| W L OT Pts GF GA |
| x-Pittsburgh 46 26 7 99 242 210 |
| New Jersey 43 28 7 93 198 189 |
| N.Y. Rangers 40 27 11 91 203 191 |
| Philadelphia 40 28 11 91 241 229 |
| N.Y. Islanders 34 37 8 76 189 235 |
| Northeast Division |
| W L OT Pts GF GA |
| x-Montreal 44 25 10 98 253 220 |
| Ottawa 42 29 8 92 252 240 |
| Boston 40 28 11 … |
ALMOST ANY handicapping schoolboy within touting distance ofLaramie Avenue--Cicero/Stickney's Basin St. South--can rattle off WarEmblem's streak from Sports-man's anonymity to thoroughbredimmortality--allowance win, Illi-nois Derby win, Kentucky Derby win,Preakness win.
Bernie Flint isn't any handicapping schoolboy. He's one of themost savvy trainers in the Midwest. But Flint not only has takenanecdotal note of War Emblem's route from gulag-to-riches, he has sethis Lx Commander on the same arc. And with Saturday's $500,000Illinois Derby (Grade II) at Hawthorne the next stop on the horizon,Flint's prodigy is already one-fourth of the way home.
SAID FLINT: It's one of …
A class move from the Clerk of the Circuit Court
Applause and plaudits to Circuit Court Clerk Aurelia Pucinski for putting the best interests of the people above politics.
But shame on some Cook County GOP operatives for their backbiting and sniping at Pucinski for simply being -- as she put it -- "a responsible public servant" who wants only the best for her constituents.
Pucinski has been briefing Clerk candidate and probable winner in November Dorothy Brown, a Democrat, on the ins and outs and perplexities of the job.
She is also providing similar information about the post to GOP nominee Mary Mynard, but some Republicans are upset because they think Mynard …
Pope Benedict XVI will bless the sick at the shrine of Lourdes on the final day of his four-day visit to France.
Benedict is to anoint about a dozen ailing pilgrims at the close of Mass in Lourdes' basilica Monday.
The Roman Catholic shrine in the foothills of the Pyrenees …
An executive at the background-check company Intelius Inc. who recently promoted an iPhone application for weeding out sleazy dates has been charged with lying to a Seattle grand jury when he denied engaging in sex acts at a strip club.
John Kenneth Arnold, a co-founder and executive vice president of business development at Intelius, pleaded not guilty Thursday in federal court following his arrest.
The indictment alleges that Arnold, who is married, lied to a grand jury when he testified he had never engaged in sex acts with dancers at Rick's.
His testimony was part of the government's racketeering investigation of the club's owner, Frank …
Time was when furniture-and clothes, bed linens and table settings-were supposed to match. Black shoes and brown purse? No way!
But, like fashion, rigid rules about what goes with what in homefashions are increasingly obsolete. And now, it seems, the time hascome for an a la carte approach even to the once-uniform dining-roomchair.
"You don't have to have eight identical Hepplewhites around yourtable anymore," says Washington interior designer Steven J. Richards."With so many wonderful styles for dining-room chairs out there, it'spredictable and boring to limit yourself to only one."
Richards says some clients are so self-confident about shopping atflea markets …
| East Division | ||||
|---|---|---|---|---|
| W | L | Pct | GB | |
| Philadelphia | 82 | 44 | .651 | — |
| Atlanta | 77 | 52 | .597 | 6 1/2 |
| Washington | 62 | 64 | .492 | 20 |
| New York | 60 | 67 | .472 | 22 1/2 |
Fast decisions on life-and-death cases are the bread and butter of hospital emergency rooms. Nowhere do doctors face greater pressures to overtest and overtreat.
The fear of missing something weighs heavily on every doctor's mind. But the stakes are highest in the ER, and that fear often leads to extra blood tests and imaging scans for what may be harmless chest pains, run-of-the-mill head bumps, and non-threatening stomachaches.
Many ER doctors say the No. 1 reason is fear of malpractice lawsuits. "It has everything to do with it," said Dr. Angela Gardner, president of the American College of Emergency Physicians.
The fast ER pace plays a …
Ask any entrepreneur what their biggest holdup to launching or growing their business is and the answer will come quick and suremoney.
There is little doubt, if any, that finding capital to finance your business venture is the most basic and important of all your business activities. It also can be the most frustrating one if you don't know where and how to look, but it doesn't need to be that way. Finding capital can be a smooth, rewarding experience provided you study diligently and plan effectively.
However obvious it may seem, the first thing you need to know before setting out in search of money is how much you need. Here, again, a thorough business plan will be …
The difference in 12 months was not hard to see.
Serena Williams, wearing hot pink bicycle shorts, a short white dress and dangling, chandelier-inspired earrings, found her form quickly and beat Jarmila Gajdosova 6-3, 6-3 Monday in the first match at the Australian Open.
Last year, she was unseeded, ranked 81 and coming off one of her worst losses on tour _ in a Tier 4 event at Hobart _ yet she beat six seeded players en route to the championship at Melbourne Park. It was her eighth, and least expected, Grand Slam title.
Expectations _ hers and the pundits _ are much higher this season.
"It's obviously a lot different _ I'm not …
Buddy Guy got a street sign.
Chicago's greatest living popular music icon came to his club, Legends, 700 S. Wabash, Sunday night to receive a proclamation from the City of Chicago, hang with his band and to catch a set from his daughter Carlise Guy's Nu Blu Band. The renaming of the stretch of South Wabash in front of his club as "Buddy Guy Way" was unexpected.
The blues guitarist, who turned 75 years old on July 30, was genuinely moved.
"This is the biggest surprise since B.B. King asked me to play with him," Guy said after the ceremony. "Things like this only come when you're gone. My mother always told me, 'Give …
FORT CAMPBELL, Ky. - A fire at Fort Campbell last month that killed a soldier's two young children was arson, law enforcement officials said Friday.
Details about what was used to start the fire were not being released, said Craig Roegner, a spokesman for the Bureau of Alcohol, Tobacco, Firearms and Explosives.
"There was enough information to say yes, in fact it is arson, but we don't have enough to give a cause yet," he said. The investigation was continuing.
Six families lived in the same two-story housing unit where the fire broke out, a housing development called Lee Village that dates to the 1940s.
In a joint release with Army Criminal Investigation Command, the ATF identified the dead children as Rebekah Smallwood, 2, and Samuel Fagan, 9.
Spc. Wayne Smallwood, 33, of the 626th Corps Support Battalion, and his wife, Billi-Jo Lee Smallwood, 32, suffered minor injuries. Their infant daughter, Nevaeh, was not injured, the release said.
Bangladesh captain Mohammad Ashraful and wicketkeeper Mushfiqur Rahim have been fined for excessive appealing during the second test against Sri Lanka that ended this week, a news release said Wednesday.
Ashraful was fined 15 per cent of his match fee, while Rahim got 50 per cent deducted for breaching the International Cricket Council's Code of Conduct.
Match referee Jeff Crowe imposed the fines after hearing charges laid by umpires Steve Bucknor and Nigel Llong at the end of the match Tuesday in southeastern Chittagong city, an ICC statement said.
Ashraful was found guilty of "excessive appealing _ a Level 1.5 breach, while Mushfiqur was charged under Level 2.5 for charging or advancing toward the umpire in an aggressive manner when appealing."
The hearing was attended by the two players, Bangladesh manager Shafique Haque, coach Jamie Siddons and the umpires.
Sri Lanka won the match by 465 runs _ and clinched the test series 2-0 _ with a day to spare.
A charter bus ran off a highway overpass north of Dallas and crashed onto its side on a roadway below, authorities said. Reports said the passengers were members of a Vietnamese church and that as many as 13 were killed.
The bus carrying 55 people to Missouri from the Texas city of Houston may have blown a tire, said Sherman police Lt. Robert Fair.
The Dallas Morning News and KHOU-TV quoted Sherman police as saying 12 adults died at the scene and another died at a Dallas hospital.
The Herald Democrat of Sherman reported the bus came to a rest on its right side in the northbound lane of an access road below U.S. 75.
The accident happened at 12:45 a.m. (0545 GMT) Friday, Fair said. Scores of injured were being rushed to Sherman and Dallas hospitals via helicopters and ambulances.
A spokeswoman at Wilson N. Jones Medical Center in Sherman said it was treating 17 patients. A spokesman with Parkland Memorial Hospital in Dallas said four people from the crash were in critical condition there. Another person brought to the hospital has died.
Northbound traffic on the highway was shut down as ambulances and helicopters used the roadway and median to ferry the injured to hospitals.
KHOU-TV quoted a Galveston/Houston archdiocese church official as saying many of those on the bus were from the Vietnamese Martyrs Church of Houston and were on their way to a religious festival in Carthage, Missouri. At least four translators were brought to the scene, the Herald Democrat reported.
The accident happened less than a mile from the spot where a trucker crossed the median and killed 10 people five years ago.
Sherman is about 65 miles (105 kilometers) north of Dallas.
PRINCIPLES OF ESTIMATING BY BRUCE BURROW
Is color match included in a refinish labor amount of 0.3 for a small filler panel? It's a simple question without a simple answer. Actually, the answer has several dimensions to consider-the most obvious being the repair situation surrounding this part. Another is the way each information provider deals with this subject.
In the most likely case, this small part is being refinished along with one or more major exterior panels. We can assume any color-match effort is spread across all those parts, as is the compensation. Occasionally, this small filler is the only part being refinished. In that case, one might question how close the color match needs to be and how much effort would actually be involved. It's probably not as critical as a fender-to-door match. Still, 0.3 is not a great deal of compensation for all that is needed when refinishing a single part by itself.
If it's a customer-pay job, explaining and recovering additional costs may be relatively easy. If this is for an insurance claim, you may need to bring out your best negotiating skills. Either way, the procedure pages will need to come into play. That widens the subject to color match in general. Is color match considered at all? If so, what part of the process? Is there a line between normal and excessive?
The following basic color match steps are relatively clear in theory:
* Obtain color information, code, paint chip and/or variance data.
* Order or mix paint.
* Check color against the vehicle.
* Spray-out test panel.
* Check test panel against vehicle.
* Tint paint and retest color.
* Repeat until satisfactory.
* Blend into adjacent panels.
All these steps could be done on any job, but they aren't necessary for every job. The decision between the extremes of everything and nothing has many variables, including the color itself, paint products, equipment, painter's skill and experience, and business decisions related to workflow.
Now how do the information providers handle this?
MOTORS' refinish labor includes the following: Retrieve accurate color information, including paint chip.The following tasks are itemized as "Does Not Include": color matching to adjacent panels, test spray-out panel, tinting to achieve color match and blending into adjacent panels.
ADP's refinish labor includes the following: get paint code, order/get paint, initial color tint and spray test panel. "Not-Included" tasks include spray additional test panel, tint primer or clear and blending into adjacent panels.
MitchelTs refinish labor includes the following: mix materials and adjust spray equipment without identifying specific steps for obtaining proper color. "Not-Included" tasks include the following: blending into adjacent panel and/or panels, or nearest break point and color match or tinting. ;
Each information provider references it a little bit differently, so there is no simple or general rule when it comes to compensation for color-match efforts. Each information provider acknowledges some initial effort for paying attention to color, and that would be reflected in their refinish labor amount.The line between expected effort and excessive effort is different among them, although they each leave a basis for negotiations.
Blending could still be needed on many jobs to hide any remaining color variation. Blending is not included by any information provider and has automatic calculations by each information provider when selected. However, a good color match doesn't always go easy and the effort, or steps involved, can quickly escalate into tinting, additional test panels and more. Some jobs can turn out quite problematic and that is difficult to predict at estimate time. Maybe with the right experience, trouble could be predicted on some jobs, but most are surprises, such as working with a new color, an unknown color variance or poor color coverage.
Being aware of this and your information provider's procedures is the first step in negotiating fair ; compensation when these situations arise. Requesting additional labor for color match on every job is probably unreasonable. Requesting additional labor for color match on no job is equally unreasonable. There is a middle ground somewhere, and good, honest negotiations are the only way to find it. Negotiate that lone 0.3 filler panel to a fair amount for all involved: the vehicle, its owner, the insurer and yourself.
"Every problem contains within itself the seeds of its own solution."
- Stanley Arnold
[Author Affiliation]
Brace Burrow, AAM, started writing estimates more than 30 years ago and has worked for most of the information providers. He has been an independent instructor for the last 10 years. Burrow is ASE Master certified and an instructor for the Automotive Management Institute (AMi) and carries the Accredited Automotive Manager (AAM) designation. Send questions, ideas and comments that you would like to see addressed to bunow@fnc.net and stay tuned.
[Author Affiliation]
BRUCE BURROW started writing estimates more than 30 years ago. Color matching can be tricky. Bruce explains why. E-mail him at burrow@mc.net.
Talk about relieving the pain of the Cubs' big hurt. ScottBullett's performance at bat and in right field made "Sammy Who?"the tongue-in-cheek catch phrase Wednesday at Wrigley Field.
Bullett (3-for-4, three runs, three RBI) singled and scored inhis first two at-bats and homered his third time up in his firstofficial start as injured Sammy Sosa's replacement. Though heoverthrew cutoff man Mark Grace on one play, he made a running catchof a Bob Natal fly ball and doubled up Kurt Abbott at first.
"I'm no Sammy Sosa," said Bullett, who raised his average to.234, "I'm at a point where this is my career right now. I'mstruggling pinch-hitting (9-for-47), but this is an opportunity toget four or five at-bats a game, do good and show that I can playhere next year."
Bullett said every Cub must perform better with Sosa out.
"Sammy always put us on the board," Bullett said. "We have topick Sammy up, and that's a big weight to carry."
KIESCHNICK RETURNS: Power-hitting outfielder Brooks Kieschnick,who hit .257 with 20 doubles, 18 homers and 64 RBI in 116 games atClass AAA Iowa, was recalled to fill Sosa's roster spot.
Kieschnick, who was 1-for-6 in seven games at the start of theseason, will be on the bench.
DAWSON SAYS GOODBYE: Former Cub Andre Dawson, who is retiringafter the season, was presented before the game with a large framedphoto of himself in a Cubs uniform, framed ivy and grass from theWrigley Field outfield and a right-field bleacher seat with his No.8.
Dawson jogged from the right-field corner along the warningtrack, bowing to fans in the bleachers.
"I want you all to know that I love you from the bottom of myheart," Dawson said. "The Cubs allowed me to rejuvenate my career.The right-field fans, you're the greatest. . . . I'll miss Chicago,I'll miss Wrigley, I'll miss (equipment manager) Yosh (Kawano) andalso the greatest fans in baseball." Dawson has been on the Marlins' disabled list since Aug. 8 becauseof right knee inflammation.
ROTATION SET: With off days today and Monday, manager JimRiggleman can stay in a four-man rotation and use Frank Castillo,Steve Trachsel and Jaime Navarro on four days' rest in the series atHouston on Tuesday-Thursday. The Cubs return home next Friday for adoubleheader with Atlanta to begin a four-game series that willrequire a fifth and possibly sixth starter. Riggleman wouldn'tspeculate that far ahead.
STREAKING: Grace (2-for-3) extended his hitting streak to 15games and raised his average to .341.
NASHVILLE, Tennessee (AP) — Congress has taken the unusual step of waiving immigration restrictions for two Japanese citizens fighting to live in the United States.
The private immigration bills passed by the House on Wednesday — they had already been passed by the Senate — are the first to be approved in more than five years. The measures now go to President Barack Obama for his signature.
One bill would clear the way for the granting of legal status to the widow of a Tennessee Marine who gave birth to their son after he was killed in Iraq in 2008. Another would provide relief to a Japanese man living in California whose mother was killed in a car crash when he was a teenager and who was never legally adopted.
"I have always seen myself as part of this whole American society, and I am American, just like my friends but without the status or papers," said the man, Shigeru Yamada, now 28. "For me to finally become, or have the potential to become a permanent resident, it means a great deal to me, it really does. I can't really express how happy I am."
Congress can vote to let individual immigrants in exceptional cases live in the country legally but hasn't done so since the 108th Congress, in 2003-04. Immigrant advocates see such bills as a last resort when other efforts to obtain a green card have failed.
"The logjam has been broken open for the first time in five years," said Brent Renison, an immigration attorney who represented Hotaru Nakama Ferschke, whose husband Marine Sgt. Michael Ferschke died in Iraq in 2008.
The couple married by telephone while she was in Japan and he was in Iraq. She was already pregnant with his child. But their marriage was not considered valid under immigration law because they did not consummate the marriage due to the fact he died in combat before they were reunited.
The Marine's mother, Robin Ferschke, was elated when Republican congressman John Duncan of Tennessee called her Wednesday morning to tell her know Congress had passed the relief measure after many legislative setbacks. Ferschke hopes the actions open the door for more private bills to be considered by Congress.
"These bills are tragedies and they shouldn't just sit there for years," she said.
A private bill is one that benefits or provides relief to specified individuals or companies, often when no other remedy is available. Subjects of private bills include immigration, taxation, public lands, medical affairs and Armed services decorations.
Anywhere from a few dozen to more than a hundred private immigration bills are introduced each session of Congress, though most don't go anywhere, according to a report by the Congressional Research Service.
Private immigration bills were more common until a corruption scandal involving payoffs for the sponsorship of legislation in the 1970s. Use of the bills declined further after the Sept. 11 terrorist attacks, with only four enacted in the 108th Congress, the report said.
But immigration attorneys say that even getting a member of Congress to introduce a private bill can help an immigrant facing deportation.
Immigration and Customs Enforcement holds off on deporting immigrants who have private bills pending in the Senate, or those whose cases have been vetted by the House immigration subcommittee and for whom an investigative report has been requested from ICE.
Carl Shusterman, an immigration attorney in Los Angeles, said he has four clients who have private bills pending in Congress — the only protection that keeps them from being deported.
The bills expire soon after the end of each session, and new ones are typically introduced when Congress starts up again. But most private bills don't pass.
"Some of our people are on their fourth private bills already," Shusterman said.
Lawmakers may use private bills to help individual citizens but also to gauge whether there's a need for broader changes in immigration policy.
Shigeru Yamada came to the United States on a visa with his mother from Japan when he was 10 years old. She was killed in a car crash three years later, in 1995, and he went to live with his aunt in Chula Vista, California, but was never formally adopted.
He finished high school and attended community college. But Yamada, known to his friends as "Shiggy," was arrested by U.S. immigration agents in 2004 while riding a bus to downtown San Diego.
Democratic Sen. Dianne Feinstein and Rep. Bob Filner introduced bills on behalf of Yamada, which prevented his deportation. On Wednesday, he planned to celebrate with friends after getting off work as a coordinator at a Lasik center, but noted that the president still must sign the bill into law.
Republican lawmakers have resisted passing private bills in recent years, arguing that immigration should be tackled on a policy, not an individual, level, said Gregory Chen, director of advocacy at the American Immigration Lawyers Association.
The Ferschke case won them over, he said, and that led to a compromise with Democrats working on Yamada's behalf.
"To break an almost six-year blockade on these cases of private bills, I think, is an example of an opportunity to work together on these," Chen said. "It certainly eliminates arguments where people have been resisting private bills, saying private bills are not, as a policy, the way such matters should be handled."
Duncan had been working for more than a year on his private bill for the Ferschke family, along with Democratic Sen. Jim Webb, a combat Marine in Vietnam and former Navy secretary. Webb was the sponsor of the version that the House passed on a voice vote Wednesday. The two measures allow the Japanese citizens to be eligible for permanent resident status upon filing an application.
"Helping people caught up in extraordinary circumstances like the Ferschkes' is one of the most basic and important jobs of Congress," Duncan said, "and I am so grateful for all the bipartisan support in the House and Senate."
Since Michael Ferschke's death, his mother has only been able to see her grandson, Mikey, during temporary visits. Nearly two years old, he's starting to look a lot like his Marine father and is speaking English and Japanese words, she said.
"Just to hold that baby means so much to me," she said. "When I hold him, I am holding my Michael."
Mennonite Church Eastern Canada
Marianne Mellinger of Kitchener, Ontario, has been appointed to a new position in Leadership Formation, beginning January 1. This is a half-time position shared by Mennonite Church Eastern Canada and Conrad Grebel University College.
The position reflects the growing partnership between MCEC and Grebel to develop resources for training and nurturing leaders.
Marlene Epp, academic dean at Grebel, says, "Marianne has already demonstrated a gift and passion for leadership development within the Graduate Theological Studies department of CGUC. We look forward to her expanded contribution."
Mellinger began pastoral ministry in the Franconia Conference of Pennsylvania. Since moving to Ontario she has served as an interim pastor, counsellor, teacher and clergy coach.
Her training includes a Doctor of Ministry degree in Pastoral Psychotherapy from Garrett Theological Seminary.
"I am excited to be working at the task of leadership development in the creative context of this shared position," says Mellinger. -From MCEC/Grebel release
The next generation of manned ground vehicles (MGVs) will represent the convergence of new operational concepts and advanced technologies, finally allowing the Army to achieve the elusive goal of commonality. A family of MGVs is evolving that will, for the first time, share operational capabilities and parts that will simultaneously revolutionize operations and logistics. The MGVs are eight of the 18 Future Combat Systems (FCSs) under development for the Army. (Four are pictured on the previous page.)
If you are a heavy brigade commander today, you lead a force with unmatched lethality but unequal operational capability, survivability and with logistical incompatibility. Your tracked vehicle fleet consists of M1A2SEP Abrams tanks, M2A3 Bradley fighting vehicles, M88A2 recovery vehicles, a family of M113s, and M109A6 Paladin self-propelled howitzers.
They all have different mobility, operational, diagnostic and repair procedures, levels of survivability, operational ranges and more. If you just consider propulsion and suspensions, each of these vehicles has different track, road wheels, road wheel arms, hub and bearing assemblies, final drives, sprockets, engines, transmissions and generators. There are innumerable line replaceable units, electronics boxes and circuit card assemblies (CCA) that provide this very capable and reliable fleet with its operational performance. Each current vehicle family contains many unique CCAs that are not only functionally different but also designed to different specifications and are totally incompatible between systems.
In the not-too-distant future, should you be fortunate enough to be a unit of employment (UE) commander, your Future Combat System MGV fleet will be radically different both operationally and logistically. All tracked vehicles will ride on the same suspension system, be powered by a single engine/generator combination, propelled by a single traction drive motor assembly and cooled by a common thermal management subsystem, making these systems true hybrid electrics. Power and energy for all vehicles will be an energy distribution and storage subsystem that has dual voltages (600/28 volts) with redundancy and is dynamically reconfigurable via software and/or soldier directed inputs.
All the forward driver/crewman's two stations will be identical in physical layout and driving operations but totally reconfigurable via flexible warrior machine interface software to meet specific mission functions. All vehicles will have the same on-board integrated computer systems, crew station displays, internal databus sub-systems and basic joint tactical radio systems, allowing each MGV to be a fully integrated component of the FCS Battle Command and Control network. All drivers will have the same day and night situational awareness sensors and be able to move their vehicles in the same terrain and environments as all other tracked vehicles in the UE with basically the same operational limits. Each vehicle will have the same levels of ballistic and nuclear, biological and chemical (NBC) protection for its crews. Endurance of the operators will be improved through ergonomic seating and uniform environmental heating and cooling in all variants.
The requirement for a new generation of C-130 deployable MGVs and the availability of technologies that allow design teams to reduce the parasitic weight of structures associated with survivability have led to this radical new way of configuring combat vehicles. The MGV team, led by the lead systems integrator team of Boeing and SAIC, has brought together the best and the brightest of America's combat vehicle makers-General Dynamics Land Systems and BAE Systems (formerly UDLP)-to create a family of combat vehicles based on a common chassis that can be configured into at least eight variants within the single architectural design.
The common chassis and variant mission module design teams integrate, but do not develop under their vehicle contracts, the unique FCS network-centric common command, control, communications, computers, intelligence, surveillance and reconnaissance (C^sup 4^ISR) subsystems. These are provided by the FCS-level C^sup 4^ISR integrated product teams (IPT). In the common chassis this includes the autonomous navigation system, integrated computer system and remote operations capability via a crewman's remote interface system and/or crewman's decision aid. For the variant mission modules there is an array of common components and subsystems from both the C^sup 4^ISR and MGV IPTs, including joint tactical radio system, multifunction radio frequency device, combat identification, close combat auxiliary weapon station, short-range and long-range active protection systems and short-, medium- and long-range electro-optical devices and infrared sensors. These are not necessarily common to all variants.
The key to the design success of the common chassis will bo the selection, development and integration of various components and subsystems in a physical and electronic architecture that serves the most demanding technical and performance needs of the variant mission modules. To do this, the General Dynamics/BAE common chassis team has evolved an initial design concept that includes:
* Single chassis design with reconfigurable armor.
* Power generation via a high density diesel engine and matched generator.
* Hybrid electric traction drive system.
* High voltage energy storage system.
* Thermal management subsystem.
* Band track.
* Hydro-pneumatic road arms.
* NBC/environmental control.
* Autonomous navigation drivers aids.
* Universal crewmen's forward stations and displays.
* Numerous other common subsystems and components.
The common chassis provides power and stored energy to meet the propulsion and electronic demands of all variants by dynamically managing its generation, storage and distribution to subsystems based on the priorities established by the states and modes software unique to each variant's operational needs and crewman's instructions. Thermal management, environmental control and NBC protection are handled in the same fashion. The subsystems are designed so that they can be altered both physically and via software to cool and protect the occupants of a vehicle.
Although a long way from design review, the concept above gives a clear indication of the design advances that have been made in achieving true commonality within the manned combat systems of the FCS program.
The goal of the commonality plan for the Future Combat Systems family of MGVs is to improve operational availability, reduce the logistics footprint and balance performance with life-cycle cost. The commonality approach is based on emphasizing maximum commonality from the beginning of the concept phase. The first 5 percent of program expenditure commits up to 70 percent of life-cycle cost of the program and this early stage is where commonality must be firmly established.
The approach is to adopt the strategy that everything in the vehicle is common until proven uncommon. Based on this strategy, for example, the nonmission-specific hardware starts out as common and must be justified to be uncommon by evaluating the impact of the suggested change on the key performance parameters, critical operational requirements and the program life-cycle costs. This evaluation highlights the true program benefit and burden associated with making the item uncommon, which will allow the standardization team (ST) to make an informed decision to balance performance with life-cycle cost. An example of this balance is the engine. Since the MGV family is composed of 24-ton class vehicles, a common engine type can provide adequate performance for each variant while minimizing the development, production, training and logistic program costs.
In evaluating whether an item can be unique, the ST requires that the integrated product team requesting a deviation substantiate the tangible benefits resulting from making the item unique and/or the unacceptable burdens that the use of a common item will cause. Parameters to substantiate consideration include:
* Development cost and schedule impact.
* Production cost (DTURC or design to unit rollaway cost) impact.
* Reduction in system space, weight, power and cooling.
* Elimination and/or consolidation of functions.
* Hardware and software interface control documents.
* Improvement in reliability, maintainability or operational/inherent availability.
* Manufacturing tools, processes and assembly procedures impact.
* Number of different spare part numbers.
* Extent of maintenance and diagnostic procedures required.
* Software lines of code required.
* Training support packages, equipment and procedures impacts.
* Operational and maintenance training impacts.
* Maintenance tools impact.
* Consumables (for example, lubricants, cleaning agents and oils) expended.
* Support equipment impact.
Data provided by the IPT will be entered into the life-cycle cost model, where applicable. This cost data will then be balanced against the improvement in performance (as well as other program impacts) to determine if the item should be granted uncommon status. The commonality customers and stakeholders have a key interest in the dynamic realm of cost and performance. Through their active role on the MGV ST, these individuals can ensure that the commonality vision for FCS is maintained.
Only those components that are absolutely mission specific and unique fall into the noncommon category. Such items as the 155 mm cannon for the non-line-of-sight cannon, the 120 mm cannon for the maneuver control system, the 30 mm autocannon on the infantry combat vehicle (which may also be used on the resupply vehicle) and the 120 mm mortar for the non-line-of-sight mortar are examples of unique items to meet specific mission requirements.
The driving technologies that have caused this level of commonality to be achieved are the changes in operational performance associated with the networked system of systems situational awareness and the introduction of advanced active and passive survivability suites in lieu of traditional heavy metal armor. What has happened is not the elimination of the protection afforded by the current Abrams and Bradley systems but instead the raising of the entire MGV fleet's level of protection to that of the next generation close-combat manned combat system and the infantry combat vehicle.
In effect, the entire FCS vehicle fleet will achieve equal protection, mobility performance and logistical supportability with the future close-combat systems. The true benefit of commonality will be in the operational and logistical advantages and flexibility it gives to the unit of employment commander.
[Author Affiliation]
By Col. Christopher V. Cardine
U.S. Army retired
[Author Affiliation]
COL. CHRISTOPHER V. CARDINE, USA Ret., is a scientist for tank technology, General Dynamics Land Systems. He is a graduate of the U.S. Military Academy and the U.S. Army War College.
Swedish forward Patric Hornqvist practiced two days after being checked in the head and is expected to play in the Vancouver Games men's hockey quarterfinal.
Coach Bengt Ake Gustafsson said "everything's good," after watching Hornqvist take part in a 75-minute practice. Gustafsson added that he's sure Hornqvist will play Wednesday when the defending Olympic champion Swedes face the winner of the playoff qualification game Tuesday between Slovakia and Norway.
Hornqvist was hurt late in the second period of Sweden's 3-0 win over Finland on Sunday, when he was checked in the head by Joni Pitkanen. Hornqvist crumpled to the ice and required help from team trainers to get off the ice.
Pitkanen was ejected from the game and was suspended from playing in Finland's quarterfinal game on Wednesday.
The United States inserted Brad Guzan in goal against Egypt, one of three changes to the starting lineup for its final Confederations Cup first-round match Sunday.
Guzan, the backup for Aston Villa in England, took over from Everton's Tim Howard, the regular starter. Ricardo Clark, who missed Thursday's 3-0 loss to Brazil while serving a one-game suspension from a red card in the opening 3-1 loss to world champion Italy, was back in midfield in place of Sacha Kljestan, who in turn was serving a one-game suspension from a red card against Brazil.
Forward Charlie Davies replaced DaMarcus Beasley, his first start since a Jan. 24 exhibition against Sweden.
The back four defenders remained the same, with Jay DeMerit and Oguchi Onyewu in central defense, and Jonathan Bornstein and Jonathan Spector at outside backs. Michael Bradley, Clint Dempsey and Landon Donovan joined Clark in the midfield, and Jozy Altidore was with Davies at forward.
After losing its opener 4-3 to Brazil, Egypt upset Italy 1-0.
The United States would join European champion Spain and host South Africa in the semifinals by beating Egypt by at least three goals, and either Italy scoring three fewer goals than the United States in a loss to Brazil or the victory margins of the U.S. and Brazil totaling seven or more.
Egypt, the African champion, played the United States just once before, winning 3-1 in June 1987 at Seoul, South Korea.
Casey Stoner claimed his sixth victory in this year's championships as he won the season-ending Valencia Grand Prix Sunday in a race he dominated from the first bend.
Stoner, who finished second in the world championships on his Ducati, crossed the finish line in 46 minutes, 46.114 seconds, followed by Dani Pedrosa on 46:49.504 and World Champion Valentino Rossi in 46:58.308.
Rossi ends the season on 373 points, Stoner has 280 and third-place Pedrosa of Spain finished on 249.
The Australian had been on pole position on the grid for the ninth time this season _ matching Rossi's 2003 record _ but he made a slower start than Pedrosa who shot off the start line in first place.
A charging Stoner, who won the 2007 championship on the Italian bike, had recovered the lead by the end of the first bend, while Jorge Lorenzo _ who finished fourth in the overall standings on 190 points _ made a poor start, falling back from seventh to 11th place.
On lap five Rossi, who clinched his sixth championship in Sepang, Malaysia, earlier this month, had risen from 10th on the grid at the start to third place behind Stoner and Pedrosa.
By lap eight Stoner, who was racing with a slight left-wrist injury, had gone to a 1.89 second lead over second-place Pedrosa who was ahead of third-place Rossi by a similar amount.
With nine laps to go Rossi recovered well from a near-fall on a circuit where he has come off in the previous two seasons' races to maintain his position behind Stoner and Pedrosa.
In the the 250cc category, Marco Simoncelli won in 43 minutes, 29.003 seconds on his Gilera, clinching the season as champion on 281 points. Alvaro Bautista took second on 244 points and Mika Kallio was third on 196.
Kallio fell on the last bend of the race, tumbling into the gravel. The Finn finished 11th. Honda rider Yuki Takahashi of Japan was second in the race in 43:34.167, while Alvaro Bautista of Spain rode his Aprilia into third place in 43:37.651.
In the 125cc class, Simone Corsi of Italy won on his Aprilia in 40 minutes, 45.715 seconds. Spain's Nicolas Terol was second for Aprilia in 40:45.821, while Mike Di Meglio of France took third on his Derbi in 40:45.938.
Di Meglio was overall champion on 264 points, followed by Italy's Simone Corsi on 225 and Gabor Talmacsi of Hungary on 206.
An explosive device damaged a military recruiting station in Times Square early Thursday, and police blocked off the area to investigate.
The explosive device caused minor damage, and no one was injured, police said. The explosion shattered a glass entryway.
Members of the police department's bomb squad and fire officials gathered outside the recruiting station.
Witnesses staying at a hotel in the area said they heard a "big bang" and could feel the building shake. A large plume of smoke was also visible after the explosion, they said.
Subway trains were not stopping at the Times Square station during the investigation, and police cars and tape blocked the streets.
Carlyle Group said Tuesday that it plans to raise $100 million through an initial public offering. The private equity firm wants to go public even as fears about the global economy have punished stock markets.
Many companies canceled their IPOs in August. Only four companies went public during the month, down from 14 in July and 13 in August of last year, according to data from Renaissance Capital. Carlyle, based in Washington, did not say how many shares it plans to sell, when it intends to go public, or predict a price for its stock. While a filing with the Securities and Exchange Commission said Carlyle plans to raise as much as $100 million, the offering's value may change as the IPO's bankers gauge demand for the stock.
Private equity firms buy companies and later try to sell them for more money. They often borrow money to fund their purchases. The business slumped during the recession, but several major firms have gone public following the revival in the stock market after the downturn. Blackstone Group's IPO was in 2007, before the Great Recession began that December, while KKR & Co. listed its stock in the U.S. in July 2010 and Apollo Global Management debuted in April.
It's a terrible time for a private equity company to go public, said IPO researcher Scott Sweet, the owner of IPO Boutique. Investors might not be hot on the stock of a private equity firm because of the declines in rivals' shares, he said. A sliding stock market will limit the firms' abilities to sell the companies in their portfolios back to the public.
Some of Carlyle's rivals have not fared well in the stock market since their IPOs. Blackstone has dropped 60 percent since its 2007 debut. Apollo has lost 36 percent. And Fortress Investment Group LLC has fallen 83 percent. KKR is up 4 percent since its U.S. listing.
There have been some notable successes for private equity firms, including Carlyle. It's Dunkin' Brands Group Inc., the parent company of the Dunkin Donuts and Baskin-Robbins chains, went public in July, and investors clamored for the stock. Carlyle's portfolio includes more than 200 companies.
The stock market decline and the lull in the IPO market may mean Carlyle won't be able to raise as much money in an IPO as it might have if it went public earlier this year.
But for private equity firms, going public isn't necessarily about raising as much money as possible to fund their business, said Colin Blaydon, professor of private equity and entrepreneurship at Dartmouth's Tuck School of Business. Instead, the IPO allows investment firms more flexibility. They can make acquisitions with stock instead of cash.
Proceeds from Carlyle's offering will be used to repay outstanding indebtedness and for general corporate purposes.
Going public also lets the private equity firm's founding partners and major shareholders cash out some of their stakes.
Carlyle was founded in 1987 in Washington by Daniel D'Aniello, David Rubenstein, and William Conway Jr. D'Aniello will serve as the chairman of the publicly traded company and Rubenstein and Conway will be co-CEOs.
The company has about $153 billion in assets under management. Carlyle said revenue for 2010 was $2.80 billion, up from $1.32 billion in 2009.
Net income attributed to the company more than doubled to $1.53 billion from $694.1 million in 2009. A measure preferred by private equity firms, called economic net income, which strips out charges related to stock-based compensation, income taxes and other charges, also more than doubled, to $1.01 billion.
TIMMONSVILLE, S.C. - Presidential hopeful Barack Obama faces two major obstacles in South Carolina, the first Democratic testing ground for black support: the popularity of the Clinton name and doubts among blacks that white America is ready for a minority president.
The candidacy of the 45-year-old Obama elicits genuine excitement in a state where blacks comprise about half of the primary electorate. Yet coupled with that emotion is a strong degree of skepticism about the freshman senator's experience and whether he can win.
Obama also is up against the formidable Hillary Rodham Clinton, the Democratic front-runner who enjoys strong support in the black community and is married to former President Clinton, who is wildly popular in the community.
The Associated Press interviewed Democratic voters across the state, including about a dozen blacks, and found evidence of excitement and doubts.
Ashley Torrence, a 27-year-old college instructor in Greenville, S.C., is torn between voting for Obama and Clinton, and considers her vote crucial because either candidate could smash barriers. Torrence has talked to Clinton and was disappointed when all she got from her encounter with Obama was a handshake.
"I wanted to ask him how he had planned to combat the feeling that unfortunately a lot of people have about just not being ready for a black male to be president and particularly a lot of people with old South mentality," she said. "How is he going to deal with that? Because you can't campaign as though it doesn't exist."
It was an issue Obama confronted on his first trip to South Carolina in February, telling those who doubted he could win because he's black: "Don't tell me I can't do something. ... I don't believe in this can't do, won't do, won't even try, style of leadership. Yes we can. Don't believe in that."
Obama's plea was directed not only at voters, who will participate in the Jan. 29 primary, but Democratic state Sen. Robert Ford of Charleston. Ford garnered headlines when he said he was backing Clinton in part because he was skeptical that Obama could win the presidency and feared that his nomination could hurt other Democratic candidates.
"Every Democrat running on that ticket next year would lose - because he's black and he's top of the ticket. We'd lose the House and the Senate and the governors and everything," said Ford, who is black.
He drew widespread criticism for his comment and later apologized.
But the AP interviews suggested the view is prevalent among blacks, along with concerns across racial lines about whether Obama has enough experience to be president. Greenville County Democratic Party chairman Andy Arnold hears it frequently among blacks, who are supporting Clinton in greater numbers in recent polls.
"A lot of the African-Americans are with Hillary because I think they don't believe white America is ready for a black president," said Arnold, who is white and uncommitted in the race. "They want to win and so in a way, I think it is a barrier to him. And it may be more so in the South where the remnants of the old South are still in the older folks mind. They just can't believe in their right mind that white folks will elect a black man president, so let's not put ourselves through that agony."
Clinton is a favorite of black women in current polls, due largely from goodwill for her husband and her lifelong focus on issues affecting families and children. Much of her lead comes from women and blacks, and it's strongest among black women. According to Associated Press-Ipsos polls taken this summer, 59 percent of black women said they support Clinton and 27 percent Obama.
The South Carolina primary, coming after heavily white Iowa and New Hampshire vote, and Nevada casts its ballots, is crucial for Obama. In 1984 and 1988, Jesse Jackson won the state's primary.
The Obama campaign argues that doubts about whether a black man can be elected is not widespread. They cite a Winthrop Poll of South Carolinians in May in which 79 percent of respondents said they think the country will be ready for a black president in the next 12 years. However, the poll did not ask whether they would be ready in 2008.
The Obama campaign launched radio ads this week with a direct appeal to black voters and has embarked on a labor-intensive effort to reach voters in their homes. Obama has about 40 staffers in the state, more than double any of his rivals. His top-ranked fundraising - he has raised nearly $60 million - allows him to invest in advertising and staff through the primary race.
Part of the campaign's strategy is to host small meetings in voter's homes, in the style of a Mary Kay or Tupperware party but where the pitch is for a candidate instead of cosmetics or plastic containers.
Iris Gladden was one of nine to attend a party this week in Timmonsville, a rural community not far from Interstate 95. Obama field organizer Ryan Cooper, 22, talked about the candidate's appeal across racial lines and showed the group a video about the candidate that focused on Obama's modest background and accomplishments, then asked to hear their concerns.
The group discussed their worries about health care costs, the quality of education, the difficulty to earn a living wage, opportunities for their young people and ending the war in Iraq. At Cooper's encouragement, they also talked about what they liked about Obama after seeing the video. Gladden mentioned that as a single mother who raised four children, she was proud to see someone from a single-parent home achieve great things.
But as she left the gathering, she was still undecided about whether to vote for Obama or Clinton. Gladden said one of her sons was in the Naval Reserves and came close to being sent to Iraq. She said her top priority is a president who will end the war, and although both Obama and Clinton are running solid campaigns, she's not convinced they could win.
"I see problems with both," she said. "Obama because he's black and Hillary because she's a woman. Are we ready? Is America ready to go there?"
Rep. Jim Clyburn, the only black member of South Carolina's congressional delegation, said he thinks concerns about the war are more prominent in voters' minds than issues of candidates' race or gender. But he said if anything, Clinton's gender might help her in that regard.
"When it comes to issues of war and peace, women hold out hope for peace more than men do," said Clyburn, who hasn't endorsed a candidate but hasn't ruled out doing so later. "If the election were held today, Clinton would carry the state. It's not going to be held today."
Another candidate, 2004 Democratic vice presidential nominee John Edwards, has been arguing that he will be more electable in the South. He denies that's because of Obama's race or Clinton's gender but says it's because they have never run in the South while he won the primary in 2004 and was elected senator in North Carolina.
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For the first time, astronomers said Thursday, they have whatthey call "irrefutable evidence" that planets exist outside the solarsystem.
The historic discovery of at least two planets, made by a teamled by Alexander Wolszczan of Penn State University, ends a string ofdisappointments in the quest to find the first extrasolar planet and,astronomers say, lends credence to the idea that planets are commonin the cosmos.
The new planets, more than 7,000 trillion miles away and eachabout three times the mass of Earth, are not likely to harbor life,scientists note. The star they orbit is distinctly unsunlike: awhirling dead sphere called a pulsar that, instead of light, emits abarrage of deadly invisible radiation.
But it is the dead star's exotic nature that enabled astronomersto detect its planetary family in the first place - by studyingirregularities in the precise pattern of radio waves the star emits.It is because of that same nature that the prized discovery fell notto optical astronomers, who traditionally have led the century-longsearch for planets around stars other than the sun, but to radioastronomers.
Wolszczan said in an interview his team was able to confirm theexistence of the planets by measurements so fine they detectedchanges in the motions of the pulsar equal to "the crawl of a snail,at 1,200 light-years."
In 1990, Wolszczan first identified the star - in theconstellation Virgo, about 1,200 light-years from Earth in the planeof the Milky Way Galaxy. (One light-year is the distance traveled bylight in a year: 5.9 trillion miles.)
In 1991, aided by Dale Frail of the National Radio AstronomyObservatory in Green Bank, W. Va., he detected the presence of thesuspected planets and, in 1992, announced the finding in the journalNature.
But astronomers had heard similar announcements before. Inevery case, the putative planets had either proved impossible toconfirm, turned out to be something else or been shown to be theproduct of an error.
Since then, the Wolszczan team has bolstered its originalfindings with three more years of even more finely nuanced datadesigned to eliminate any explanation other than planets.
Wolszczan reports on how he confirmed the claim in today's editionof the journal Science. "I'm really glad I don't have to take itback," he said.
The pulsar, known as PSR1257+12, is believed to be the collapsedhusk of a formerly huge star that most likely died in a cataclysmicexplosion called a supernova. Once more massive than the sun, it isnow a dense, lightless object only about 12 miles in diameter.
As it spins on its axis 161 times a second, the pulsar sends outradio waves that sweep Earth in regular pulses, like the beam from alighthouse beacon. This action provides the most precise "clock" inthe cosmos.
But Wolszczan and Frail, in 1991, noticed that the pulses wereslightly irregular, in a regular and predictable way. The teamdetected variations of plus or minus 0.003 second in the arrivaltimes of the radio pulses.
This variation was caused by a wobble, in which the pulsar moved560 miles toward and away from Earth as it spun on its axis.
ABSTRACT
A number of channel-forming peptides derived from the second transmembrane (TM) segment (M2) of the glycine receptor [alpha]^sub 1^ subunit (M2GlyR), including the 22-residue sequence NK^sub 4^-M2GlyR p22 wild type (WT) (KKKKPARVGL-GITTVLTMTTQS), induce anion permeation across epithelial cell monolayers. In vitro assays suggest that this peptide or related sequences might function as a candidate for ion channel replacement therapy in treating channelopathies such as cystic fibrosis (CF). The wild-type sequence forms soluble associations in water that diminish its efficacy. Introduction of a single substitution S22W at the C-terminus, NK^sub 4^-M2GlyR p22 S22W, eliminates the formation of higher molecular weight associations in solution. The S22W peptide also reduces the concentration of peptide required for half-maximal anion transport induced across Madin-Darby canine kidney cells (MDCK) monolayers. A combination of 2D double quantum filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating frame nuclear Overhauser effect spectroscopy (ROESY) data were recorded for both the associating WT and nonassociating S22W peptides and used to compare the primary structures and to assign the secondary structures. High-resolution structural studies were recorded in the solvent system (40% 2,2,2-Trifluoroethanol (TFE)/water), which gave the largest structural difference between the two peptides. Nuclear Overhauser effect crosspeak intensity provided interproton distances and the torsion angles were measured by spin-spin coupling constants. These constraints were put into the DYANA modeling program to generate a group of structures. These studies yielded energy-minimized structures for this mixed solvent environment. Structure for both peptides is confined to the 15-residue transmembrane segments. The energy-minimized structure for the WT peptide shows a partially helical extended structure. The S22W peptide adopts a bent conformation forming a hydrophobic pocket by hydrophobic interactions.
INTRODUCTION
Peptide-based channel replacement therapy has been proposed as a novel treatment modality for correcting genetically based diseases caused by defective channel proteins. Defective ion channels are becoming increasingly implicated in a variety of human diseases, such as episodic ataxia, diabetes, epilepsy, cystic fibrosis (CF), and Alzheimer's dementia. Numerous channel-forming peptides have been designed, synthesized, and tested to generate a peptide sequence that is deliverable from aqueous solution, binds quantitatively with membranes, and forms a de novo anion conductive pathway.
Peptides derived from the second transmembrane (TM) segment of the brain glycine receptor (M2GlyR, PARVGLGITTVLTMTTQSGSRA) have been studied extensively for their ability to form anion channels in lipid bilayers, single cells, and epithelial monolayers. The wild-type (WT) sequence, shown above, displays poor aqueous solubility and the propensity to aggregate in solution leading to reduced bioavailability and inconsistent channel forming potentials (Reddy et al., 1993; Wallace et al., 1997). It was found that by adding at least three lysines to either termini of the peptide (CK^sub x^-M2GlyR and NK^sub x^-M2GlyR) solubility was increased (Tomich et al., 1998). Nuclear magnetic resonance (NMR) data showed some soluble peptide-peptide associations remained for those sequences, indicating that there was a transition from highly associated forms to less associated forms including monomer (Tomich et al., 1998). Electrophysiological analyses were performed on a series of these synthesized peptides by studying the increase in I^sub SC^ (short-circuit current, an indicator of anion secretion). The results indicated that both the CK^sub 4^- and NK^sub 4^-modified M2GlyR peptides did, in fact, induce a de novo anion-select current in lipid bilayers and whole cell membrane patches, as well as in epithelial monolayers (Mitchell et al., 2000; Broughman et al., 2001).
Subsequent truncation studies on both the CK^sub 4^- and NK^sub 4^modified M2GlyR peptides were performed to find the minimal, fully active, peptide length (Broughman et al., 2002a). The truncations were performed on the nonlysine adducted ends of the CK^sub 4^- and NK^sub 4^-M2GlyR peptides. The N-terminal modified peptides showed anion transport activity (I^sub SC^) even after as many as 11 C-terminal residues were deleted. The 22-residue NK^sub 4^-M2GlyR p-22 ([Delta]S^sub 23^[arrow right]^A^sub 27^) displayed channel properties that were indistinguishable from the full-length NK^sub 4^-M2GlyR p27. Removal of two and five residues from the N-terminus of CK^sub 4^-M2GlyR resulted in reduced channel forming activity. Complete loss of channel activity was seen with removal of more than five residues in CK^sub 4^-M2GlyR.
Peptide-peptide solution associations were evaluated for each of the truncated peptides using chemical cross-linking with Bis[sulfosuccinimidyl]suberate (BS^sup 3^). The results of these experiments showed that peptide-peptide solution associations of the NK^sub 4^-M2GlyR peptide decreased with each truncation. The CK^sub 4^-M2GlyR peptide, on the other hand, showed consistent amounts of monomer, dimer, and trimer throughout all truncations (Broughman et al., 2002a). NMR structural studies on NK^sub 4^-M2GlyR and CK^sub 4^-M2GlyR revealed substantial differences between the interactions of the lysine adducted segments with the core M2GlyR sequence. The lysines of NK^sub 4^-M2GlyR p-27 do not interact with each other and are unstructured. In CK^sub 4^-M2GlyR, however, the lysines fold back on the sequence to form a capping structure that is stabilized by the formation of two hydrogen bonds. The capping structure effectively reduces the overall length of the peptide. Therefore, CK^sub 4^-M2GlyR truncations with deletions greater than five residues were modeled and appeared to be too short to span the bilayer (Broughman et al., 2002b). A major conclusion from this study was that the peptide-peptide association sites that lead to 1), soluble solution associations and 2), membrane bound oligomeric channel assemblies are different and separable.
Restricting our designs to NK^sub 4^-M2GlyR p22, we began substituting residues to replace the deleted arginine and add an aromatic residue to help anchor the C-terminus of the TM segment. In this article, the introduction of a tryptophan residue at the C-terminus of M2GlyR peptide, NK^sub 4^-M2GlyR p22 S22W, is shown to have a dramatic effect on both solution peptide-peptide association and the kinetics of channel assembly.
To better understand the mechanism by which the introduction of a single tryptophan at the C-terminus reduces solution associations, we determined and compared the solution structure of the associating NK^sub 4^-M2GlyR p22 WT (KKKKPARVGLGIT-TVLTMTTQS) and the monomeric NK^sub 4^-M2GlyR p22 S22W (KKKKPARVGLGITLTMTVTTQW) peptide in 40% 2,2,2-Trifluoroethanol (TFE) in water. This environment stabilizes intermediates in the folding pathway by inducing partial structure (Roccatano et al., 2002). The TFE molecules aggregate around the peptide, which helps to displace water. This displacement prevents hydrogen bonding of the CO and NH groups of the peptide backbone to water molecules providing a low dielectric environment that encourages the formation of intramolecular hydrogen bonds.
MATERIALS AND METHODS
Peptide synthesis and purification
For this study the following sequences were chemically synthesized: NK^sub 4^-M2GlyR-p22 WT (NH^sub 2^-KKKKPARVGLGITTVLTMTTQS-CONH; molecular weight (MW) = 2356.9) and NK^sub 4^-M2GlyR-p22 S22W (NH^sub 2^-KKKKPARVGLGITTVLTMTTQW-CONH; MW = 2456.0). The peptides were prepared by solid-phase peptide synthesis by an Applied Biosystems model 431A peptide synthesizer (Foster City, CA) using 9-fluorenylmethoxycarbonyl (Fmoc) chemistries. CLEAR amide resin (0.3 mmol/g) was purchased from Peptides International (Louisville, KY) and N^sup [alpha]^-9-fluorenylmethoxycarbonyl amino acids were purchased from Anaspec (San Jose, CA), Bachern (Torrance, CA), and Peptides International. All peptides were purified using a Phenomenex (Torrance, CA) reversed-phase C-18 column on a System Gold high performance liquid chromatography (HPLC) system (Beckman Instruments, Fullerton, CA). Peptides were eluted from the column using a linear gradient of 3.0% min^sup -1^ of 1-90% acetonitrile containing 0.1% trifluoroacetic acid (TFA) at 2 ml/min. High performance liquid chromatography-purified peptides were characterized by matrix assisted-laser desorption time-of-flight mass spectroscopy (MALDI-TOF). After characterization, peptides were lyophilized and stored as dry powders until use.
Cell culture
Madin-Darby canine kidney cells (MDCK) cells were a generous gift of Dr. Lawrence Sullivan (KUMC, Kansas City, KS) and were cultured as described previously (Wallace et al., 1997). 1-Ethyl-2-Benzimidazolinone (1-EBIO) (Acros, Morris Plains, NJ) was prepared as a 1-M stock solution in dimethyl sulfoxide (DMSO).
Transepithelial ion transport measurements
Transepithelial ion transport was evaluated in a modified Ussing chamber (model DCV9, Navicyte, San Diego, CA). For electrical measurements, MDCK cells were bathed in a modified Ringer solution (120 mM NaCl, 25 mM NaHCO^sub 3^, 3.3 mM KH^sub 2^PO^sub 4^, 0.8 mM K^sub 2^HPO^sub 4^, 1.2 mM MgCl^sub 2^, and 1.2 mM CaCl^sub 2^ prepared fresh daily) (Broughman et al., 2001) maintained at 37�C and continuously bubbled with 5% CO2/ 95% O2 to maintain pH, provide aeration, and mix fluid in the chambers. The transepithelial membrane potential (V^sub TE^) was clamped to zero and the I^sub SC^ was measured continuously with a voltage clamp apparatus (model 558C, University of Iowa, Dept. of Bioengineering, Iowa City, IA). Data acquisition was performed at 1 Hz with a Macintosh computer (Apple Computer, Cuppertino, CA) using Aqknowledge software (ver. 3.2.6, BIOPAC Systems, Santa Barbara, CA) with an MP100A-CE interface. I^sub SC^ data are presented at steady-state flux levels.
Ion transport data analysis
The data points represent the mean I^sub SC^ stimulated by the peptides at concentrations of 10, 30, 100, 200, 300, and 500 �M, and the error bars indicate the SE of the mean. The Differences between control and treatment data were analyzed using analysis of variance (ANOVA) and Student's t-test. The probability of making a type I error less than 0.05 was considered statistically significant. The lines are a best fit of a modified Hill equation to the data, I^sub SC^ = I^sub max^ � (x^sup n^/(k^sub 1/2^^sup n^ + x^sub n^); where k^sub 1/2^ is the concentration of peptide that provides a half-maximal I^sub SC^ and n represents the Hill coefficient.
Circular dichroism
Measurements of peptide secondary structure in water, TFE, and sodium dodecylsulfate (SDS) were performed by circular dichroism (CD). CD spectra measurements were recorded on a Jasco-720 spectropolarimeter (Jasco, Tokyo, Japan) using a 1.0-mm path length rectangular quartz cuvette (Uvonic Instruments, Plainview, NY) for the deionized (DI) H2O, TFE, and SDS studies. All spectra were recorded from 260 to 190 nm using a 1.0-nm spectral bandwidth, 0.2-nm step resolution, 20-nm/min scan speed, and 2-s response time. A baseline measurement was performed every day to check instrument stability. The peptide spectra shown are an average of eight scans with the background subtracted. Peptide stock solutions were prepared fresh daily to a concentration of 1 mM in water. Peptide stock concentrations were determined using either tryptophan fluorescence at 278 nm or the Bicinchoninic Acid protein assay (Pierce, Rockford, IL). Bovine serum albumin (BSA) served as the protein standard and all peptide samples were corrected for dye binding differences between BSA and the peptides as previously determined (Broughman et al., 2002a). Peptide concentrations in all CD studies were matched at 50 �M. DI-distilled H2O peptide samples were prepared by diluting 50 �l peptide stock solution with 950 �l DI H2O. TFE peptide samples were prepared by vorlexing 50 �l peptide stock solution with appropriate volumes of 2,2,2-trifluoroethanol (Acros) and DI-distilled H2O to obtain the desired % TFE. SDS peptide samples were prepared by adding 50 �l peptide stock solution with appropriate volumes to yield 10 mM SDS, 10 mM NaCl, and 10 mM phosphate buffer at pH 7.0. The detergent containing samples were prepared using a 2-min sonication step.
BS^sup 3^ chemical cross-linking reactions
The Bis[sulfosuccinimidyl]suberate cross-linking reactions were carried out as described previously (Broughman et al., 2002a). Cross-linking reactions were carried out at either 150 �M or 3 mM peptide using a 20-fold molar excess of the BS^sup 3^ cross-linker. The peptide/cross-linker reaction was terminated after 30 min by the addition of 1 N HCl. Two microliters of the terminated reaction samples of 3 mM were added to 118 �L distilled water/ lricine SDS sample buffer (50:50, v/v) to prepare for SDS-polyacrylamide gel electrophoresis (PAGE). A noncross-linked sample was prepared as a control. All samples were heated at 100�C in loading buffer for 5 min, then 5 �L of each SDS boiled sample was loaded on to the precast 10-20% gradient tricine gel (Novex division of Invitrogen, Carlsbad, CA). The reference well contained 1 �L of either a Mark 12 unstained standard (Novex division of Invitrogen) or a MultiMark prestained standard (Novex division of Invitrogen). The electrophoresis was carried out at a constant 110 V for 90 min. The gel was then visualized using silver stain (Invitrogen).
NMR spectroscopy
The NMR spectroscopy was performed as described previously (Kimarsky et al., 2000). Briefly, all high-resolution one- and two-dimensional ^sup 1^H-NMR experimental data were acquired with an 11.75 T Varian UNITYplus spectrometer (Varian, Palo Alto, CA), operating at 499.96 MHz for ^sup 1^H, with a 5-mm triple-resonance inverse detection probe. Using peptide concentrations of 3 mM, spectra were recorded at 10�C in 60% H2O/40% TFE-d^sub 3^, and were processed and analyzed using Varian software VNMR 6.1b on a Silicon Graphics Indigo2 XZ workstation (Mountain View, CA). The invariant nature of NMR chemical shifts and line widths upon 10-fold dilution indicated that the peptide was monomeric in solution at the 3-mM concentration used for 2D-NMR analysis. Standard NMR pulse sequences were used for 2D double quantum filtered correlation spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY), and rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiments. Water peak suppression was obtained by low-power irradiation of the H2O resonance at 4.91-ppm peak during relaxation delay (1.2 s). A total of 512 increments of 8-K data points were collected for the DQF-COSY spectra, and a total of 256 increments of 4-K data points were recorded for all other experiments. All data sets were obtained in hypercomplex phase-sensitive mode. Proton resonance assignments were confirmed by comparison of cross peaks in a NOESY spectrum with those in a TOCSY spectrum (Wiithrich, 1986) acquired under similar experimental conditions. NOESY experiments were performed with 100-, 200-, and 400-ms mixing times. ROESY spectra were acquired with a mixing time of 300 ms. TOCSY spectra were recorded using the MLEV-17 sequence (Bax and Davis, 1985) for isotropic mixing for 90 ms at a B^sub 1^ field strength of 8 KHz. The TFE peak (3.88 ppm at 10�C) was considered as reference for chemical shift assignment. The temperature dependence of amide proton chemical shifts was measured by collecting data from 5 to 35�C in steps of 5�C using a variable temperature probe. Coupling constants (^sup 3^J^sub H[alpha]NH^) were measured directly from 1D- and 2D-DQF-COSY (Piantini et al., 1982) NMR spectra. All experimental data were zero-filled to 4-K data points in t^sub 1^ dimension and, when necessary, spectral resolution was enhanced by Lorenzian-Gaussian apodization.
Structure calculation
The NOE spectra of mixing time 200 ms was evaluated to provide distance constraints for the structure calculations. The volume of every unambiguously identified cross peak was calculated within the VNMR 6.1b program. These volumes were converted into interproton distances by setting the volume of the peak from the two [beta]-protons of Proline to 2.20 [Angstrom] and using our program called calDist which calculates the NOE volume, which is equal to 1/R^sub 6^. The program HABAS (Peter Guntert Scientific Software, Institute of Molecular Biology and Biophysics, Zurich, Switzerland) was used to convert coupling constant values to dihedral angle constraints. The torsion angle dynamics calculations were performed using the distance and dihedral angle constraints within the program DYANA (DYnamics Algorithm for NMR Applications, Peter Guntert Scientific Software) on a Silicon Graphics O2 workstation. The resulting 100 DYANA conformations were clustered with the software Sybyl version 6.7 (Tripos, St. Louis, MO). The groups were based on the pairwise root mean-square deviation (rmsd) values that were less than 1 [Angstrom] deviation in the C-terminal region (from Gly-9 to Gln-21). The cluster containing the majority of the structures and lowest target function values was subjected to 100 steps of energy minimization with the software Insight II (Accelrys, San Diego, CA) utilizing the constant valence force field. The resulting structures were evaluated with the program Procheck v.3.5.4. (European Bioinformatics Institute, Cambridge, UK). The lowest-energy structure was rendered with the program MIDAS (Midas Plus Computer Graphics Laboratory, University of California, San Francisco, CA).
RESULTS
Introduction of tryptophan at C-terminus
Fig. 1 shows the concentration dependence on I^sub SC^ for the full length and the truncated M2 peptides when applied to the apical membrane of MDCK cells in the presence of 1-ethyl-2-benzimidazolinone. The solid lines represent the best fit of a modified Hill equation to each data set. In paired monolayers both NK^sub 4^-M2GlyR p27 and NK^sub 4^-MZGlyR p22 peptides (previously characterized; Broughman et al., 2001, 2002a) display similar concentration dependency curves and induce similar anion currents at each concentration tested. The summarized ion transport data are presented in Table 1. NK^sub 4^-M2GlyR p27 provides benchmark values for I^sub max^ at 26.2 � �A/cm^sup 2^, K^sub 1/2^ of 270 � 70 �M, and a Hill coefficient of 1.9, with the p22 WT displaying 23 � 1.8 �A/cm^sup 2^, K^sub 1/2^ of 210 � 70 �M, and a Hill coefficient of 2.2 � 1.0. Introduction of the tryptophan in NK^sub 4^-M2GlyR p22 S22W yields a curve with a reduced I^sub max^ (13.0 � 1.0 �A/cm^sup 2^) but more importantly a significantly reduced k^sub 1/2^ (44 � 6) with a significantly greater Hill coefficient of 5.4 � 2.9. The sum effect of changes in the three kinetic parameters (I^sub max^, k^sub 1/2^, and Hill coefficient) is a significant reduction in the peptide concentration required to yield maximal anion secretion.
Fig. 2 A represents a silver-stained gel of cross-linking reactions for the sequences presented in Table 1. Lane 1 contained a MultiMark prestained standard to provide relative molecular weights. Lanes 2 and 3 show the wild-type sequence, NK^sub 4^-M2GlyR p27, in the absence and presence of a 20-fold excess of BS3, respectively. In the presence of the cross-linker, numerous higher molecular weight homooligomers are observed. Without the addition of cross-linker and after boiling the sample in SDS containing loading buffer, only monomer is observed. Lanes 4 and 5 contained NK^sub 4^-M2GlyR p22 WT in the absence and presence of excess cross-linking reagent. The cross-linked lane revealed the presence of higher molecular weight oligomers formed in solution, although somewhat reduced over that seen for the full-length sequence (lane 3). Lanes 6 and 7 contain NK^sub 4^-M2GlyR p22 S22W, again in the absence and presence of excess cross-linking reagent. The substituted sequence population was observed to be predominantly monomeric in solution even in the presence of the cross-linking reagent. Results indicate that NK^sub 4^-M2GlyR p22 S22W (150 �M) forms few higher molecular weight associations, especially when compared to the WT M2GlyR truncated form.
A second cross-linking study employed a higher concentration of NK^sub 4^-M2GlyR p22 S22W (3 mM) (Fig. 2 B). The cross-linking patterns suggested that, even at higher concentration, this peptide remains monomeric in both water and in TFE (40%) solution. The NK^sub 4^-M2GlyR p22 WT peptide was similarly unaffected at 3 mM and in the presence of TFE. Note that a different standard is used in this gel. Identical peptide concentrations were loaded into each lane. These data indicate that NMR can be performed on these peptides without the complication of further peptide-peptide associations affecting the spectra.
Circular dichroism analysis was performed on the two p22 peptides to make preliminary structural comparisons. Fig. 3 shows the CD spectra of M2GlyR p22 wild type and the NK^sub 4^-M2GlyR p22 S22W measured in water, 40% TFE/ water, and 10 mM SDS at matched peptide concentrations of 50 �M. The CD spectra of NK^sub 4^-M2GlyR p22 WT (Fig. 3 A) indicated that the peptide in water was somewhat structured. The spectrum has a minimum at ~195 nm, which is a hallmark for random coil. However it is missing the random coil's characteristic positive maximum at 215 nm. The spectra measured for the WT peptide in 40% TFE and 10 mM SDS were similar to one another and showed some helical content showing the characteristic [alpha]-helix double minima at 208 and 222 nm and a maximum at ~192 nm. The NK^sub 4^-M2GlyR p22 S22W peptide in water, however, showed considerable [beta]- or extended structure (Fig. 3 B). The same peptide dissolved in 40% TFE showed a transition to a more helical secondary structure. Although NK^sub 4^-MZGlyR p22 S22W (in 40% TFE) does not include the second minimum at 222 nm the spectrum had a similar shape to that of the p22 WT sequence indicating retention of some helical structure. The spectral data from 185 to 260 nm of the S22W peptide in TFE indicate that the sequence contained 23.4% helix and 30.3% [beta]-structure as determined by a CD analysis program (CD Spectra Deconvolution v.2.0c, Gerald Bohm Institute fur Biotechnologie, Martin-Luther-Universitat, Halle-Wittenberg, Germany, http://bioinformatik.biochemtech.uni-halle.de). In SDS micelles, the peptide spectrum matches that seen for the p22 WT sequence in SDS. The S22W peptide in SDS had significantly less [beta]-structure (10.0%) than the TFE/water sample (30.0%). These data also indicated that the helical content was slightly greater in the SDS sample (30.8% vs. 23.4% in the TFE solution by the same analysis). CD experiments were also carried out with higher peptide. concentrations (3 mM) to ensure that the data was relative to our NMR samples. The results were indistinguishable from the spectra recorded at 50 �M.
NMR spectra
As observed by CD studies, TFE stabilized the secondary structure of the peptides, so a 3-mM solution of the peptide was made using 60% water and 40% TFE-d^sub 3^. The pH of the samples was ~2.8, which minimizes the rate of amide proton exchange.
Proton resonance assignments were made using 2D ^sup 1^H-^sup 1^H DQF-COSY and TOCSY for intraresidue spin systems and NOESY and ROESY spectra for interresidue connectivities by standard techniques (Wuthrich, 1986). The chemical shift assignments are summarized in Table 2. TOCSY spectra were used to identify NH-C^sub [alpha]^H, NH-C^sub [beta]^H, NH-C^sub [gamma]^H, and NH-C^sub [delta]^H intraresidue spin systems to determine residue assignments (Fig. 4). NOESY spectra provided d^sub [alpha]N^(i, i + 1) protons connectivity data for sequential assignments and multiple residue assignments such as lysines 1-4, leucines 10 and 16, valine 8 and 15, and threonine 13, 14, 17, 19, and 20.
The chemical shift dispersion for the NH and C^sub [alpha]^H proton resonances supported the presence of a folded structure as predicted from the CD spectra recorded in TFE. An estimate of both peptides' secondary structures was made based on the [alpha]-proton chemical shift values as compared with the random coil values in TFE, termed the chemical shift index (CSI). It has been shown that these values can provide a valuable approximation of secondary structure (Wishart et al., 1992; Merutka et al., 1995). The deviation, in ppm, of each residue from the expected random coil values observed in TFE are shown in Fig. 5. A negative value (<-0.10 ppm, upfield shift) suggested a turn in the structure and several consecutive residues with negative values can mean a helical conformation exists, whereas a comparatively positive value (downfield shift) suggested a [beta]-sheet type conformation. As visible in the figure, the deviation of [alpha]-proton chemical shift values of residues 12-16 and 18-19 of both sequences were, in fact, heavily shifted upfield. Residues 5-11 and 20-21 were shifted slightly upfield. These shifts suggest that a large percentage of the peptides could be made up of helical regions or a series of turns. The main difference between the two sequences is that the chemical shift of the Thr-17 C^sub [alpha]^proton resides in the heavily negative region in the WT
sequence but is only slightly negative in the S22W sequence. This chemical shift difference suggests a continuous helix from Ile -12 to Thr-19 in the WT sequence but a split in the helical structure in the S22W peptide, which could result in separated helical portions.
The amide regions (NH-NH) of the NOESY spectra are overlayed in Fig. 6 to display the differences. Other connectivities found including d^sub NN^(i, i + 1), d^sub [alpha]N^(i, i + 1), d^sub NN^(i, i + 2), and d^sub [alpha]N^^(i, i + 3) by the NOE spectra provided some structural information about the peptides. The observed NOEs were summarized graphically in Fig. 7. The observation of sequential d^sub NN^(i, i + 1) proton NOEs for the Gly(9)-Thr-(17) strongly indicated a continuous stretch of backbone [phi]- and [psi]-values in the helical regions of conformational space (Wuthrich,1986). In addition, the observation of d^sub [beta]N^(i, i + 1) proton connectivities and particularly the presence of d^sub agr;N^(i, i + 3) protons NOE between Gly-9 and Ile-12, Gly-11 and Thr-14, Ile-12 and Val-15, Thr-14 and Thr-17, and Met-18, Leu-16 and Thr-19, and Thr-17 and Thr-20 confirmed that this region of the peptide was in ahelical conformation. The S22W sequence displayed many of these same interactions but lacked the Ile-12-Val-15 and Thr-14-Thr-17 connectivities. This would suggest a difference in the backbone conformation between the two peptides in this region.
Structural calculation
Using the NOE cross peaks, 136 for WT and 141 for S22W, distance-constraints were calculated. Due to a large number of overlapping peaks in C^sub [alpha]^H regions of the DQF-COSY spectra, only a limited number of angle constraints could be calculated for these 22 amino-acid peptides. The distance constraints and the ^sup 3^J^sub [alpha]HNH^ coupling constants were submitted to HABAS for a local conformational analysis, which resulted in dihedral angle constraints on five residues and four residues for WT and S22W peptides, respectively. With the distance constraints and the dihedral angle constraints, 100 conformations were obtained by torsion angle dynamics calculations using SYBYL/DYANA. The resulting structures had no significant violations of the input constraints (<0.66 [Angstrom]), and they had a target function value of 1.15 � 0.78 [Angstrom]^sup 2^ for the WT sequence and 0.45 � 0.42 [Angstrom]^sup 2^ for the S22W peptide. All one hundred structures were clustered into groups based on rmsd values by SYBYL. The WT peptide structures were clustered into 20 groups. A group containing 11 conformations was examined based on its target function values. The structure of these conformers was limited to the C-terminal region. This structure included a continuous stretch of helix from Ile-12 to Thr-19. The rmsd values for the backbones of the 11 structures was 1.95 � 0.60. The S22W peptide structures were clustered into 11 groups and only one major group was observed, containing 30 conformations.
These structures were energy-minimized in Insight II with all the constraints, and the resulting structures' [psi]- and [phi]angles were evaluated against the Ramachandran Plot values by the program PROCHECK (Table 3). The plot of the WT peptide cluster showed that >99.9% of the [psi]- and [phi]-angles of the peptide backbone reside within the allowed regions. The analysis of S22W showed that 91.7% of residues were in the favored region, 8.3% were in other allowed regions, and no residues were in disallowed region.
The structures of the S22W peptide, like the WT sequence, could be broken up into two conformationally distinct parts. The first eight residues of the NT-terminal, including the four capping lysines were highly variable in conformation. Even with angle and NOE constraints pertaining to this area, it was not limited to a strict conformation by structure simulation. The results indicate this portion does not have a strict structure and can be free in solution. Throughout the group of structures, Gly-9-Gly-11 were found to create a turn that led into the [alpha]-helix-like C-terminal region. The rmsd values for the backbones in the region from Gly-9 to Thr-20 between these 30 conformations and the corresponding mean coordinates of this group is 0.40 � 0.20 [Angstrom] (Table 3).
The structures with the lowest total energies are shown in Fig. 8 created in Qmol (J. D. Ganes, Shalloway Labs, Cornell University, http://www.mbg.cornell.edu/shalloway/shalloway.html). Fig. 9 shows a representative modeled structure of the two sequences presented as an aid to this discussion.
The WT peptide structure is linear with a continuous helix in the C-terminus. The backbone of this region agrees with the NOE data. The helix structure allows the [alpha]- and amide protons to come in close contact with other protons on the same side of the helix. Although the S22W peptide, like the WT sequence, has a flexible, extended region from Lys-1 to Val-8, a turn stabilized by the presence of hydrogen bonding between the amide group of Thr-14 and Val-15 to the carbonyls of Gly-11 and Ile-12, respectively, redirects the C-terminal portion of the chain forming a turn that looks like a pocket. The three segments, from Val-8 to Gly-10, from Gly-11 to Leu-16, and from Thr-17 to Trp-22, formed the three sides of the pocket. Gly-9 and Thr-19 came close at the opening of the pocket with NH of Gly-9 and the [gamma]-oxygen of Thr-19 forming a hydrogen bond. PROCHECK reported that the C-terminal region contained two [alpha]-helix domains. Residues Ile-12, Thr-13, Thr-14, and Val-15 formed a onecycle [alpha]-helix in that Thr-13 and Thr-14 extruded outside of the pocket. The seven residues at the C-terminal end formed a two-cycle [alpha]-helix. It was observed that all the residues facing out of the pocket are hydrophilic residues, and most of the residues facing inward are hydrophobic.
DISCUSSION
The results presented in this study suggest important properties about the solution structure of transmembrane peptides derived from the M2GlyR sequence in aqueous environments. The sequences are derived from an anion selective glycine receptor channel found in the spinal cord. These sequences have been proposed as possible therapeutic agents for treating disease conditions that are the result of an absent or defective channel (Wallace et al., 1997). When applied to membranes the peptides insert and then assemble into helical bundles to give ion transport activities that can be measured in synthetic bilayers or cells. Although this peptide displays its biological activity in a membrane environment, delivery from an aqueous solution is of paramount importance. The self-assembling native M2GlyR sequence has been shortened and modified through an iterative process to improve water-solubility and reduce solution associations of the peptide. Based on aggregation studies (Tomich et al., 1998) it appears that only the lower molecular weight forms of the peptide are active. In solution, the hydrophobic surfaces of these amphipathic peptides apparently self-associate to form soluble oligomers. With the hydrophobic portions of the molecules sequestered from and the hydrophilic surfaces exposed to solvent, there is little driving force for the soluble peptide oligomers to associate with membranes. Therefore we hypothesize that monomer is the most bioactive species. Increasing the amount of monomer in solution should enhance membrane affinity thereby increasing efficacy.
The profiles for the ion transport data in Fig. 1 appear similar except for the lowest concentrations tested for the full-length NK^sub 4^-M2GlyR p27 WT and the truncated NK^sub 4^M2GlyR p22 WT sequences. As judged by cross-linking patterns shown in Fig. 2 A, the two peptides show different propensities to form higher molecular weight associations and NK^sub 4^-M2GlyR p22 WT appears to have more monomer present.
Replacement of the C-terminal serine with a tryptophan affects both the kinetics of assembly of the functional channel pore and the free peptide's solution properties. The peptide NK^sub 4^-M2GlyR p22 S22W displayed anion conductance activity (I^sub max^ = 13.0 � 1.0 �A/cm^sup -1^) with increased affinity for channel assembly (K ^sub 1/2^ - 45 �M) relative to the WT sequences. The observed reduction in maximal anion current is postulated to be the result of an alteration in the pore structure imposed by the introduction of the tryptophan.
The cross-linking studies reveal an absence of higher soluble molecular weight peptide-peptide associations for the S22W peptide. In fact, most of the soluble material appears as monomer. Increasing the amount of bioactive material could explain the improved efficacy of the compound, which is important in the design of membrane active drugs that need to be delivered from buffered aqueous solution. The abundance of soluble monomer in the presence of cross-linking agent clearly indicates that the tryptophan containing peptide must reside primarily in conformations that favors only intramolecular interactions, whereas the WT sequences adopts multiple conformations that stabilize monomer, dimer, and the other oligomers by allowing both intra- and intermolecular interaction.
CD studies showed that both peptides displayed different secondary structures in water and 40% TFE. NK^sub 4^-M2GlyR p22 S22W appears to adopt a structure that was intermediate to those observed in water or SDS micelles. On the other hand, the structures for NK^sub 4^-M2GlyR p22 WT in 40% TFE and SDS micelles were quite similar. Since NK^sub 4^-M2GlyR p22 WT had little structure in water and the NK^sub 4^-M2GlyR p22 WT and NK^sub 4^-M2GlyR p22 S22W had similar CD spectra in SDS micelles, high-resolution structural studies were undertaken in the solvent system that gave the largest structural difference, preserved some of the soluble characteristics of the peptide, and provided samples that could be analyzed by NMR. CD studies and cross-linking studies performed at the higher concentrations required for NMR analyses showed no concentration-induced structural or aggregation effects.
Chemical shift values of C^sub [alpha]^H protons indicative of secondary structure existed predominately in the C-terminal end and were found to be accurate predictions in the final structure. The overall shapes of the two molecules are substantially different. The overlapping of the minimized structures showed that the region spanning from residue 11 to 22 was highly structured (KKKPARVGLGITTVLTMTTQW). All of the 11 structured residues (underlined) are located in the putative transmembrane segment (bolded).
Both peptides were found to have an unstructured N-terminus and a structured C-terminal portion. The wild-type peptide is made up of a continuous helix with an extended, linear segment at the N-terminus. The S22W peptide, unlike the WT peptide, adopts a more closed structure due to a turn in the region separating the structured and unstructured sections. The two glycines in both peptides may limit interresidue interaction ensuring that the segment containing Val8-Gly 11 can be flexible enough to extend the N-terminal of the peptide into a linear structure or to fold the backbone so that a closed structure like the S22W peptide is formed. Although the proline residue in both of these sequences lies within the unstructured portion of the peptide, the absence of [alpha]- to [alpha]-proton connectivity in the NOESY spectra between the preceeding lysine and the proline of both peptides rules out the presence of a cis isoform.
A measurement of the linear WT peptide structure shows that the length of the entire TM segment (from valine 8 to the anchoring tryptophan 22) could be greater than 32 [Angstrom], more than enough to span the hydrophobic core of the membrane. This structure is similar to the results shown in micelle studies done on the wild-type glycine receptor [alpha]^sub 1^ subunit (Tang et al., 2002; Yushmanov et al., 2003). Their sequences are identical to our WT p22 sequence except for the inclusion of other WT residues at the termini. An identical region was found to be helical when incorporated in SDS micelles. The rest of the peptide was found to be unstructured and flexible, including the longer C-terminus. The results of this article suggest that the NMR solution structure of the WT peptide in 40% TFE is only slightly different, if at all, from the structure the peptide assumes in SDS micelles. These findings also indicate that the structured portion of the peptide is restricted to residues 8-22 regardless of the length of the peptide. The WT structure resembles other TM segments observed in the x-ray crystal structure of a CLC chloride channel. In that structure, the Cl- binding TM segments are made up of shorter helices that do not completely span the width of the membrane (Dutzler et al, 2002; Estevez and Jentsch, 2002).
The structured C-terminus of the S22W peptide was made up of a single-turn helix (residues 11-14), a stretched [beta]-like turn (residues 14-17), and then another two-turn helix (residues 15-21). The observed structure indicated a helical content of ~40%, which was in good agreement with CD data for the tryptophan containing 22-residue peptide. These backbone structures allow the peptide to loop over into a closed structure. This fold gathers the hydrophobic residues such that the acyl side chains have a reduced exposure to water. Clearly, this structure has little relevance to a TM segment where the aliphatic side chains would be fully exposed and interacting with the lipid acyl chains in the membrane bilayer. On the other hand, this may explain why the peptide remains in the monomeric form in solution, since the hydrophobic groups are not exposed and able to associate with other hydrophobic groups from other peptides. Based on the CD data obtained using 10-mM SDS micelles (Fig. 3 B), the peptide is able to adopt a more helical structure, much like the WT peptide.
After closer examination of the side chains of the structures it was determined that the polar threonines are exposed on a single side of the structure. This suggested that this side of the structure might be involved in the pore lining face of the assembled channel. Once assembled in the membrane these side chains would face toward the lumen of the pore and would be available to interact with either water or ions passing across the membrane.
In this study we report the structural consequences of incorporating a tryptophan at the C-terminus of an amphipathic channel-forming peptide. The tryptophan reduces solution aggregation thereby increasing the concentration of monomer. Realizing that producing detailed structures of TM sequences in a TFE/water solvent system is not ideal, nonetheless, the structural findings have provided remarkable insight into the possible membrane structure of the membrane-bound forms of the peptides. The structural data presented here has helped to explain how a 15residue TM segment can and does span the bilayer to form functional channels. Aggregation is reduced due to the peptides ability to adopt a solution conformation in water that shields the hydrophobic residues from solvent. Future experiments are planned for further structural determinations of this peptide in micelles and oriented bilayers using both solution and solid-state NMR techniques.
The two structures in water containing 40% TFE show differing degrees of helical, extended [beta]-like, and random coil structures. The segments displaying secondary structure are confined to those residues believed to form the transmembrane segments for the two sequences. The WT sequence is linear in 40% TFE, whereas the S22W peptide shows a fold that is stabilized by hydrophobic interactions. The structure of the S22W peptide in SDS micelles shows an increase in helical content as well as a dramatic decrease in [beta]-structure. This suggests a significant alteration in secondary structure upon entering a membrane. A structural transition upon membrane insertion would reposition the hydrophobic residues to maximize interaction with the fatty acid acyl chains.
The authors are grateful to Gary Radke and Robert Brandt for technical support.
The work is supported by the National Institute of General Medical Sciences (NIGMS) grant GM43617 (J.M.T.) and a structural supplement for the same grant. This work is contribution 03-179-J from the Kansas Agricultural Experiment Station (J.M.T., B.D.S., and O.P.).
[Reference]
REFERENCES
Bax, A., and D. G. Davis. 1985. MLEV-17 based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson. 65:355-360.
Broughman, J. R., K. E. Mitchell, R. L. Sedlacek, T. Iwamoto, J. M. Tomich, and B. D. Schultz. 2001. NH^sub 2^-terminal modification of a channel-forming peptide increases capacity for epithelial anion secretion. Am. J. Physiol. Cell Physiol. 280:C451-C458.
Broughman, J. R., L. P. Shank, W. Takeguchi, T. Iwamoto, K. E. Mitchell, B. D. Schultz, and J. M. Tomich. 2002a. Distinct structural elements that direct solution aggregation and membrane assembly in the channel forming peptide M2GlyR. Biochemistry. 41:7350-7358.
Broughman, J. R., L. P. Shank, T. Iwamoto, O. Prakash, B. D. Schultz, J. M. Tomich, and K. E. Mitchell. 2002b. Structural implications of placing cationic residues at either the NH^sup 2^- or COOH-terminus in a poreforming synthetic peptide. J. Membr. Biol. 190. 93-103.
Dutzler, R., E. B. Cambell, M. Cadene, B. T. Chait, and R. MacKinnon. 2002. X-ray structure of a CIC chloride channel at 3.0 [Angstrom] reveals the molecular basis of anion selectivity. Nature. 415:287-294.
Estevez, R., and T. Jentsch. 2002. CLC chloride channels: correlating structure with function. Curr. Opin. Struct. Biol, 12:531-539.
IUPAC-IUB Commission on Biochemical Nomenclature. 1970. Abbreviations and symbols for the description of the conformation of polypeptide chains. J. Mol. Biol. 52:1-17.
Kirnarsky, L., O. Prakash, S. M. Vogan, M. Nomoto, M. A. Hollingsworth, and S. Sherman. 2000. Structural effects of O-glycosylation on a 15residue peptide from the mucin (MUC1) core protein. Biochemistry. 39:12076-12082.
Merutka, G., H. J. Dyson, and P. E. Wright. 1995. 'Random coil' ^sup 1^H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG. J. Biomol. NMR. 5:14-24.
Mitchell, K. E., T. Iwamoto, J. M. Tomich, and L. C. Freeman. 2000. A synthetic peplide based on a glycine-gated chloride channel induces a novel chloride conductase in isolated epithelial cells. Biochim. Biophys. Acta. 1466:47-60.
Piantini, U., O. W. Sorensen, and R. R. Ernst. 1982. Multiple quantum filters for elucidating NMR coupling networks. J. Am. Chem. Soc. 104: 6800-6801.
Reddy, L. G., T. Iwamoto, J. M. Tomich, and M. Montal. 1993. Synthetic peptides and four-helical bundle proteins as model systems for the poreforming structure of channel proteins. III. Transmembrane segment M2 of the brain glycine receptor channel is a plausible candidate for the porelining structure. J. Biol. Chem. 268:14608-14615.
Roccatano, D., G. Colombo, M. Fioroni, and A. E. Mark. 2002. Mechanism by which 2,2,2-trifluoroethanol/water mixtures stabilize secondarystructure formation in peptides: a molecular dynamics study. Proc. Natl. Acad. Sci. USA. 99:12179-12184.
Tang, P., P. V. Mandal, and Y. Xu. 2002. NMR Structures of the second transmembrane domain of the human glycine receptor [alpha]^sub 1^ subunit: model of pore architecture and channel gating. Biophys. J. 83:252-262.
Tomich, J. M., D. Wallace, K. Henderson, K. E. Milchell, G. Radke, R. Brandt, C. A. Ambler, A. J. Scott, J. Grantham, and T. Iwamoto. 1998. Aqueous solubilization of transmembrane peptide sequences with retention of membrane insertion and function. Biophys. J. 74:256-267.
Wallace, D. P., J. M. Tomich, T. Iwamoto, K. Henderson, J. J. Grantham, and L. P. Sullivan. 1997. A synthetic peptide derived from glycine-gated CP channel induces transepithelial CP and fluid secretion. Am. J. Physiol. 272:C1672-C1679.
Wishart, D. S., B. D. Sykes, and F. M. Richards. 1992. The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry. 31:1647-1651.
Wuthrich, K. 1986. NMR of Proteins and Nucleic Acids. Wiley Inc., New York.
Yushmanov, V. E., P. K. Mandal, Z. Liu, P. Tang, and Y. Xu. 2003. NMR structure and backbone dynamics of the extended second Iransmembrane domain of the human neuronal glycine receptor [alpha]^sub 1^ subunit. Biochemistry. 42:3989-3995.
[Author Affiliation]
Gabriel A. Cook,* Om Prakash,* Ke Zhang,* Lalida P. Shank,* Wade A. Takeguchi,* Ashley Robbins,* Yu-Xi Gong,* Takeo Iwamoto,* Bruce D. Schultz,[dagger] and John M. Tomich*
* Department of Biochemistry and [dagger] Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506
[Author Affiliation]
Submitted August 5, 2003, and accepted for publication October 13, 2003.
Address reprint requests to Professor John M. Tomich, Dept. of Biochemistry, 103 Willard Hall, Kansas State University, Manhattan, KS 66506. Tel.: 785-532-5956; Fax: 785-532-6297; E-mail: jtomich@ksu.edu.
� 2004 by the Biophysical Society
0006-3495/04/03/1424/12 $2.00
