Caption Only [Color Photo: CASH STASH: $19.1 million. Hillary Rodham...]

Caption …

21 deaths in US linked to listeria in cantaloupe

WASHINGTON (AP) — U,S. health authorities say a nationwide outbreak of listeria in cantaloupe is now responsible for 21 deaths and the number may continue to grow.

The Centers for Disease Control and Prevention on Friday reported new deaths in Indiana and New York. The CDC also confirmed a death in Wyoming that state officials reported last week. CDC said 109 people have been sickened in …

Aides: Zelaya to return soon to Honduras

TEGUCIGALPA, Honduras (AP) — Former President Manuel Zelaya will likely return to Honduras within a month, ending his exile nearly two years after the coup that ousted him, an aide and a key supporter said Wednesday.

Conditions are right for Zelaya to return this month from the Dominican Republic after the Honduran Supreme Court dropped corruption charges against him, said Rasel Tome, a top aide of the former president.

"He has the will and desire to return to his homeland," Tome told The Associated Press.

Zelaya's return could pave the way for Honduras to be reincorporated into the Organization of American States, which suspended the Central American country following …

Hong Kong: Recent Movements of Japanese-Affiliated Firms

In response to the changes in China such as a unified corporate income tax for domestic and foreign firms, preferential treatment for the high-tech industry and special emphasis on the environment, Hong Kong has continued to change its position. Japanese-affiliated firms operating in Hong Kong are continuing a transformation of their operational nature to improve the quality and ensure profits against a backdrop of the scale being on a downtrend.

Drive to Improve Quality Against Shrinking Sales

Six years have passed since the start of the 21st century. During that period, China's accession to the WTO (World Trade Organization), the lifting of import quotas by Europe and the …

Whitman on Hot Seat Over 9/11 Aftermath

WASHINGTON - Ex-EPA chief Christie Whitman was bombarded by boos and a host of accusations Monday at a hearing into her assurances that it had been safe to breathe the air around the fallen World Trade Center.

The confrontation between the former head of the Environmental Protection Agency and her critics grew heated at times. Some members of the audience shouted in anger, only to be gaveled down by Rep. Jerrold Nadler, D-N.Y., who chaired the hearing.

For three hours Whitman faced charges from Nadler and others that the Environmental Protection Agency's public statements after the Sept. 11, 2001, attacks gave people a false sense of safety.

Whitman maintained …

Wozniacki moves into Ponte Vedra quaterfinals

Second-seeded Caroline Wozniacki moved into the quarterfinals of The MPS Group Championships by beating France's Virginie Razzano 6-3, 7-5 on Thursday.

Wozniacki's sixth quarterfinal in seven tournaments this year will be against Slovakia's Daniela Hantuchova, who beat No. 8 Bethanie Mattek-Sands of the United States 6-3, 7-5 in the late match.

Razzano led 40-0 on serve in the eighth game when Wozniacki reeled off five straight points to break for 5-3 then hold for the first set.

In the second, Razzano again missed game points serving at 5-5 and was broken. She conceded the match to the 18-year-old Dane on a long serve return.

…

56 dead, 240 hurt in Cuban train crash

MEXICO CITY A train in central Cuba derailed early Saturday,killing at least 56 people and injuring more than 240, Cuba'sofficial news agency reported.

At least six cars derailed, just after passing Santo Domingo ona trip from Havana to Guantanamo, said the Prensa Latina news agency,monitored in Mexico City. The accident site is about 155 milessoutheast of Havana.

A Prensa Latina journalist said by telephone that two radiostations reported six cars derailed, …

TCU moves to No. 3, Auburn No. 2 in AP poll

NEW YORK (AP) — TCU's impressive victory over the weekend not only lifted the Horned Frogs to No. 3 in The Associated Press poll, it caused Auburn to move up behind top-ranked Oregon and Boise State to fall two spots to No. 4.

The Horned Frogs won 47-7 at Utah on Saturday in a matchup of unbeaten Mountain West Conference teams. With that thoroughly dominant performance, TCU drew so much support away from Boise State, it caused movement in the top four for the first time in three weeks.

Oregon received 49 first-place votes and 1,484 points, only three points less than last week. Auburn received the exact amount of first-place votes (two) and points as last week.

TCU …

Britain, Germany level at Hopman Cup

Britain's Andy Murray has beaten Philipp Kohlschreiber of Germany 6-4, 6-1, to level their Hopman Cup clash 1-1.

Sabine Lisicki beat 15-year-old Laura Robson 7-6 (5), 6-3 to give Germany a 1-0 lead earlier Wednesday. The No. 22-ranked Lisicki also beat …

Ryan confronts health issue as he weighs a Senate bid

As Illinois Attorney General Jim Ryan contemplates a run for theU.S. Senate, he looks in the mirror at a head gone bald fromchemotherapy and wonders how much of an issue his health would be.

He hopes, he said, it would be no issue at all. As long as heis making progress toward recovery from his cancer, large-celllymphoma, Ryan said he hopes the voters will support him, or notsupport him, for reasons other than his health.

"I do think it's a legitimate issue, something people have aright to know about," Ryan said Friday. "But my doctor tells me he'svery optimistic about a full recovery. He thinks I'm in remission orsoon will be, and I should be able to …

Democrats encouraged about Obama in 2012

WASHINGTON (AP) — After last summer's fight over government borrowing, many rank-and-file Democrats say they are growing more optimistic about President Barack Obama's prospects for re-election in 2012.

They cite encouraging signs from Obama's more populist tone and what they view as a messy …

Jazz forward Kyle Korver has surgery on wrist

Utah Jazz forward Kyle Korver has had surgery to remove scar tissue from his right wrist.

Jazz general manager Kevin O'Connor says Korver had the operation Monday in New York. The wrist will …

Gold down

NEW YORK (AP) — Gold for current delivery closed at $1,480.90 per troy ounce Thursday on the New York Mercantile Exchange, down from $1,514.90 late Wednesday.

PGA Tour Schedule

Jan. 6-9 — Hyundai Tournament of Champions (Steve Stricker)

Jan. 12-15 — Sony Open (Johnson Wagner)

Jan. 19-22 — Humana Challenge (Mark Wilson)

Jan. 22-29 — Farmers Insurance Open (Brant Snedeker)

Feb. 2-5 — Waste Management Phoenix Open (Kyle Stanley)

Feb. 9-12 — AT&T Pebble Beach National Pro-Am (Phil Mickelson)

Feb. 16-19 — Northern Trust Open, Riviera CC, Los Angeles

Feb. 22-26 — WGC-Accenture Match Play Championship, Ritz-Carlton GC at Dove Mountain, Marana, Ariz.

Feb. 23-26 — Mayakoba Golf Classic, El Camaleon GC, Maya, Mexico.

March 1-4 — Honda Classic, PGA National GC, Palm Beach Gardens, Fla.

March 8-11 — WGC-Cadillac Championship, TPC Blue Monster at Doral, Doral, Fla.

March 8-11 — Puerto Rico Open, Trump International GC, Rio Grande, Puerto Rico

March 15-18 — Transitions Championship, Innisbrook Resort (Copperhead Course), Palm Harbor, Fla.

March 22-25 — Arnold Palmer Invitational, Bay Hill Club & Lodge, Orlando, Fla.

March 29-April 1 — Shell Houston Open, Redstone GC (Tournament Course), Houston.

April 5-8 — Masters Tournament, Augusta National GC, Augusta, Ga.

April 12-15 — RBC Heritage, Harbourtown GL, Hilton Head Island, S.C.

April 19-22 — Valero Texas Open, TPC San Antonio (AT&T Oaks Course), San Antonio

April 26-29 — Zurich Classic, TPC Louisiana, New Orleans

May 3-6 — Wells Fargo Championship, Quail Hollow Club, Charlotte, N.C.

May 10-13 — The Players Championship, TPC Sawgrass (Players Stadium Course), Ponte Vedra Beach, Fla.

May 17-20 — HP Byron Nelson Championship, TPC Four Seasons Resort, Las Colinas, Texas.

May 24-27 — Crowne Plaza Invitational at Colonial, Colonial CC, Fort Worth, Texas

May 31-June 3— Memorial Tournament, Muirfield Village GC, Dublin, Ohio

June 7-10 — FedEx St. Jude Classic, TPC Southwind, Memphis, Tenn.

June 14-17 — U.S. Open, The Olympic Club (Lake Course), San Francisco

June 21-24 — Travelers Championship, TPC River Highlands, Hartford, Conn.

June 28-July 1 — AT&T National, Congressional CC (Blue Course), Bethesda, Md.

July 5-8 — The Greenbrier Classic, The Greenbrier (The Old White TPC), White Sulphur Springs, W.Va.

July 12-15 — John Deere Classic, TPC Deere Run, Silvis, Ill.

July 19-22 — British Open, Royal Lytham & St. Annes, Lytham, England

July 19-22 — True South Classic, Annandale GC, Madison, Miss.

July 26-29 — RBC Canadian Open, Hamilton Golf & CC, Ancaster, Ontario

Aug. 2-5 — WGC-Bridgestone Invitational, Firestone CC (South Course),Akron, Ohio

Aug. 2-5 — Reno-Tahoe Open, Montreaux Golf & CC, Reno, Nev.

Aug. 9-12 — PGA Championship, Kiawah Island (Ocean Course), Kiawah Island, S.C.

Aug. 16-19 — Wyndham Championship, Sedgefield CC, Greensboro, N.C.

Aug. 23-26 — The Barclays, Bethpage State Park (Black Course), Farmingdale, N.Y.

Aug. 31-Sept. 3 — Deutsche Bank Championship, TPC Boston, Norton, Mass.

Sept. 6-9 — BMW Championship, Crooked Strick GC, Carmel, Ind.

Sept. 20-23 — Tour Championship, East Lake GC, Atlanta

Sept. 28-30 — The Ryder Cup, Medinah CC (No. 3), Medinah, Ill.

Oct. 4-7 — Justin Timberlake Shriners Hospital for Children Open, TPC Summerlin, Las Vegas.

Oct. 11-14 — Frys.com Open, CordeValle GC, San Martin, Calif.

Oct. 18-21 — The McGladrey Classic, Sea Island Resort (Seaside Course), St. Simons Island, Ga.

Oct. 25-28 — CIMB Asia Pacific Classic, The Mines Resort & GC, Selangor, Malaysia

Nov. 1-4 — WGC-HSBC Champions, TBD, China

Nov. 8-11 — Children's Miracle Network Classic, Walt Disney World Resort (Magnolia, Palm), Lake Buena Vista, Fla.

Greeley `mystery' a soggy sermon

A Hollywood superstar - a Chicago-born Irish Catholic - comes hometo visit. In her North Side hotel, she is bludgeoned and burned withcigarets, then hovers near death in Northwestern Memorial Hospital.

Did her accountant husband, an unimaginative, shy man, do thedeed? Or her tough, loyal, foul-mouthed childhood girlfriend? Herpompous, malicious, cardiologist brother? Her slimy agent? Apsychopath off the street?

You won't much care by the time Andrew Greeley winds up HappyAre the Clean of Heart (Warner, $3.95), his second paperback-originalmystery. His sleuth, Msgr. John Blackwood ("Blackie") Ryan of HolyName Cathedral, is more into preaching and philosophizing thanprying.

Greeley uses the old gimmick of viewing the victim from thedifferent viewpoints of the principal characters. Is she a seducer,as her husband says, or a woman who doesn't need much physicalaffection, as her friend thinks? Was she pure of heart or did shelive the high life? Was she aggressive in business or meek and mild?

You'll never know, for Greeley clogs his plot. He begins it as asermon on envy, then endlessly interrupts the action with opinions onsundry matters such as a World War II battle, the Chicago Cubs'pennant loss in 1984, the sociology of a middle-class Roman Catholicneighborhood, childhood memories, sexual gropings, feminism, thehorrors of Water Tower Place ("an insult to the eye of all who mustwalk down the Magnificent Mile") and even Sun-Times movie criticRoger Ebert (who does not smoke) happily puffing a cigar.

His sentences sag: "The fog that hung over the city, some sort of meteorlogical accident accompanying the dastardlyinjustice worked by Steve Garvey and his friends in taking theNational League championship away from our Cubs, was closing inagain."

Incidentally, Greeley's first science fiction novel, God Game(Warner, $16.95) was recently published. His biography, Confessionsof a Parish Priest, is being brought out in hardcover next month bySimon and Schuster. His third Blackie Ryan mystery is due nextsummer.

Meanwhile, there are some decent mysteries in softcover:

Flood, by Andrew H. Vachss (Pocket Books, $4.50). Asuper-cynical private eye named Burke helps a woman find the man whomolested and killed a friend's child.

Murder at the FBI, by Margaret Truman (Fawcett, $3.95). Hersixth Washington whodunit: Who killed an agent in front of touristsvisiting FBI headquarters? NEW ORIGINALS

Bed & Breakfast Coast to Coast, by Bernice Chesler (Penguin,$12.95). The new guide to B & Bs gives descriptions and names andaddresses to write for information and reservations of private roomsand apartments all over the United States. NEW REPRINTS

Elvis and Me, by Priscilla Beaulieu Presley with Sandra Harmon(Berkley, $4.50). The actress's revelations of life with Elvis.

Amyloid-beta peptide assembly: A critical step in fibrillogenesis and membrane disruption

ABSTRACT Identifying the mechanisms responsible for the assembly of proteins into higher-order structures is fundamental to structural biology and understanding specific disease pathways. The amyloid-p (AP) peptide is illustrative in this regard as fibrillar deposits of AP are characteristic of Alzheimer's disease. Because AP includes portions of the extracellular and transmembrane domains of the amyloid precursor protein, it is crucial to understand how this peptide interacts with cell membranes and specifically the role of membrane structure and composition on AP assembly and cytotoxicity. We describe the results of a combined circular dichroism spectroscopy, electron microscopy, and in situ tapping mode atomic force microscopy (TMAFM) study of the interaction of soluble monomeric AP with planar bilayers of total brain lipid extract. In situ extended-duration TMAFM provided evidence of membrane disruption via fibril growth of initially monomeric A/beta1-40 peptide within the total brain lipid bilayers. In contrast, the truncated A/beta1-28 peptide, which lacks the anchoring transmembrane domain found in Abeta1-40, self-associates within the lipid headgroups but does not undergo fibrillogenesis. These observations suggest that the fibrillogenic properties of Abeta peptide are in part a consequence of membrane composition, peptide sequence, and mode of assembly within the membrane.

INTRODUCTION

Amyloid-beta (Abeta) peptide is derived by proteolytic processing of the extracellular and transmembrane regions of the membrane-anchored amyloid precursor protein (APP) and has been causally linked with the formation of diffuse and senile plaques (Esch et al., 1990; Haass et al., 1992; Masters et al., 1985; Roher et al., 1986). The overproduction and deposition of Ap are characteristics of Alzheimer's disease, Down's syndrome, and hereditary cerebral hemorrhagic disease, HCWA-D (Dutch type; Cai et al., 1993; Citron et al., 1994; Iwatsubo et al., 1995; Suzuki et al., 1994). In vivo, AP is released as a monomeric peptide (Esch et al., 1990; Haass et al., 1992), but during normal aging or as a result of a disease process, AP self-associates into fibers that precipitate as plaques in the brain parenchyma. This process is enhanced by increased peptide concentrations, altered pH, and non-Abeta nucleation seeds. In vitro studies have demonstrated that synthetic AP can assemble spontaneously into beta-sheet-containing fibrils (Halverson et al., 1990; Kirschner et al., 1986; Lomakin et al., 1996; Walsh et al., 1997), that fibrillar AP can induce neuritic dystrophy (Lorenzo and Yankner, 1994; Pike et al., 1993; Simmons et al., 1993; Soto et al., 1995), and that Abeta-induced cellular degeneration occurs due to stimulation of inappropriate cell signals (Behl et al., 1994; Giulian et al., 1996; Mattson et al., 1997; Paradis et al., 1996). These observations suggest that defining the role of Abeta in mitigating specific disease pathways requires careful characterization of both the biological activity and the active conformation of the protein.

Because Abeta bears a portion of the APP transmembrane domain, it has been proposed that it may modulate membrane function by altering the physicochemical properties of various membrane constituents. These perturbations may include disruption of leaflet structure, an increase in bilayer curvature, or formation of membrane channels (Arispe et al., 1993; Choo-Smith and Surewicz, 1997; Mason et al., 1996, 1999; McLaurin and Chakrabartty, 1996; McLaurin et al., 1997, 1998; Mirzabekov et al., 1994; Pillot et al., 1996, 1997; Terzi et al., 1994, 1995). Although recent studies have demonstrated that lipids, and in particular acidic phospholipids, can induce a conformational transition in AP from random to beta-structure, a process that enhances fibril formation and initiates destabilization of the associated membrane structure, the fundamental mechanisms associated with these events remain unknown (Arispe et al., 1993; Choo-Smith and Surewicz, 1997; McLaurin and Chakrabartty, 1996, 1997; McLaurin et al., 1997, 1998; Terzi et al., 1994, 1995).

These questions prompted us to investigate the membranolytic action of Abeta peptides on planar bilayers of total brain lipid extract by circular dichroism (CD) spectroscopy, electron microscopy, and in situ solution tapping mode atomic force microscopy (TFAFM). This latter technique has proven to be a powerful approach for deciphering biomolecular assembly and protein structure under near-native conditions (Hansma et al., 1994; Karrasch et al., 1994; Moller et al., 1999), including Ag-fibrillogenesis (Blackley et al., 1999; Harper et al., 1997; Kowalewski and Holtzman, 1999; Oda et al., 1995; Roher et al., 1993, 1996; Stine et al., 1996). We have applied TMAFM to study protein assembly into the crystalline solid state (Yip and Ward, 1996; Yip et al., 1998a,b, 2000), role of chemical chaperones in modulating Abeta aggregation (Yang et al., 1999), and the oriented association of proteins at lipid interfaces (Plaskos and Yip, 2000).

The present studies provide evidence that the mechanism of Abeta-induced membrane disruption is strongly dependent on both lipid composition and peptide structure and conformation. Our in situ observation by TMAFM that Abeta fibril formation occurs in association with the lipid bilayer only in the presence of acidic lipids and only when the transmembrane domain of Abeta is present suggests that membrane anchoring is important for fibrillogenesis whereas amorphous aggregation is favored in the absence of the acidic lipid and/or the anchoring Abeta29-40 transmembrane domain. These results illustrate that peptide structure and sequence and membrane composition dramatically influence protein assembly (or misassembly) at membrane interfaces.

MATERIALS AND METHODS

Peptides

Abeta1 -28 and Abeta1 -40 were synthesized by solid-phase Fmoc chemistry by the Hospital for Sick Children's Biotechnology Centre (Toronto, Ontario, Canada). Peptides were purified by reverse-phase HPLC on a C18 mubondapak column. Peptides were initially dissolved in 0.5 ml of 100% trifluoroacetic acid (Aldrich Chemicals, Milwakee, WI) to ensure that the peptide remained monomeric and free of fibril seeds, diluted in distilled H2O, and immediately lyophilized (Jao et al., 1997). Peptides were then dissolved at 1 mg/ml in 40% trifluoroethanol (TFE, Aldrich Chemicals) in H2O and stored at -20degC until use. Alternatively, the lyophilized peptides were dissolved at 10 mg/ml in PBS.

Lipids

Dimyristylphosphatidylcholine (DMPC) and total brain lipid extract were purchased from Avanti Lipids (Alabaster, AL). To prepare the bilayer suspensions, the lipids were dried under a stream of nitrogen, lyophilized, and resuspended in PBS (pH 7.4) at a concentration of 1 mg/ml. Lipid suspensions were carried through five cycles of freeze-thaw in an acetone-- solid CO. bath, which forms both bilayer and multilayer lipid sheets. The lipid suspensions were then bath sonicated for 15-20 min to ensure formation of vesicles.

Circular dichroism spectroscopy

CD spectra were recorded on an Aviv CD spectrometer model 62DS (Lakewood, NJ) at 25degC. Spectra were obtained from 200-260 nm, with a 0.5-nm step, 1-nm bandwidth and 10-s collection time per step. The effects of the lipids on peptide conformation were determined by adding an aliquot of stock peptide solutions to lipid suspended in PBS (pH 7.4) at a 1:20 ratio (by weight). Final peptide concentration was 10 (mu)M. CD spectra were examined immediately after addition of Abeta and over a 24-h time course. The contribution of lipids to the CD signal was removed by subtracting the lipid-only spectra (McLaurin and Chakrabartty, 1996). Spectra were deconvoluted using Prosce (Yang et al., 1986).

Electron microscopy

Abeta1 -40 was incubated in the presence and absence of total brain lipid extract bilayers at a final peptide concentration of 0.1 mg/ml. The Abeta-to-- lipid ratio was maintained at 1:20 (by weight) matching the solutions sampled by CD. Preformed Abeta1 -40 fibrils were prepared by incubating a 10 mg/ml solution of Abeta1-40 for 3 days at room temperature. For negative-stain electron microscopy, carbon-coated pioloform grids were floated on aqueous solutions of peptides. After grids were blotted and air-dried, the samples were stained with 1% (w/v) phosphotungstic acid. The peptide assemblies were observed in a Hitachi 7000 electron microscope operated at 75 V.

Atomic force microscopy

Solution TMAFM images were acquired on a Digital Instruments Nanoscope IIIA MultiMode scanning probe microscope (Digital Instruments, Santa Barbara, CA) using 120-em oxide-sharpened silicon nitride Vshaped cantilevers installed in combination contact/tapping mode liquid flow cell. The AFM images were acquired using the E scanning head, which has a maximum lateral scan area of 14.6 (mu)m X 14.6 (mu)m. All imaging was performed at tip scan rates from 1.25 to 2 Hz, using cantilever drive frequencies of ~8.9 kHz (Yip et al., 1996). All images were captured as 512 X 512 pixel images and were low-pass filtered. Feature size and volumes were calculated using the Digital Instruments Nanoscope software (version 4.21) and shareware image analysis program National Institutes of Health-Image (version 1.62). Lateral calibration of the AFM piezoscanner was effected by resolving the crystallographic lattice of the basal plane of freshly cleaved mica and graphite whereas the vertical calibration was performed by measuring mica and graphite step heights in fluid. In situ TMAFM experiments were performed by sequential addition of the solutions to the AFM fluid cell. Planar bilayers were formed by directly injecting -500 (mu)l of the 1 mg/ml lipid suspension into the AFM fluid cell, previously sealed against a piece of freshly cleaved mica and allowing the vesicles to fuse in situ (Plaskos and Yip, 2000). The cell was flushed with neat buffer and reference TMAFM height and phase images acquired in PBS (pH 7.4) to confirm formation of stable lipid bilayers. Approximately 500 (mu)1 of a 0.1 mg/ml PBS solution of the peptide of interest (API -40 or Apt-28) was injected directly into the AFM cell and imaging initiated 30 min after Abeta addition.

RESULTS

Secondary structure of Abeta in the presence of lipid bilayers

Recognizing that Abeta-fibrillogenesis (Halverson et al., 1990; Lomakin et al., 1996; Walsh et al., 1997) and Abeta-lipid interactions (McLaurin and Chakrabartty, 1996) are dependent on initial Abeta structure, the Abeta1-40 and Abeta1-28 peptides were dissolved in 100% trifluoroacetic acid and immediately lyophilized to ensure that they were monomeric, in a random conformation, and free of fibril seeds (Jao et al., 1997). Secondary structure of Abeta1-40 and Abeta1-28 in the presence and absence of lipid bilayers was determined using CD spectroscopy (Fig. 1). Total brain lipid extract is ideally suited for these studies as it provides a physiologically relevant ratio of membrane components, including acidic and neutral phospholipids, gangliosides, cholesterol, sphingolipids, and isoprenoids. For our control experiments, planar bilayers of the zwitterionic lipid DMPC were used as the interactions of AP with this lipid have been reported previously in the literature (Arispe et al., 1993; McLaurin and Chakrabartty, 1996, 1997; McLaurin et al., 1997, 1998; Terzi et al., 1994, 1995).

Time-course CD spectroscopy performed over a 24-h period on Abeta1-40 in PBS pH 7.4 revealed that the initially randomly structured peptide, when mixed at a 1:20 peptide-- to-lipid ratio to a final peptide concentration of 10 gM underwent a partial conformational change in the presence of total brain lipid but remained random in the presence of DMPC vesicles (Fig. 1, A-GC). In the presence of total brain lipid vesicles, the time-course CD spectra revealed the gradual development of a spectral change indicative of both random and beta-structure in the Abeta1-40 peptide (Fig. 1 B). Deconvolution of CD spectra indicates a 77% random and 21% beta-structure in the presence of total brain lipid extract in comparison with Abeta40 alone, 82% random and 15% beta-structure. Because CD represents an average of all species present in solution, it is not possible to discern a single population associated with lipid vesicles. CD measurement accuracy is limited by the methodology used to determine peptide concentrations. Previous studies have demonstrated that amino acid analysis and ninhydrin techniques for peptide quantitation typically lead to an underestimation of peptide concentration by -10%, which leads to 10-20% higher mean ellipticity values (Marquess et al., 1989). Alternatively, the use of tyrosine absorbance in trifluoroethanol, as is used routinely in our laboratory, is accurate within 2%, representing a more accurate and more precise method (Marquess et al., 1989). It should also be noted that beta-structure is inherently underestimated by current deconvolution programs. Nevertheless, our CD data are consistent with the random structure Abeta1-40 peptide undergoing a beta-structure transformation upon binding to the total brain lipid vesicles. We cannot rule out the possibility that random structured Abeta1-40 peptides are in fact associated with the lipid (as well as being in solution); however, previous studies have demonstrated that acidic lipids can induce a beta-structural transition (McLaurin and Chakrabartty, 1996, 1997; McLaurin et al., 1997, 1998; Terzi et al., 1994, 1995). Control experiments performed with the zwitterionic lipid DMPC revealed that the Abeta1-40 peptide retains mostly a random conformation, 88%, over a 24-h time period (Fig. 1 C). Time-course CD studies performed with the N-terminal peptide Abeta1-28 revealed that its predominantly random structure was unchanged in the presence of total brain lipid extract and DMPC (Fig. 1, D-F). CD spectra at 24 h were superimposable on the spectra collected within 30 s of mixing Abeta1-28 with lipid vesicles.

Structural details of A13-lipid interactions

The relationship between the spectroscopically observed conformational transition of Abeta1-40 in the presence of total brain lipid extracts, fibril formation, and membrane disruption is largely unknown. Although various models of Abeta fibril formation have been proposed, they all agree that the beta-sheet regions are defined by the hydrophobic internal residues 17-21 and C-terminal residues 29-40/42. Fibrillogenesis is viewed as a two-step process involving a slow lag phase associated with the thermodynamic barrier to seed formation and fibril nucleation and a more rapid fibril growth and aggregation stage. These phases are known to be mediated by various factors including peptide concentration, changes in the AP peptide primary sequence, pH, and interactions with other elements that stimulate AP aggregation (reviewed in McLaurin et al., 2000).

Traditionally, electron microscopy has been used to evaluate Abeta fibrillar structures and peptide-lipid interactions. However, the sample preparation requirements of negative-- stain electron microscopy are problematic due to issues related to sample drying and staining procedures. These limitations restrict EM interpretation to the physical characteristics (shape/size) of the fibrils and rule out real-time interpretation of aggregation kinetics. With these limitations in mind, negative-stain electron microscopy revealed mature Abeta1-40 fibers extending along the peripheral surface of total brain lipid extract vesicles after a 3-day incubation (Fig. 2 A). However, these images are acquired ex situ and thus can provide only indirect evidence of association between the Abeta1-40 fibers and intact vesicles. Therefore, to study the real-time kinetics of Abeta-lipid interactions, in situ solution TMAFM was used.

In intermittent-contact, or tapping-mode, AFM, the scanning tip is vertically oscillated during scanning, which reduces frictional loads on the sample and thus the potential for sample distortion. Although TMAFM height imaging provides details on surface topography, phase imaging measures the relative phase shift between the applied and detected tip oscillation during TMAFM imaging and thus can provide information regarding intrinsic local surface modulus and viscoelasticity (Winkler et al., 1996). In fluid, the observed phase shift reflects differences in the amount of energy dissipated during tip contact with the surface and may correlate with specific adhesive interactions between the tip and surface, with the degree of phase shift increasing with increasing adhesion and energy dissipation (Brandsch et al., 1997; Cleveland et al., 1998; Czajkowsky et al., 1998; Noy et al., 1998). For our purposes, this mode of imaging can distinguish between lipid islands and aggregated protein patches on top of the lipid bilayers and has the potential for identifying structurally dissimilar domains within a lipid bilayer that may not be detectable by topographical contrast.

In situ formation of lipid bilayers within the AFM fluid cell was accomplished by continuously imaging the fusion of a 1 mg/ml lipid vesicle suspension onto the mica surface (Brian and McConnell, 1984). Previous studies have shown that supported bilayers are typically separated from the solid support by a thin buffer film and retain many of the properties of free membranes, including lateral fluidity (Bayerl and Bloom, 1991; Salafsky et al., 1996; Groves et al., 1997). AFM imaging was used to monitor the deposition of lipid vesicles from solution to the mica surface and their subsequent spreading and lateral fusion into molecularly flat bilayers populated by small amorphous globules and disc-- shaped islands. We note that bilayers were not used unless they were found to be essentially defect-free (i.e., no exposure of the underlying mica) over randomly selected >50-- Am 2 scan areas (Fig. 3 A).

In situ TMAFM revealed Abeta1-40 protruding -10 Angstrom above the smooth -40-Angstrom-thick total brain lipid extract bilayers, -30 min after the addition of 0.1 mg/ml Abeta1-40 (Fig. 3 B). We were able to distinguish the Abeta1-40 molecules from the lipid globules and islands by both height and phase imaging as the lipid islands and globules typically extended ~35-40 Angstrom above the underlying ~40-Angstrom-thick bilayer whereas the phase contrast of the lipid globules and islands was intermediate to that of the peptide and the supporting bilayer. The lipid islands were also morphologically distinguishable from the Abeta molecules. We attribute the variations in phase contrast to local differences in the energy dissipated by these structures as they interact with the oscillating AFM tip (Winkler et al., 1996, Brandsch et al., 1997; Cleveland et al., 1998; Czajkowsky et al., 1998; Noy et al., 1998).

The physiological structure of Abeta has yet to be determined as its tendency to aggregate and polymerize has limited the use of classical techniques such as crystallography. Previous in situ AFM imaging has determined that two-stranded beta-sheet structured Abeta1-42 has an approximate volume of 10,000 Angstrom (Yang et al., 1999) and Abeta40 has dimensions of 1-2 nm in diameter (Walsh et al., 1997). Alternatively, NMR studies of Abeta1-40 and Abeta1-28 peptides have determined that the Abeta1-28 domain is ~35 Angstrom in length whereas the full 40-residue peptide has a length of ~48 Angstrom (Sticht et al., 1995; Talafous et al., 1994). Using these two divergent techniques as an indicator of size, the extent of Apl-40 exposure as determined by TMAFM suggests partial insertion of the peptide into the bilayer, likely through the 29-40 hydrophobic domain. At 3 h after peptide addition, small Abeta fibers were barely visible on the surface of or within lipid headgroups using TMAFM height imaging (Fig. 4 A). To elucidate the presence of Abeta fibrils, TMAFM phase images were examined. The use of phase imaging to differentiate lipid and fibers that appear to be in the same plane topographically is based on local surface differences in viscoelasticity and differential adhesive interactions between the lipid and peptide with the tip. Although not easily resolvable by topographical contrast, phase imaging provided evidence of Abeta1-40 fibrils growing within the bilayer (Fig. 4 B). Examination of the height and phase images revealed the presence of small fibers not associated with disrupted lipid regions. This indicates that the lipid headgroups can initially accommodate fibril growth. Larger fibers could be detected and appear adjacent to the initiation of small lipid disruptions. These images represent an intermediate phase in AP fibrillogenesis (Fig. 4).

The gradual growth of 120-180-Angstrom-wide Abeta1-40 fibrils in the bilayer was revealed by in situ TMAFM and phase imaging performed over 24 h (Fig. 3 C). The fibrils are in the plane of and extend parallel to the bilayer surface, suggesting that fibril nucleation and growth occur within the lipid headgroups (Fig. 3 C, inset). We cannot discount lateral diffusion of Abeta1-40 through the bilayer although we did not observe this process on the time scale of the TMAFM imaging (minutes). The absence of Abeta1-40 aggregates indicate that, for total brain lipid extract, Abeta1-40 interaction with lipid domains facilitates formation of the fibril. The lack of AP aggregates suggests that off-pathway intermediates, reported for solution-grown fibrils (Huang et al., 2000), are not formed in the presence of total brain lipid extract bilayers.

In situ TMAFM imaging revealed extensive destabilization of the total brain lipid extract bilayer after Abeta1-40 fibril growth had initiated (Fig. 3 C). We found no evidence of bilayer defects before the introduction of monomeric Abeta1-40 (Fig. 3 A), nor did we observe any incidence of tip-induced bilayer damage, either before or after fibril formation. In the disrupted membrane regions, Abeta1-40 fibrils were associated with bilayer edges and extended across the exposed mica substrate (Fig. 3 C). Both membrane-bound and exposed fibrils were branched, and the degree of branching appeared to be greater than that reported for solution-grown fibrils (Blackley et al., 1999; Harper et al., 1997; Kowalewski and Holtzman, 1999; Oda et al., 1995; Roher et al., 1996; Stine et al., 1996). This increased branching may result from growing fibrils encountering membrane patches that are energetically impermeable to fiber growth, either due to specific lipid composition or tighter packing of the lipid headgroups. To rule out tip interference over the time course of our experiment, randomly chosen scan areas were examined. Although massive membrane disruption was detected after 15 h, less disrupted areas also were present (Fig. 5 A). High-resolution phase imaging of the less disrupted areas revealed small AP fibers within the intact bilayer headgroups in regions that were not associated with bilayer defects (Fig. 5 B).

We considered the possibility that the fibrils we observed were a consequence of solution fibril growth and deposition to the bilayer surface. To address this concern, the same 0.1 mg/ml Abeta1-40 solution used in the bilayer studies was left to incubate at room temperature while we conducted our bilayer studies. In situ TMAFM imaging and negative-stain electron microscopy performed on aliquots of this solution after 24 h did not reveal any fibrillar aggregates although small peptide monomers or oligomers were found (Figs. 2 B and 5 C). These observations would suggest that fibril nucleation is facilitated by the lipid bilayer and that the structures observed by in situ TMAFM are in fact not due to fibril formation in the surrounding fluid.

We propose that by displacing lipid from the bilayer as it forms, the growing fibril weakens the stabilizing lipid-lipid lateral interactions within the bilayer and initiates the release of small bilayer patches. We did on occasion find small fibrils that were not associated with a particular bilayer edge or that appeared to extend away from a bilayer island (data not shown). We propose that these are fibrils left behind once the membrane patch has been destabilized and released into the surrounding buffer.

Lipid specificity of Abeta interactions

To investigate the role of lipid composition in Aa-induced membrane disruption, planar DMPC bilayers were employed as model substrates. As described above, AFM imaging was used to monitor the fusion of lipid vesicles onto the mica and subsequent spreading to form an intact bilayer. Bilayers were not used unless they were found to be essentially defect-free over randomly selected scan areas. In situ TMAFM imaging revealed Abeta1-40 extending ~12-25 Angstrom above the DMPC bilayer surface (Fig. 6 A). Abeta1-40 was typically located adjacent to small ~20-Angstrom-wide holes, suggesting that the insertion process facilitated local disruption of the bilayer. These holes extend through the bilayer to expose the underlying mica substrate. TMAFM imaging performed over a 15-h time period revealed the gradual expansion of irregular patches of Abeta-40 aggregates throughout the DMPC bilayer (Fig. 6 B). Initially, we found no evidence of the Abeta-40 peptide on the exposed mica surfaces or within the bilayer holes, which suggests that the peptide prefers to self-associate or associate with the zwitterionic lipid. In these patches, the Abeta1-40 aggregates protrude up to 100 Angstrom above the bilayer surface, not only indicating that Abeta1-40 interacts with the bilayer but also confirming self-association (Fig. 6 B). In contrast to our total brain lipid extract studies, only globular aggregate masses of Abeta1-40 were found in the DMPC bilayers; no fibrils formed over a 24-h time period. These results suggest that the critical step in membrane destabilization is Abeta1-40 self-assembly into fibers and aggregates on the bilayer surface.

This model was examined by studying the interaction of preformed Abeta1-40 fibrils or aggregates with planar bilayers (Fig. 6). Negative-stain EM demonstrated that fibrils formed by incubation of Abeta1-40 at 10 mg/ml for 3 days had the characteristic amyloid fibrillar structure (Fig. 2 C). To examine the effects of both protofibril and aggregates on the stability of the planar bilayers, we examined Abeta1 -40 after short and long incubation periods. On total brain lipid extract bilayers, small preformed Abeta1-40 fibrils formed after short incubation were found on the bilayer surface but did not extend into, or disrupt, the bilayers (Fig. 6 C). The fibers detected by TMAFM extended 20-30 Angstrom above the bilayer surface. Upon interaction with the bilayer, all fibrils appeared to circle back on themselves to form enclosed ring-type structures rather than the extended fibrils detected in the absence of lipid. These dimensions and fibril characteristics are similar to those previously examined by AFM in the presence of synthetic phosphatidylethanolamine and phosphatidylserine vesicles (Lin et al., 1999). Even after 24 h of incubation, both the AP fibrils and the bilayer remained intact. Additional incubation of these fibrils results in lateral aggregation into large AP masses in which individual fibers are not readily distinguishable. Addition of these aggregates onto the bilayer surface results in massive peptide aggregates extending 50-100 A above the bilayer but does not result in bilayer disruption (Fig. 6 D). These results reinforce our proposal that self-assembly of AP on the surface of the bilayer is critical for membrane disruption. Abeta sequence requirements for

membrane disruption

To investigate the relationship between AP sequence and the extent of membrane insertion, we examined the propensity of Abeta1-28, the extracellular domain of Abeta, to induce membrane disruption. Abeta1-28 lacks the hydrophobic C-- terminal domain of Abeta1-40 but still assembles into 3-sheet fibrils, with the conversion between a-helix, random coil, and beta-structure in Abeta1-28 being highly dependent on solution conditions (Barrow and Zagorski, 1991; Brooksbank and Marinez, 1989; Fletcher and Keire, 1997). TMAFM height and phase imaging revealed aggregation of API-28 on both total brain lipid and DMPC bilayers (Fig. 7). In both cases, extended duration TMAFM indicated that membrane destabilization resulted from lateral expansion of aggregated Abeta1-28 patches on the bilayer. DMPC bilayers formed within the AFM fluid cell demonstrate random membrane defects and a few lipid globules sitting on the surface of the bilayer (Fig. 7 A). The lipid globules can be distinguished from peptide using both height and phase imaging. At initial time points, small aggregates of Abeta1-28 were associated with membrane holes of varying size or on the surface of the bilayer (Fig. 7 B). As in our results with the Abeta1-40 peptide, the absence of obvious aggregates on the exposed mica surface demonstrates that the peptide prefers to associate with the lipid bilayer. The amount of peptide associated with the bilayer could not be directly correlated with the size of the hole, although membrane defects did not form in the absence of peptide (Fig. 7 C). Similar results wherein the extent of membrane disruption cannot be directly correlated to the extent of the perturbation have been observed for other bilayer systems and been described as spinodal decomposition (Sackman and Feder, 1995; Tsai and Torkelson, 1989). Phase imaging confirmed the presence of Abeta peptide at the bilayer edges and demonstrated the association of AP with pre-existing AP aggregates, suggesting that Abeta-Abeta interactions increase with time (Fig. 7 D). Because membrane destabilization occurred in the complete absence of fibril formation, we propose that self-aggregation of Abeta at the lipid surface is sufficient to cause local disruptions in membrane integrity.

DISCUSSION

Our CD and in situ TMAFM investigations of Abeta-lipid interactions have revealed that Abeta membranolytic action depends on Abeta structure and lipid environment. Our observations by in situ TMAFM that suggests that Abeta1-40 undergoes partial insertion into lipid membranes contradicts previous x-ray diffraction and fluorescence data that reported complete Abeta1-40 insertion into the hydrocarbon region of the bilayer (Mason et al., 1996, 1999; Mirzabekov et al., 1994; Pillot et al., 1996). However, in contrast to our approach where monomeric Abeta1-40 was added to preformed lipid bilayers, these earlier studies were performed on lipid bilayers prepared in the presence of Abeta. Energetically, preparing lipid bilayers by the latter method favors burying the hydrophobic Abeta peptide within the lipid acyl chains to generate a stable bilayer structure. Moreover, because it is known that in vivo the Abeta peptide is released in a monomeric form, it is therefore appropriate to investigate how the freely soluble peptide will interact with membrane-mimetic surfaces under analogous conditions (Esch et al., 1990; Haass et al., 1992).

Previous studies have demonstrated that acidic phospholipids and phosphoinositides promote a conformational transition in Abeta from random to beta-structure (Arispe et al., 1993; Choo-Smith and Surewicz, 1997; Mason et al., 1996; 1999; McLaurin and Chakrabartty, 1996, 1997; McLaurin et al., 1997, 1998; Mirzabekov et al., 1994; Pillot et al., 1996; Terzi et al., 1994, 1995). We now have evidence suggesting that this lipid-induced conformational transition is facilitated by Abeta's C-terminal hydrophobic domain. By forming a beta-sheet scaffold structure, the sterically compact Abeta peptide can reside within or on the surface of the lipid head-- groups and self-associate to form the critical fibril nucleus. After nucleation, fibril growth through the lipid bilayer ultimately results in destabilization of the membrane through excision of bilayer regions (Fig. 7). These results are in agreement with Kremer and colleagues (2000), who reported that Abeta-induced changes in membrane fluidity were dependent on insertion of Abeta into the fatty acyl chains of the bilayer. In the absence of acidic lipids or a C-terminal hydrophobic domain, Abeta fibril formation is abolished; however, membrane disruption still occurs via expansion of aggregated Abeta through the bilayer. Our observation that aggregation of the truncated Abeta1-28 peptide occurred on the total brain lipid and DMPC. Comparison of the lateral size and thickness of the aggregated Abeta1-28 patches on the total brain lipid bilayers with those found on the neutral DMPC bilayers demonstrate that patches on total brain lipid bilayers were smaller laterally but thicker than those found on the DMPC bilayers. We attribute these observations to preferential binding of Abeta1-28 to small, possibly acidic, lipid domains within the heterogeneous total brain lipid bilayers followed by Abeta1-28 self-association within these domains. Based on these observations, we propose that the steric bulk of randomly structured Abeta peptide cannot be readily accommodated by the lipid headgroups and gradual enlargement of the Abeta1-28 patches facilitates local disruption of the bilayers (Fig. 8).

Based on our observations, we propose the following two-stage model for the membranolytic action of Abeta. Assembly of Abeta leads to highly localized disruption of the membrane. This disruption may enable ions to pass freely between the intra- and extracellular space and is consistent with reports that Abeta enhances ion fluxes, thereby altering cellular homeostasis (Arispe et al., 1993; Mirzabekov et al., 1994). Increasing accumulation of Abeta and the initiation of fibril formation through the membrane results in destabilization of larger regions of the membrane. These openings may facilitate the passage of proteins, as has been demonstrated for endosomal-lysosomal vesicles, resulting in the protein compartmentalization errors characteristic of Alzheimer's disease (Avdulov et al., 1997; Blanc et al., 1997; Cataldo et al., 1996; Yang et al., 1998). The presence of cell surface fibril formation was eloquently demonstrated on cerebrovascular smooth muscle cells using EM (Van Nostrand et al., 1998), and plasma-membrane-bound Abeta is the initiation site for deposition of diffuse plaques (Yamaguchi et al., 2000). Finally, the inability of preformed AP fibrils to induce membrane disruption correlates well with the lack of in vitro toxicity observed when neuronal and cerebrovascular smooth muscle cells are incubated with preformed Abeta fibrils (Davis-Salinas and Van Nostrand, 1995; May et al., 1992). Recent studies detailing the importance of the Cterminal region of Abeta for its neurotoxic properties have demonstrated that nonfibrillar AP induces an apoptotic death in neurons whereas aggregated AP induces a necrotic death (Pillot et al., 1999). Our TMAFM studies have provided structural evidence of how these processes may take place and, in particular, how membrane disruption can be triggered by alternate structures of the same peptide. This and related approaches to studying protein assembly (or misassembly) at membrane interfaces will facilitate realtime investigations of the mechanisms by which specific protein-lipid interactions are driven by either sequence, structure, or compositional differences and will be of particular applicability for investigating other amyloidogenic pathways.

We thank Dr. N. Wang at the Hospital for Sick Children's Biotechnology Centre for the synthesis of all peptides used in this study, Dr. A. Chakrabartty for use of the Aviv Circular Dichroism Spectrometer, and the Electron Microscopy Suite at the University of Toronto for use of Hitachi 7000 electron microscope.

C.M.Y. gratefully acknowledges the generous support of the Medical Research Council of Canada (MT-14769), the Natural Sciences and Engineering Research Council of Canada (194435-99), the Ontario Research and Development Challenge Fund, and the University of Toronto's Connaught Foundation grant (413578). J.M. acknowledges support from the Kevin Burke Memorial Amyloid Fund (407966), the University of Toronto Dean's Fund (414744), the Banting Research Foundation (415961), and the Bickell Foundation (416157).-32767

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[Author Affiliation]

Christopher M. Yip* and JoAnne McLaurin^

[Author Affiliation]

*Institute for Biomaterials and Biomedical Engineering and Centre for Studies in Molecular Imaging, Centre for Research in Neurodegenerative Diseases, *Departments of Chemical Engineering and Applied Chemistry, *Biochemistry, and ^Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5S 3G9 Canada

[Author Affiliation]

Received for publication 15 May 2000 and in final form 15 December 2000.

Address reprint requests to Dr. JoAnne McLaurin, Centre for Research in Neurodegenerative Diseases, Tanz Neuroscience Building, 6 Queen's Park Crescent West, Toronto, Ontario, Canada MS 3H2. Tel.: 416-978-1035; Fax: 416-978-1878; E-mail: j.mclaurin@utoronto.ca.

A new day at Capitol: Change coming _ right now

WASHINGTON (AP) — Old and new Washington collided on Capitol Hill Monday, and new won.

Within moments of flicking on the Senate lights, Sen. Mitch McConnell announced that when it came to pork barrel politics he had changed his mind. The Senate's staunchest fan of so-called earmarks reversed course and supported a ban on those special spending requests, a bow to the tea partiers and others in the populist, antiestablishment wave that gave the GOP control of the House and six more seats in the Senate.

"Old habits aren't easy to break, but sometimes they must be," McConnell said on the Senate floor.

His announcement put an exclamation mark on the return of lawmakers to Capitol Hill Monday for a "lame duck" session before the newcomers take their places officially in January. There's still major business for the current Congress, from tax cuts that will or won't be preserved to possible special Social Security checks to spending bills to keep the government going.

Monday was an extraordinary day that blended Congresses past, present and future as the fading Democratic majority began to yield.

It wasn't going quietly.

For the more than 100 rookies dining and orienting around campus, there was no starker lesson than the spectacle of Rep. Charles Rangel, a once-mighty committee chairman now facing ethics charges — four decades after his arrival was supposed to herald the shake-up of an old, corrupt political order.

"My reputation — 50 years of public service! — has to suffer," the New York Democrat cried out before stalking out of his ethics trial.

There were plenty of other lessons, pedestrian as well as profound for the new folks: not only how to be an employer, a first for some of them, and how to avoid Washington's ethical traps, but also where to eat, how to vote, how to get to the subway beneath the Capitol, even which elevators to use.

As for politics — in case any politicians had missed the message of the Nov. 2 elections — triumphant conservative activists, many of them tea partiers, rallied on the Capitol lawn with signs urging Congress to heed their call for smaller government and greater accountability.

"Phase One, Nov. 2010. Complete," read one sign. "Phase Two. Nov. 2012. We are watching you."

Acutely aware of that, longtime lawmakers began to let change flow through the corridors of power, already heavy with the cold-weather scent of fireplaces ablaze in the Capitol's grand parlors.

President Barack Obama, just back from a 10-day trip to the other side of the globe, said he would be ready to talk policy when the Republicans were finished celebrating. He said of his upcoming meeting with congressional leaders: "I'm sure it will be very relaxing."

"Campaigning is very different from governing. All of us learn that. And they're still flush with victory," he said. "We're going to have a whole bunch of time next year for some serious philosophical debates."

Obama, who earlier had endorsed a crackdown on earmarks, praised McConnell's announcement, but not all Democrats were on the anti-earmarks bandwagon.

Senate Majority Leader Harry Reid's spokesman, Jim Manley, said, "From delivering $100 million in military projects for Nevada to funding education and public transportation projects in the state, Sen. Reid makes no apologies for delivering for the people of Nevada."

Change was evident at the lunch table, too. In the Senate, 16 newly elected senators — 13 of them Republicans — were invited to dine with veterans, including some not returning next year. Two rookies, Democratic Sens. Chris Coons of Delaware and Joe Manchin of West Virginia, were sworn in to fill empty seats in their states.

And change took hold of House Speaker Nancy Pelosi's world, which was suspended between the old Congress and new.

She's still speaker, second in line to the presidency but not yet the Democratic leader in the new Congress. Those elections are to be held on Wednesday.

She still has chores, including spending time with the very Republicans who won big gains in Congress in part by vilifying her.

On Sunday, she left the comfort of a visit with her grandchildren for a speech welcoming the incoming freshmen. Officials who attended the closed-door session said Pelosi stuck to stock advice: represent your district, follow the Constitution.

Monday night, she was hosting an open house in what is her turf for just a little longer — the speaker's suite of offices under the Capitol dome.

In the House, the old guard reconvened for the first votes since the Democrats' shellacking. Democrats Suzanne Kosmas and Ron Klein, both of Florida and both defeated, shared a warm embrace as Kosmas wiped a tear.

_______

Associated Press writers Andrew Taylor and Julie Hirschfeld Davis contributed to this report.

(This version corrects first name of Rep. Kosmas to Suzanne.)

EPA sets water pollution limits for Florida

TALLAHASSEE, Fla. (AP) — The Environmental Protection Agency has set legal limits for farm and urban runoff polluting waterways in Florida, the first time the federal agency has set standards for a state.

The EPA set the limits Monday after it settled a lawsuit with environmental groups last year. The numeric limits on runoff are designed to reduce pollution from sewage treatment plants and runoff choking lakes, rivers and other Florida waters with algae blooms.

Agriculture, business interests and some politicians, including Gov.-elect Rick Scott, say that meeting the new standards will be too costly.

EPA officials say critics' cost estimates are wildly exaggerated. The EPA puts the cost at $130 million to $200 million a year, not the billions claimed by opponents.

NATO warns Kosovo Serbs to remove road barricades

MITROVICA, Kosovo (AP) — The commander of NATO-led peacekeepers in Kosovo said Tuesday he is disappointed by the Serb refusal to remove roadblocks in the north of the country and warned of action if the blockade is not lifted soon.

Serbs defied a Tuesday deadline to remove the barriers and gathered by the hundreds to protect them from removal by the peacekeeping troops. Two NATO troop supply convoys were stopped by the Serbs manning the barricades on Tuesday.

For nearly three months, Kosovo Serbs have been blocking main roads to stop Kosovo's ethnic Albanian leadership from extending their control over the Serb-run territory. Serbs reject Kosovo's 2008 declaration of independence from Serbia.

The 5,500-strong peacekeeping force, known as KFOR, had requested that the 16 barriers of rocks, mud and logs be taken down by early Tuesday.

"I am disappointed with this outcome," NATO's top commander in Kosovo, Maj. Gen. Erhard Drews, said in a statement. "The north did not comply with the request to remove the roadblocks."

Drews said he will wait for the outcome of a meeting of Kosovo Serb leaders who will discuss on Wednesday whether to lift the blockade for the passage of the peacekeepers.

"KFOR is ready and resolved to take action on behalf of freedom of movement, if the municipality meeting on Wednesday does not have satisfactory results," Drews said. "Our focus is on enabling civilians to lead normal lives."

On Tuesday, hundreds of Serbs gathered at the barriers to protect them from forced removal by the peacekeepers who say they want to establish freedom of movement in the region and reopen supply routes for their troops.

Kosovo Prime Minister Hashim Thaci urged the international peacekeepers to apply the rule of law and ensure freedom of movement on "all territories of Kosovo."

"We want to build a better Kosovo, for all citizens of Kosovo, no matter what nationality they are," Thaci told AP Television News in Kosovo's capital, Pristina. "These actions are coordinated between the international community and the government of Kosovo."

In July, ethnic Albanian authorities deployed their security forces to two border checkpoints in northern Kosovo to enforce a trade ban with Serbia. Serbs reacted by blocking roads and triggering clashes with Kosovo police that left one police officer dead.

___

Associated Press writers Dusan Stojanovic in Belgrade and Slobodan Lekic in Brussels contributed to this report.

SOUND OFF: WHAT YOU SAID

Last week's question:

Should cigars and smokeless tobacco be taxed? Why or why not?

YES: "Such tax exemptions are prime examples of a special interest buying the votes of incumbent legislators. Only a moron or a congressman could rationalize the taxation of one sort of tobacco product and not another."

-Roland Henry, Cumberland County

YES: "Absolutely yes; there is no valid reason to not tax tobacco in any form."

-John Klinedinst, York County

YES: "If cigarettes are taxed, then any other tobacco product should be taxed as well. Some lawmakers must really like their chew!"

-Ken Mark, Cumberland County

YES: "Do they cause cancer? Do they increase health care costs?"

-Anne Quist, Cumberland County

YES: "I'm dumbfounded there is even a question on this issue! Absolutely!"

-Arty Beahm, Cumberland County

NO: "I am a nonsmoker and feel that they are taxing cigarettes to death! They tax things that are bad for you: cigarettes, tobacco, alcohol. Why not go after the fast food that is (bad) for everyone - thatwould bring in plenty of revenue."

-Jackie Dubs, York County

NO: "... A cigar smoker treats smoking as celebratory, fraternity or social networking.

Also, we only have three stores in Lancaster County that sell a wide variety of cigars. The product - thus revenue from a tax - is not available everywhere.

I can't find projections of revenue from a tax. This is not a significant revenue stream and we have two of the largest mail-order cigar companies: Cigar International & Famous Cigar shop in Pennsylvania. That said, they will leave (taking their jobs, income and real estate taxes out of state if a tax happens).

Simply put: The politics is that this is a palatable tax to voters with the acceptance of the nonsmoking ban in restaurants/ bars."

-Mark A. Vergenes, Lancaster County

2012 election weighs on GOP agenda

WASHINGTON - The Republican agenda for the new Congress thatconvenes Wednesday may have a greater impact on the 2012 electionsthan on the lives of Americans in the next two years.

Republicans promise to cut spending, roll back President BarackObama's health care overhaul and prevent unelected bureaucrats fromexpanding the government's role in society through regulations thattell people what they must or can't do. Getting this agenda throughthe House may be easier than in the Senate, given the GOP's 241-194majority in the House. Getting the Senate to act will be achallenge. Democrats still hold an edge there, though smaller thanthe one Obama had during his first two years in the White House.

Even if the next two years end in gridlock, Republicans will havebuilt a record for the next election that they hope will demonstrateto voters that they can get it right.

House Republicans also pledge to hold tough investigations andhearings on the president's programs and policies, ending the freepass that Democratic committee chairmen gave the Obamaadministration the past two years.

Republicans insist they'll bring key administration officialsbefore congressional microphones and that the public can watch thewebcasts. The friendly tone of inquiry from Democratic chairmen willbe replaced by Republicans demanding answers to these questions:What's the purpose of this program? Is this the best use of thetaxpayers' money?

The chief Republican investigator, Rep. Darrell Issa ofCalifornia, is raring to get started, and he's not alone. Issa, theincoming chairman of the House Oversight and Government ReformCommittee, has been especially critical of what he calls waste inObama's economic stimulus spending.

"The sooner the administration figures out that the enemy is thebureaucracy and the wasteful spending, not the other party, thebetter off we'll be," he told "Fox News Sunday."

Rep. Harold Rogers of Kentucky, incoming leader of the HouseAppropriations Committee, says he wants top officials from all majorgovernment agencies to appear and justify their spending.

The next chairman of the House Energy and Commerce Committee,Republican Fred Upton of Michigan, says he'll work to stop over-zealous government regulators. A big target for him is theEnvironmental Protection Agency, which is writing rules to limitgreenhouse gases blamed for global warming after Obama's effort toget Congress to do it stalled in the Senate last year.

"We are not going to let this administration regulate whatthey've been unable to legislate," he told Fox.

Upton, like Issa, will have a large investigative staff.

"Republicans need to make sure they bring forward solutions, eventhough it may be difficult to get them accomplished," Rep.-electKristi Noem, R-S.D., said in an interview. She said the lesson fromthe November election is, "The American people will replace peopleif they're no longer in touch or listening."

Noem benefited from that view, defeating Democratic Rep.Stephanie Herseth Sandlin. Noem has risen to the forefront of thefreshman class; she was chosen to serve in the GOP leadership.

In the Senate, there's a chance the Democrats will replaceRepublicans as the party of "no," assuming the House GOP passes muchof its agenda. Democrats will control the Senate 51-47 with twoindependents, and only need 41 votes to block initiatives thatarrive from the House.

Among the reasons that the Republican agenda will likely have abigger impact on the next election than on the day-to-day lives ofmost Americans are:

n Much of the government spending has been politicallyuntouchable. About 60 percent goes for entitlement programs,including Medicare, Medicaid and Social Security. The nation also ispaying for wars in Iraq and Afghanistan and major reconstructionprojects in those countries. Both parties have considered itpolitically foolish to mess with Medicare and Social Security. Also,Republicans don't have a clean record as budget cutters.

"Spending restraint on the Republican side is a theory yet to beproven," said Robert Bixby, executive director of the budget-watching Concord Coalition. He noted that Democratic President BillClinton's budget surplus turned into deficit under Republican GeorgeW. Bush.

* Obama may be more willing to compromise with Republicans thanin his first two years, but he will fight repealing the health carelaw. Senate Democrats will almost certainly stop major revisions. Iffor some reason they don't, Obama will use his veto to stop them.

n Republican attempts to overturn regulations on issues such asglobal warming also could falter in the Senate. When the EPAannounced just before Christmas that it planned to set greenhousegas emissions standards for power plants and oil refineries, Uptonsaid, "We will not allow the administration to regulate what theyhave been unable to legislate." Senate Democrats may have adifferent view.

Many eyes in the new session will focus on Issa, who will havesubpoena power and can investigate any government program.

Issa has played good cop and bad cop. He criticized Obama's mostimportant programs, including the economic stimulus. But less than amonth after the Republicans won big in November, he had a privatepeace meeting with Vice President Joe Biden. Neither is shy aboutentering a political brawl, but initially they have pledged to worktogether against waste and for openness in tracking governmentspending.

Issa has not discouraged articles suggesting he will send theadministration subpoenas by the trainload. But he also wants to givesubpoena power to nonpolitical government watchdogs - inspectorsgeneral - and let them use that authority to uncover fraud, wasteand abuse. With a degree of political cover, Issa could then usethose findings to conduct his own investigations.

If the peace pact between Biden and Issa holds, there are otherissues where the Obama administration and congressional Republicanscan compromise - as they did on extending Bush-era tax cuts for all,coupled with an extension of unemployment benefits sought by thepresident.

The incoming House Ways and Means Committee chairman, Rep. DaveCamp of Michigan, favors overhauling tax laws. So does Obama.

"The tax code is longer than the Bible, but without the happyending," Camp has said. "What we need is a comprehensive reform ofthe tax code that expands the tax base and lowers rates by beingfairer, simpler and conducive to growth."

That's not too far, in theory, from Obama's desire to "simplifyconfusing provisions in the tax code, encouraging saving andcreating a tax system that works for all Americans." The challengewill be in reaching agreement on the details.

There could be times when Obama will be closer to Republicansthan to liberal Democrats, who were furious that Obama agreed tocontinue tax cuts for the wealthy - and to levy inheritance taxesonly on the very richest Americans.

Maryland Rep. Steny Hoyer, who is being replaced as the House'smajority leader, was asked by a reporter near the end of the lastCongress how much trust exits between Obama and liberals.

"On a scale of one to 10 I'm not going to give you how much," hesaid. "As you know, I'm not willing to kind of create or affirm abreach between the White House and the Congress. I think there'salways tension and there should be."

Starbucks revamps Seattle's Best Brand

Coffee chain Starbucks Corp. says it will revamp its Seattle's Best brand, giving it a new logo and expanding its availability through franchises and a big expansion at other retailers.

Starbucks plans to increase availability of the brand from 3,000 locations to 30,000 by the end of the year.

That includes a new agreement with AMC Theaters, which will serve Seattle's Best Coffee in 300 AMC Theaters nationwide beginning in July.

Seattle's Best will also be offered at all Burger King restaurants by September.

Starbucks has faced increased competition as lower-priced rivals such as McDonald's Inc. tout their own brews. But it has responded by cost-cutting and other moves and the company posted an eight-fold increase in its second-quarter profit.