CANBERRA, Australia (AP) — Danish Crown Prince Frederik and his Australian-born wife, Crown Princess Mary, have arrived in Sydney with their four children for an official weeklong visit.
The family landed Saturday and will spend two days in Sydney, where they met in a nightclub during the 2000 Olympics. The couple will then visit Melbourne, Canberra, western …
The death of Nelson Mandela's great-granddaughter cast a shadow over the opening day of the World Cup on Friday, dampening the spirits of a nation proud and excited to be hosting the world's most popular sporting event.
Zenani Mandela, 13, was killed in a car accident on the way home from a World Cup concert in Soweto on Thursday night, where tens of thousands of people had sung and danced with headline music stars Shakira and the Black Eyed Peas.
The Nelson Mandela foundation said later Mandela would not attend Friday's World Cup opening ceremony and game in Johannesburg, dashing South Africans' hopes the frail 91-year-old former president would make a rare …
According to the Hong Kong sources, the Intertextile Pavilion organised by Messe Frankfurt, CCPIT-Tex and the Shenzhen Garment Industry Association will return to Shenzhen from 25 - 27 July 2006. The fair is recognised by overseas and domestic textile manufacturers as an ideal marketing platform from which to tap into the South China market, one of the country's largest garment manufacturing centers.
"The pavilion was created to provide a professional meeting point for suppliers and buyers in this booming textile region," says Ms. Annie Ma, Group Manager for Messe Frankfurt (HK) Ltd. "We believe the time is right for us to help textile manufacturers explore new markets outside the …
Two suspected Islamic rebels and one soldier died as scores of government troops ended a four-day hotel siege in Indian Kashmir on Sunday, officials said.
Two paramilitary soldiers and two policemen were also wounded in the fighting in Sopore, a town 50 kilometers (30 miles) north of Srinagar, the summer capital of India's Jammu-Kashmir state, said B. Srinivasan, a senior police official.
One rebel fighter was killed Saturday night while the paramilitary soldier and another rebel died in the fighting Sunday before the standoff ended, Srinivasan said.
The siege _ punctuated by sporadic automatic gunfire and grenade blasts _ began when suspected …
President George W. Bush and congressional leaders seized on the latest grim unemployment data Friday to try to fire up lukewarm support in Congress for bailing out U.S. automakers. They clashed again, however, over terms of the rescue plan and the source of any aid.
As the Big Three auto chiefs pressed their case for $34 billion in a second day of hearings, Bush said in the White House Rose Garden that the loss of 533,000 jobs in November was even more reason to help the companies.
"I am concerned about the viability of the automobile companies" along with workers and their families, Bush said. "And likewise, I am concerned about taxpayer …
Longtime New York Philharmonic Orchestra conductor LeonardBernstein received the prestigious Edward MacDowell Medal on Sundayfor his lifetime achievements as a composer.
Bernstein was the 28th recipient of the award given at theMacDowell Colony, a 450-acre artists' …
SEOUL, South Korea - South Korea's president walked across North Korea's border Tuesday on his way to Pyongyang for a summit with Kim Jong Il, pledging to foster peace on the divided peninsula in the second-ever such meeting between its leaders.
Roh Moo-hyun and his wife Kwon Yang-sook stepped across a yellow plastic strip marked with the words "peace" and "prosperity" and laid across the Military Demarcation Line that divides the Koreas in the middle of the heavily fortified Demilitarized Zone.
Crossing near the North Korean city of Kaesong, the South Korean delegation was greeted by North Korean officials and women in traditional Korean hanbok dresses bearing bouquets. …
Rows of missiles and tanks rumbled through Moscow's Red Square and dozens of combat jets streaked overhead in the Victory Day parade Saturday in the largest display of military might since the Soviet times.
President Dmitry Medvedev warned sternly that Russia was ready to respond to any challenge and said its military has proven that in real action _ a clear reference to the war with neighboring ex-Soviet Georgia.
While Medvedev didn't specifically mention the war, he alluded to the five-day conflict, saying that the World War II taught a lesson which "remains acute today when again there are those who engage in military adventurism."
…
When Angela Porcelli was 8, she picked up a bat and hit asoftball across her yard. At 12, she learned how to spike avolleyball.
Porcelli ran everywhere she went to develop her track skills.By the time she was 14, she knew she had exceptional athleticskills.
So when Porcelli enrolled at Morton in Cicero, she had the pickof several sports. But rather than limit herself to a single sport,she chose them all.
"I actually enjoy playing every sport there is," she said."It's just that I can only play three sports in school."
Porcelli is a two-time all-conference volleyball player, holdsthe school long-jump record of 17-4 1/4, the conference record …
HARARE, Zimbabwe (AP) — New Zealand beat Zimbabwe by 34 runs in the second Twenty20 international Monday to seal the two-match series 2-0.
Openers Martin Guptill (67) and Brendon McCullum (64) both struck half-centuries as New Zealand reached 187-3 in a game reduced to 18 overs per side after rain stopped play three overs in.
Guptill faced 46 balls, hitting seven boundaries and two sixes while McCullum smashed two fours and six sixes, having faced 37 balls.
Jesse Ryder was undefeated on 30 runs.
Captain Ross Taylor (8) was run out on the last ball of the innings from Charles Coventry's throw to wicketkeeper Forster Mutizwa.
Chasing a daunting total, Zimbabwe …
Greek unions will stage their second nationwide strike in a week on Thursday, shutting down all public services, closing schools, customs and tax offices, halting public transport and grounding flights for 24 hours.
Unions are protesting the Socialist government's harsh austerity measures, designed to curb the country's massive debt and pull it out of an unprecedented financial crisis that has hammered the euro. The measures have cut civil servants' salaries, frozen pensions and increased taxes, including on fuel and general sales tax.
"They are trying to make workers pay the price for this crisis," said Yiannis Panagopoulos, leader of Greece's …
China's biggest national news agency launched its global, English-language television news network on Thursday as part of efforts to expand the communist government's influence abroad.
Experts say China's media expansion also results from unhappiness with much of the international coverage of sensitive events in China such as Tibet and human rights. The government has accused international media organizations of being biased and focused on negative news.
China Xinhua News Network Corp. (CNC) began broadcasting its English TV service, CNC World, around the clock, including news segments, feature stories, weather updates and special bulletins in Asia, Europe, North America and Africa, the official Xinhua News agency said.
"CNC will present an international vision with a China perspective. It will broadcast news reports in a timely way and objectively and be a new source of information for global audiences," Xinhua quoted its President Li Congjun as saying.
In recent years, China has announced multibillion-dollar plans to raise the country's profile by expanding Xinhua, state broadcaster China Central Television and the People's Daily, the flagship newspaper of the Communist Party.
"As the country gains more prominence and confidence on the world stage, China naturally wants to spread its message to the world," said Yu Guoming, a journalism professor at People's University in Beijing. "This expansion abroad will help outsiders better understand China."
CNC World will be broadcast internationally by satellite, cable, cell phone and the Internet, and its U.S. service will be available on cable networks beginning Oct. 1, Xinhua said.
Last year, CCTV began a 24-hour channel airing in 22 Arabic-speaking countries, reaching a total population of nearly 300 million people.
Xinhua, a ministry-level body under the administration of the State Council, China's Cabinet, said it is transforming itself into a multimedia, worldwide news agency.
All three state media outlets enjoy top-level party support and funding, along with virtual monopolies in certain sectors of their domestic markets.
Despite China's rapid economic growth and rising global influence, there has not been a freeing of the media in the country. China has retained its authoritarian one-party political system with strict limits on freedom of speech and civil and political life.
PGA TOUR: Reno-Tahoe Open
Site: Montreux Golf & Country Club (7,552 yards, par 72), Reno,Nev.
TV: Pax-38 (5 p.m. today) and CNBC (2 p.m. Saturday-Sunday).
1999 winner: Notah Begay.
Notes: Begay is competing in the NEC Invitational. . . . PGAChampionship runner-up Bob May is in the field.
LPGA TOUR: Oldsmobile Classic
Site: Walnut Hills Country Club (6,191 yards, par 72), EastLansing, Mich.
TV: ESPN2 (noon Friday; 3 p.m. Saturday; 2 p.m. Sunday).
1999 winner: Dottie Pepper.
Notes: Pepper is sidelined with a back injury. . . . MichiganState grad Barb Mucha won the inaugural event in 1992.
SENIOR PGA TOUR: FleetBoston Classic
Site: Nashawtuc Country Club (6,787 yards, par 72),
Concord, Mass.
TV: ESPN (10 a.m. Friday; 4:30 p.m. Saturday-Sunday).
Defending champion: Tom McGinnis.
Note: It has had six names, but this is the third-oldest event onthe tour after the PGA Seniors and U.S. Senior Open.
USGA: U.S. Amateur
Site: Baltusrol Golf Club, Upper Course (6,887 yards, par 71),Springfield, N.J.
TV: ESPN (2 p.m. today-Friday) and Ch. 5 (3 p.m. Saturday-Sunday).
1999 winner: David Gossett.
Notes: Gossett turned pro last month. . . . Match play beganWednesday. . . . Stroke-play qualifying also was held on the LowerCourse, a regular U.S. Open site.
TOKYO - The Bank of Japan's tankan survey Monday showed that business sentiment improved slightly in the three months through June amid signs of the Japanese economy's steady and balanced expansion.
The tankan index, which measures large manufacturers' business conditions, rose in the April-June quarter of 2006 to 21 from 20 in the January-March period.
The index measures the percentage of companies reporting that conditions are better minus those reporting conditions are worse.
by Calvin Forbes
Louisiana State University Press
April 2002, $15.95, ISBN 0-8071-2666-7
This collection of poetry is divided into four different sections, and begins with reflective poems that speak of a "forty something" man's experience with language, which moves through nostalgic moments with clarity and music that is rooted in jazz and blues traditions. In these three lines from the poem "Three Folk Songs," Forbes sums up the relationship of human-to-human, and words- to-poetry:
"Good poems are like some people
sometimes I can't remember their
names only how they make me feel"
In the second section, Forbes combines poetry that reflects his inner voice with insight from others' stories, in a narrative style that not only captures the color and timing of a moment but also the inherent surprise that is real life. Poems such as "Two" or "More Conversations" are incredulous, innocent, playful, inquisitive, quietly angry, erotic, and truthful.
"let me get this straight
first you lied to me
now you mad at me
because I believed you"
In the third and fourth sections, Forbes joins in a universal dialogue of poets and philosophers who challenge the construct and confines of language. In this world, nothing is ever what it seems, and any thinking to the contrary is at best enlightening, usually humbling and occasionally dangerous. In this section, poems such as "Oreo," "Picture of a Man" and "Facts" reach like a coal miner into a world where language collides with reality to form new paradigms.
"Survey says: people consume more
FACTS
than are good for them."
The final section introduces the resurrected "Shine," an African-American folk character. In his new life, Shine meets Glow, a lady DJ. Each of these poems is a sensual portrait of his love for her. Here, Shine grapples with the specter of his death, his travels through time and a ghost life.
"Outward he was mostly
Only red-eyed
And just the same Shining."
At the end of the collection is "A Little Poem," which stands in as Shine's reprieve in the guise of his love for his son Shade. There is nothing for Shine to negotiate here but hope, through the eyes of his son: "A man can live up/to what he's bringing up/a man can be a child/a child can't be a man/ this is why the son/had shine for his father."
A deadly B-52 bomber crash off Guam last year was caused by part of the plane's tail assembly being set in the wrong position, a U.S. Air Force investigation report released Friday said.
The plane's stabilizer trim was improperly set between 4.5 and 5.0 degrees nose-down at impact, indicating the aircraft had been in a nose-down descent at low altitude, according to a report by the Air Combat Command in Langley, Virginia. The stabilizer trim is used in conjunction with the aircraft's elevator to control the pitch of the aircraft.
The unarmed bomber was on a training mission that included a flyby in support of the Guam Liberation Day celebration when it crashed in July off Guam, a U.S. territory located 3,700 miles southwest of Hawaii. All six crew members on board were killed.
The report said the reason why the stabilizer trim was improperly set could not be determined because there were no survivors or emergency radio calls from the plane. Only a minimal amount of aircraft control systems or instruments were recovered.
The investigation also determined that the combination of a low altitude and a descending left turn, and the crew recognizing too late the severity of the situation, contributed to the crash.
But the board said any experienced crew could have found it difficult to recognize, assess and recover from the rapidly developing situation involving the stabilizer trim setting.
The B-52 was assigned to the 20th Bomb Squadron at Barksdale Air Force Base in Louisiana.
THE tightest of finishes is in prospect in the Hope & AnchorSummer Singles Darts League in Cardigan with five players incontention for the title. Results -- Stuart Robinson 6 Will Evans 6;Sion Thomas 7 Peter Bagley 0; Michael Blake 7 Gemma Bowen 0; JohnLocke 5 Steve Kinsella 7; Jamie Lewis 3 Lee Chenery 7; Peter John 7Lee Jones 2; Mandy Lewis 2 Guy Jeremiah 7; Craig Pope 7 LeightonJones 2; Kevin Phillips 6 Jordan Phillips 6; Jordan Lewis 3 WillEvans 7; Stuart Robinson 7 Gemma Bowen 4; Clive Lewis 7 AdamPhillips 2; Guy Jeremiah 7 Paul Thomas 2; Lee Chenery 5 SteveKinsella 7; Michael Blake 7 Mandy Lewis 1; Peter Bagley 0 Lee Jones7; Sion Thomas 7 John Locke 4; Peter John 7 Jordan Lewis 0; WillEvans 6 Leighton Jones 6; Gemma Bowen 0 Jamie Lewis 7; JordanPhillips 3 Stuart Robinson 7. 180: Jamie Lewis x 3; Sion Thomas,Craig Pope, Steve Kinsella, Will Evans x 2; Lee Chenery; Peter John;Kevin Phillips; Guy Jeremiah; Lee Jones; Peter John; StuartRobinson. High out: Stuart Robinson 137; Jamie Lewis 122; Will Evans120; Stuart Robinson 114.
Low darts: Jamie Lewis 13; Lee Chenery, Sion Thomas 14; SteveKinsella, Peter John, Lee Chenery, John Locke, Jamie Lewis, SionThomas 15.
Positions: Clive Lewis, Jamie Lewis 33; Peter John 31; Mark Lewis30; Sion Thomas 29; Guy Jeremiah 28; Steve Kinsella 26; MichaelBlake 24; Lee Jones 21; John Locke 19.
KOLKATA, India (AP) — England captain Alastair Cook won the toss Tuesday and elected to bowl first against India in the final one-day international of a five-game series at Eden Gardens.
India, which is looking to complete a whitewash after winning the first four games, brought in batsman Manoj Tiwary for Parthiv Patel.
England had to bring in Ian Bell for batsman Kevin Pietersen, who was ruled out due to a thumb injury.
India has used its homes conditions well to return to winning ways despite the absence of key players like Sachin Tendulkar, Virender Sehwag, Yuvraj Singh and Zaheer Khan, all of whom were injured during a recent tour of England where it failed to win a single game.
India was blanked 4-0 in the test series, lost a five-game one-day series 3-0 and also lost the solitary Twenty20 game on the tour of England.
After the one-day series, the teams play a one-off Twenty20 game in Kolkata on Saturday.
_____
India: Ajinkya Rahane, Gautam Gambhir, Manoj Tiwary, Virat Kohli, Suresh Raina, Mahendra Singh Dhoni (captain), Ravindra Jadeja, Ravichandran Ashwin, Vinay Kumar, Praveen Kumar, Varun Aaron.
England: Alastair Cook (captain), Craig Kieswetter, Jonathan Trott, Ian Bell, Ravi Bopara, Jonny Bairstow, Samit Patel, Stuart Meaker, Graeme Swann, Tim Bresnan, Steven Finn.
Iceland's president offered Thursday to give India clean energy technology to help it move away from its dependence on coal and oil to generate electricity.
President Olafur R. Grimsson also invited Indian businesses to invest in his country and set up joint ventures in information technology, banking, pharmaceutical and other sectors to strengthen India's presence in the U.S. and European markets.
Grimsson, who met Prime Minister Manmohan Singh and other Indian leaders, said that 100 percent of Iceland's electricity and space heating now comes from clean energy.
"One of the great opportunities for cooperation is that we can bring our geothermal and hydro expertise to India," he said.
Energy-starved India is largely dependent on coal and oil for electricity generation. It gets just 3 percent of its energy from nuclear power. It plans to increase its nuclear capacity to 63,000 megawatts by 2030 from 4,120 megawatts now.
Grimsson is scheduled to visit Bangalore, India's information-technology hub, on Friday.
For many jazz fans, Peterson (born on Aug. 15, 1925, inMontreal) and Evans (born on Aug. 16, 1929, in New Jersey) are thebe-all and end-all of modern piano trio practitioners - and, perhaps,the yin and yang. In Gene Lees' notes to Evans' "Complete FantasyRecordings," issued in 1989, Peterson is likened to Liszt for"conquering" the instrument and Evans to Chopin for "seducing" it.
But as different as they were, these prolific, mutually admiringartists were linked by a relentless pursuit of beauty and perfection.Three new CD collections of live performances, most of thempreviously unreleased, reveal that they also shared a love ofcommunica-tion - of playing for the people.Peterson has long said that his best albums are his live ones.He'll get no argument from anyone sampling the five-CD "London HouseSessions" (Verve), drawn from a long engagement at the defunctChicago club in the summer of 1961. The definitive set comprisesfour previously issued albums by "The Trio," including bassist RayBrown and drummer Ed Thigpen, and more than two hours of materialleft off them.No one will ever accuse Peterson of profundity. He frequentlysubstitutes flash for insight and adornment for lyrical depth. He isnot out to reveal himself as much as entertain with readilyaccessible effects. Listening to "The London House Sessions," youdon't get the sense of unfolding emotion or deepening intensity thatyou get from McCoy Tyner or Tommy Flanagan - to name two masters ofthe trio format who can be called profound.But when you're Oscar Peterson, and you can play with suchhairpin precision, awesome speed and perfect touch, those traitscreate their own heightened emotion. Hearing him dip into hismassive vocabulary on songs such as "Sometimes I'm Happy," which hecrowns with blues warmth while cutting ribbons of abandon, is to haveany quibbles swept off the carping-room floor.Peterson fans sometimes have difficulty choosing between hisprevious, drummerless trio with guitarist Herb Ellis and thisthreesome. Hearing him ride Thigpen's immaculate clean strokes tohigher rhythmic ground clinches the argument in favor of the lattergroup.In a recent interview (see accompanying article), Peterson saidhis producers decided which of the London House performances torelease, a statement that contradicted previously published reportsthat the pianist had the final say. In either case, some bets werehedged in leaving his sparkling bebop treatments such as CharlieParker's "Scrapple From the Apple" off the original albums in favorof more easily digested popular standards."The London House Sessions" is packaged in a coolly attractivebut problematic spiral-booklet format - the discs fit too snugly inthe pockets for easy extraction. But in immersing us in thesestate-of-Oscar performances - and offering us a vital piece ofChicago jazz club history to go with Miles Davis' "Complete Live atthe Plugged Nickel 1965" - it makes such an inconvenience a smallone.The market is so flooded with tracks by the incessantlystudio-driven Evans, including the 11-CD "Complete RiversideRecordings" and nine-CD "Complete Fantasy Recordings," some listenersmay be put off by the prospect of diving into two more box sets: thesix-CD "Turn Out the Stars: The Final Village Vanguard Recordings,June 1980" (Warner Bros.) and the eight-CD "The Secret Sessions"(Milestone/Fantasy), recorded at the Vanguard between 1966 and 1975.And that won't be all, folks: Verve's long-in-the-releasing18-CD opus is due out next year. That adds up to a lot of disc froma musician who infrequently departed the trio setting or altered hisapproach except to deepen it.Only the most diehard Evans fans really need "The SecretSessions," recorded by a fanatic named Mike Harris, who snuck a taperecorder into New York's treasured Vanguard on 26 occasions tocapture his hero. Unlike obsessive Parker chronicler Dean Benedetti,who recorded only the immortal saxophonist's solos on the tapes thatbecame Mosaic Records' 1990 compendium, "The Complete BenedettiCharlie Parker," Harris kept his machine running for entireperformances by various Evans trios.While the sound of "The Secret Sessions" is better than youwould expect, its canned, muffled quality is still something to getpast. And though there are some choice moments - the great bopdrummer Philly Joe Jones powers Evans to some of his hardest-edgedplaying, and you can hear longtime Evans bassist Eddie Gomez settlinginto his mastery - they are generously answered by ones that find thepianist in a sorting-out phase.Evans and many of his followers were still measuring his workagainst that of his legendary 1959-61 trio with young bass virtuosoScott La Faro (whose death in a car crash devastated Evans) anddrummer Paul Motian. It proved an impossible standard to meet - oravoid."Turn Out the Stars," on the other hand, finds Evans on firm,long-term footing with bassist Marc Johnson and drummer JoeLaBarbera. He was writing new songs, reinventing old ones andotherwise pushing the group into new directions. These performances,taken from multiple sets over four nights, amount to a valuable finalstatement by an artist whose life light would be extinguished threemonths later.A barometer of his rejuvenation - and his trio's - can be foundin three expansive versions of "Nardis," a composition credited toMiles Davis. Evans had been playing the song for years ("SecretSessions" includes a rough and tumble 1967 reading with Gomez andJones) and had previously recorded it - unlike Davis, to whoselegendary album, "Kind of Blue," Evans made a crucial contribution.Here, "Nardis" takes the shape of a dark, exploratory epic thatdraws as much from modern classical concepts as modal and otherpost-bop innovations. Up and down the scales Evans goes on oneversion, creating thick storm clouds with his left hand whilesearching for an opening in them with his lyrically beaming right.When he finds that opening, the song explodes into melody, propelledso fiercely that it should give pause to those who underrate Evans'capacity to swing, and swing hard.(Inexplicably, the single-disc sampler of "Turn Out the Stars,"dubbed "The Artist's Choice" because it approximates the double albumthat Evans had in mind but was never released, edits a 16-minuteversion of "Nardis" down to a six-minute excerpt. There's room onthe CD for the entire piece, so why the abridgement? That wouldhardly have been Evans' choice.)Even when he played pretty, as on the infectious, richlymelodic "Bill's Hit Tune" and Johnny Mandel's wistfully beautiful"Theme From MASH," Evans never allowed his notes the luxury ofrelaxation. Digging into the unrooted chords he patented, he openedboth them and his emotions to new possibility.Are "The Final Village Vanguard Recordings" the equal of Evans'Vanguard albums with La Faro and Motian? Such was the shock ofsurprise that emanated from that trio, any subsequent group would befated to secondary consideration.But when he hooked up with Johnson and LaBarbera, Evans had adeeper reservoir of pained experience and wisdom to draw on, and thatcertainly comes through. For all the records he made, you never geta sense that he had said all he had to say. If ever a musician wasdriven to explore himself in the name of art, relentlessly, it wasthis one. One can only regret not being able to hear him go down thepaths on which he was headed."London House Sessions": (STAR)(STAR)(STAR) 1/2; "Turn Out theStars: The Final Village Vanguard Recordings, June 1980":(STAR)(STAR)(STAR)(STAR); "The Secret Sessions," (STAR)(STAR)(STAR).In Tuesday's Jazz Etc. column, Lloyd Sachs discusses another boxset, the complete Dexter Gordon on Blue Note.
TOKYO (AP) — Chilean miner Edison Pena completed the Tokyo Marathon on Sunday, shaving more than 30 minutes off the time he set in New York in his second such race since he was among the 33 men rescued in October.
The 35-year-old Pena had been training more than 6 miles a day and it showed, as he finished the race through the streets of downtown Tokyo in 5 hours, 8 minutes, 19 seconds. In the New York City Marathon, he ran and walked on bad knees to finish in 5 hours, 40 minutes on Nov. 7.
"It was not like the New York Marathon," Pena said. "I didn't walk, I ran the entire time."
Pena jogged through tunnels while trapped underground, and word of his dedication inspired New York City Marathon organizers to invite him to watch the race. Instead, he asked to run it.
Pena said there may be more marathons in his future.
"It's just a big challenge for me," Pena said. "It's an incredible thing to do and it's just amazing to be able to do something like this for a second time. I felt very good about my result today."
Executives from Remo System, a Japanese maker of shoe inserts and other training products, were in New York for the marathon and were moved to invite him to Tokyo. They worked with Fuji TV to get him into Sunday's race.
Remo plans to donate 2,000 pairs of shoes to needy Chileans in his honor.
Today is Tuesday, Dec. 23, the 358th day of 2008. There are 8 days left in the year.
Highlights in history on this date:
1588 - Henry, Duke of Guise and leader of militant Catholics who wanted a Spanish princess on the French throne, is assassinated on King Henry III's orders at Blois, France.
1601 - Irish rebels Tyronne and O'Donnell are routed near Kinsdale by British forces.
1698 - George Lewis succeeds as Elector of Hanover on death of Ernest Augustus.
1728 - Holy Roman Emperor Charles VI and Frederick William of Prussia sign Treaty of Berlin.
1783 - George Washington resigns as commander in chief of the U.S. Army and retires to his home at Mount Vernon, Virginia.
1832 - The French take Antwerp, forcing Holland to recognize Belgium's independence.
1861 - Sultan of Turkey agrees to unification of Moldavia and Wallachia as Romania.
1876 - The first constitution in a Muslim country is passed in Turkey, but power remains largely in the hands of the sultan.
1920 - French and British approve convention fixing boundaries of Syria and Palestine.
1941 - U.S. forces on Wake Island in the Pacific surrender to Japanese in World War II.
1947 - The transistor is invented at the Bell Telephone Laboratories in New Jersey. This made equipment miniaturization possible and ushered in a tidal wave of electronic miracles including the personal computer.
1948 - Japanese Premier Hideki Tojo and six other Japanese World War II leaders are executed in Tokyo.
1950 - A treaty in which Vietnam becomes a sovereign nation within the French Union is signed in Saigon.
1968 - Eighty-two crew members of the U.S. intelligence ship Pueblo are released by North Korea, 11 months after they were captured.
1972 - Earthquake strikes Managua, Nicaragua, killing 10,000.
1977 - Eight French nationals kidnapped in May by guerrillas fighting for the independence of the former Spanish Sahara, are handed over to U.N. Secretary General Kurt Waldheim in Algiers.
1986 - The experimental U.S. airplane, Voyager, lands in California's Mojave Desert after becoming the first aircraft to circumnavigate the globe nonstop without refueling.
1989 - United States sends 2,000 reinforcement troops to Panama to combat unexpectedly stiff resistance from Panamanian troops loyal to ousted General Manuel Antonio Noriega.
1990 - Slovenes vote overwhelmingly in favor of secession from Yugoslavia.
1993 - The Rhine sweeps to its greatest height in 67 years, flooding Cologne's old town and menacing the new Parliament building in Bonn. The death toll in European flooding reaches six.
1994 - Bosnia's Serbs and the Muslim-led government agree to a cease-fire in their war.
1995 - A fire in Dabwali, India, kills 540 people, including 170 children, during a year-end party being held near a children's school.
1996 - Tajikistan's President Emomali Rakhmonov and the top opposition leader Sayed Abdullo Nuri sign a cease-fire agreement aimed at ending the four-year civil war.
1997 - Gunmen charge into a village of rebel sympathizers in Chiapas, Mexico, and kill 45 people, including 15 children.
1998 - South Korean police use water cannon and tear gas to evict a group of monks from the Chogye temple, after the monks threw firebombs and rocks at them. The clash ends a 40-day standoff between rival monks at the spiritual home to eight million Buddhists.
2000 - U.S. and British warplanes in Baghdad strike targets in southern Iraq. Iraqi officials say the attack killed one person and injured two others. The U.S. Central Command headquarters reports that the warplanes, patrolling the no-fly zone in southern Iraq, attacked a radar system and anti-aircraft sites with precision-guided weapons.
2001 - San Luis Governor Adolfo Rodriguez Saa takes over as interim president of Argentina, immediately halting payments on the nation's $132 billion debt. He resigns a week later saying "an attitude of pettiness and haggling" within his Justicialist party has left him unable to govern.
2003 - An explosion at a natural gas field near Chongqing in Sichuan province of China kills at least 198 people. The explosion reportedly occurred when a pocket of natural gas and hydrogen sulfide was accidentally drilled open.
2005 - South Korean scientist Hwang Woo-suk resigns from his university after he was found to have faked stem-cell breakthroughs that kindled worldwide optimism of revolutionary cures for disease.
2006 - U.N. Security Council votes unanimously to impose the first set of sanctions on Iran for refusing to suspend uranium enrichment amid suspicions Tehran wants to build nuclear weapons.
2007 - The world's last Hindu monarchy is to be swept aside under an agreement signed between Nepal's former communist rebels and its major political parties that sets the stage for the country to become a republic.
Today's Birthdays:
James Gibbs, Scottish architect (1682-1754); Richard Arkwright, English inventor (1732-1792); Sarah Breedlove Walker, U.S. philanthropist (1867-1919); Joseph Smith, U.S. founder of the Mormon Church (1805-1844); Jose Greco, Spanish dancer-choreographer (1918-2000); Silvia, queen of Sweden (1944--); Akihito, emperor of Japan (1933--); Eddie Vedder, U.S. musician (1964--).
Thought For Today:
Christmas is the season when you buy this year's gifts with next year's money _ Anonymous.
ABSTRACT
Proapoptotic phosphorothioate oligonucleotides such as G3139 (an 18-mer) induce Bc1-2-independent apoptosis, perhaps partly via direct interaction with VDAC and reduction of metabolite flow across the mitochondrial outer membrane. Here, we analyzed the interactions at the molecular level. Ten micromolar G3139 induces rapid flickering of the VDAC conductance and, occasionally, a complete conductance drop. These phenomena occur only when VDAC is in the "open" conformation and therefore are consistent with pore blockage rather than VDAC closure. Blockage occurs preferentially from one side of the VDAC channel. It depends linearly on the [G3139] and is voltage-dependent with an effective valence of -3. The kinetics indicate at least a partial entry of G3139 into VDAC, forming an unstable bound state, which is responsible for the rapid flickering (~0.1 ms). Subsequently, a long-lived blocked state is formed. An 8-mer phosphorothioate, polydeoxythymidine, induces partial blockage of VDAC and a change in selectivity from favoring anions to favoring cations. Thus, the oligonucleotide is close to the ion stream. The phosphodiester congener of G3139 is ineffective at the concentrations used, excluding a general polyanion effect. This shows the importance of sulfur atoms. The results are consistent with a binding-induced blockage rather than a permeation block.
INTRODUCTION
VDAC is the major metabolite pathway across the mitochondrial outer membrane (1-3). It is composed of a single 30-32 kDa polypeptide chain forming a barrel-like channel (4-6) with a molecular mass cutoff at ~5 kDa for nonelectrolytes (7). In the open state, VDAC is weakly anion selective and permeable to multivalent anionic metabolites such as ADP, ATP, and NADH (8-10). At high positive or negative voltages (>25 mV), the positivelycharged voltage sensor region of VDAC tends to move out of the channel, reducing the diameter of the channel from ~2.5 mil to 1.8 nm and reversing the ion selectivity (11,12). This forms the closed states of VDAC. Even though the closure of VDAC results in only a 40-60$ reduction of conductance, and the size of the channel is still larger than a single ATP molecule (13), closed VDAC actually becomes impermeable to ATP (9). Thus, in addition to lhe channel size, the distribution of the electric charge in the channel wall is very important for controlling the How of anionic metabolites between the cytosol and mitochondria (14).
This regulation of metabolite permeation through the mitochondrial outer membrane plays a significant role in controlling cell death and apoptosis (15). Recent studies on phosphorothioate oligonucleotides, such as G3139, have strengthened the linkage (16). Phosphorothioate oligonucleotides have each phosphodiester linkage modified such that one nonbridging oxygen is replaced by a sulfur atom. This makes the oligonucleotide resistant to nuclease action. G3139 is an 18-mer phosphorothioate oligonucleotide that is antisense to the initiation region of Bcl-2 mRNA (17). It was designed to sensitize cancer cells to chemotherapy agents by downregulating Bcl-2 levels (18.19). However, when G3139 was introduced into cells, cytochrome c release preceded the downrcgulation of Bc1-2. This and the lack of strict sequence dependence (20) indicated that another mechanism must be responsible for the proapoptotic effect of G3 1 39. Experiments with isolated mitochondria showed that G3139 interferes with metabolite flow through VDAC, and this may partly account for the ability of G3139 to induce apoptosis (16.21).
Interactions between oligonucleotides and protein channels have been intensively studied, mainly using the a-hemolysin channel (22-24). α-Hemolysin forms a heptameric channel with a diameter of 2.6 nm (25). Unlike VDAC, the �-hemolysin channel is very asymmetric with a mushroom-like domain on one membrane surface (25). However, its channel size is very similar to the open conformation of VDAC. The permeation of oligonucleotides through α-hemolysin channels (22-24) suggests that they may enter VDAC in a similar manner. This entry may account for blockage of the VDAC channel.
In this work, we investigated the molecular basis for the interaction between phosphorothioate oligonucleotides and VDAC. We propose that partial entry of G3139 into and subsequent binding to VDAC channels account for the apparent result that G3139 induces VDAC closure.
MATERIALS AND METHODS
Planar phospholipid membrane studies
Planar phospholipid membranes were generated according to standard methods (26.27). The membrane was formed from phospholipid monolayers consisting of diphytanoyl phosphatidylcholine, polar extract of soybean phospholipids (both from Avanti Polar Lipids. Alabaster, AL). and cholesterol (Sigma, St. Louis, MO) in a 1:1:0.1 mass ratio.
VDAC was purilied from mitochondria isolated from rat liver (5.28). A 0.1-�l aliquot of the VDAC-containing solution (2.5% Triton XlOO, 50 mM KCl. 10 mM Tris. I mM EDTA. 15% DMSO. pH 7.0) was stirred into 4-6 ml of aqueous solution containing 1.0 M KCl. 5 mM CaCl^sub 2^. 1 mM EDTA. and 5 mM HEPES (pH 7.2) on the cis side of the chamber. The trans side, containing the same aqueous solution, was held at virtual ground by the voltage clamp.
After a single channel insertion, a triangular voltage wave at a frequency of 3 mHz from -52 mV to +52 mV was applied to the eis side. The current was recorded at a rale of 100 �s per point with no filtering unless mentioned otherwise. All experiments were performed at ~-23�C.
The data were analyzed using the QuB program downloaded from the web site (http://www.qub.buffalo.edu/wiki/index.php/Main_Page) of the State University of New York. Buffalo.
Ion selectivity measurements
These were periormeii in a similar way as above except that KCl concentration gradients (0.50 M KCl vs. 0.10 M KCl; other components are: 0.5 mM CaCl^sub 2^. 1 mM HEPES, pH 7.2) were used and recordings were made at 1 ms per point with 2-kHz filtering. The salt activity coefficients of 0.5 M KCl and 0.1 M KCl are 0.651 and 0.770, respectively. The reversal potential was measured by a linear fit to the current-voltage curve. The ion selectivity was estimated using the Goldman-Hodgkin-Katz theory.
Oligonucleotides
G3139 was kindly donated by Dr. R. Brown, Genta (Berkeley Heights, NJ). Phosphorothioate homopolythymidine 8 or 14 nucleotides in length was synthesized using standard automated phosphoramidite chemistry on an ABI Model 3400 DRNA/RNA synthesizer. ^sup 3^H-1,2-benzodithiol-3-one 1,1-dioxide was used as the sulfurizing agent. The 5'-dimethoxytrityl-derivatized oligonucleotides were released form the support by treatment with 400 �l of a solution containing concentrated ammonium hydroxide and 959? ethanol (3:1 v/v) for 3.5 h at 55�C. The dimethoxytrityl derivatized oligonucleotide was dissolved in 50 mM sodium phosphate (pH 5.8), and the solution was then loaded onto a C- 18 reversed-phase SEP PAK cartridge (Watters, Milioni. MA). The cartridge was washed sequentially with 10% acetonitrile in 50 mM sodium phosphate buffer, water, 2% trifluoroacetic acid and water. The detritylated oligonucleotide was then eluted from the cartridge with 50% aqueous acetonitrile. The purity of the oligonucleotide was confirmed by reversed-phase HPLC.
The phosphodiestcr congener of G3139 was synthesized by standard automated phosphoramidite chemistry. The oligonucleotide was deprotected as described above and purified by strong anion-exebange HPLC.
RESULTS
G3139 induces complete conductance loss of VDAC
VDAC is a voltage-dependent channel. When reconstituted into planar membranes, it is open at low voltages and tends to enter low-conducting, closed states at high voltages (>25 mV)(1). There are two independent sets of closed states, one at positive and the other at negative potentials. The normal VDAC closure results in only a partial conductance loss (Fig. 1 A), with the remaining conductance generally between 40% and 60% ofthat of the open state (Fig. 2). When 10 �M G3139 was added to the eis side of the planar membrane (same side as the addition of VDAC), rapid flickering of VDAC conductance occurred in the millisecond time scale. However, flickering was observed only when negative voltages were applied (Fig. 1, B and C). This is consistent with the held driving the negatively charged G3139 toward the lumen of the channel. Each time-resolved conductance drop during the flickering process reaches a conductance level that is close to zero. This is clearly evident in the very long-lived events (Fig. 1 B).
The G3139-induced closure of VDAC is clearly different from voltage-induced closure. Although both processes are voltage dependent, the kinetic properties are quite different. G3139 induces rapid gating accompanied by long-lived closing events. Without G3139, channels close with slow kinetics. The extent of closure is much greater (Fig. 2) in the presence of G3 1 39. This closure often represents a complete loss of conductance. This complete closure requires direct interactions between G3139 and VDAC and suggests that G3139-induced VDAC closure is distinct from the normal VDAC closing process.
The closed states of VDAC have been shown to be favored by a variety of polyanions (29). In these cases, the closure is not distinguishable from normal VDAC closure. Indeed, the evidence strongly indicates that polyanions act on the normal gating process by interacting electrostatically with the positively charged voltage sensor forming part of the wall of the aqueous pore and favoring its translocation to the membrane surface (30). However, what appears to be G3139-induced VDAC closure may actually be blockage of VDAC by G3139. This mechanistic distinction is reinforced by the fact that the equally charged phosphodiester congener of G3139 does not induce VDAC flickering or conductance loss (16). It also has little effect on VDAC at the concentrations used Io observe lhe effects of G3I39.
G3139 directly binds to a specific conformation of VDAC
There are several possible molecular mechanisms that could account for the observed conductance fluctuations after the addition of G3139 to VDAC channels. G3139 could induce a conformational change in VDAC resuliing in lhe occlusion of the pore. This seems unlikely because VDAC does not normally close completely. Alternatively. G3I39 could permeate through VDAC channels. Its large size and multiple negative charges might block the channel completely. Thus, the flickering would reflect the transit time of G3I39. However, one could also propose that G3139 simply binds to the mouth and inner wall of the channel but does not translocate through lhe membrane lo lhe other side. Longlived occlusions would result from conformers of G3 1 39 that bind tightly to the walls of the channel. A particularly pertinent observation that addresses this issue was made by varying the location of G3139 addition. Addition of G3139 to the same side of the membrane as VDAC addition (eis side) resulted in rapid flickering in -90 of all experiments. Addition of G3139 to the opposite (trans) side, resulted in very little flickering ( 10 events per second in 2-kHz filtering). When G3139 was added to both sides, flickering was almost exclusively observed when the eis side was held at a negative voltage (Fig. 3 A). Recall that the G3I39 side must be made negative to induce flickering. The difference in the rate of flickering was quantilied by measuring the time constant for channel closure (τ^sub on^, or mean duration of the open state) obtained using the QuB program (see Methods). When the cis side was at -50 mV compared to the trans side, τ^sub on^ = 9.8 � 0.8 ms, whereas when the trans side was at -50 mV compared to the cis side. τ^sub on^ [asymptotically =] 100 ms. This asymmetric effect could arise from a permeation blockage bul is more consistent with an asymmetric binding site.
The asymmetric nature of the G3139-induced llickering reveals the asymmetric insertion of VDAC (from ral liver) into planar membranes. This observation agrees with recent findings about the asymmetric insertion pattern of VDAC from mammalian sources (31), which is different from observations made with yeast VDAC (32).
These observations also strengthen the hypothesis thai G3139 specifically interacts with one area of the channel's surface. The asymmetry reveals specificity for a region of the protein. There is also specificity for the ligand because the phosphodiester congener does not induce channel blockage. The focus is clearly on the phosphorothioate backbone of G3139 because lhe specific sequence of bases is unimportant (a phosphorothioate composed of just thymidine also induces flickering and full blockage, see Fig. 6). Thus, there must be some specific affinity between the phosphorothioate and some region or domain of the VDAC molecule.
The existence of a specific interacting domain on VDAC is further supported by the observation that G3139 induces flickering almost exclusively when VDAC is open. When VDAC is closed (i.e., in a low-conducting state), there is nearly no flickering (Fig. 3 B). This suggests some specific interactions between G3139 and the mobile region of VDAC, the voltagc-scnsor region (12). This is a positively charged region and thus ideal for interacting with G3I39. When that positively charged region moves out of the channel (the gating process of VDAC (12)). G3139 is likely to still bind to this region, which is mostly outside the pore. This binding would not induce VDAC flickering or complete closure.
The kinetics of the interaction
Because G3139 is a charged molecule, the mechanism just presented would predict the existence of a voltage dependence to the blocking action of G3139. The QuB program was used to analyze the kinetics of the flickering events. For these experiments, G3139 was added only to the cis side.
Clearly, one could Ht the data with more blocked states. There may be a variety of ways for G3139 to bind to VDAC: different conformers of G3139 and different ways of binding to the site on VDAC. It is possible that, with time, G3139 may adapt to the VDAC surface, increasing the strength of the interaction and thus decreasing the rate constant of dissociation. This latter process is what is modeled in Eq. 1. where k^sub 1^ and k^sub -1^ are the forward and reverse rate constants of the first reaction, respectively; k^sub 2^ and k^sub 2^ are the forward and reverse rate constants of the second reaction, respectively.
Fig. 5 B shows that k^sub 1^ is voltage dependent. This is not surprising because the applied voltage changes the activation energy between the open state and the �rsl blocked state. The energy change is equal to nFV (n is the effective valence of the block, F is the Faraday constant, and G is the applied voltage). Thus, k^sub 1^ = k^sub 0^exp(nFV/RT) (k^sub 0^ is the rate constant of the same reaction with no applied voltage. R is the gas constant, and T is the temperature). Fitting the results to this equation (Fig. 5 B) yields an effective valence of -2.9 � 0.1 (n = 3, mean � SE) (T = 295 K). Even though, formally, G3139 bears a net charge of 18. experiments indicate that an oligonucleotide composed of 18 bases has a net charge of 9 because of the screening by counlerions (33). In the narrow confines within the channel, the screening effect may be quite different. In any event, the voltage dependence suggests that at least part of G3139 moved through the electric field by entering the pore of the channel.
Oligonucleotide binding induces changes in selectivity
If the oligonucleotide enters the pore of the channel, its negative charge should alter the ion selectivity. However, G3139 results in a virtually complete block to ions. Thus, we tested shorter phosphorothioate oligonucleotides to achieve incomplete block and thus detect any change in ion selectivity. Phosphorothioate oligonucleotides composed entirely of the base, thymidine, are readily made, and those of comparable lenglh are as effective on VDAC as G3139. Even the 14-mer. dT^sub 14^ has similar abilities to induce rapid flickering and complete conductance loss of VDAC channels (Fig. 6). However, the shorter version. dT^sub 8^, only reduces the conductance to ~40-60% ofthat of an open VDAC channel.
The residual conductance remaining after the binding of dT8 is cation selective (P^sub K^/P^sub CI^ = 2) with a reversal potential of -14 mV � 2 mV (mean � SE, n = 9). This is substantially different from the selectivity of the open VDAC channel (P^sub K^/P^sub CI^ = 0.5). This change in ion selectivity supports the proposed model that the flickering may be a result of the interaction of the phosphorothioate oligonucleotides and the inner wall of VDAC. dT^sub 8^ may not be long enough to fully occlude the pore. dT^sub 10^ causes ~70-80% of full blockage, and dT^sub 12^ causes ~80-90% of full blockage.
DISCUSSION
Attempts to make cancer cells sensitive to chemotherapy agents by knocking down the antiapoptotic protein Bcl-2 have resulted in the generation of a phosphorothioate oligonucleotide that blocks VDAC channels. This blockage does not depend on the sequence but on the phosphorothioate backbone. These molecules are the most specific VDAC blockers available to date. They will likely be useful tools to study the role of VDAC in cellular function.
The blockage of VDAC by G3139 is linearly dependent on the |G3139|. indicating a 1:1 complex. It is also voltage dependent, indicating that G3139 is entering the lumen of the channel at least to some extent. dT^sub 8^, which is eight nucleotides long, produces only a partial block. Full blockage requires a length of ~14 nucleotides. From a molecular dynamics simulation of a single-stranded DNA oligonucleotide inside a 2.4-nm hole (which is similar in size Io the VDAC pore), the oligonucleotide has a conformation similar to that of a B -form helix, which has a distance of 0.34 nm bet w ecu each base and pitch of 10 bases per turn (34). However, in the simulation the oligonucleotide had no affinity for the wall of the pore, resulting in a helix with an outside diameter on 2.0 nm. Affinity for the wall and the larger size of the VDAC pore (2.5-3 nm) should result in a helix with a greater pitch, perhaps 12. A minimum of a full turn of the helix would be needed to achieve complete block, so this explains why dT^sub 8^ achieves only an incomplete block. This also explains the almost linear increase in blockage with oligonucleotide length.
Our analysis indicates that blockage results in the effective translocation of three charges through the entire potential difference across the membrane. If the oligonucleotide were located entirely within the pore, the IS charges would be moving an average of half way through the transmembrane potential difference. This should result in an effective valency of -9 if there is no charge screening or ~ -5 with charge screening (33). Thus, it appears that only part of the oligonucleotide is located inside the channel.
The ability of phosphorothioate oligonucleotides such as G3I39 to block VDAC channels seems to arise from their ability to bind to VDAC preferentially from one side of the channel. The substantial asymmetry of the rapid flickering of conductance (Fig. 3 A) argues against the possibility of a permeation block, blocking in transit. VDAC is functionally and structurally rather symmetrical ( 1.35). forming a simple cylindrical pore. Thus. VDAC would be expected to allow oligonucleotides to permeate equally well in both directions. Single-stranded phosphodiester oligonucleotides of 30-200 bases have been shown to block the current How through a-hemolysin (22-24) as they flow through the channel. The flickering arising from this transient blockage is asymmetric (24. 3d). However, ihis asymmetrv is understandable considering the highly asymmetric structure of α-hemolysin (25). Indeed α-hemolysin is also functionally asymmetric, rectifying even in KCl solutions (36). VDAC normally shows essentially no rectification in its open state (37). Despite all this structural and functional asymmetry, α-hemolysin shows a lower leve) of asymmetric nucleotide block (only 6-fold) as compared to over 10-fold for VDAC exposed to G3 1 39.
An additional difference between the G3I39 block of VDAC and the observations with a-hemolysin is that much longer oligonucleotides (200 bases) must be added to a-hemolysin to obtain blockage lifetimes similar in magnitude to those reponed here (23). This ind�cales that transit times of short oligonucleotides are too short to accounf fotthe blockage lifetimes we observe without substantial slowing of the transit by interactions with the walls of the pore.
A variety of experimental observations provide further evidence for a "binding block"* as opposed to a permeation block:
1. Phosphodiester oligonucleotides of similar si/c and charge should have a similar ability to permeate VDAC channels as the phosphorothioate congener, yet these fail to cause tlickering (16). This is easily explained by the binding but not the permeation model.
2. The voltage independence of the unbinding rate for the rapid flickering suggests that dissociation of the oligonucleotide is the rate-limiting step. Diffusion away from the site against the held is not rate limiting. In the permeation blockage model, unblockage should be voltage dependent. Indeed, for �-hemolysin there is an inverse voltage dependence of the polyj U l-induced blockage time (23). demonstrating that unblockage is voltage dependent.
3. The long-lived complete closure of VDAC induced by G3139 cannot be cleared by reversing the polarity Of the Iransmembrane potential. When permeation of poly'U) through α-hemolysin results in long-lived blockage, the channels can be unblocked by reversing the sign of the potential, consistent with permeation block (23).
4. The shorter phosphorothioate oligonucleotides, such as dT8, cause only a partial blockage. Note that the permeation blockage model would predict that the length of the oligonucleotides should determine only the blockage time (23). not the extent of blockage. Therefore, the binding of 03 1 39 to the inner wall of VDAC and subsequent conformational changes appear to be the mechanisms for the observed flickering and complete conductance loss.
One observation that remains mysterious is the long delav between addition of G3 1 39 and onset of tlickering. The rapid llickering of conductance occurs after ~3-5 min after the addition of the phosphorothioate oligonucleotide. For G3139. which has a diffusion coefficient of 2 � 10^sup -6^ cm^sup 2^/s (33), it takes ~6 s to diffuse through a 50-�m unstirred layer next to the planar membrane. Thus, it seems that G3I39 needs to adapt to VDAC before flickering occurs. Indeed, when the concentration of G3I39 is increased, it takes about another 3-5 min for the tlickering effect to increase. These results suggest that there might be some conformational change of G3139 after addition to the buffer solution bathing the membrane. We have tried to incubate G3139 with the buffer solution and heat it before adding it to the system, but there was still a delav. Another explanation for the delav might be a slow process of interaction between G3139 and the phospholipid membrane, which may sensitize VDAC to G3139 by changing, for example, the surface potential. Regardless of the fact that, at this stage, we don't have a good explanation for this phenomenon, this delay does not affect the proposed mechanism by which G3I39 acts on VDAC.
For a VDAC channel, the transient blocked state (first step in Eq. 2) is clearly unfavorable because the k^sub -1^ is 2000-3000 s^sup -1^ . The on rate, k^sub 1^ is concentration and voltage dependent: 0.1 1 [G3139|exp(0.11 Voltage) �M^sup -1^s^sup -1^. At 1 �M G3139 and no applied voltage, the on rate is 0.11 s^sup -1^. Thus, the equilibrium constant is 4 � 10^sup -5^ with an unfavorable free energy change of 25 kJ per mole or 10 kT. The high off rate can be compensated for by a higher on rate. A higher concentration and the application of a negative voltage obviously favor binding and make the process feasible. Rapid unbinding of G3139 from VDAC suggests the instability of the transient G3I39-VDAC complex. However, the occurrence of longer complete closures clearly shows that the intermediate transient state can be stabilized. The analysis shows a slow on rate k^sub 2^ and off rate k^sub -2^ of the second interaction step in Eq. 2. suggesting that both of those interactions may involve some slow conformational change of VDAC and/or G3139. These structural changes make the bound state more energetically favorable and lead to complete, long-lasting VDAC closure.
Thus, the G3l39-bloeked VDAC channel is composed of an "open" VDAC channel and G3139 binding from one aqueous surface. Good candidates lor surfaces forming the binding site are the positively charged voltage sensor and some adjacent region outside the pore. This explains why the lower-conducting, closed states, whose pore size was estimated to be 1.8 nm (35.38.39). show virtually no flickering despite being large enough to accommodate the oligonucleotides. However, the unfavorable net charge in the pore of the closed state of VDAC may also explain the lack of flickering. Another possible binding site is the nucleotide binding site in the inner wall of VDAC (13,40). This site may not be distinct from the voltage sensor region. The importance of phosphorothioate may be a greater affinity for the binding site on VDAC. allowing the formation of a long-lived block to metabolites. In any case, the binding of G3I39 to the inner wall forms the completely blocked state, and the shorter oligonucleotides do not have the capacity to fully block the channel (Fig. 6). Obviously, the sulfur group is very important for the binding, as the phosphodiester congener does not cause VDAC flickering and complete closure (16).
Phosphorothioate oligonucleotides are. presently, the most specific inhibitors of VDAC channels. Their potency of blockage/closure of VDAC channels in isolated mitochondria is 50-fold higher than that observed for the experiments on pure VDAC reconstituted in planar membranes (as presented here). Perhaps a VDAC-associated protein or lipid increases the affinity of VDAC for the phosphorothioate. In any case, this work provides a mechanistic foundation for understanding effects in cells and organelles.
[Sidebar]
doi: 10.1529/biophysj.107.105379
[Reference]
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[Author Affiliation]
Wenzhi Tan,* Yue-Hin Loke,* C. A. Stein,[dagger] Paul Miller,[double dagger] and Marco Colombini*
* Department of Biology, University of Maryland, College Park, Maryland 20742; [dagger] Albert Einslein-Montefiore Cancer Center,
Department of Oncology, Montefiore Medical Center, Bronx, New York 10467; and [double dagger] The Johns Hopkins University,
School of Public Health, Baltimore, Maryland 21287
[Author Affiliation]
Submitted January 26. 2007. anil accepted for publication April Zh. 2007.
Address reprint requests to Marco Colombini. Dept. of Biology. University of Maryland. College Park. MD 20742. Tel.: 301 405-6925: Fax: 301 314-9358: E-mail: colombini@umd.edu.
Editor: Tzyh-Chang Hwang.
� 2007 by the Biophysical Society
0006-3495/07/08/1184/08 $2.00
AVON A 41-12 triumph at Midsomer Norton saw Avon climb to thirdspot in Somerset Premier.
In doing so, they clocked up their fourth win from five matchesthis season, scoring six tries in the process. Skipper Kai Harwoodopened the scoring when he finished off a break from fly-half DesBowden to power over the line with a trademark charge for a tryconverted by Sam Rowlands.
Norton struck back strongly with a try from their pack after alineout catch and drive.
Avon then started to revel in the excellent conditions throughthreatening runs from Ben Dilloway, Dan Mutlow and Jack Moon beforeRowlands finished off some close quarter approach work from the packto steal over the line for another try which he coolly converted.
Immediately after the break, Avon engineered the try of the matchas they spread the ball left, right and left again to bemuse theiropponents before Bowden plucked the scoring pass out of the air witha horizontal dive over the tryline. Rowlands again added theconversion.
After a lengthy injury break, Avon continued where they had leftoff, with Harwood collecting his second try in the corner from awell rehearsed front lineout peel, while Rowlands also slotted apenalty.
Norton, to their credit, responded with a converted try from aforward drive but this simply spurred Avon on to greater heights asthey built on their excellent team-work with two further scores.
First, predatory man of the match Scott Book charged down anattempted clearance before pouncing on the loose ball for anotherconverted try.
Rowlands then capped an excellent display with a mazy run fromthe halfway line to expose the tiring home defence and go over forthe final try.
Avon 2nds went down 40-17 to a powerful Clevedon 3rds outfit butplayed a full part in an entertaining game.
Ollie Harding Wyatt, who is improving in every game, scored twotries, with another being added by Andy Mead. Brother Pete Meadkicked one conversion.
RUGBY TEAMS Saturday Stotherts 2nds v Old Culverhasians 2nds,home, meet 1pm. R Hart, R Morgan, P Thurston, M White, C Drew, CWhite, B Moorelands, T Drew, T Wannel, C Carter, M Smart, DRichards, A Wicks, L Roman, M Solomon, T Clarke, G Cooke, T Vincent,S Croogan.
President Bush is being pushed by a newly forming consensus ofhis advisers to prepare a dramatic means of getting supplies toLithuania if President Gorbachev orders a boycott, just as HarryTruman beat Joseph Stalin's Berlin blockade in 1948.
That runs counter to the timidity of other NATO members,especially the Germans. Nor is that what George Bush really wants.Concerned that Soviet hard-liners are using Lithuania to undermineMikhail Gorbachev, Bush's policy is to ease the pressure on his newsuperpower partner.
But warnings from his advisers are bolstered by congressionalleaders of both parties, who want concrete language from thepresident that would bind U.S. support to Lithuania. SenateDemocratic Leader George Mitchell agrees with his Republicancounterpart, Sen. Robert J. Dole, that the United States may have totry helping Lithuania through the other two Baltic states, Estoniaand Latvia. On NBC's Meet the Press, Dole suggested extending themhard-currency credit that "might supply these vital needs toLithuania" cut off by Gorbachev.
In the House, insiders predict a torrent of demands forpostponing or canceling the summit scheduled next month inWashington.
The administration labored late last week - without decision -over exact wording to be used in a letter from Bush to Gorbachev, butthe consensus was clearly moving toward a harder line. Worried thatanother dose of equivocation will only embolden Gorbachev, theseofficials privately argued for a declaration that the United Stateswill not accept the slow strangulation of Lithuania, just as Trumanrefused to knuckle under to the Berlin blockade.
Despite stark differences in the Lithuanian case, memory ofTruman's response runs strong as a symbolic undercurrent inside theadministration's policy talks. During the 18-month Berlin airlift,Stalin did not permit a single U.S. aircraft to be attacked as tonsof food and fuel were flown to beleaguered West Berlin. Bushadvisers now are urging similar methods to be used by their chief toreduce Gorbachev's pressure on Lithuania, much as Stalin's was onBerlin.
The situations, over 40 years apart, are dissimilar. Lithuanianairfields and Baltic ports are in Soviet hands, while the UnitedStates and its allies controlled West Berlin. There is one newfactor: U.S. allies, lusting for business with Moscow, have become sostrong that they think they can tell Uncle Sam where to get off.
That is particularly true of soon-to-be-reunified Germany.Behind the scenes, Bonn lobbied furiously to bring the Soviets intothe new $12 billion European Bank for Reconstruction and Development.The United States yielded, in effect retreating from Bush's NationalPublic Radio interview March 17 that opposed loans to Moscow.
Bush will face heavy going if he converts handwringing into atrenchant U.S. policy toward Lithuanian independence.
But Bush is undisputed leader of the West. This is also onecrisis he could enter enjoying a united home front. Bipartisancongressional demands for an end to ambiguity are steadily pulling insupporters from across the ideological spectrum.
These politicians want George Bush to lay down a marker for allthe world to see, no matter how much discomfort it brings Gorbachevand his trading pals in Bonn. With the ghost of Harry Trumanstalking the Oval Office, Bush may be ready to act.
Evans & Novak are nationally syndicated columnists of theChicago Sun-Times.
Sitting in a small room, 250 km from Pune in the heart of ruralMaharashtra, farmer Satish Jagtap swears by the daily price andweather updates that he gets from Nokia's new MajhaNokia service. Itsaves him time and money. But can he rely on those numbers? I trustthe brand. Nokia is the Hero Honda of mobile phones, reliable. Ibought their mobile phone because of this service.
A thousand kilometres away, Aircel is launching its mobileservices in Delhi. Sandip Das, CEO of parent Maxis Cellular, isclear about focus on service. Aircel's TV promo features Indiancricket captain Mahendra Singh Dhoni using his handset to do dealson Yahoo and MakeMyTrip. Dhoni never puts the handset to his ear.Offering reliable data services is how we will distinguish ourselvesfrom the pack, says Das.
Concurs D. Shivakumar, MD, Nokia India, which controls about 65per cent of the handsets market here. It does not matter if theservice does not make too much money, it helps me put my product inthe consumer's hand. Across India, the mobile revolution is passe bynow and is just a matter of tracking the millions. (By the time youwill be reading this, the number of mobile subscribers in India willhave crossed 400 million, making it the world's second-largestmarket.) But this very growth has put the fear of commoditisationinto the hearts of the players. They need a differentiator. Thatdifferentiator is services.
Sending an SMS to a special number or downloading callertunes.Accessing e-mail or Googling with your phone. All these are termedservices.
Voice will certainly remain the main revenue generator foroperators, who are expected to earn a total of Rs 1,50,000 crore in2008-09. Of this, services or non-voice revenues SMSes, datasubscriptions, caller ring-back tones et al will fetch only aroundRs 10,000 crore. Yet, services will be the driver by which handsetvendors and operators will try and rise to the top of a very cloudymixture.
Rural raga, urban pop
Nokia's MeraNokia (Majha Nokia in Marathi) is actually a NokiaLife Tools (NLT) application coded into the 2300 and 2323 handsetsbeing used in the pilot. Farmers and villagers pay around Rs 2 perday, every 10 days, for the latest on crop pricing, weather, farmingtips, among other things. All this is freely available on the netfor those with PCs and Internet access. For the farmers, the mobileis the PC.
Jagtap, a cereal farmer with a large landholding, explains thatpaying Rs 2 a day makes sense for him: It saves me the hassle ofmaking three, four, five phone calls that cost more, andoccasionally even a bus journey. The prices are accurate .... Buthaving health tips would be a nice touch.
Jawahar Kantilal, Nokia's Head for Emerging Market Services, isextremely bullish about these services. The urban population is wellrepresented in services, usually around the entertainment area, saysKantilal. The fastest growing market across India, Africa, China andAsia was in rural or non-urban areas and these subscribers neededunique services.
He says entertainment is not the first concern of the ruralconsumer. The rural user wants to know: can the device help him inhis livelihood?
That is what we aimed at, says Kantilal, who led the push forNLT. He says the Maharashtra pilot can be easily taken global,particularly in the next major growth area for mobility sub-SaharanAfrica. People want...the device to improve their quality of life,he says. And it isn't just in rural areas that services are takingoff. Ashok Thapa, a Delhi taxi-driver from Nepal who has spent thelast decade in India, wonders why the mobile phone can't be used forelections just like how they vote in the singing contests on TV. Aliftman in a commercial building shows off his latest Samsunghandset, which comes with the soundtrack of Aamir Khan's Ghajini.
The mobile phone has moved from being a simple communication toolto an all-round entertainment and information device thanks to suchservices. People will pay for a service which they find convenientand one that adds value to their device, says Anshul Gupta,Principal Research Analyst for Mobiles at Gartner.
Balika Vadhu? Information Services Mobile Farmer
There could be problems for Mobile TV, because the rules aroundthis are very fuzzy
Several rival technologies exist and one is even being piloted inDelhi (DVB-H)
Other systems where you can download and watch TV clips have hadlimited popularity
Broadcasters would also want a piece of the pie and make moneyoff mobile TV
You can get everything from cricket scores to Bollywood gossip torandom jokes with all these services costing between Rs 10-50 amonth
These will be incremental revenues for all the operators andeasily accessible by users, particularly the tech-savvy ones
The problem is that most of this information is available freefor the users who know how to use the mobile Internet
Agricultural services may not be high-tech but can potentially bea big device sales driver
Farmers will get info on agricultural prices, local weather infobased on their location
This service will also allow agri scientists to get crucialmessages across to farmers on weather systems or practices
These services can be bundled with other services such as healthtips, education (English learning) and entertainment (ringtones,videos, etc.)
Serve or Die
But how many and what types of services can the present mobiletechnology both handset and network support? Services are beingenabled increasingly by more and more powerful processors onboardmobile devices. While numbers from Gartner classify only around 10per cent of the 1.2 billion handsets sold in 2008 as smartphones ,Shivakumar believes that there are no more dumb phones . When peoplesay that the mobile is a computer, it is. It is a computer, acamera, an alarm, everything.... These are immensely capabledevices.
Service provider Idea Cellular sees psychographics insubscribers. There are three psychographics under which you canclassify all mobile users productivity, convenience and safety,argues Rajat Mukerji, Corporate Director, Idea. We have backedNokia's service because we feel that that it is the second aspect,convenience, which will be the big selling point in rural India infuture. And once that is established, people will see that mobilesare productive also as they can enhance livelihood.
The Differentiator
The rush to get into services cannot be explained by the need tomake money, says Gupta, but to increase sales of core offerings.According to Simon Beresford-Wylie, CEO, Nokia-Siemens Networks, thebusiness of pipes , i.e., plain vanilla products, will exceed $1.2trillion (Rs 60 lakh crore) this year globally. The current estimateof $100 billion (Rs 5 lakh crore) in mobile services annually is adrop in this ocean but it is not a number to be scoffed at,particularly since India is expected to lead the servicesrevolution.
Services are helping push sales at Nokia. Obviously, saysShivakumar when asked about increasing handset sales. Thedifferentiator I can offer on my N-series devices will be NGagegaming, on XpressMusic phones will be Comes With Music (CWM) and onlow-end rural handsets it will be NLT. The service is what will makepeople buy my device. The same applies for operators, as exempliedby Aircel. I don't feel that there can ever be a onesize-fits-allapproach to services, says Das. I do not think that is what Nokia,other operators or third-party players are doing. We have been verysuccessful with data services in Malaysia and almost a third of ourrevenues are from services and in India we want to play a role theretoo.
Mera Nokia is not unique; similar services are offered byoperator Airtel and news agency Thomson Reuters. Airtel runs it in adeal with a fertiliser company, while Reuters Market Light alreadyhas 100,000 subscribers in Maharashtra and Punjab. But thiscompetition highlights the problem in services. Every stakeholder isfighting for a share of the pie.
Cost conundrum
Hugo Barra, Group Product Manager, Google Mobile, believes thatit will be a healthy ecosystem where everyone will co-exist.Operators, vendors and service providers will need to work together.
There is no point having services for free, like we do, if itcosts a lot to access them over a network like it does in India. Henotes that in most parts of the world, cheap access plans havepowered data usage from mobile devices, and says that India, too,needs to move to such a regime. In the US, the inflection point fordata usage came in 2007, when unlimited data access plans tumbled to$5 a month (in India fully unrestricted data plans cost Rs 499 withmost carriers).
Barra brings up the biggest bugbear of mobile services: theservices that have done well until now have been low-data ones. EvenNLT is a service offered over SMS rather than the Internet. Typicaldata charges on mobile networks are 10 paise for 10 kilobytes, so itcould cost Rs 40 to download the typical four megabyte MP3 musicfile.
But operators have not cut rates: according to them, it has lessto do with fleecing the customer and more with the network itself.The fact is that today voice still accounts for 90 per cent ofrevenues and subscribers are being added at a fantastic pace and youcan't refuse to take on a subscriber. So, in order to ensure voicequality we have to keep data access limited, says a spokesperson forone of India's largest mobile operators.
Wait for 3G, Sir-ji!
Until now, services that have done well have usually beenindependent of the requirement for a data network. Caller ring backtones (CRBT) and SMS updates do not clog the network. However,Vineet Taneja, Head of Marketing, Nokia, is betting big that 3Gnetworks will give services a fillip, none more so than Nokia's CWM.The idea is simple: the cost of music is built into the handset andusers can choose the music they wish to download. But the servicewill require a decent download experience, and that means 3G. Iguess that if such a service comes along, Nokia will be and istalking to operators so as to enhance the download experience, saysthe spokesman for an operator.
Taneja has his hands full launching a whole host of otherservices, those that will integrate the mobile even more thoroughlyinto people's lives. However, there have been some hiccups. Nokia,which acquired maps provider NAVTEQ in 2007, offers free navigationfor a few months with its high-end devices but has not been able tolaunch a paid service for navigation or for its games platform, N-Gage.
Taneja admits there have been issues of pricing and distribution.You have to understand that one cannot just have one system inIndia. Many of the kids who will use N-Gage may not have creditcards, so to buy the licence we would like them to walk into mobileoutlets and buy the game over the counter. With navigation we haveto figure out pricing as well as get more comprehensive maps. ButTaneja did admit that something big was around the corner. I stillfeel that navigation and location services can be a major driver inIndia, he says.
Not all of Nokia's competitors have a strong services-led ploy:The No. 2 in the Indian market, Samsung Mobile, believes thatdevices will still be sold on the strength of their features. SunilDutt, Managing Director, Samsung Mobile India, points out that untildata costs come down and reliability improves, India may not be astrong services market. We have moved into offering services inmature markets such as Korea and the US. I still believe that Indianconsumers want their devices to have top-class features. I am notruling out services, I just do not think the time is right just yet.
Pied Piper's plight
That is an opinion echoed by Google's Barra. People will not wantto pay for services that they can get for free, and the serviceswill be free because there is a massive opportunity for advertisersto come onto the mobile platform. This is still untapped, thanks tothe proliferation of location information, specific advertising, andI mean non-intrusive advertising can easily come onto the mobile.The mobile will also lead to a whole host of unique services. We areabout to roll-out a voice-activated mobile search.
But the real reason Nokia is getting into services is not aboutmaking consumers lives easier or earning incremental revenue. Nokiahas pushed into services in a big way across the world with its Ovi(Finnish for Door ) platform, and the amount Nokia can suffer whencompetitors launch services globally is highlighted in its massiveerosion of smartphone market-share as competitors launch new,services-heavy devices. Apple's success in the smartphone space injust three years it has grabbed an eight per cent share almostentirely at Nokia's cost rides on its Application Store, whichsells, you guessed it, services.
The stand-alone product era is over. The new game is product plusservices. The devices will be the rock on which we will build oursolutions, says Nokia's Shivakumar.
The Music Is Free
Where Are You?
Mummy! Pizza!
Users will be able to download unlimited music tracks directlyfrom their phone for 'free'
The price of the music rights would be included in the price ofthe handset
Consumers could also buy music tracks at mobile retail outletsfor
Rs 5-25 per song
Users could listen to music on demand by dialling a specialnumber
'Digital Rights Management' might be an issue
Devices already come with maps and navigation; applications pre-installed but maps will get more accurate
NAVTEQ is launching maps for 50 Indian cities shortly from theeight cities currently covered
Maps and navigation will not only help lost drivers, but alsooffer advertisers new avenues to reach to consumers
The maps will be free, but navigation services will cost Rs 50-250 a month
Imagine calling a number saying 'Pizza' and the device tellingyou where the closest pizza parlours are. It is coming soon,courtesy Google
You can also use your handset to access your e-mail for free
You could also stream videos live to the Internet from yourmobile device or watch them on services such as YouTube
But data plans aren't cheap at Rs 299-999 per month
