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Rachael Ray knows the way to a person's heart is through the (pet's) stomach

In the latest expansion of her culinary empire, celebrity chef Rachael Ray has launched a charity-driven line of dog foods based on recipes she has created for her pit bull, Isaboo.

Ray, who came to fame with her "30 Minute Meals" Food Network show, said all of her proceeds from sales of Rachael Ray Nutrish pet foods will go to Rachael's Rescue, which she founded to help at-risk animals. The outspoken dog lover regularly features recipes for pet food in her magazine, Every Day With Rachael Ray.

"It seemed like not a lot of extra time to donate to something that could potentially raise millions of dollars for championing these little creatures …

Starr under new attack from Dems

WASHINGTON - House prosecutors and the White House legal teamquestioned Monica Lewinsky under oath today in the impeachmenttrial,while President Clinton's lawyer renewed his complaints in courtthatIndependent Counsel Kenneth Starr was violating grand jury secrecylaws.

Rep. Ed Bryant, R-Tenn., the lone House prosecutor to quizLewinsky, and presidential lawyers Cheryl Mills, Nicole Seligman andDavid Kendall arrived this morning at the hotel where the eight-hour,videotaped deposition was being conducted.

Before turning to the extraordinary question-and-answer session,Kendall announced he was taking legal actions against Starrconcerning a weekend story in The New York …

INDEX: Big NGOs (BINGOs)

* By far the largest NGOs of any kind (not just international ones) are in the United States, where they are usually called "non-profits." Some of the biggest-YMCAs in particular-generate large revenues from the services they provide. The $248.3 billion donated to all NGOs in the U.S. in 2004 was 20 times larger than official U.S. overseas aid and almost half as much again as the annual income of all 215 million people living in Indonesia. More than one-third of these U.S. charitable donations went to religious NGOs.

* Out of the total U.S. donations to NGOs in 2004, more than three out of four came from individuals and only 4.8% from corporations. Government funding also …

Obama: New tax on oil drilling

Offshore oil drilling would become more expensive, but scientists pursuing clean energy could get a windfall under President Barack Obama's proposed budget.

The proposal imposes new excise taxes on oil and gas pumped offshore. And oil companies would have to pay a fee on drilling leases they own but are not using.

While the budget summary gives no specific numbers, …

The dynamics of growing up in a political dynasty

In his book The Pecking Order, sociologist Dalton Conley looks atfamilies like the Kennedys, in which several members excel atsociety's highest level, in part because of extraordinary experiencestheir parents give them at an early age.

In Chicago, the Daleys are such a family -- seven children, two ofwhom have a high profile even on the national stage.

The oldest boy, Richard M., picked up from dad Richard J. asChicago's mayor; William, the baby of the family, was the U.S.commerce secretary in Bill Clinton's administration and is now thepresident of SBC Communications. The middle boys are highly visiblein the community as well, John as finance chairman of the Cook …

Blaze at Roane County store closes down Spencer streets

A structure fire engulfed a carpet and floor covering business inSpencer and blocked off traffic on surrounding roads today, accordingto a Roane County 911 dispatcher.

Several fire departments arrived at the Floor & More on East MainStreet at around 6 a.m. and reported large plumes of smoke coming outof the business, which is near a Laundromat and Foodland.

It was not known if the fire spread to any of the otherbusinesses, said the dispatcher, as fire crews were still battlingthe flames at the Floor & More a couple of hours after the initialreport came in.

The dispatcher said no one knew what might have …

Schwarzenegger Seeks New Budget Process

Saying "the wolf is back" at the door, Gov. Arnold Schwarzenegger proposed a constitutional amendment in his State of the State address Tuesday that would force automatic cuts to the state budget to avoid large deficits like the one he is now facing.

The governor wants cuts triggered when state finances start falling into the red and a rainy-day fund to start building when revenues come in above average. The surplus would be limited to 15 percent of the budget and could be used only to help with the next downturn.

Schwarzenegger got the idea from former President Bill Clinton, who had similar budget powers as governor of Arkansas. He said it would put …

Elgar's 'Dream' comes true for Kalmar, orchestra

"The Dream of Gerontius,'' a deeply felt meditation on death and the afterlife written in 1900 by English composer Edward Elgar, is a big, demanding work for orchestra, chorus and three vocal soloists. Popular in the United Kingdom, it's done more rarely in the United States, and only by orchestras with sufficient musical resources to do it right. The Chicago Symphony Orchestra presented memorable performances of the work in 2002 conducted by Daniel Barenboim.

Over the weekend, it was the Grant Park Orchestra's turn, and a finely tuned turn it was. At the Saturday night performance led by principal conductor Carlos Kalmar, the orchestra, chorus and soloists --mezzo-soprano Allyson …

What I learned in Friesland

This report was presented to the Vancouver First United Mennonite Church congregation by Peter Dueck after he and his wife Dina visited Friesland, where she was born.

I want to talk about Friesland and what I think we can learn about- and from- the colonies. After a four-day visit, I obviously qualify as an expert!

Our visit was very interesting and enjoyable. We stayed with Dinas cousin, and I immediately renewed my appreciation for farm life, roads (or lack thereof) and dislike for roosters. I offered to help make soup if we could use the rooster, who got us up at 4 in the morning.

I found the visit to Friesland very thought-provoking and relevant to my interest in …

American League Leaders

BATTING_HMatsui, New York, .331; Mauer, Minnesota, .330; Youkilis, Boston, .322; Ordonez, Detroit, .315; Grudzielanek, Kansas City, .312; Bradley, Texas, .311; Jeter, New York, .308.

RUNS_Crawford, Tampa Bay, 30; Ellsbury, Boston, 29; Kinsler, Texas, 28; Youkilis, Boston, 27; Damon, New York, 27; Quentin, Chicago, 26; ISuzuki, Seattle, 26.

RBI_Hamilton, Texas, 39; EBrown, Oakland, 33; Youkilis, Boston, 30; Quentin, Chicago, 29; Ortiz, Boston, 28; Morneau, Minnesota, 27; Upton, Tampa Bay, 25; Blake, Cleveland, 25; Ordonez, Detroit, 25; Ibanez, Seattle, 25.

HITS_Pedroia, Boston, 51; Youkilis, Boston, 47; Hamilton, Texas, 47; ISuzuki, Seattle, 47; …

Judge says young terror defendants won't be identified

TORONTO - The judge in the trial of a youth charged with plottingterror attacks in Ontario has ruled that a publication ban on theidentities of his co-accused is necessary to ensure their fairtrials.

Superior Court Justice John Sproat said in a ruling thatpublicity in the case could influence potential jurors. Sproat putan interim ban on the publishing of the defendants' identities lastweek and made it permanent on Tuesday.

The ban applies to the identities of 14 adults who were arrestedin a massive police sweep in 2006 after they allegedly tried toobtain three tons of ammonium nitrate, an explosive materialcommonly used in fertilizer. Officials accused the suspects …

Actualidades: Transmite Radio Marti hacia Cuba simposio sobre el futuro de la Isla

Actualidades: Transmite Radio Marti hacia Cuba simposio sobre el futuro de la Isla

"Cuba, los cubanos y su futuro", fue el tema analizado el jueves por un panel de expertos en el tema cubano, que fue organizado y moderado por Frank Calzon, director ejecutivo del Centro por una Cuba Libre --basado en Washington-- en la LUniversidad Internacional de la Florida (FIU). El simposio fue transmitido integramente a Cuba por las ondas de Radio Marti, de manera que el pueblo de Cuba recibio este 26 de julio una inyeccion de esperanza, al darsele un abanico de opiniones sobre la inevitable necesidad de una transicion a la democracia en la Isla. Ya sea con transicion o sucesion, el periodo de …

Louw on weather watch

The rains of the last few weeks have left Stoneywood-Dyce with amountain to climb if they are to entertain any hope of gaining apromotion place.

"It's hard to recall when we last completed a game,'' said captainMichael Louw, who admitted to feeling frustrated by the elementswhich have seen his side slide to fifth place in National LeagueTwo, 23 points behind leaders Stirling County and 14 in arrears ofsecond placed Dunfermline.

Louw has not given up on the People's Park side and said: "Givena break we can haul back the situation, but it's the very devil whenthe weather is the winner.

"I feel sorry for my guys who are as good as any in the leaguewhen we get the chance to play. ''

Louw's men have lost five of their 13 games to the rain, but havea chance to leapfrog Weirs when the two clash at Dyce tomorrow.

''It's win or bust,'' said a defiant Louw.

Today in History

Today is Tuesday, Nov. 2, the 306th day of 2010. There are 59 days left in the year.

Today's Highlight in History:

On Nov. 2, 1783, Gen. George Washington issued his Farewell Orders to the Armies of the United States near Princeton, N.J.

On this date:

In 1795, the 11th president of the United States, James Knox Polk, was born in Mecklenburg County, N.C.

In 1865, the 29th president of the United States, Warren Gamaliel Harding, was born near Marion, Ohio.

In 1889, North Dakota and South Dakota became the 39th and 40th states.

In 1917, British Foreign Secretary Arthur Balfour issued a declaration expressing support for a "national home" for the Jews in Palestine.

In 1920, radio station KDKA in Pittsburgh broadcast returns from the Harding-Cox presidential election.

In 1930, Haile Selassie (HY'-lee sehl-AH'-see) was crowned emperor of Ethiopia (his reign lasted nearly 44 years).

In 1947, Howard Hughes piloted his huge wooden flying boat, the Hughes H-4 Hercules (derisively dubbed the "Spruce Goose" by detractors), on its only flight, which lasted about a minute over Long Beach Harbor in California.

In 1948, President Harry S. Truman surprised the experts by winning a narrow upset over Republican challenger Thomas E. Dewey.

In 1959, former game show contestant Charles Van Doren admitted to a House subcommittee that he'd been given questions and answers in advance when he appeared on the NBC's "Twenty-One."

In 1979, black militant JoAnne Chesimard escaped from a New Jersey prison, where she'd been serving a life sentence for the 1973 slaying of a New Jersey state trooper, Werner Foerster. (Chesimard, who took the name Assata Shakur, is believed to be living in Cuba.)

Ten years ago: An American astronaut and two Russian cosmonauts became the first residents of the international space station, christening it Alpha; their arrival marked the beginning of a decade of continuous habitation of the orbital outpost.

Five years ago: The Bush administration released details of its potential flu pandemic strategy, saying a pandemic that hit the United States would force cities to ration scarce drugs and vaccine and house the sick in hotels or schools if hospitals were to overflow. A Detroit church packed with 4,000 mourners celebrated the life of Rosa Parks in an impassioned, song-filled funeral.

One year ago: A suicide bomber killed 35 people outside a bank near Pakistan's military headquarters in Rawalpindi. Afghanistan's election commission proclaimed President Hamid Karzai the victor of the country's tumultuous ballot, canceling a planned runoff. The Philadelphia Phillies staved off elimination from the World Series with an 8-6 victory over the New York Yankees in Game 5. World Boxing Hall of Famer Lou Filippo, 83, died in Downey, Calif.

Today's Birthdays: Rhythm-and-blues singer Earl "Speedo" Carroll (The Cadillacs; The Coasters) is 73. Singer Jay Black (Jay and the Americans) is 72. Political commentator Patrick Buchanan is 72. Actress Stefanie Powers is 68. Author Shere (shehr) Hite is 68. Rock musician Keith Emerson (Emerson, Lake and Palmer) is 66. Country-rock singer-songwriter J.D. Souther is 65. Actress Kate Linder is 63. Rock musician Carter Beauford (The Dave Matthews Band) is 53. Singer-songwriter k.d. lang is 49. Rock musician Bobby Dall (Poison) is 47. Pulitzer Prize-winning playwright Lynn Nottage is 46. Actress Lauren Velez is 46. Actor David Schwimmer is 44. Christian/jazz singer Alvin Chea (Take 6) is 43. Rock musician Fieldy is 41. Rock singer-musician John Hampson (Nine Days) is 39. Rhythm-and-blues singer Timothy Christian Riley (Tony Toni Tone) is 36. Rapper Nelly is 36. Prodigy (Mobb Deep) is 36. Actor Danny Cooksey is 35. Rock musician Chris Walla (Death Cab for Cutie) is 35. Country singer Erika Jo ("Nashville Star") is 24.

Thought for Today: "The amount of sleep required by the average person is just five minutes more." — Anonymous.

(Above Advance for Use Tuesday, Nov. 2)

Copyright 2010, The Associated Press. All rights reserved.

Dubai still considering Olympic bid

Dubai is still considering a bid to host the Olympics in 2020, said the ruler of the Arab emirate Sunday as leaders and executives from the sports industry gathered in the city for an international convention.

Dubai has spent billions of dollars to build sport venues and host high profile golf and tennis tournaments. Sheik Mohammed bin Rashid Al Maktoum praised Dubai's sports infrastructure and said Dubai has "a lot to offer" to the Olympic movement.

"There is a strong 'can do' culture here," Mohammed said in comments, released Sunday by the ruler's media office. Dubai continues to evaluate "the costs and benefits" of a bid, Mohammed also said and added, that no formal paperwork has been filed yet.

Dubai is the second Gulf state trying for the Olympics. Doha, the capital of Qatar, failed to make the list of finalists for the 2016 Games.

Dubai has run far ahead of its Gulf competitors on the sports front by linking investment with a decade-long effort to attract tourists and entertain the Asian and Western expatriates that make up most of the emirate's 1.5 million inhabitants.

The Gulf emirate has earned high marks for hosting ATP and WTA tour tennis events, the Dubai Desert Classic golf tournament and horse racing's Dubai World Cup.

Since Dubai has been hammered by the global financial crisis in the past months, the ruling Al Maktoum family has scaled back some of its development plans, but sports events have not been a causality in Dubai's struggle to pay off its massive debt.

Last month a $2 billion racing complex was inaugurated. Managers of Dubai Sports City said progress has been undeterred on the cluster of mega-venues that will stage sports events and set up training facilities and specialized academies for golf, tennis, soccer, rugby, cricket and swimming.

Sheik Mohammed has relied on such large-scale global ambitions to help drive his territory from little more than a patch of sand 15 years ago to the Middle East's business, sports and tourism hub.

The harsh economic reality has caused Mohammed to include some caution in his comments on Dubai's possible Olympic bid.

"We will have to take an honest look at our weaknesses as well as our strengths," Mohammed said Sunday. "I can assure you of this, though: if we decide to make a bid for the Olympics, we will be in it to win."

Your views: Bobby Wesley was always a class act, and his loss is a tragedy

Thanks for the recent article on Bobby Wesley. Indeed, Charlestonhas lost one of its treasures. How many lives he touched.

What a tragedy, but what a wonderful life. Bobby was a tremendoushusband, father, brother and son - a great family man.

Those of us who played basketball with him loved him. Those of uswho guarded him, or tried to, knew how impossible it was. Having himguard you wasn't any picnic either.

He was a fierce competitor - so talented. He knew how to play andhow to win. He was a winner in basketball and in life.

He was so quick - could stop on a dime and then jump out of thegym. And even though you knew he was going to shoot, you couldn'tstop him. His strange-looking jump shot came from way behind hishead, and then he arched it high out of your reach, and it was alwayssoft and usually accurate.

I'd like to say we were mature enough over the years to haveplayed without ever arguing about fouls, but I can't.

Oh, what the heck, Bobby, I admit it. I fouled you more than youdid me. But no matter how intense we got while playing, we werealways friends afterwards.

All the guys who regularly played with Bobby over the yearsrespected him as much as a person as we did as a player. Bobby wasalways a class act.

He was a great referee in the youth leagues. More than once hehelped me calm down while I was coaching. He was not only a blessingto all the kids I coached over the years, but also to my ownchildren, who appreciated his kind and encouraging words, hisfriendly teasing and his infectious smile.

Bobby's shocking and sudden death reminds us of just how fragilelife is. It's crucial that we don't put off what's important in thislife: getting right with the Lord; letting loved ones know how muchwe care about them and appreciate them; and taking steps to mendwounded or broken relationships.

Bobby, we'll miss you, but we'll never forget you.

Tim DiPiero

Charleston

HK agents find $2.2M in illegal rhino horns, ivory

HONG KONG (AP) — Hong Kong customs agents say they have confiscated a shipment of rhino horns and ivory worth about 17.4 million Hong Kong dollars ($2.2 million), their biggest such seizure to date.

Officials said Tuesday that they seized 33 rhino horns, 758 ivory chopsticks and 127 ivory bracelets concealed in a shipping container that arrived at Hong Kong's port from Cape Town, South Africa.

They would not name the container's final destination.

Customs agents found the hidden haul by X-raying the container and then peeling away layers of foil, paper and plastic wrapped around the items.

The seizure tops one in August of $1.6 million worth of African ivory.

Several rhino subspecies are believed to have recently become extinct.

Biomass Energy Outlook

RECYCLING CARBON EMISSIONS INTO THE ECONOMY

IN February in this space, I discussed the economic recession and the new Administration's efforts to stabilize our failing economy. Now it is time to take that a bit further. Climate laws are being written with conflicting objectives that are adding unnecessary costs. Even though a climate law has an environmental focus, the bottom line depends on economic growth that is greater in value than the total costs incurred by consumers, industry and the government. Economic growth means the pie gets bigger for everyone. The quality of life goes up while using fewer resources. We learn to live more efficiently.

Part of the problem with the emerging climate law is how politicians and government agencies view carbon emissions. Essentially, carbon emissions are biomass resources that are in the wrong place. We should all be working together to pull leftover carbon (emissions) back into the economy as more biomass for energy, compost, organic nutrients, and biobased fibers. The goal is a higher value of life using fewer resources. By intensively managing our resources, there are fewer emissions. The result (not the goal) is zero waste.

BioCycle readers understand that adding value to waste stream organics is about getting more from less. This is the 50-year legacy of BioCycle. Solid waste recycling and composting pulls solid waste "emissions" back into the economy. Now the same thing needs to be done with greenhouse gas emissions. Our new carbon policies must make recycling emissions easy and relatively costless.

Unfortunately, that is not the direction we are going in at the moment. The Energy Independence and Security Act of 2007 (EISA) is the enabling statute for the revised Renewable Fuel Standards (RFS2) regulations. EISA was legislatively written to create an economic demand for biofuels. The motivation was to increase the supply and demand for a locally grown, cleaner liquid fuel. Between 2007 when the statute was written by the legislators and now, EPA switched the priority from increasing demand to restricting greenhouse gas emissions. As proposed, the rule makes it difficult for biofuels to qualify for the RFS economic benefits. While EPA can legally do this, proceeding as the agency has proposed will halt economic growth in biofuels production. The additional emission criteria EPA has added to biofuels make other non-RFS uses like making electricity from biomass more compelling.

In June, the Waxman-Markey climate legislation passed the U.S. House of Representatives by a narrow margin of 7 votes: 219-for and 212-against. Just days before this vote took place, House Agriculture Committee Chairman Collin Peterson announced a "deal" had been struck in which USDA would provide oversight instead of EPA for agricultural offsets. USDA is able to tie environmental performance to program payments. It would also put on hold significant portions of the emissions-based RFS2 rule. In short, to ensure passage of the climate bill, the House Agriculture Democrats placed a priority back on economic growth.

BITING THE CAPITAL COST BULLET

The infrastructure that got us into this climate "mess" relies on large, centrally located food, energy and waste collection and distribution systems. The new one based on small, locally grown biomass and renewable energy systems will cost trillions of dollars. In addition to these large physical system capital costs, we must add in interest on the trillions of dollars of federal stimulus money that has been borrowed against our children's future.

If the commercial interest on this new production and distribution network costs 7 percent, we can set the interest on the federal stimulus funding at a cost of living of 3 percent. If everything runs smoothly, we pay an effective 10 percent interest on the new infrastructure until the trillions of dollars of borrowed capital are paid off. This is workable as long as the new bioeconomic infrastructure is returning more. If the average of all renewable energy projects is a 20 percent return before paying the debt and half of that (10 percent,) goes to service the collective debt, we could still be ahead.

Unfortunately neither the legislative (Congress) nor administrative (agency) lawmakers are very good at writing costless laws. Serious policy collisions between poorly crafted oversight regulations and funding programs are very costly to industry, government and consumers. For years now, the USDA and the Department of Energy have been funding biomass energy projects and research in which industry and government have invested billions of dollars. If the biomass utilization/carbon emission laws are written to now exclude those investments, all those false-start dollars get added to the debt with no hope of a return.

Any economic friction added to the cost of building the bioeconomy will threaten the success of this risky, bold approach. For instance, if the policy implementation cost increases the debt servicing fees described above by 5 percent, then the new bioeconomy must pay an effective debt service cost of 15 percent before we have economic growth. If the policy implementation cost increases the effective interest by 10 percent, the breakeven economic bar now requires more than a 20 percent return. In this last example, the additional implementation costs have consumed all economic growth. This is not sustainable.

Substantive and sustainable change is achievable, but the risks are real. Policies can be written that promote enhanced quality of life and more efficient use of resources. The biomass industries need to guide the policies.

[Author Affiliation]

Mark Jenner, PhD, operates Biomass Rules, LLC and has over 25 years of biomass utilization expertise. Burning Bio News is Jenner's scorecard of bioenergy project adoption, available at www.biomassrules.com.

Northeast power outages hit many businesses hard

HARTFORD, Conn. (AP) — Businesses big and small have taken a beating from the power outages caused by the record-setting October snowstorm and the losses are only beginning to be tallied, owners and experts said Monday as tens of thousands of Connecticut homes and companies entered a second week without electricity.

"I think there's going to be a huge trickle-down effect and we may not know the results for several months," said Andy Markowski, Connecticut director for the National Federation of Independent Business. "I don't know of any small business that can afford to lose a week or more of sales. ... We're just literally and figuratively beginning to pick up the pieces."

The Oct. 29-30 storm dumped heavy snow across the Northeast and downed scores of trees and utility wires. Three million homes and businesses lost power at the height of the storm.

Connecticut was hit the hardest, racking up more than 830,000 outages, and more than 37,000 utility customers remained in the dark Monday. New Jersey utilities said everyone was back on line, while Massachusetts power companies were working to restore electricity to about 300 customers.

The storm also is affecting municipal elections Tuesday in Connecticut, where nine cities and towns were moving and consolidating their polling places. Secretary of the State Denise Merrill said some polling locations do not yet have electricity while others have been damaged or are being used as shelters and warming centers.

Gov. Dannel P. Malloy has called the duration of the outages unacceptable and has launched an independent probe of the utilities' response. He said the state is keeping its legal options open in case there are grounds for recourse in the courts once the circumstances are examined.

When asked how he planned to hold the utilities accountable for the slow recovery response, as he has promised, the governor said it will be through the state's regulatory process.

"We can bring dockets and ask for things to happen with respect to how they conduct their business and what way they conduct their business and what they recover losses for, for instance," said the governor, who said he presumed that the investigation of Connecticut Light and Power, the state's largest utility, would uncover "some degree of malfeasance" and could lead to legal action by the state.

Some homes and businesses weren't expected to get their power back until Wednesday night.

No power and no water meant no work for Angela Campetti, who runs a small house-cleaning business in Simsbury, Conn. All 10,100 electricity customers in the town were without power for several days and nearly half still weren't restored by Monday.

Campetti said she lost a significant amount of money because of the outages and hasn't been able to pay herself or her two employees at First Class Housekeeping.

"I'm not very happy," she said. "All the houses I was supposed to clean the power was out and my employees weren't able to go out and clean. You can't go in there with your vacuum."

The outages have affected a wide variety of companies, Markowski said, including small machine shops, home-based businesses and restaurants that were hit with the double whammy of losing sales and having to throw out food.

Peter Gioia, an economist at the Connecticut Business and Industry Association, said large companies also took big hits to their revenues. Though some businesses such as tree trimming and gas stations did well during the extended outages, many others were forced to shut, he said.

"The net effect is a minus," Gioia said.

He added that the CBIA's incoming email and website visits were down 50 percent during the week when power was out, reflecting that many businesses were not open.

Insurance agents, meanwhile, are reporting higher volumes of claims for business interruption insurance than what was submitted during hurricanes Irene and Lee, said Dan Corbin, director of research at Glenmont, N.Y.-based Professional Insurance Agents.

Business interruption insurance could include payments for lost profit and extra expenses such as moving to a different site that has electricity. To make a claim, the insured must prove property damage.

Insurance typically pays for continuing expenses such as rent or mortgage payments, payroll to avoid laying off workers and replacements of perishable goods, Corbin said.

One place that was not affected was the mall in Manchester, Conn.

General Manager Nancy Murray said that because The Shoppes at Buckland Hills are served by underground wires, they did not lose power and became a magnet for people in search of food, heat and power to charge cellphones and other equipment.

"It especially affected people the first Sunday," she said. "We were packed that day."

___

Associated Press Writer Susan Haigh contributed to this report.

Pupils are left seeing stars

Four-hundred youngsters at a school have been enjoying sciencelessons with a difference.

A planetarium was brought into Cheadle Primary School as part ofNational Science Week.

Headteacher Michael Prescott said: "The pupils took part in arange of unusual and exciting experiments.

"They learned a lot and have been thoroughly engrossed by theactivities."

Left, scientist Stephen Blight inside the inflatable planetariumand, inset from left, pupils Eloise Shaw, aged seven, CarlaEcclestone, eight, Samuel Valentine, eight, and Luke Webster,seven.

Picture: Malcolm Hart

Navistar to trim work force, reduce some health benefits

Navistar International will cut 20 to 25 percent of itswhite-collar sales and administrative staff over the next threeyears, the Chicago-based heavy-truck maker said yesterday.

In addition, the firm will scale back health benefit plans forits non-union employees, adding deductibles and co-payment provisionsto the plan that formerly covered all medical expenses. Unionemployees, who are covered under a three-year contract that expiresin October, 1987, are not affected.

Savings will total $40 million over the three-year period, thecompany said.

Donald D. Lennox, Navistar chairman and chief executive officer,said the cost-cutting moves are part of the company's plan to "keepNavistar profitable and competitive during a period of reducedproduct demand and intense pressure on margins resulting from arestructuring of the trucking industry."

In the company's third-quarter earnings report, released earlierthis month, Lennox said the problems that have plagued the heavytruck industry - price discounting, continued weak demand andindustry overcapacity - "show no signs of abating."

The latest round of staff reductions will affect between 350 and450 of Navistar's 1,800-person white-collar work force. Most ofthose jobs are in the Chicago headquarters.

About 150 of the positions will be phased out by Oct. 31through a combination of early retirements and voluntary terminationswith deferred pension benefits. Approximately 275 employees areeligible for early retirement and another 600 qualify for thedeferred retirement plan, the company said.

One industry observer suggested that Navistar already has cutcosts so severely that this latest round of cuts "will be cuttinginto muscle. I find it hard to believe there are 400 moredispensable people."

But Barry A. Mannis, analyst with Shearson Lehman Bros., said:"I have confidence they wouldn't cut into the muscle at this point."Mannis is the co-author of a report recommending that "risk-oriented"investors purchase Navistar stock, a conclusion based on a beliefthat industry demand will pick up in the near future.

Navistar's common stock fell 25 cents after its announcementyesterday to close at $7.75.

White House Ponders NG Nuclear Warheads

LIVERMORE, Calif. - The scientists who crack open the nation's nuclear weapons for a living are never quite sure what they will find inside.

Many of the warheads were designed and built 40 years ago, and their plutonium and other components are slowly breaking down in ways that researchers do not fully understand. With no new bombs in production, the government spends billions of dollars each year tending to its aging stockpile.

The Bush administration wants to revamp the entire arsenal with a weapon now on the drawing board named the Reliable Replacement Warhead.

The redesigned weapon is needed to ensure "a safe, secure, reliable and effective nuclear deterrent for the indefinite future," said Linton Brooks, chief of the National Nuclear Security Administration.

The administration ordered up a competition between Lawrence Livermore National Laboratory near San Francisco and Los Alamos National Laboratory in New Mexico. The two laboratories submitted their proposals for the weapon in March. The White House plans to pick a winner by November.

As envisioned, the next-generation nuclear weapon would have the same destructive power as existing ones, but be durable enough to last for decades.

The next bomb is also meant to be so secure that it has jokingly been dubbed the "nuclear doorstop" - useless for any other purpose, should it fall into the wrong hands.

The government and the labs refuse to discuss details of the two designs, citing national security. But they describe both proposals as "conservative" blueprints meant to assure reliability without violating a moratorium on full-scale nuclear testing in place since 1992.

"We're not going to come up with anything cutting-edge and stick it in the stockpile without testing," said David Schwoegler, spokesman for Lawrence Livermore's nuclear weapons program.

The United States has not built a nuclear warhead since 1991. The government spends about $5 billion a year maintaining the weapons, and engineers have patched problems by opening up warheads that were never meant to be opened. The accumulation of tiny engineering changes meant the bombs moved incrementally away from their original designs, with unknown effects.

The White House believes designing a replacement warhead is vital to preserving the nation's nuclear edge, particularly amid looming questions about North Korea, which reportedly possesses several nuclear weapons, and Iran, which the administration fears wants them.

The redesign project "means making sure that aging phenomena don't cause us any questions about nuclear reliability," Brooks said in a telephone interview with The Associated Press. "It means making sure that we incorporate safety and security and use-control in a way we didn't know how to do when we designed the stockpile."

Critics, including some former nuclear weapons scientists, question the need to resume nuclear weapons production, at a cost of billions of dollars, when they believe the current stockpile is safe and reliable and can remain so for years.

They also question whether a next-generation bomb can improve reliability and safety if it cannot be tested. Congress has financed the research on the condition that the redesigned weapon reduce the need for testing.

Opponents fear the project could send the wrong signal to the world at a time when the United States and its allies are trying to curb the spread of nuclear technology.

Brooks said North Korea and Iran play into the project only "indirectly," explaining that the administration would press for the program anyway. "We didn't sit down and say, 'Look, there's problems in Iran. Let's go and invent a new design,'" he said.

The project also aims to improve safeguards against accidental detonation or use of the weapons by terrorists, Brooks said. It marks the first time that an American nuclear bomb has been designed with those goals as the top priority.

Proponents say a revamped weapon could help the United States to reduce the number of warheads held in reserve in case other weapons are found to be faulty.

A new weapons production line would be needed to produce the bomb. For instance, the Rocky Flats, Colo., plant that once made plutonium triggers for nuclear warheads was shuttered in 1989. Los Alamos can only build a handful per year; the administration is aiming for 10 next year.

The Livermore and Los Alamos labs set aside bomb-designing more than a decade ago in favor of maintaining the current stockpile.

Each year, the nation's nuclear arsenal loses about a half-dozen bombs from its reserve of several thousand as the Livermore and Los Alamos teams rip them apart in what is called "destructive analysis." Others are painstakingly dismantled and refurbished with new parts.

On Thursday, engineers gathered at a high-security plant near Amarillo, Texas, to toast a milestone: the first rebuild of a B-61 nuclear bomb. It's the oldest warhead in the arsenal, having been designed in the early 1960s and built into the 1970s.

The government is spending $470 million over nine years to refurbish the B-61s. That's money the Bush administration would rather channel into an overhaul of the entire arsenal and the mostly dormant nuclear-weapons complex.

Brooks sees the bomb-redesign project as making that complex more adaptable.

"Any weapon we have will sooner or later go through some type of modernization or have (some) problem to repair, and right now that takes a very long time," he said.

Formation and Reversible Dissociation of Coiled Coil of Peptide to the C-Terminus of the HSV B5 Protein: A Time-Resolved Spectroscopic Analysis

ABSTRACT

An understanding of the molecular mechanisms of the newly characterized herpes simplex virus (HSV) B5 protein is important to further elucidate the HSV cell entry and infection. The synthetic peptide of B5 (wtB5) was functionalized with the nonlinear optical chromophore cascade yellow and its molecular dynamics was probed at physiological and endosomal pH (pH 7.4 and 5.5, respectively). Steady-state CD spectroscopy was utilized to characterize the peptides at different pH. These spectra showed structural changes in the peptide with time measured over several days. Nonlinear optical measurements were carried out to probe the interactions and local environment of the labeled peptide, and the increase in the two-photon cross section of this system suggests an increase in chromophore-peptide interactions. Time-resolved fluorescence upconversion measurements reflected changes in the hydrophilic and hydrophobic local environments of the labeled peptide-chromophore system. Ultrafast depolarization measurements gave rotational correlation times indicative of a reversible change in the size of the peptide. The time-resolved results provide compelling evidence of a reversible dissociation of the coiled coils of the wtB5 peptide. This process was found to be pH-insensitive. The data from this unique combination of techniques provide an initial step to understanding the molecular dynamics of B5 and a framework for the development of novel imaging methods based on two-photon emission, as well as new therapeutics for HSV.

INTRODUCTION

Herpes simplex virus (HSV) types 1 and 2, are widespread human pathogens that infect a wide range of animal tissue. They establish lifelong latency in cells and recurrent infections result from periodic viral reactivation causing oral and genital lesions (1-3). HSV infection of cells requires fusion of the viral and cell membranes through the synchronized action of specific viral (gB, gC, gD, gH, and gL) and cell-surface proteins (heparin sulfate (HS) proteoglycans, nectin-1 and nectin-2, and herpes virus entry mediator (HVEM)) (1-7). The current working model for HSV entry into cells proposes that in the initial step, gB and gC interact with HS proteoglycans, facilitating virus-cell attachment. gD then binds to a receptor, facilitating membrane fusion mediated by gB and the gH/gL heterodimer (4-7). Recent reports outlined the structural characteristics of the C-terminus of gD and revealed that this region plays an important role in HSV entry (2,7). Paralleling this finding is the characterization of the 43-kDa protein B5, which is believed to be one of the cell-surface receptors that mediate HSV entry into cells via gD activation (7). Computational analysis showed that the C-terminus (amino acids 344-374) of B5 has a high probability of forming an α-helical coiled coil (7-9). This is particularly interesting, since computational modeling has also shown that it is highly possible that gH and gL form coiled coils (8-10).

Coiled coils are the oligomerization domains essential to the fusion of a number of viral and cell membranes. The coiled coil results from α-helices possessing a seven-residue (denoted a-g) repeat motif (8-11) of hydrophobic and hydrophilic residues. The hydrophobic residues occupy the a and d positions and generate a hydrophobic seam along which two or more helices align in parallel or antiparallel mode. The helices are stabilized by hydrophobic and ionic interactions (8-11). One of the criteria that established B5 as an HSV cell receptor is the inhibition of viral activity by synthetic peptides identical to its active region (8). Well-documented studies utilizing synthetic peptides to the coiled coil domains of viral proteins include those of human immunodeficiency virus retroviruses (12,13), parainfluenza viruses (14), Sendai virus (15) and paramyxov irus (16), and a 30-mer synthetic peptide to the B5 C-terminus, denoted wtB5, is reported to inhibit HSV activity (8,9). Current therapeutic strategies against HSV are aimed at viral DNA replication and are prone to inactivity due to the evolution of mutated drug-resistant virus strains. For the viruses that contain coiled coils, as in the case of human immunodeficiency virus, therapeutics that work at the site of action of the inhibitory synthetic peptides are being developed as antiviral agents. The functional C-terminus of the B5 or functional regions of its viral ligand, therefore, provide excellent antiviral targets (8). The ultimate development of effective therapeutics demands an in depth understanding of the structure and function of B5 and its associated viral ligand/s. Although mutagenesis and computer simulations have indicated that B5 affects infection of the cell by the HSV, and some structural properties have been elucidated (8), to date, a viral ligand for B5 is still undefined and the atomic structure of B5 or functional regions such as the C-terminus is unknown. There have been no reports of direct verification (biochemical or biophysical) that the B5 actually forms coiled coils and the molecular dynamics and mechanism of interactions are unclear (8,9).

Molecular dynamics of proteins and peptides are paramount to understanding their structure-function relationships (17-19). To probe the detailed interactions of the C-terminus and ultimately the function of the B5 protein, time-resolved and nonlinear optical measurements of wtBS labeled with a fluorophore were carried out. These are sensitive methods that provide valuable information regarding fluorophore and peptide dynamics (20-25). Time-resolved fluorescence anisotropy, for instance, has been used to probe the rotational dynamics of peptides and proteins, providing insight into biophysical mechanisms through global and local motions of peptides via side-chain or extrinsic fluorophores (17-25). Analyses of the physical parameters associated with the motion and nonlinear optical properties of the fluorophore and fluorophore-labe led peptide allow for the elucidation of the molecular mechanisms associated with these changes. Herein, we report results from a detailed analysis of the wtB5 using a unique combination of steady-state, ultrafast spectroscopic, and nonlinear optical techniques, and we provide compelling evidence for reversible dissociation of the coiled coils at physiological and endosomal pH.

MATERIALS AND METHODS

Synthesis end characterization

The 30-amino-acid residue wtB5 peptide (8,9) was synthesized at the Protein Structure Facility at the University of Michigan.

Peptide labeling with fluorescent dye

We dissolved 5 mg of the wtB5 peptide in 0.8 mL of 0.1 M sodium bicarbonate buffer solution at pH 8.2 and 0.2 mL dry dimethylsulfoxidc. The solution was gently stirred and kept on ice. We added 8.2 mg of cascade yellow succinimidyl ester (CY) in 0.1 mL dry dimethylsulfoxide to the peptide solution, and the reaction mixture gradually warmed to room temperature. This was then incubated overnight. Using MALDI-TOF mass spectrometry, 20-�L volumes wens analyzed at 0, 2, and 24 h to follow the labeling process. We then added 65 �L of a 1.5 M hydroxylamine solution at pH 8 to the reaction mixture and stirred it for 2 h. The degree of labeling under these conditions was 63%.

Purification of conjugates

Low-molecular weight compounds and excess dye were separated from the labeled peptide by gel filtration chromalography. Bio-Gel P-6DG get with an exclusion limit of 6000 D was used as the stationary phase on an econopac 10DG column from Bio-Rad (Hercules, CA). The fluorescent fractions were collected. Those showing absorbance at 280 nm and 405 nm were pooled and concentrated on a pretreated Microsep centrifugal device with a molecular-weight cut-off of 1K (Pall Life Sciences, East Hills, NY). The concentrated sample was freeze-dried and reconstituted with 500 �L acetic acid for HPLC analysis. HPLC purification was carried out at the Protein Structure Facility and MALDI-TOF mass spectromeiry confirmed the identity of the peptide. The monolabeled conjugate was used in this study.

Steady-state spectroccopy

Far-UV CD spectra were recorded in a quartz cell (1-mm path length) on an AVIV 202 series spectrophotometer in the range 190-250 nm at 298 K. At pH 7.4, samples were recorded in PBS buffer with 150 �M NaCl and pH 5.5 in sodium citrate buffer. Experiments were done in duplicate, and the concentration-dependent studies were performed in order both of increasing and of decreasing peptide concentration. The raw data were corrected for the buffer contributions by subtracting the buffer spectra. Ellipticity values (θ, deg) were converted to molar-residue ellipticity values ([θ], deg cm^sup 2^ dmol^sup -1^), and molar-residue ellipticity ratios ([θ]^sub 222 nm^/[θ]^sub 208 nm^] were calculated.

Time-resolved spectroacopy

Time-resolved fluorescence measurements were carried out using a fluorescence upconversion set-up that has been described in detail in (28-30). The sample solution was excited with frequency-doubled light from a mode-locked Ti:sapphire femtosecond laser (Tsunami, Spectra Physics, Mountain View, CA) pumped by a Nd-YLF laser (Millenia X, Spectra Physics). This produces pulses of ~100 fs duration in a wavelength range of 385-430 nm. The polarization of the excitation beam for the anisotropy measurements was controlled with a Berek compensator. The horizontally polarized fluorescence emitted from the sample was unconverted in a nonlinear crystal of β-barium borate using a pump beam at ~800 nm that was first passed through a variable delay line. This system acts as an optical gate and enables the fluorescence to be resolved temporally. Spectral resolution was achieved by dispersing the upconverted light in a monochromator and detecting it using a photomultiplicr tube (R1527P, Hamamatsu, Hamamatsu City, Japan).

Raw fluorescence anisotropy R(t) was calculated from the decay curves for the intensities of fluorescence polarized parallel 1part(t) and perpendicular Iper(t) to the polarization of the excitation light, according to the expression R(t))(1par - Glper)(Ipar + 2G/per). The factor G accounts for the difference in sensitivities for the detection of emission in the perpendicular and parallel polarized configurations. It was measured using perylene in methanol as a reference, tn the real experiment, the G-factor has been found to be essentially unity (1.02 (0.02)).

Time-correlated single-photon counting (TCSPC) was performed using the second harmonic of the Kapteyn Mumane laser described below.

Two-photon absorption cross-sectional measurements

Two-photon cross-sectional methods were calculated using the two photon excitation fluorescence method as outlined in (2831.32). We used 10^sup -4^M coumarin 307 in methanol as the standard reference, and a Kapteyn Mumane mode-locked Ti:sapphire laser with a bandwidth of 47 nm at 800 nm and pulse duration of 30 fs. A polarizer was used to vary the laser input power and an iris placed before the polarizer ensured a circular beam. A lens with focal length of 11.5 cm focused the laser beam on the sample cell. The fluorescence was collected perpendicular to the incident beam. A planoconvex lens directed the collected fluorescence into a monochromator. The output from the monochromator was coupled to a photomultiplier tube. Collected photons were converted to photon counts by a photon counting unit. A logarithmic plot between collected fluorescence photons and input intensity gave a slope of 2. This confirmed a quadratic relationship, and the two-pholon-absorption cross section was calculated from the intercept.

RESULTS

Steady-state analysis

Absorption spectra of the free dye CY, wtB5, dye-peptide mixture (CY-wtBS), and labeled conjugate (CYwtBS) at pH 7.4 and pH 5.5 were recorded in the range 200-700 nm. The wtB5 showed characteristic maxima at 220 nm and 280 nm (data not shown). The spectrum of CYwtBS showed a peak at 405 nm compared to 398 nm for CY and CY-wtB5 (Fig. 1). The bathochromic shift of the low energy peak, along with the increased intensity for CYwtBS, confirm conjugation of the fluorophore to the peptide (33). These effects result from the change in geometry of CY upon conjugation. The aromatic backbone of CY becomes more planar (22) and allows for greater π conjugation and substantial electron delocalization over the entire molecule. There are no such geometrical modifications in the case of the peptide-dye mixtures and therefore no significant perturbation of the electronic absorption spectra. The decrease in intensity of the absorption bands at low pH is most likely due to the acidification of residues in the peptide backbone and/or the fluorophore, which could result in mitigation of the intermolecular interactions.

The fluorescence intensity of CY, CY-wtB5 mixture, and labeled CYwtBS is shown in Fig. 2. The fluorescence intensity of CY decreases upon interaction with wtB5 and the decrease is more pronounced when it is covalently bound to wtB5. This may result from the interaction of CY with quenchers such as the aromatic amines of the peptide backbone. The high internal flexibility of the peptide as well as the resultant geometry change in CY could facilitate these interactions. The fluorescence intensity of the systems (CYwtB5 and CYwtBS) changed over time, and the rate of the change was concentration-dependent. The labeled peptide (50 �M) and a 1:5 dye/peptide mixture (50 �M peptide) showed a nonmonotonic change in fluorescence intensity over a period of approximately 90 days. The mixture at a 1:200 molar ratio (2 mM peptide), however, showed the nonmonotonic change over only 14 days. For instance, the fluorescence of CY decreases upon the initial mixing with wtB5 followed by a gradual increase up to 4 days, after which the intensity of the fluorescence decreases over 10 days.

To study the structural characteristics of the peptide and the effect of covalent and noncovalent binding of the dye, wtB5, CY-wtB5, and CYwtBS were analyzed by far-UV CD speclroscopy. The far-UV CD spectra show minima centered at ~222 nm and 208 nm. characteristic of the presence of a- helices (Fig. 3). and indicate that the presence of the dye does not alter the secondary structure of the peptide. The shape of the spectra al pH 7.4 and pH 5.5 are similar, with a slight dampening of signals at lower pH. The ellipticity ratio (9,26,27) for wtB5, CY-wtB5, and CYwtBS changed over time, and the rate of change depends on the peptide concentration. The rate of change for 2 mM wtB5 is shown in Fig. 4 and represents the common trend observed in all cases. It shows an interchange of the ellipticity ratios between average values of 1 and 0.85. The molar ellipticity, [θ]^sub 222 nm^, is concentration-dependent, and results are given in Figs. 5 and 6.

Time-resolved fluorescence and two-photon absorption measurements

Time-resolved lifetime measurements

Even though CD spectroscopy has been extensively used to study α-helices and coiled coils of peptides, the technique by itself is inconclusive (34-39). Thus, we used ultrafast upconversion and TCSPC techniques, along with twophoton absorption measurements, to further analyze the behavior of the wtB5 peptide. These techniques provide sensitive and real-time information (17-25) that cannot be determined using CD spectroscopy (40,41). The fluorescence intensity decays (Fig. 7) determined from upconversion measurements were fitted with two exponentials and the lifetimes are given in Table 1. The short lifetime of ~2 ps was common to all the systems studied and the long lifetime (~400 ps) was resolved using TCSPC. These results gave intensity decays that were also fitted with a biexponential function. The longer lifetime of ~3 ns was unchanged in all the flourescent system and reveals insensivity to the environment. The shorter lifetim, on the other hand, revealed some degree of sensitivity to the local environment, as is evident from the resulting decrease upon binding (covalently and noncovalently) to the peptide (Table 1).

The change in quantum yields for CY, CY-wtB5, and CYwtB5 (Table 1) implies significant differences in the fluorescence lifetimes of the free dye and the labeled peptide. However, this was not observed, as the approximately eightfold decrease in the quantum yield of CYwtB5 (0.05) compared to CY (0.39) and CY-wtB5 (0.36) was not reflected in their lifetimes. This observation hints at the operation of complex excited-state interactions, and the disparity could arise from either of two scenarios (42). In the first instance, fluorescence quenching occurs before relaxation to the ground state. After excitation, there is rapid relaxation to a prefluorescent excited state, and the depopulation of this state is rather slow. A dye-peptide interaction strong enough to compete with the excited-state relaxation would result in quenching of the excitation energy before the prefluorescent state is reached. In the second case, it could result from a heterogenous mode of quenching. This involves a dark species that absorbs energy but relaxes to the ground state via an efficient nonradiative process (42).

Tables 2-4 summarize the time-dependent fluorescence lifetime data for CYwtB5 and CY-wtB5. The lifetimes changed nonmonotonically, at a concentration-dependent rate, and samples at millimolar concentrations changed faster than those at micromolar concentrations. The lifetime of the free dye in the PBS buffer did not change as a function of time, and within the limits of experimental accuracy, the average lifetimes determined at pH 5.5 and 7.4 were similar (Tables 2-4).

Time-resolved anisotropy measurements

The rotational correlation times of the fluorophore determined from time-resolved anisotropy experiments relate to overall rotational diffusion of the peptide and intramolecular motions of the peptide and attached fluorophore (43-46). The time-resolved anisotropy decays were best fitted with single exponentials (Fig. 8), and Table 1 shows values for the rotational correlation times at pH 7.4. There is a slight increase in the rotational correlation time of CY-wtB5 (254 ps) and CYwtB5 (220 ps) compared to the free dye (200 ps). Based on the timescale s of the observed motions, the longer rotational correlation time for CY-wtB5 and CYwtB5 can be attributed to the intramolecular motions associated with the peptide backbone. In the case of CYwtB5, this may result from restricted movement about the lysine side chain (43,46).

The rotational correlation time changed significantly over time (Tables 2-4). The nonmonotonic change is concentrationdependent, and possibly due to a change in the local unwinding of the coiled coil of the peptide (47). The initial decrease may be a result of the dissociation of the coiled coil of wtB5. whereas the increase in rotational correlation time may be due to the association or recoiling of the individual �-helices. The timescales and the trend in the changes of the rotational correlation time were similar for CYwtB5 and CYwtB5, and it can therefore be inferred that the same changes are being detected in both scenarios. Collectively, these results strongly suggest a reversible dissociation of the coiled coils of the wtB5 peptide at pH 7.4 and pH 5.5.

Two-photon absorption measurements

The two-photon measurements allow for the probing of detailed molecular interactions that cannot be done with one-photon measurements (48,49). The fluorescence from two-photon excitation is similar to that from one-photon excitation, with a maximum centered at 540 nm (data not shown). For all the systems studied, a logarithmic plot of the photon flux versus input intensity gives a slope of 2 and implies a two-photon excitation mechanism (31,50). A representative plot is shown in Fig. 9. Two-photon absorbance cross sections for CY, CY-wtB5, and CYwtB5 are given in Table 1. The drastic increase in the two-photon cross section of the labeled peptide results from the covalent interaction of the wtB5 and the CY. Conjugation of the dye to the peptide causes the low-energy band in the electronic absorption spectrum to be red-shifted due to an increase in the electronic delocalization over the system. This leads to an increase in intramolecular interactions such as charge-transfer reactions and/or transition dipole moments (27,31,50) as will happen when the aromatic rings of CY are made more planar upon conjugation to wtB5.

DISCUSSION

The wtB5 forms colled coils with very weak interactions that dissociate reversibly at physiological and endosomal pH

Recent reports have shown that the B5 protein has a high propensity to form coiled coils and favors the formation of dimers, trimers, and tetramers at physiological pH (8,9). Although the cell receptors of the HSV, along with their many viral ligands, have been extensively studied, the molecular interactions governing the actual fusion and entry of the virus into cells are still not clearly understood.

The circular dichrotsm spectra of wtB5, CY-wtB5, and CYwtB5 reveal that the peptide forms coiled coils with weakly associated units. Results of the concentration-dependent studies of wtB5 are shown in Figs. 5 and 6. The increase in helical content, indicated by the increase of [#!222 nm. is consistent with an increasing number of coiled-coil structures formed at higher peptide concentrations (51-53). The sigmoidal relationship (Fig. 6) is consistent with the formation of very weak (51,52) intermolecular associations at higher concentrations (53). The dependence of the helical content on concentration was similarly observed in the case of CY-wtB5. The coiled-coil stability depends on such factors as helical propensity, hydrophobicity of the core residues, packing of residues in the core, electrostatic interactions adjacent to the core, and chain length (51). Particular amino acid residues have been shown to destabilize the coiled coil. These include tyrosine (Tyr), tryptophan (Trp) and asparagine (Asn) (52), and the destabilizing effect of these residues (8,9) in the core of wtB5 (Fig. 10) is confirmed from our results. Changes in the time-dependent spectra were surprising and implied an interchange between an α-helix and a coiled coil motif. The molar ellipticity ratio, [θ]^sub 222^ ^sub nm^/[θ]^sub 208^nm tells whether the helix is monomeric or forms a coiled coil (9,26,27). Our results showed that the ellipticity ratio was interchangeable between average values of 0.85 and 1, reflecting a transition between these two forms. The relative structural simplicity of the coiled coil belies its varied functions in numerous proteins (34,54), and their molecular function is associated with the unwinding and unfolding of the coiled coil. For instance, Wang et al. (55) showed that the nanomechanical operation of myosin is associated with the reversible and irreversible dissociation and unwinding of the coiled coil structure. Carr and Kim (56) and Leikina et al (57) highlighted coiled coils uncoiling in the membrane fusion activities of hemagglutinin of the influenza virus, Hodges and colleagues (51) demonstrated that the unzipping or unwinding of coiled coils in kinesin is associated with its molecular function. Woehlke et al. (47) also alluded to the alternating of a coiled coil structure and a molten conformation in the kinesin function. The molecular mode of action of wtB5 may therefore rely on the observed reversible dissociation of its coiled coil. Reversible and irreversible dissociation of coiled coils as a function of pH, temperature, and denaturant concentration have been widely studied. The behavior of the wtB5, however, is most fascinating, reflecting a concentration-dependent, spontaneous, reversible dissociation process.

HSV infects a variety of cell types and the entry of the viral components into the host cells may operate via multiple cellular pathways. One of the most important factors of biological systems is pH, and both pH-dependent and pH-independent mechanisms of cell entry have been proposed (8,9,11). The effect of pH on the spectroscopic properties of CYwtB5 and CY-wtB5 was investigated. The systems were studied at pH 7.4 (physiological) and pH 5.5 (endosomal). The steady-state and time-resolved measurements of the labeled peptide and dye mixtures at pH 7.4 are tantamount to the results at pH 5.5, which reveals that the behavior of wtB5 is insensitive to the pH studied. This is consistent with the hypothesis put forward by Fuller et al. (8,9), and points to the operation of a pH-independent mechanism.

Under all the conditions studied, the rotation of the fluorophore is independent or uncoupled from the rotation of the peptide molecule. This is evident from the absence of a long rotational correlation time, reflecting the rotational diffusion of the wtB5 peptide, and may be due to the limited spectroscopic properties of CY. Global motion of the wtB5 peptide containing 30 amino acids is expected to take place on a timescale >20 ns. The lifetime of the fluorescent probe (~3 ns) may be too short to detect and report the expected global motions. The rotational diffusion of the peptide is therefore uncoupled from the dye (43) and the reorientational dynamics of the dye is dominated by contributions from intramolecular motions. Even though the fluorescence lifetime of CY is insufficient to facilitate the accurate determination of the rotational correlation time of the whole peptide, its time-resolved anisotropy decay can be used as a measure of peptide interaction, that is, coiled coil dissociation and association through the determination of local motion (58). The nonmonotonic change in the rotational correlation times observed for the wtB5 during 14 days indicates reversible changes in the size of the peptide with time. This is further corroborated by results from the CD spectra. Over time, the CD spectrum changes (Figs. 6 and 7) to reflect changes in the ellipticity ratios that are consistent with the dissociation and association of coiled coils (58,59). This trend was observed for peptide concentrations in the micromolar and millimolar range. At millimolar concentration, the peptide is initially aggregated. It is therefore tempting to infer that the changes in rotational correlation time may be due to a reversible aggregation process. The results from the studies done at micromolar concentrations (Tables 2 and 3) dispel this view, however, as the change in the rotational correlation time paralleled that at micromolar concentrations. Additional proof also stems from the comparable timescales. A noteworthy feature is the absence of the very long rotational correlation time (>400 ps) in the case of the peptide at micromolar concentration. At micromolar concentrations, the peptide is nonaggregated and the changes are most likely due to an interplay between the coiled coil and monomeric forms. It is also interesting to note that after the initial dissociation of the aggregated species, the peptide does not revert to the aggregated state. Since aggregation was shown to be concentration-dependent, it is tempting to conclude here that the concentration changes after the initial aggregate dissociation. This was not the case, however, since the UV-visible spectrum confirmed no concentration changes. This locking of the peptide in a state where it dissociates reversibly between a coiled coil and a monomer even at millimolar concentrations that promote aggregation is surprising and might be paramount to the molecular function of B5. It therefore warrants further investigation. No such changes were observed in the case of me free dye. Taken together, these results show that the rotational correlation time of -450 ps is due to the motion from the aggregated peptide. The ftuorophore is in a restricted environment and does not have much freedom of movement. The motion of the coiled coil and α-helical monomer accounts for the rotational correlation limes of -300 ps and -200 ps, respectively. These findings are consistent with the expectation that there is more freedom of movement in the monomeric species compared to the coiled coit and aggregates. Again, the kinetics of dissociation of wtB5 may be linked to its biochemical function and the mechanism of HSV infection, and it requires more detailed study.

The time-dependent results of the time-resolved lifetime measurements of CYwtB5 and CY-wtB5 (Tables 2-4) show changes in the microenvironment of CY. Since the lifetime of the fluorophore depends on the hydrophobicity of its local environment, the nonmonotonic change in the fluorescence lifetimes may be due to the fluorophore experiencing an interchange between a hydrophilic and hydrophobic environment (25,33). This may result from the association and dissociation of the wtB5 α-helix. At 50 �M, the wtB5 is monomeric and forms aggregates at 2 mM. The initial decrease in the fluorescence lifetimes (Table 4) is perhaps the result of the gradual transition from a "closed" peptide structure to a more "open" configuration, as can occur upon deaggregation of wtB5. The increase in the lifetime would then be due to unwinding of the coiled coil.

There is a rapidly increasing trend in the use of novel microscopic techniques based on nonlinear optical phenomena such as two-photon excited fluorescence in biological imaging (48). Molecules that are ideal for use in these imaging techniques are those with a large two-photon absorption cross section (27,31,49,50,60-62) and this two-photon absorption process is confirmed by the quadratic dependence of the two-photon fluorescence intensity on the laser intensity. A representative plot for CYwtB5 is shown in Fig. 9. The significantly larger two-photon cross section of CYwtB5 therefore indicates that CYwtB5 is a very good specimen for in vitro two-photon-excitation fluorescence imaging. With higher resolution and 3-D imaging coupled with the ability to image at greater sample depths, this technique far exceeds the conventional confocal microscopic methods. Also, the problem of photodamage that is frequently encountered in conventional microscopic techniques is greatly reduced, since the excitation radiation used in twophoton excitation fluorescence microscopy is in the nearinfrared region. Added to this, CY is biocompatible with wtB5: the CYwtB5 is water soluble and does not form aggregates at physiological conditions.

Implications for viral fusion and entry

We have used a unique combination of steady-state and ultrafast spectroscopic and nonlinear optical techniques to study the wtB5 peptide of the recently characterized HSV B5 protein. These types of molecular dynamics studies compliment bioassays and steady-state biophysical studies and can give information on the molecular mechanisms of B5 even in the absence of detailed structural information. The existence of reversible conformational changes may be critical in HSV viral fusion and entry. The mechanism of HSV entry into the cell proposed by Fuller at al. (8,9) occurs at physiological pH and is pH-independent, These same conditions also promote the reversibility of the coiled coil formation of B5.

Since coiled-coil domains normally reflect the oligomerization state of their full-length proteins, they present the advantage of structure and function analysis without obstacles such as supramolecular assembly, which is associated with full-length proteins. The main function of coiled coils is the oligomerization of individual α-helices, and they possess hydrophobic seams along which two or more helices align in parallel or antiparallel mode (53,63-65). Oligomerization is essential in a myriad of cellular functions since it generates a high local concentration of functional sites and allows the concerted operation of clustered domains (57). The observed reversible dissociation may imply the regeneration of wtB5 after fusion of the viral and cell membranes. A number of scenarios can now be envisaged. The interaction of the viral ligands containing coiled coils with the coiled coils of the BS brings about conformational changes that concentrate the B5 protein at the contact zone. This is similar to the mechanism predicted for hemagglutinin of the influenza virus (56,66). Conversely, the interaction of gD with one of its cell receptors could elicit the conformational changes that concentrate B5 protein at the contact zone through self-association of the B5 coiled coils. After fusion, B5 reverts to a monomeric α-helix, allowing for further interactions. These propositions further lend credence to a pH-independent mode of action of the HSV. Even though these studies provide much needed information on the newly discovered B5 protein, there are still a number of questions to be answered and mechanisms to elucidate to fully understand the molecular dynamics of the B5. These include, for instance, information on the mutation-related inhibition mechanism, details on the kinetics of the reversible dissociation, the specific viral ligands that B5 engages, and the process by which this is done. Work continues in our laboratories with the aim of answering these questions. Notwithstanding this, however, we have provided exciting new insights into the molecular dynamics of the wtB5 of HSV B5.

CONCLUDING REMARKS

Using very sensitive ultrafast spectroscopic methods, we have shown the unusual behavior of wtB5. Photophysical parameters determined from nonlinear optical measurements also highlight the in vitro fluorescence imaging potential of labeled wtB5. The question of whether or not B5 forms coiled coils (8,9) has been answered, but even more interestingly, we have provided evidence that the coiled coils reversibly dissociate at physiological and endosomal pH. We believe that these results are important in the elucidation of the overall mechanism of HSV fusion and entry into host cells. They provide the initial critical details of the molecular dynamics of wtB5. They also concur with a pH-independent mechanism for viral fusion and entry and indicate that the coiled coils of B5 may operate in such a way as to concentrate the protein at the point of contact of the virus. This may occur through a combination of processes that involve fusion of the viral and cell membranes via the self-association and/or interaction of B5 coiled coils with the coiled coils of the viral ligand. A recent study showing that the wtB5 has a relatively high probability of fusion (67) further supports these propositions.

[Reference]

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[Author Affiliation]

Ordel J. Brown,* Santiago A. Lopez,[dagger] A. Oveta Fuller,[dagger] and Theodore Goodson III*

* Department of Chemistry, University of Michigan; and [dagger] Department of Microbiology and Immunology, School of Medicine,

University of Michigan, Ann Arbor, Michigan

[Author Affiliation]

Submitted November 15, 2006, and accepted for publication April 9, 2007.

Address reprint requests to Theodore Goodson III, Dept. of Chemistry, University of Michigan, Ann Arbor, MI 48109. E-mail: tgoodson@umich.edu.