Vol. 55, No. 3INFECTION AND IMMUNITY, Mar. 1987, p. 616-6200019-9567/87/030616-05$02.00/0Copyright © 1987, American Society for Microbiology

Antigenic Differences between Mannoproteins of Germ Tubes andBlastospores of Candida albicans

PAULA M. SUNDSTROM,t EVERETT J. NICHOLS, AND GEORGE E. KENNY*Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle,

Washington 98195

Received 18 August 1986/Accepted 1 December 1986

To determine the nature of germ tube-specific antigens of Candida albicans, procedures for intrinsicallylabeling cell wall antigens metabolically were developed. Blastospores or germ tubes labeled either in theirproteins with L-[35S]methionine or in mannose-containing carbohydrates with D[2-3H]mannose containedsurface components similar to those found previously with 125I-labeled organisms. Germ tube-specificdeterminants, were found on a 200-kilodalton protein in digests from germ tubes, whereas a component ofsimilar molecular size in blastospore extracts reacted weakly or not at all with germ tube-specific antibody. Inaddition, a glycan fraction prepared from germ tubes reacted with the unadsorbed anti-C. albicans polyvalentantibody but not with the germ tube-specific antibody, suggesting that the germ tube-specific determinants arenot carbohydrates.

Our previous results comparing 1251I-labeled blastosporeand germ tube cell wall digests strongly suggested thatmannoprotein cell wall molecules contained germ tube-specific determinants (22), which may be responsible for thesurface differences between blastospores and germ tubesseen by immunofluorescence. However, questions about thechemical differences between blastospores and germ tubesremained unanswered. In addition, a criticism of studieswith 125I-labeled material is that protein structures may bealtered in the labeling procedure, leading to spurious results.In the present study, we radiolabeled cell wall antigensmetabolically to identify and characterize the germ tube-specific antigens in cell wall mannoproteins. In addition, aglycan fraction from cell walls of metabolically labeled germtubes was prepared and found to be precipitated by theanti-Candida albicans serum but not by the germ tube-specific antiserum. This strongly suggests that germ tube-specific determinants are not carbohydrates, but most likelyproteinaceous in nature.

MATERIALS AND METHODSOrganisms and culture. C. albicans 441B and B311 were

obtained from K. J. Kwon-Chung, National Institutes ofHealth, and maintained as described previously (21).

Antisera. Rabbit antiserum to blastospores bearing germtubes of C. albicans 441B was prepared as described previ-ously (21). Germ-tube-specific antiserum was prepared byadsorption of this antiserum with C. albicans blastospores aspreviously described (21).

Radiolabeling. A homogeneous population of blastosporesin the stationary phase was prepared by inoculating a loopfulof organisms from a Sabouraud agar slant into 100 ml ofmodified Lee medium without amino acids and with sucroseinstead of glucose, containing (in grams per liter):(NH4)2SO4, 5; MgSO4- 7H20, 0.2; K2HPO4, 2.5; NaCl, 5;sucrose, 10; and biotin, 0.04. This mixture was incubated on

* Corresponding author.t Present address: Department of Microbiology and Molecular

Genetics, College of Medicine, University of California, Irvine,Irvine, CA 92717.

a gyrorotary shaker (New Brunswick Scientific Co., Inc.,Edison, N.J.) at 200 rpm for 48 h at 25°C. Organisms werecentrifuged and washed twice in 0.15 M NaCl containing 10mM TES [N-tris (hydroxymethyl)methyl-2-aminoethane-sulfonic acid, pH 7.4] (TES-saline) and held at room temper-ature overnight. For radiolabeling of germ tubes, washedblastospores (8 x 106/ml) were suspended in the abovemedium containing 1% bovine serum albumin (Sigma Chem-ical Co., St. Louis, Mo.) and 5.5 ,uCi of L-[35S]methionineper ml in tricine buffer (1,126 Ci/mmol; New England Nu-clear Corp., Boston, Mass.) or 11 ,uCi of D-[2-3H]mannoseper ml (22 Ci/mmol) (ICN Radiochemicals, Irvine, Calif.) (1,uCi = 37 kBq). When radiolabeled mannose was used, coldmethionine (0.01 g/liter) was added. Organisms in labelingmedium were incubated at 37°C for 4 h until germ tubes twoto three times the length of the blastospore diameter hadformed. Over 95% of organisms produced germ tubes underthese labeling conditions. Pseudohyphae were not produced.Labeled blastospores were prepared in the same medium butwere inoculated at 107/ml and incubated at 25°C with shakingfor approximately 7 h. Under these conditions, blastosporesincorporated approximately 90% and germ tubes 80% of theradioisotopes.

Cell digests. Cells were digested enzymatically as in aprevious study (22), except that the time of Zymolasedigestion was reduced from 2.5 h to 45 min and the digestwas ultracentrifuged at 25,000 x g for 40 min, the resultingsupernatant being stored at -70°C. In general, 10 to 20% ofthe radioisotope was recovered in cell wall digests. Incorpo-ration of L-[35S]methionine per ,ug of protein (15) was 53,000and 120,000 cpm for labeled blastospores and germ tubes,respectively, and 23,000 and 40,000 cpm for D-[2-3H]man-nose-labeled blastospores and germ tubes, respectively.Radioimmunoprecipitation was done as described previ-ously (22) except that 5 x 106 cpm of blastospore cell walldigest and 3 x 106 cpm of germ tube digest were used perimmunoprecipitation.SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel

electrophoresis (SDS-PAGE) was done as described previ-ously (13, 22), except that all experiments were done with10% gels and approximately 15,000 cpm per lane was used.

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2t '

200K -97.4-

68 -

43- -

3 4

3

25.7-

i84-

7 85 6 lo «f-

i -200 "^'-200. -97.4

- - - 97.4 _-3_68-68 -

- -43

-43-25.7

-18.4

-184

Antigen -* B GBG BG 81 BG B

Antibody -* bg g bg g g goFIG. 1. SDS-PAGE electrophorograms of L-[355]methionine-

labeled cell wall Zymolase digests from blastospores (B) (5 x 106cpm) (lanes 1, 2, and 8) or germ tubes (BG) (3 x 106 cpm) (lanes 3,4, 5, 6, and 7) of C. albicans 441B were immunoprecipitated witheither unadsorbed anti-C. albicans antibody (bg) (5 ,ul) (lanes 1 and3), germ tube-specific antibody (g) (10 ,lI) (lanes 2, 4, and 5), or germtube-specific antibody which had been adsorbed with germ tubes(ga) (10 pA) (lane 6) before electrophoresis. Lanes 7 and 8 are cellwall digests not precipitated with antibody. Sizes are indicated (inkilodaltons).

Gels were treated with En3Hance (New England Nuclear) asdescribed by the manufacturer, dried, placed next to KodakXRP-1 film (Eastman Kodak, Rochester, N.Y.), and incu-bated at -70°C for 3 to 5 days before being developed.

Endoglycosidase H digestion of immunoprecipitates. A pro-cedure similar to that of Anderson et al. (1) was followed forendoglycanosidase H digestion of immunoprecipitates. Im-munoprecipitates were given an additional wash in 50 mMsodium citrate buffer, pH 5.6, and suspended in 20 ,ul ofcitrate buffer with or without 0.3 U of endoglycosidase H(Miles Scientific, Naperville, Ill.) per ml and incubatedovernight at 37°C. Suspensions were boiled after the additionof 2x SDS sample buffer and electrophoresed as usual (13).

Proteolytic digestion of cell wall digest. To obtain im-munoreactive cell wall carbohydrates free from proteins, aZymolase digest of [2-3H]mannose-labeled germ tubes wasexhaustively degraded with Pronase (Calbiochem, SanDiego, Calif.) by a method similar to that described by Arimaet al. (2). Pronase, dissolved in 0.15 M Tris acetate buffer,pH 7.8, containing 15 mM CaC12 was preincubated at 60°Cfor 30 min to destroy any glycosidic enzymes and added (1mg/ml final concentration) to 1 ml of a [2-3H]mannose-labeled Zymolase cell wall digest of germ tubes (strain441B). This mixture was incubated overnight at 37°C withconstant stirring, followed by two additions of fresh pronaseat 24 and 48 h, resulting in a total digestion time of 72 h, andthen lyophilized.

Gel ifitration of pronase digest. To separate free aminoacids from carbohydrates, the pronase digest was dissolvedin 0.5 ml of 0.1 M pyridine acetate buffer, pH 5.0, andfractionated on Biogel P-6 (Bio-Rad Laboratories, Rich-mond, Calif.) in a column 1 in. (ca 2.54 cm) in diameter andpacked to a height of 60 in. (ca. 152.4 cm). Elution wasachieved with the 0.1 M pyridine acetate buffer at a rate of0.2 ml/min. Fractions (2.2 ml) were collected, counted forradioactivity, and analyzed by the fluorescamine assay (24)for protein and the orcinol assay for carbohydrate. Thecarbohydrate-containing fractions which contained minimalamino acids were pooled and termed the glycan fractions.

RESULTS

Identification of germ tube-specific antigens in L-[35S]methi-onine-labeled C. albicans 441B. When Zymolase digests ofblastospores or germ tubes from strain 441B which had beenlabeled with L-[35S]methionine were reacted with unad-sorbed, polyvalent serum raised against strain 441B, approx-imately 12 proteins were precipitated (Fig. 1, lanes 1 and 3),representing about one-third of the labeled proteins detectedin unprecipitated digests (Fig. 1, lanes 7 and 8). A compo-nent which comigrated with the 200-kilodalton (kDa) markerwas the most strongly precipitated component from bothdigests, although it appeared heavier and broader in germ.ube digests (a result consistently seen in the 10 experimentsconducted). Germ tube-specific antiserum (antiserum whichhad been exhaustively adsorbed with intact blastospores[21]) reacted strongly with the 200-kDa component fromgerm tubes (the upper part of the band was usually precipi-tated), but weakly with this component from blastospores(Fig. 1, lanes 2 and 4). This indicated that the 200-kDacomponent from blastospores is not only located on the cellsurface, but is also immunologically different from that ofgerm tubes. Attempts to further resolve the 200-kDa com-ponent from germ tube digests by using different percentagegels resulted in a smear (not shown). The 200-kDa compo-nent from germ tubes was also located on the surface, asshown by its inability to be precipitated by germ tube-specific antiserum which had been adsorbed with germ tubes(Fig. 1, lane 6). Most of the other precipitated componentswere present in the wall but not on the surface, as shown bytheir ability to react with both adsorbed and unadsorbedantiserum. A weakly labeled component of approximately 32kDa appeared to be surface associated because it did notreact with blastospore-adsorbed serum.

Results with Zymolase digests of strain B311 were similarto those with strain 441B in that a 200-kDa surface compo-nent from both digests was precipitated with the unadsorbedserum (Fig. 2, lanes 3 and 5), but only the component fromgerm tube digests reacted strongly with the germ tube-

| 2 3 4 5 6

*4 * -i200K

- 97.4-68

43

- 25.7

-18.4

Antigen-B BG

Antibody

B B BG BG

bg g bg g

FIG. 2. SDS-PAGE electrophorograms of L-[355]methionine-labeled cell Zymolase digests from blastospores (B) (5 x 106 cpm)(lanes 1, 3, and 4) or germ tubes (BG) (3 x 106 cpm) (lanes 2, 5, and6) of C. albicans B311 were immunoprecipitated with either unad-sorbed anti-C. albicans antibody (bg) (5 ,ul) (lanes 3 and 5) orgerm-tube-specific antibody (g) (10 ,ul) (lanes 4 and 6). Sizes areindicated (in kilodaltons).

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1 2 3 4

U * I .4 - 200

-97.4-68

- 43

-25.7

2 3 4

200K - U_ b97.4 - _ __

68 -

423-

25.7 -

184 --18.4

Antigen -- B B BG BG

Antibody -- bg 9 bg 9

FIG. 3. SDS-PAGE electrophorograms of D-[2-3H]mannose-labeled cell wall Zymolase digests from blastospores (B) (5 x 106cpm) (lanes 1 and 2) or germ tubes (BG) (3 x 106 cpm) (lanes 3 and4) of C. albicans 441B were immunoprecipitated with either unad-sorbed anti-C. albicans antibody (bg) (5 [LI) (lanes 1 and 3) or germtube-specific antibody (g) (10 ,ul) (lanes 2 and 4). Sizes are indicated(in kilodaltons).

Antigen-B B BG BSAntibody-bg bg g gEndo H - + -

FIG. 5. Endoglycosidase H treatment of immunoprecipitates.L-[35S]methionine-labeled cell wall Zymolase digests from blasto-spores (B) precipitated with the unadsorbed anti-C. albicans anti-body (bg) (lanes 1 and 2) or germ tubes (BG) precipitated with germtube-specific antibody (g) (lanes 3 and 4) were incubated overnight at37°C with (lanes 1 and 3) or without (lanes 2 and 4) endoglycosidaseH (Endo H) treatment. Sizes are indicated (in kilodaltons).

specific antibody (Fig. 2, lanes 4 and 6). In addition, acomponent of approximately 155 kDa which reacted with thegerm tube-specific antibody was found in digests from germtubes but not from blastospores (Fig. 2). Also, the upad-sorbed antibody precipitated a very high molecular weight,weakly labeled component from both blastospore and germtube digests which did not react with germ tube-specificantibody (Fig. 2, lanes 3 and 5). This very high molecularweight component was also seen in strain 441B in someexperiments. In all cases, no labeled components wereprecipitated in control experiments without antibody (Fig. 2,lanes 1 and 2).

Identification of germ tube-specific antigens in D-[2-3HJman-nose-labeled C. albicans 441B. When organisms were labeledwith [2-3H]mannose under conditions in which the label isincorporated into mannoprotein (10), the 200-kDa compo-nent appeared prominently labeled while other internal com-ponents were not labeled (Fig. 3). As with L-[35S]methionine-labeled digests, the 200-kDa component from germ tubesreacted with the germ tube-specific antiserum while theblastospore component did not (Fig. 2, lanes 2 and 4). Inaddition, faint components at approximately 100 kDa weredetected which had not been seen before.

Endoglycosidase treatment of immunoprecipitates. To com-pare carbohydrate structures on the 200-kDa componentsfrom blastospores and germ tubes, washed L-[35S]methio-nine-labeled immunoprecipitates bound to Formalin-fixedstaphylococci bearing protein A were treated with endogly-

Man a 1 2 Man a 1_&Man a 1

,6/A

Man aI3

Man a I-- 2 Man a

A

Manp1-_ 4 GlcNAc II---* 4 GIcNAc-ASN

I~~~~~~~~~~~~~~~~~~~~~~~Site of Endo H

Cle4vage

FIG. 4. Schematic diagram showing the location of the

Endoglycosidase H (Endo H) cleavage site in glycoproteins fromSaccharomyces cerevisiae and other organisms (19). Abbreviations:Man, Mannose; GlcNAc, N-acetylglucosamine; ASN, asparagine.

cosidase H, an enzyme which cleaves N-linked high-mannose oligosaccharides from glycoproteins (19) (Fig. 4).Precipitates of the blastospore digest and unadsorbed anti-body were compared with those of the germ tube digest andthe germ-tube-specific antibody for endoglycosidase H sen-sitivity. The 200-kDa component from germ tube digests butnot from blastospore digests was sensitive to enzyme diges-tion, indicating that the germ tube component but not theblastospore component has exposed N-linked high-mannosecarbohydrate cleavage sites (Fig. 5).

Preparation of glycan fractions. Gel filtration of the prote-ase-degraded cell wall digests resulted in two peaks contain-ing [2-3H]mannose-labeled carbohydrates, eluted just afterthe void volume, which were well separated from the aminoacid-containing peak (Fig. 6). Those two peaks contained

vi

100 . + + orcinolstain 25

80 20

70 I

60 Il1

50 104020~~~~~~~~~I1

30

10..

I

0 20 40 60 80 100 120 140Volume (ml)

FIG. 6. Gel filtration on Biogel p-6 of pronase-degraded,[3H]mannose-labeled cell wall digest of C. albicans 441B germtubes. The column (1 by 60 in.) was equilibrated and eluted with 0.1M pyridine acetate buffer, pH 5.0. The elution diagram showscarbohydrate, as detected by cpm of [3H]mannose (left-hand scale[kcpm] and solid line) and the orcinol assay (positive fractions areindicated by +), and peptide as detected by the fluorescaminemethod (right-hand scale [relative fluorescence (24)] and dashedline). VO, Void volume; V1, inclusion volume. Fractions designatedby the bars were pooled to obtain glycan peaks I and II.

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TABLE 1. Radioimmunoprecipitation of anti-C. albicans andgerm tube-specific antibody and oligosaccharide fractions

Immunoprecipitation (cpm) with antibody:

Glycan peak BlastosporeI antigen (p.g Unadsorbed Blastospore and germ None

[dry wt]) (5 IL) adsorbed tube(5i.1) (10 ~i1) adsorbed

(10 ii1)

1 1,460 88 76 NDa10 5,575 104 102 ND30 7,048 113 123 49

a ND, Not done.

only small amounts of amino acid, as shown by their lack ofreaction in the fluorescamine assay, and thus are referred toas glycan peaks I and II. Of the radiolabel placed on thecolumn, 85% was contained in peaks I and II.

Antibody binding of the glycan fractions. The unadsorbedantibody reacted strongly with glycan in peak I comparedwith controls consisting of assays without antibody (Table 1)or assays with preimmune serum or rabbit antiserum tobovine serum albumin (not shown) instead of anti-C. albi-cans serum. In contrast, the germ tube-specific serum re-

acted no better than germ tube-specific serum which hadbeen adsorbed with germ tubes, indicating that germ tube-specific epitopes were not present in the glycan fraction.None of the antibodies reacted with peak II glycan (notshown).

DISCUSSION

The presence of surface cell wall components found withmetabolic labeling in this study confirms the results obtainedpreviously for digests from cells surface-labeled with 1251(22) in that germ tube-specific components were found toreside on 200- and 155-kDa components isolated form germtubes but not from blastospores. The finding of theseepitopes on components of similar size by three differentlabeling methods considerably strengthens the argument thatthese components are responsible for germ tube-specificreactivity and are specific for filamentous growth. The recentdiscovery of heat shock proteins in C. albicans (6) raises thepossibility that the germ tube-specific epitopes are merelyassociated with a temperature change and not with the germtube form of growth. This is unlikely, however, since ourearlier studies showed that blastospores grown at 370C didnot react with the germ-tube-specific antisera by immunoflu-orescence (21).A minor difference between the results was noted in the

surface components seen with metabolic labeling comparedwith 1251 surface labeling. In 125I surface labeling (22), themajor surface component from blastospores had a highermolecular weight than this component produced under met-abolic labeling conditions. With 1251 labeling, this componentbarely migrated in SDS-PAGE gels, whereas it comigratedwith the 200-kDa molecular size marker with metaboliclabeling conditions. Importantly, this major blastosporecomponent did not react with the germ-tube-specific anti-body under any conditions used for radiolabeling. It ispossible that differences seen in the molecular weights ofwall mannoproteins after Zymolase cleavage might havebeen the result of the different growth media used. One studyon cell wall mannoproteins of Saccharomyces cerevisiaedetected additional mannoproteins when cells were grown inmedium containing sucrose as the carbon source rather than

glucose (11). We used sucrose in the metabolic labelingmedia and glucose in the growth media for cells labeled with1251I. It is interesting that the mannoprotein we detected wasmetabolically labeled with L-[35S]rnethionine, since all deter-minations of amino acid composition of mannoprotein iso-lated by chemical methods from cell walls of C. albicans orS. cerevisiae have noted the absence of sulfur-containingamino acids (7, 11, 12). The reasons for this are unknown,but perhaps such amino acids are destroyed or lost by theextraction procedures used.Few other studies have examined mannoproteins from C.

albicans cell walls by SDS-PAGE. One study (4) used SDSand heat to extract proteins from isolated cell walls of strainB311, one of the strains which we used, and detectedmaterial at the top of the gels (10 to 11.5%) which stainedwith the periodic acid-Schiff reagent but not with Coomassieblue. We noticed similar results with the mannoproteins inour studies (not shown). Components with similar propertieswere found when dithiothreitol was used to extract cell wallproteins of the same strain (18), and in this case a mycelium-specific component was detected on 7% gels which had anestimated molecular size of 235 to 250 kDa. The differencesin molecular weight between their component and ours couldbe explained by different extraction procedures or by dif-ferent media used to grow the organisms. Zymolase diges-tion of isolated walls from a different strain (9) released apolydisperse material with an apparent molecular massgreater than 150 kDa which bound concanavalin A and didnot stain with Coomassie blue. Zymolase digestion of cellwalls from S. cerevisiae also releases mannoproteins of highmolecular weight, although different investigators havefound different protein profiles (3, 11, 16, 17, 20).

Binding of the glycan fraction, which is essentially aminoacid free as indicated by the fluorescamine assay, by anti-C.albicans antibody and not by preimmune serum or rabbitantibody to bovine serum albumin strongly suggests thatsome of the antibodies present in the anti-C. albicanspolyvalent serum bind to the mannosaccharide portion ofmannoprotein. In addition, germ tube-specific antibody didnot bind this fraction, suggesting that germ tube-specificdeterminants are protein and are degraded by pronasedigestion. This result is supported by our earlier immunoflu-orescence (21) studies, showing that germ tube-specificdeterminants on whole cells were sensitive to heat andchymotrypsin, whereas common determinants were resist-ant to these treatments. Thus it appears that germ tube-specific epitopes are not the result of modification of apreexisting carbohydrate structure, but are the result ofnewly exposed protein determinants.The lack of binding of the germ tube-specific antibody to

the glycan fraction from germ tubes suggests that blasto-spores and germ tubes have similar surface carbohydratestructures. To compare carbohydrates, immunoprecipitatesfrom the two forms were treated with endoglycosidase H, anenzyme which cleaves asparagine-linked high-mannoseoligosaccharides from glycoproteins. Although the germtube 200-kDa component was decreased in molecular size,indicating the presence of asparagine-linked carbohydrates,it is unlikely that blastospore mannoprotein lacksendoglycosidase H-sensitive clevage sites, as evidenced bythe fact that mannosaccharides on mannoprotein from S.cerevisiae are approximately half asparagine-linked and halfserine- or threonine-linked (11). It is more likely that theblastospore endoglycosidase H sites were not exposed inthis experiment. These sites were possibly hidden by theway the protein is folded, as in the case for some of the

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endoglycosidase H sites in invertase and carboxypeptidaseY, which are only exposed after SDS denaturation of theprotein (23). However SDS pretreatment did not render theblastospore 200-kDa component sensitive to endogly-cosidase H (not shown). Alternatively, endoglycosidase Hsites could have been obscured by the antibody in theimmunoprecipitate, just as antibodies can protect antigencleavage by proteases if the protease site is obscured byantibody binding (7). Therefore, the difference in endogly-cosidase H sensitivity was a result of different carbohydratestructures or spatial differences in antibody binding to thetwo components and is probably not responsible for theimmunological differences between the 200-kDa componentsof the two forms. However, differences in carbohydratecompositions of the two growth phases should not be com-pletely ruled out. Characterization of mannoproteins fromthe yeast and mycelial forms of Mucor rouxii has revealeddifferences in both carbohydrate composition and linkagebetween the two growth forms (25), although the influence ofcarbohydrates on surface antigenic differences is unknown.To compare peptide portions of mannoproteins, gel pieces

containing these components were cut out of untreated gelsand rerun in the presence of staphylococcal V8 protease asdescribed by Cleveland et al. (5). Even after 2 h of in situdigestion with 30 ,ug of protease per sample, the quantity offragments resulting from degradation of mannoproteins ob-served was so small that very long exposure times (10 daysor more) were needed for detection. Although several frag-ments were similar between the two components, one frag-ment was released from the germ tube component and notfrom the blastospore component, indicating a possible aminoacid difference between them (not shown). Although man-noprotein of S. cerevisiae is sensitive to digestion withpronase, it is resistant to digestion with specific proteases(11).

Although other studies have detected mannoproteins fromisolated cell walls (4, 9), we have shown that this material isstrongly precipitated by antibodies which react with the cellsurface and that it possesses epitopes specific for germtubes. In addition, we have radiolabeled this material byboth metabolic and cell surface labeling methods. Furtherpurification of this material would allow compositional stud-ies which would provide a better understanding of theimmunogenic surface of C. albicans.

ACKNOWLEDGMENTS

This study was supported in part by Public Health Service grantsRR-051407 and DE-02600 from the National Institutes of Health.

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