonging to genus Pediococcus and genus Streptococcus.
Comparison of these strains with the identification manual of the above references is as follows:
______________________________________ genus genus Strain Strain Pedio- Strepto- B-0667 B-0668 coccus coccus______________________________________Growth at 45� C. - - + dTolerance at 60� C.for 30 min. - + - dGlycerol(acid formation) - - - dArabinose(acid formation) - - + dHalotolerance(NaCl 10%) + - + -______________________________________ In table: + = positive more than 85%; - = negative more than 85%; d = varies among strains or species.
Hence the strain B-0667 will be referred to as genus Pediococcus or Streptococcus. Consulting the above "Manual for the Identification of Medical Bacteria" and J. Gen. Microbiol., 26, 185-197 (1961), the taxonomic properties of the strain B-0667 were almost identical with those of Pediococcus urina-equi, however the characteristics described in "Bergey's Manual of Determinative Bacteriology", 8th Ed., 1974 were slightly different therefrom. Therefore the strain B-0667 is referred to as genus Pediococcus and designated as Pediococcus sp. B-0667.
The strain B-0668 resembles genus Streptococcus rather than genus Pediococcus. Further consulting the "Manual for the Identification of Medical Bacteria", the strain B-0668 resembles Streptococcus faecium var. durans, however no taxonomic properties were described in "Bergey's Manual" and therefore it is impossible to make a detailed comparison. The strain B-0668, therefore, is referred to as Streptococcus sp. B-0668.
The strains B-0667 and B-0668 were deposited for permanent collection in the Institute of Microbial Industry and Technology, Agency of Industrial Science and Technology, M.I.T.I., Japan, as deposition numbers FERM-P No. 4438 and FERM-P No. 4439, respectively. IFO-12219 and IFO-12317 were deposited for permanent collection in the Institute for Fermentation, Osaka, Japan, under those numbers.
In an embodiment of the present invention, the above Pediococcus sp. B-0667, Streptococcus sp. B-0668, Aerococcus viridans IFO-12219 or Aerococcus viridans IFO-12317 are cultured in a conventional medium for enzyme production. Cultivation can be by conventional liquid culture and submerged aeration culture is preferable for industrial production.
A conventional medium for culturing microorganisms can preferably be used. For the carbon sources, assimilable carbon sources such as glucose, sucrose, lactose, maltose, fructose, molasses, pyruvic acid or the like can preferably be used. Assimilable nitrogen sources such as peptone, meat extract, yeast extract, casein hydrolyzate or the like can be used. Various inorganic salts such as phosphates, carbonates, sulfates, salts of magnesium, calcium, potassium, divalent iron, manganese or zinc can be used.
The culturing temperature can be selected within the range for growth of microbial cells and production of pyruvate oxidase, and is preferably 25�-37� C. The culturing time can be altered depending on conditions and is terminated when the pyruvate oxidase production is substantially complete, and is usually 18-48 hours.
To separate pyruvate oxidase from the culture, the cultured mass is filtered or centrifuged to collect the cells, which are disrupted by treatment with mechanical means or enzymes such as lysozyme. Further if necessary pyruvate oxidase is solubilized by adding ethylene-diaminetetraacetic acid (EDTA) and a surfactant such as Triton X-100 (trademark) or Adecatol SO-120 (trademark) to separate the enzyme. The thus-obtained solution of pyruvate oxidase is treated with or without concentration, and thereafter the enzyme is precipitated by salting out with the addition of a soluble salt such as ammonium sulfate or sodium chloride. Low molecular weight impurities are removed by dialysis. Furthermore impurities in the solution of pyruvate oxidase are preferably removed by adsorption chromatography, ion-exchange chromatography or gel filtration. The enzyme solution thus obtained is treated by vacuum concentration and lyophilization to produce powdered pyruvate oxidase. Further purification can be achieved by conventional purification methods for proteins and enzymes such as adsorption chromatography, ion-exchange chromatography or gel filtration.
Pyruvate oxidase produced by the present invention has the following physico-chemical properties, in which abbreviations are used as follows:
______________________________________Pediococcus sp. B-0667: abbreviated as B-0667;Streptococcus sp. B-0668: abbreviated as B-0668;Aerococcus viridans IFO-12219: abbreviated as IFO-12219;Aerococcus viridans IFO-12317: abbreviated as IFO:12317.______________________________________
(1) Enzyme action:
The enzyme catalyzes the oxidative reaction of pyruvic acid, inorganic phosphate and oxygen to form acetylphosphate, carbon dioxide and hydrogen peroxide:
(2) Optimum pH:
Effect of pH on pyruvate oxidase activity is measured. Phosphate buffer solutions of pH 6-8 are used for assay. The results are shown in FIG. 1 in which optimum pH is as follows:
______________________________________B-0667: pH 6.3-7.5B-0668: pH 7.5-8.5IFO-12219: pH 7.0-8.0IFO-12317: pH 6.8-7.5______________________________________
Slight variation is observed according to phosphate concentration and kind of metallic ion.
(3) Heat stability:
Heat stability of the enzyme is determined by incubating in 10 mM phosphate buffer (pH 6.5) containing 10 μM FAD at 0�, 40�, 50�, 60� and 70� C. for 10 minutes according to the method of enzyme assay. As shown in FIG. 2 the enzymes obtained from B-0667, IFO-12219 and IFO-12317 are slightly activated at 40� C. and denatured above 60� C. The enzyme obtained from B-0668 is not activated at 40� C. and almost denatured above 60� C.
(4) pH stability:
To each enzyme solution (0.1 ml) is added 0.2 M phosphate buffer for pH 6-8 (0.9 ml) or 0.2 M of Tris-HCl buffer for pH 7-9 (0.9 ml) each containing 10 μM FAD, and allowed to stand for 10 minutes at 40� C. 20 μl of enzyme solution are taken and enzyme activity is determined. As shown in FIG. 3, the enzyme obtained from B-0667, IFO-12219 and IFO-12317 is most stable at about pH 7 and that of B-0668 is stable at an acidic pH.
(5) Effect of several substances:
(a) Effect of several substances on the enzyme activity is examined by adding 5 mM of the substance indicated below instead of MgCl2 in the assay system.
______________________________________ Relative activity (%)Substance added B-0667 B-0668 IFO-12219 IFO-12317______________________________________No addition 25.4 72.0 42.0 50.3EDTA 0 0 0 0MgCl2 100 100 100 100CaCl2 69.4 75.0 83.4 78.7MnCl2 129.1 102.7 116.2 111.0CoCl2 81.3 81.1 85.0 84.0BaCl2 20.6 58.8 23.9 28.8ZnCl2 16.0 38.2 14.9 22.8______________________________________
As shown hereinabove, the enzyme is inhibited by EDTA and activated by Mg++, Ca++, Mn++ and Co++.
(b) Effect of elimination of the following substance from the assay system on the enzyme activity is shown below. 0.1 M dimethylglutarate-NaOH buffer is used in the case of phosphate elimination.
______________________________________Substance Relative activity (%)eliminated B-0667 B-0668 IFO-12219 IFO-12317______________________________________No elimination 100 100 100 100Thiaminepyro-phosphate 0 0 0 0FAD 33.9 100 32.7 41.7Thiaminepyrophos-phate and FAD 0 0 0 0Phosphate 0 0 0 0______________________________________
As a result, the enzyme requires thiaminepyrophosphate and FAD as cofactor and phosphate as substrate.
Further, oxygen consumption during enzyme reaction is measured by oxygen-electrode, and the oxygen is consumed in proportion to an enzyme activity (formation of hydrogen peroxide). Results are shown in the following:
______________________________________ Reaction product (μ mole/min.)Oxygen Consumption(μ mole/min.) H2 O2 Acetylphosphate______________________________________B-0667 0.042 0.042 0.038B-0668 0.022 0.0213 0.020IFO-12219 0.040 0.038 0.037IFO-12317 0.035 0.036 0.034______________________________________
Assays are performed as follows:
Oxygen consumption: dissolved oxygen meter (tradename; YSI-dissolved oxygen meter Model-53).
Acetylphosphate: F. Lipman et al., J. Biol. Chem., 134, 463-464 (1940).
Hydrogen peroxide: method using N,N-dimethylaniline, 4-aminoantipyrin and horseradish peroxidase.
As hereinabove explained, the enzyme obtained from the above four strains is referred to as pyruvate oxidase and flavine protein.
The assay method of pyruvate oxidase of the present invention uses a reaction mixture as follows:
______________________________________0.5 M potassium pyruvate 0.1 ml0.5 M phosphate buffer (pH 7.9) 0.2 ml0.2% 4-aminoantipyrin 0.1 ml0.2% N,N-dimethylaniline 0.2 ml0.2 M MgCl2 50 μl10 mM thiaminepyrophosphate 20 μlperoxidase (45 U/ml) 0.1 ml1 mM FAD 10 μldistilled water 0.22 ml______________________________________
The above reaction mixture (1.0 ml) is pre-incubated at 37� C. for 3 minutes. To this solution is added the enzyme solution (20 μl) and incubated at 37� C. for 10 minutes. 0.1 M citrate buffer (pH 6.0, 2 ml) containing 0.1 M EDTA is added to stop the reaction. The violet color formed is measured by colorimetric method at 565 nm.
A unit (1 unit, 1 U) of enzyme activity is defined as the activity which generates 1 μmole of hydrogen peroxide per minute.
In order to activate the pyruvate oxidase reaction system, FAD, thiaminepyrophosphate and phosphate are added. Further for activation of the enzyme, an ion-liberating salt which liberates calcium ions, cobalt ions, magnesium ions or manganese ions, in the form of chloride is preferably added thereto. An indicator such as a coloring indicator or fluorescent indicator for hydrogen peroxide can preferably be used.
The amount and ratio of components in the enzyme reaction system can be selected for substantial enzyme reaction and will be varied according to the amount of pyruvate, temperature and time of enzyme reaction. For example, 1-20 U of pyruvate oxidase, 0.1-20 μmoles of FAD, 0.05-0.5 μmole of thiaminepyrophosphate, 1-10 μmoles of inorganic phosphate and 0.05-10 μmoles of ion liberating salt per test can preferably be used. Pyruvate oxidase can be in a microcapsulated form or in an immobilized form of covalent linkage with an organic or inorganic carrier or adsorbed on a carrier. The molar ratio of indicator for hydrogen peroxide is at least an equimolar or excess amount of generated hydrogen peroxide. In the case of the peroxidase, 0.5-20 U per test is preferably used. These components of the enzymatic reaction mixture are preferably used by dissolving in the buffer of suitably adjusted pH.
The thus-prepared enzymatic reaction system is used for the analysis of pyruvic acid. Any samples which contain pyruvate can be analyzed. For example, pyruvic acid itself, pyruvic acid in serum or urine and pyruvic acid-forming enzyme reaction systems such as lactic acid and lactate dehydrogenas (LDH), ADP and pyruvate kinase, and glycerol, glycerol kinase and pyruvate kinase can be mentioned. Further detailed examples of the enzymatic reactions which form pyruvic acid and so can be assayed are as follows:
(1) Assay of lactic acid or LDH activity: ##STR1## (2) Assay of ADP or pyruvate kinase (PK) activity: ##STR2## (3) Assay of glutamate-pyruvate-transaminase (GPT) activity or α-ketoglutarate: ##STR3## (4) Assay of glutamate-oxaloacetate-transaminase (GOT) activity: ##STR4## (5) Assay of glycerol or glycerophospho kinase (GK) activity: ##STR5## (6) Assay of triglyceride: ##STR6## (7) Assay of creatinine or creatinine phosphokinase (CPK): ##STR7## (8) Assay of myokinase: ##STR8## (9) Assay of fatty acid or thiokinase activity: ##STR9##
These enzyme reactions are only for illustration and these pyruvate-forming reactions can be performed with the combination of enzyme and its substrate, for example a biological sample. As exemplified hereinabove, the assay can be applied not only for assay of pyruvate but also for assay of enzyme, enzyme activity or substrate.
Assay is performed by incubation with the sample and a reagent mixture. The reagent mixture is preferably a kit of necessary reagents. For assaying, consumed component or generated component is measured. Measuring the amount of oxygen consumption by dissolved oxygen meter is preferable as an assay method. In this case no indicator for hydrogen peroxide is necessary. As for the assaying of a generated component, measurement of the amount of hydrogen peroxide is preferable, for example by a hydrogen peroxide electrode meter such as a YSI-oxidase meter or by colorimetric or fluorometric assay with an indicator for hydrogen peroxide. The assay can be performed preferably for 10-60 minutes and at 20�-40� C., preferably at 35�-37� C.
The indicator for hydrogen peroxide is a combination of one or more chromogen or fluorescents, which is effected by coupling with hydrogen peroxide. Examples of such indicators are combinations of tetravalent titanium compounds and xylenol orange which couples with hydrogen peroxide to form a stable red color, or a combination of phenol or N,N-dimethylaniline or homovanillic acid, 4-aminoantipyrin and peroxidase for measuring color or fluorescence. 4-aminoantipyrin can be replaced by 4-aminophenazone. A combination of 2,6-dichlorophenol indophenol and peroxidase and of guaiacol and peroxidase can also be used. The indicator can be previously prepared as a solution. Colorimetric or fluorometric assay is performed by measuring the absorption at a suitable wave length such as 565 nm.
The amount of pyruvic acid can be measured by calculation from corresponding standard curves of consumed oxygen or generated hydrogen peroxide.
Phosphate as a consumed component or acetylphosphate as a generated component can also be assayed by any conventional method.
As hereinabove explained, a kit for analysis, especially quantitative analysis, comprises pyruvate oxidase. More particularly, as illustrated hereinbefore, diagnostic analysis such as analysis of pyruvate in pyruvate-containing reagents or in serum or urine, assay of enzyme activity of LDH, pyruvate kinase, GPT, GOT, glycerol kinase, lipase, lipoprotein lipase, creatinine phosphokinase, myokinase or thiokinase, and analysis of biological components such as lactate, ADP, glycerol, triglyceride, creatinine or fatty acids can advantageously be carried out with the kit and by the method of the present invention.
The following examples illustrate the embodiments of the present invention but are not to be construed as limiting the invention.
Four samples of culture medium (each 100 ml, pH 7) comprising glucose (1%), peptone (1%), yeast extract (0.5%), NaCl (0.2%), KH2 PO4 (0.1%), K2 HPO4 (0.1%), MgSO4 (0.05%) and CaCO3 (0.3%) in a 500 ml Erlenmyer flask were sterilized at 120� C. for 20 minutes. To each medium was inoculated a strain of Pediococcus sp. B-0667 FERM-P No. 4438, Streptococcus sp. B-0668 FERM-P No. 4439, Aerococcus viridans IFO-12219 or Aerococcus viridans IFO-12317, respectively and shake-cultured at 30� C. for 24 hours, at 300 r.p.m. Thereafter cultured cells centrifugally collected were washed with 10 mM phosphate buffer (pH 6.5) and again centrifuged to collect bacterial cells. The thus-obtained cells were suspended in 10 mM phosphate buffer (10 ml, pH 7.0) containing 0.02% lysozyme and 0.1% Triton X- 100 and incubated at 37� C. for 60 minutes. The supernatant obtained centrifugally which contains pyruvate oxidase was collected. Enzyme activity of the supernatant is shown in the following table.
______________________________________Strain Enzyme activity (U/ml)______________________________________B-0667 0.60B-0668 0.38IFO-12219 0.52IFO-12317 0.46______________________________________
A medium (20 l.) consisting of the same components as described in Example 1 in a 30 l. jar-fermenter was sterilized by steam. Cultured broth (200 ml) of Pediococcus sp. B-0667 FERM-P No. 4438 cultured the same way as in Example 1 was transferred thereto, and cultured at 30� C. for 24 hours. Bacterial cells centrifugally collected (about 100 g) were suspended in lysozyme solution (0.2 mg/ml, 4 l.), and there was further added Triton X-100 (trademark, 4 g), EDTA (3 g) and 1 M phosphate buffer (pH 6.5, 40 ml) and the mixture was stirred at 37� C. for 60 minutes. To the supernatant obtained centrifugally was added ammonium sulfate and the precipitate at 0.54-0.73 saturation was collected by centrifugation. The precipitate was dissolved in 10 mM phosphate buffer (pH 6.5, 1000 ml) (5160 U, recovery: 86%), then cold acetone (0.65 volume) was added thereto and the impure precipitate was separated. Acetone (0.3 volume) was then added and the precipitate, which was collected by centrifugation, was dissolved in 10 mM phosphate buffer (pH 6.5, 70 ml) (4750 U, recovery: 79.2%).
To the solution was added ammonium sulfate and the precipitate at 0.54-0.70 saturation was collected centrifugally.
After dissolving the precipitate in 10 mM phosphate buffer (pH 6.5), the solution was charged on a column of Sephadex G-25 (trademark) (6.0�70 cm) and the fraction was collected showing absorbency at 280 nm. The active fractions were pooled and freeze dried to obtain powdered pyruvate oxidase (3940 U, 758 mg. recovery: 65.7%).
A composition for pyruvate analysis by oxygen electrode comprises the following:
______________________________________pyruvate oxidase obtained in Example 2(the same as in the following examples) 300 UFAD 0.5 μ molethiamine pyrophosphate 10 μ molesMnCl2 25 μ moles0.2 M phosphate buffer (pH 7.5) 1.0 mlsucrose 0.5 g0.2 M dimethylglutarate-NaOH buffer(pH 7.5) 5 ml______________________________________
The above mixture is lyophilized for use in a kit for assay of pyruvic acid (by oxygen electrode measurement, 50 tests).
A kit composition for pyruvate analysis by colorimetric assay comprises the following:
______________________________________Reagent (I)pyruvate oxidase 200 UFAD 0.5 μ molethiamine pyrophosphate 10 μ moles0.2 M phosphate buffer (pH 7.5) 1.0 mlsucrose 0.5 g0.2 M dimethylglutarate-NaOH buffer(pH 7.5) 5 mlperoxidase (100 U/mg, horseradish) 2.5 mg0.3% 4-aminoantipyrin 5 ml______________________________________
The above mixture is lyophilized to prepare the reagent (I) of the kit for pyruvate analysis (by colorimetry, 50 tests).
Additional reagent (II) consisting of
0.2% aqueous N,N-dimethylaniline containing 25 μmoles MnCl2 (50 ml) and stop reagent (III) consisting of 0.1 M citrate buffer (pH 6.05, 100 ml) containing 0.1 M EDTA were the other ingredients of the kit.
The composition of Example 3 was dissolved in distilled water (50 ml) and an aliquot solution (1.0 ml) thereof was put in reaction vessels. Thereto was added 5.0 mM pyruvate solution (each 0-100 μl), or human serum 50 μl and 5.0 mM pyruvate solution (each additionally 0-100 μl), and the mixture was incubated at 37� C., then the oxygen consumption was measured by Galvanic oxygen electrode. Results are shown in FIG. 4.
Further, the composition of Example 3 was dissolved in distilled water (25 ml) and an aliquot solution (0.5 ml) thereof was put in reaction vessels and then incubated with the addition of an aqueous solution (0.5 ml) containing human serum (0-0.5 ml) at 37� C. In FIG. 5 is shown the result of assay of oxygen consumption measured by Galvanic oxygen electrode.
As shown in FIGS. 4 and 5, good linear relations were observed.
The lyophilized reagent (I) prepared in Example 4 was dissolved by addition of reagent (II) (50 ml), and plural aliquot solutions (1 ml) in small test tubes were incubated at 37� C. Thereto was added 5 mM potassium pyruvate solution (each 0-50 μl), or human serum (50 μl) along with 5 mM potassium pyruvate solution (each 0-50 μl), and the mixtures were incubated at 37� C. for 10 minutes. Stop solution (2 ml) was added to each and the absorbency at 565 nm was measured. As shown in FIG. 6, good linear relations and quantitative results were observed in the above assays and this results also coincided with the calibration curve by H2 O2 (2.5 mM H2 O2, 0-50 μl).
Further aliquot samples of human serum (0-0.5 ml) were put into small test tubes and adjusted to 0.5 ml by adding distilled water. Each solution (1.0 ml) of reagent (I) prepared with added reagent (II) in Example 4 was added thereto, and the mixtures were incubated at 37� C. for 10 minutes. Reaction was stopped by adding stop reagent (III) (1.5 ml) and the mixtures were colorimetrically assayed at 565 nm. As shown in FIG. 7, good quantitative results were obtained.
______________________________________Reaction mixture:0.2 M dimethylglutarate-NaOH buffer(pH 7.5) 0.2 ml10 mM MnCl2 50 μl0.2% N,N-dimethylaniline 0.2 ml0.3% 4-aminoantipyrin 0.1 mlperoxidase (45 U/ml) 0.1 ml10 mM thiamine pyrophosphate 20 μl0.2 M phosphate buffer (pH 7.5) 25 μl20 mM phosphoenolpyruvate 0.1 mlpyruvate kinase (4000 U/ml) 5 μl5 mM ADP 0-50 μ______________________________________
The above reaction mixture was adjusted to 1.0 ml by adding distilled water, pre-incubated at 37� C., then a solution of pyruvate oxidase (200 U/ml, 20 μl) was added thereto, and the mixture was incubated at 37� C. for 10 minutes. After stopping the reaction with the addition of 0.1 M EDTA in 0.1 M citrate buffer (pH 6.0, 2.0 ml), the absorbency at 565 nm was measured. As shown in FIG. 8, good quantitative results of ADP assay were observed by assaying hydrogen peroxide generated from the reaction mixture of ADP, pyruvate kinase, phosphoenolpyruvate and others.
Also as shown in FIG. 8, good linearity was observed when 2.5 mM hydrogen peroxide was used instead of 5 mM ADP.
______________________________________Triglyceride assay kit:Reagent (I)0.2 M dimethylgultarate-NaOH buffer(pH 7.5) 10 ml0.3% 4-aminoantipyrin 5 mlperoxidase (100 U/mg) 2.5 mgthiamine pyrophosphate 10 μ moles0.2 M phosphate buffer (pH 7.5) 1.25 mlphosphoenolpyruvate 100 μ molespyruvate kinase (4000 U/ml) 0.1 mlpyruvate oxidase (200 U/ml) 2 mllipoprotein lipase (3000 U/ml) 0.5 mlglycerol phosphokinase (300 U/ml) 1.0 mlATP 100 μ molesThe above reagent was lyophilized.Reagent (II)25 μ moles of MnCl2 in 0.2% dimethyl-aniline 50 mlReagent (III) (stopper solution)0.1 M EDTA in 0.1 M citrate buffer(pH 6.0) 100 ml______________________________________
Reagent (I) in Example 8 was dissolved in reagent (II) and each aliquot solution (1.0 ml) thereof was put in test tubes.
Each aliquot sample (0-50 μl) of human serum containing 1.02 μmole/ml of triglyceride, 5 mM glycerol solution, 4.2 mM triolein in 0.1% Triton X-100 solution or 2.5 mM hydrogen peroxide solution, respectively, was added thereto and the mixtures were incubated at 37� C. for 10 minutes. As shown in FIG. 9, good linearities were observed.
______________________________________A kit for assay of serum transaminase (for 100 tests):(1) A kit for GTP assay (for 100 tests):Reagent (I): lyophilized reagent consisting of thefollowing:pyruvate oxidase 400 UFAD 500 n molesthiamine pyrophosphate 150 μ molesL-alanine 20 m molesα-ketoglutarate 1 m molesucrose 1 g4-aminoantipyrin 150 μ molesperoxidase 450 U0.2 M phosphate buffer (pH 7.5) 2.5 ml0.2 M dimethylglutarate-NaOH buffer(pH 7.5) 30 mlReagent (II) (100 ml are used for Reagent (I)):42 μ moles MnCl2 in 0.2% dimethylanilinesolution 210 mlReagent (III) (stopper solution; 200 ml for Reagent(I), 2.0 ml per one test):0.1 M EDTA in 0.2 M citrate buffer(pH 5.0) 420 ml(2) A kit for GOT assay (for 100 tests):Reagent (I): lyophilized reagent consisting of thefollowing:pyruvate oxidase 400 UFAD 500 n molesthiamine pyrophosphate 150 μ molesL-aspartic acid 20 m molesα-ketoglutarate 1 m moleoxaloacetate decarboxylase 200 Usucrose 1 g4-aminoantipyrin 150 μ molesperoxidase 450 U0.2 M phosphate buffer (pH 7.5) 2.5 ml0.2 M dimethylglutarate-NaOH buffer(pH 7.5) 30 mlReagent (II) and Reagent (III):The same as (1) above.______________________________________
Reagents (I) prepared in Example 10, (1) (for GPT activity assay) and (2) (for GOT activity assay), were dissolved by adding reagent (II) (100 ml) to each. Each aliquot amount (1.0 ml) of the solution was separately put into small test tubes and pre-incubated at 37� C. for 5 minutes (pre-incubated solution of reagent (I)). The standard serum solution (20 μl) (Calbiochem Co., trademark Maxitol, containing GPT 700 K U/ml and GOT 1000 K U/ml) diluted with constant ratio was added thereto and incubated at 37� C. for 10 minutes. Reaction was stopped by adding stopping reagent (III) (2.0 ml) and the absorbency at 565 nm was measured. As shown in FIG. 10, both enzyme activities have linearity up to the optical density of about 1.0 (enzyme activity: about 750 K U/ml).
To each pre-incubated solution of reagent (I) obtained in Example 11 were added 20 μl of human serums (45 samples) and the mixtures were incubated at 37� C. for 20 minutes. Stopping reagent (III) (2.0 ml) was added therein and the absorbency at 565 nm was measured.
Also the same samples of human sera were assayed by LKB method (ultra violet absorption method, GOT assay kit and GPT assay kit, made by LKB Corp.) and the correlation was plotted with activity of GPT and GOT.
As shown in FIG. 11, the correlation coefficient: r=0.998 and regression equation: y=0.00295�+0.0032 for GPT assay were observed.
For GOT assay, the correlation pattern is shown in FIG. 12, in which the correlation coefficient: r=0.966 and regression equation: y=0.00287�+0.0180 resulted.
From a consideration of the foregoing disclosure, therefore, it will be evident that the initially recited objects of the present invention have been achieved.
Although the present invention has been described and illustrated in connection with preferred embodiments, it is to be understood that modifications and variations may be restored to without departing from the spirit of the invention, as those skilled in this art will readily understand. Such modifications and variations are considered to be within the purview and scope of the present invention as defined by the appended claims.
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Monday, October 31, 2011
Link is fixed!
Halloween card link is fixed!! you can download it HERE too!!
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The program comes with full warranty 60 days money back,
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Dufftown 2000 will again be arranging events to support the Spirit of Speyside Whisky Festival 30 April to 4 May 2015.
During the festival, whisky connoisseurs will be able to tour and taste an incredible array of fine single malts from a number of the Speyside Distilleries.
Gordon Haughton will host the famous "Seven Stills Tour" that visits all of Dufftown's distilleries past and present on a single trip with a dram at each; This is definitely not a bus tour for the faint hearted!
But there will be more - music, dancing, the Whisky Fair, the Dufftown Pipe Band and visits to the outlying distilleries of Speyside. The best of Scotland's culture will be on show alongside the celebration of its national drink.
The festival will start with the Mates of the Whisky Museum event on Thursday evening. The Seven Stills Bus Tour will depart the Square as usual at 12 noon on Saturday. We will also be arranging our usual complement of bus tours to the distilleries not normally open to the public on each day of the festival.
The full programme of events can be accessed through the Spirit of Speyside Website.
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You'll find plenty to do in West Palm Beach, and we can help you find excellent services and resources.
There are plenty of great beaches and places to shop nearby, and we'll be glad to help you find a local church or a great restaurant during your visit.
While you are here, you might like a leisurely sight-seeing trip down the Intracoastal Waterway. Or maybe you'd like to spend an afternoon visiting some our local attractions near the PBA campus like the Flagler Museum or the Norton Museum of Art. We can also assist you in renting a car, or finding the right flight at Palm Beach International Airport.
Contact us in the Community Services office at (561) 803-2013 for more information.
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Herd confident after loss to Memphis
HUNTINGTON -- Another defeat, but a better loss than the one before.
Losing is unacceptable in Marshall University men's basketball head coach Tom Herrion's program, but following a 56-point meldown in a mid-week game at Southern Miss, the Thundering Herd regained some of its pride Saturday in a 73-72 loss at Memphis. A crowd of 16,386 spectators watched in 18,400-capacity FedExForum.
"We came in here with every expectation to win and we're disappointed with the loss, but I'm proud of my kids," Herrion said after the game. "They were resilient. They did a great job to just keep battling."
Memphis (16-3 season and 5-0 Conference USA) had leads of 10 and nine points, and Marshall kept coming back.
The Herd never led, but was within 70-69 when Dennis Tinnon grabbed an offensive rebound and scored with 1:18 remaining. Some misses from close range and a missed 3-point try by Elijah Pittman that was contested with five seconds left halted the comeback bid.
Pittman was being guarded by D.J. Stephens who might have made contact with the ball, the shooter's hand or both. Herrion said he'll look at the tape to see whether there was contact.
Marshall (9-11, 2-3) played well overall, outrebounded the Tigers by six and had just 13 turnovers.
Dre Kane and D.D. Scarver fired in 22 and 20 points respectively to lead Marshall. Tinnon had nine points and nine rebounds. Pittman also scored nine. Rob Goff contributed six points, seven rebounds and three blocked shots from off the bench.
Scarver's fourth 20-point game of the season came before a group of about 30 or 40 friends and family members from his hometown of Birmingham, Ala.
"He was terrific," Herrion said. "Playing in front of his family is not an easy thing to do."
Memphis guard Joe Jackson scored 16, forward Adonis Thomas had 15 and forward Shaq Goodwin netted 11. Forward Tarik Black had eight points and seven rebounds as a reserve.
Marshall had more field goals (27) than Memphis (24) and both teams made seven 3-pointers. The one-point difference was created by Memphis making 18 of 28 foul shots and Marshall going 11-for-17.
"I thought we played nose-to-nose, punch-for-punch with one of the best teams in America today," Herrion said. "Now our challenge is to do that night-in and night-out through our games left in the conference."
The Herd made the long bus trip home after the game. A two-game homestand against SMU (Wednesday) and UCF (Saturday) awaits.
Hall of Fame coach Larry Brown leads the SMU Mustangs (11-10, 1-5) into Cam Henderson Center. Brown was hired in April when Matt Doherty was relieved of his duties after 11 seasons.
Brown is the only head coach with an NCAA championship and NBA title on his resume. He led the University of Kansas with first-year Tulsa University head coach Danny Manning as the star to the national championship in 1988. He won the 2004 NBA title with the Detroit Pistons.
He is also the only American male to play and coach in the Olympics. He won Olympic gold in the 1964 Tokyo Games and was head coach of the bronze medal-winning team at the 2004 Athens Games.
SMU's losing streak reached three games Saturday with a loss at UCF, 74-65. The Mustangs cut an 11-point deficit to 66-65 on Ryan Manuel's dunk with 1:49 remaining. UCF (14-5, 4-1) stopped the rally with layups by Isiah Sykes and Kasey Wilson before making all four of its foul shots in the final 31 seconds.
Jalen Jones, a 6-foot-7 sophomore forward who scored 13, leads SMU with averages of 14.9 points and 8.1 rebounds.
Nick Russell had 16 points and the 6-4 junior averages 13.7. Manuel is a 6-4 sophomore scoring 11.9 per game.
SMU doesn't score much, but doesn't give up much either. An average score for the Mustangs is 64.0 to 61.6.
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Very Beautiful Gloves
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Comments about Women's Addison Wool Gloves:
The gloves appear double knitted, so I expect them to be warm. They fit nicely and the velvet band and floral stitching are an added plus. I ordered another pair. Don't hesitate to buy these.
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Berry Craig: One of the strangest—and for me, most annoying—perversions of politics in the Obama era is the meme pursued by so many on the right suggesting that this president is a raging socialist who seeks to install a permanent welfare state in America—despite all evidence to the contrary
Vietnam Is Back in Town
Is Capitalism on Trial?
Libertarians, Watch Out!
The Case for a Woman as Vice President, and then President
Although the office of vice president carries little inherent power, it positions its occupant to be the natural frontrunner to succeed a successful president. And because no political party plans for its presidential nominee to not be successful, almost every time a party holds a convention, it is in effect nominating not just one presidential […]


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Gear Review
Chris jenney
Koka Moka!
oh man... the koka moka... I cannot get enough. For early morning tours / times Im too lazy to make breakfast, these keep me going. They are also great to throw in a backpack and forget about until that one day where you are "dying" on the trail. The koka moka really has no coffee taste at all in my opinion... just great chocolate.
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Whats going on out there? Awful quiet... I can hear the birds singing.
Whats going on out there? Awful quiet... I can hear the birds singing.
Bill Davis
Yea, all dissenting opinions have been run off!
The freedom to discriminate is essential to personal Liberty. Life in a free country is about being free to make choices based on your own criteria rather then one mandate by the government...Libertarian blogger
FWIW, I agree with Franco.
"For everyone to whom much is given, of him shall much be required." -- Luke 12:48
Raven - Moneybird's Black Magic Marker***
(Esprit's Power Play x Trumarc's Lean Cuisine)
Mick - Moneybird's Jumpin' Jack Flash***
(Clubmead's Road Warrior x Oakdale Whitewater Devil Dog)
Peerless - Moneybird's Sole Survivor
(Two River's Lucky Willie x Moneybird's Black Magic Marker)
Quiet during the day on my end because happy days the worst of tax season is behind me. The weather was great today and I got to play and so did the dogs. Hopefully the extension fools will get their stuff together soon so I don't get totally buried again in Oct.
Next crunch which means I'm stuck a computer is late June early July.
Hihope Hiland Heathen of Perth CD, RE, CGC, TDI