Studies on the Antigenic Activities of Yeasts

In order to identify the antigenic determinant groups of the mannan of C. albicans serotype A, six kinds of manno-oligosaccharides of up to 7 units in chain-length connected by α1→2 linkages were prepared from the partial acetolysate of the parent mannan. In the precipitation-inhibition test of anti-C. albicans serotype A serum with its homologous mannan, inhibitory power of the oligosaccharides was of the following order: heptaose→: hexaose>pentaose>tetraose>triose> biose, and the amounts for 50%-inhibition of the former four oligosaccharides were 0.08, 0.10, 0.50 and 3.0 μmole respectively, and the inhibitory power of the latter two oligomers at 0.5 μmole were 8 and 5% respectively. On the other hand, the cross-inhibition test of anti-C. albicans serotype A serum with the heterologous mannan of C. albicans serotype B afforded the result that the order of inhibitory activities was hexaose>heptaose>pentaose>tetraose>triose> biose, and that the amounts for 50%-inhibition were 0.05, 0.08, 0.1, 0.45, 0.50 and 3.0μmole respectively. Furthermore, the results of inhibition test on the anti-C. albicans serotype A serum absorbed with the mannan of C. albicans serotype B revealed that the biose, triose and tetraose did not show significant inhibitory power in the range employed, whereas the pentaose, hexaose and heptaose did not significantly affect the inhibitory activities. Thus, it was concluded that the antigenic determinants of the mannan of C. albicans serotype A are α1→2 linked hexaose or heptaose moieties. Based on the above facts, the serological differences between two antigenic mannans of C. albicans serotype A and B may reside at least in the length of the antigenic determinants in which the former is longer than the latter considering the length of the α-D-manno-pyranosyl residue.     

Studies on the Antigenic Activities of Yeasts

Five antigenic mannans isolated from the cells of Candida albicans serotype A, C. albicans serotype B, C. tropicalis, C. stellatoidea and Saccharomyces cerevisiae were examined for their reactivities against concanavalin A, the size of the combining site has been estimated to be relatively small, up to 4 hexopyranosyl residues. In the quantitative precipitation reaction, all mannan-concanavalin A systems afforded nearly the same amounts of nitrogen or mannan precipitated, and the ratios of precipitation-inhibition with α1→2 linked manno-oligosaccharides, from biose to tetraose, were also equal regardless of the structural differences of these mannans. Furthermore, in agar-gel double diffusion analysis, all the systems gave a corresponding precipitation arc which completely fused with the adjacent ones. These behaviors of mannan-concanavalin A systems resemble those of antigen-antibody systems consisting of the same mannans and anti-C. albicans serotype B serum. It also provided evidence that the previous interpretation on the lesser serologic specificity of this serum compared to that of anti-C. albicans serotype A was due to the smaller size of combining sites for antibodies of the former than of the latter serum.     

Studies on the Antigenic Activities of Yeasts

In order to determine the antigenic determinant groups of the mannan of Candida albicans by the precipitation-inhibition test, several oligosaccharides were prepared by acetolysis of the polysaccharide. The manno-oligosaccharides, from biose to heptaose were separated by a charcoal-Celite chromatography and a subsequent cellulose column chromatography. The oligosaccharides thus separated were examined on the degrees of polymerization and the mode of the linkages, and evidence was obtained that the biose and triose were joined through α1→2 linkage only, while the tetraose, pentaose and hexaose contained α1→3 linkage in addition to α1→2 linkages. Heptaose was joined entirely through α1→2 linkage. In the precipitation-inhibition test, the inhibitory power of the oligosaccharides of acetolysis product was found to be the following order: hexaose>heptaose>pentaose>tetraose>triose>biose, and the amount for the 50% inhibitions were 0.025, 0.09, 0.12, 0.60, 3.96 and 5.84 μmoles respectively. On the other hand, the biose, triose and tetraose, which were isolated from the acid-hydrolysate of the mannan of Saccharomyces cerevisiae and joined through α1→6 linkage, showed poor or nearly no inhibitory power. The above facts provide an evidence that the consecutive α1→6 linkages were not located in a position that is responsible for antigenic specificity of the mannan of C. albicans.     

Studies on the Antigenic Activities of Yeasts

The antigenic mannan of Candida albicans was degraded by acid-hydrolysis and the resultant oligosaecharides were fractionated by a carbon-Celite and a subsequent cellulose-powder chromatography to yield four oligosaecharides, pentaose, hexaose, heptaose and octaose, which involved 2,6-di-0- and 6-0-substituted mannopyranosyl residues as the common. feature. These oligosaccharides showed lower precipitation-inhibition activity than that of the hexaose of acetolysate, the strongest inhibitor among the oligosaccharides described in the preceding study. The order of inhibitory powers of oligosaccharides was as follows: hexaose of acetolysate>heptaose>pentaosez=octaose>hexaose. The μmoles requiring for 50%-inhibition were 0.025, 0.15, 0.20, 0.20 and 0.50 respectively. The results clearly indicate that the determinant groups of the mannan of C. albicans employed this study are the hexaose moieties which constitute the branching parts of polysaccharide.     

Immunochemical Studies on Bacterial Blood Group Substances

Two oligosaccharides were isolated from a partial hydrolysis of the lipopolysaccharide from Shigella dysenteriae which had a common antigen with human red blood cells (RBC). A disaccharide sh-2 had a structure of O-β-D-galactosyl-(l→2)-D-galactose, and was active in the hemagglutination inhibition test between human O RBC and anti-S. dysenteriae chicken serum. A tetrasaccharide sh-12 was composed of galactose and rhamnose and was a more effective inhibitor than sh-2. Both oligosaccharides were assumed to be derived from the determinant structure of a common antigen. Serological specificity of the hemagglutinin in chicken serum was examined using these oligosaccharides and human milk oligosaccharides, and the 1→2 linked galactosyl residue was supposed to be important for determination of the heterophile RBC antigenicity.     

Detection of anti-<Emphasis Type="Italic">Candida</Emphasis> antibodies in neonates from a neonatal intensive care unit

Anti-Candida antibodies were determined in a group of preterm neonates from a neonatal intensive care unit with serious diseases including candidemia. Antibodies toC. albicans blastospores,i.e. antibodies toC. albicans surface mannan and toC. albicans germ tubes were detected. Higher titers of antibodies to blastospores (1:320) occurred in all patients examined while antibodies toC. albicans germ tubes (with the highest titer of 1:160) were present in 32 out of 66 neonates examined. The highest titers of both anti-C. albicans blastospore antibodies and anti-C. albicans germ tube antibodies were detected in neonates with candidemia and disorders of saccharide metabolism.     

Enzymatic fragmentation of carbohydrate moieties of radish arabinogalactan-protein and elucidation of the structures

We investigated the structures of L-arabino-galactooligosaccharides released from the sugar moieties of a radish arabinogalactan-protein (AGP) by the action of exo-β-(1→3)-galactanase. We detected a series of neutral β-(1→6)-linked galactooligosaccharides forming branches of one to up to at least 19 consecutive Gal groups, together with corresponding acidic derivatives terminating in 4-O-methyl-glucuronic acid (4-Me-GlcA) at the non-reducing end. Some oligosaccharide chains of degree of polymerization (dp) higher than 3 for neutral, and 4 for acidic oligomers were modified with L-Araf residues. The acidic tetrasaccharide 4-Me-β-GlcA-(1→6)[α-L-Araf-(1→3)]-β-Gal-(1→6)-Gal was detected as an abundant L-Araf-containing oligosaccharide among these neutral and acidic oligomers. A pentasaccharide containing an additional L-Araf group attached to the L-Ara in the tetrasaccharide through an α-(1→5)-linkage was also found. We observed L-arabino-galactooligosaccharides substituted with single or disaccharide L-Araf units at different Gal residues along these neutral and acidic β-(1→6)-galactooligosaccharide chains, indicating that these side chains are highly variable in length and substituted variously with L-Araf residues.     

Studies on the Antigenic Activities of Yeasts

In order to provide further information on the chemical nature of the antigenideterminants of the mannan of Saccharomyces cerevisiae, the mannan was digested by Arthrobacter α -mannosidase, and 9, 21, 35, 59 and 62%-partially degraded mannans were prepared in the present study. Acetolysis of each degraded mannan showed that only a small amount of the tetrasaccharide was detectable in the 35%-digested mannan, whereas the predominant product of the 59 and 62%-digested mannan was mannose. The result of a quantitative precipitation reaction with the degraded mannans showed that the precipitation activities were partially or completely destroyed by the action of the enzyme. The lack of the tetrasaccharide moieties of the mannan were noticeable by a decrease in the precipitating ability. It was observed that the decreasing ratio of either the maximum amount of the antibody N precipitable by the mannan or per cent degradation of the mannan were essentially equal and yielded nearly a straight relationship between 0 and 2.0 hr digestion. However, the 59 and 62%-digested mannans, containing trace amounts of di- and trisaccharides in the branching parts, showed no significant antigenic activities. Furthermore, the molar ratio of the tetrasaccharide relative to the trisaccharide also gradually decreased. These observations confirm that the tetrasaccharide moiety, Man α1→3Man α1→2Manα1→2Man, plays an important role as the antigenic determinant. The core mannan moiety completely lost both the precipitating ability and inhibitory activity in ranges employed up to 1500 μg. These findings offer a direct proof that the core mannan moiety of mannan is not responsible for antigenic activity, and functions merely as the “carrier” of the antigenic determinants which dominate the immunological specificity.     

Studies on the Antigenic Activity of Yeasts

In order to determine the determinant antigenic group of the mannan of Saccharomyces cerevisiae, a series of inhibition tests were carried out employing oligosaccharides which separated from the acetolyzate and the hydrolyzate of the mannan. Tetraose, Man α1→3 Man α1→2 Man α→2 Man², corresponding to the structure of the longer branching moieties of the mannan showed the strongest inhibition, while the isomer, Man α1→6 Man α1→6 Man, corresponding to the core moiety, produced only one-tenth the inhibition of the former. This provides evidence that the branching moieties of the mannan play important role in combining with antibody. The fact that the disaccharide, Man α→3, showed significantly stronger inhibition than those of the other disaccharides, Man α1→2 Man and Man α1→6 Man, indicates that the most important part of the determinant group of the mannan is α1→3 linked D-mannose residue. The antigenic inactivity of the periodate-oxidized mannan containing unoxidized mannose residues indicates that the presence of 3-O-substituted-D-mannose residues adjacent to the D-mannose residues and joined with α1→d2 linkages, are essential to fit the combining site of the antibody.     

Structural analysis of cell wall mannan of <Emphasis Type="Italic">Candida sojae</Emphasis>, a new yeast species isolated from defatted soybean flakes

We investigated the structural and immunochemical characteristics of cell wall mannan obtained from Candida sojae JCM 1644, which is a new yeast species isolated from defatted soybean flakes. The results of a slide-agglutination test and of an enzyme-linked immunosorbent assay using anti-factor sera to the pathogenic Candida species indicated that the cells and the C. sojae mannan were cross-reactive to the specific anti-factor sera against Candida albicans serotype A (FAb 6) and Candida guilliermondii (FAb 9). Two-dimensional homonuclear Hartmann–Hahn analysis indicated that the mannan consisted of various linked oligomannosyl side chains containing α-1,2-, α-1,3-, α-1,6- and β-1,2-linked mannose residues. However, although the determinants of antigenic factors 6 and 9 could be not found in this mannan, branched side chains, Manβ1-2Manα1-3[Manα1-6]Manα1-(2Manα1-)n2Man and a linear α-1,6-linked polymannosyl backbone, which are cross-reacted by FAbs 6 and 9, respectively, were identified. The mannan was subjected to acetolysis in order to determine the polymerization length of the α-1,2-linked oligomannosyl residue in the side chains. The result of ¹H-nuclear magnetic resonance analysis of the released oligosaccharides showed that the remarkable regularity in the length of α-1,2-linked oligomannosyl side chains, which were previously found in mannans of other Candida species, is not observed in this mannan.     

A facile and effective synthesis of alpha-(1-->6)-linked mannose di-, tri-, tetra-, hexa-, octa-, and dodecasaccharides, and beta-(1-->6)-linked glucose di-, tri-, tetra-, hexa-, and octasaccharides using sugar trichloroacetimidates as the donors and unprotected or partially protected glycosides as the acceptors

Reaction of 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl trichloroimidate with allyl alpha-D-mannopyranoside in the presence of TMSOTf selectively gave allyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranoside through an orthoester intermediate. Benzoylation of 3, followed by deallylation, and then trichloroimidation afforded the disaccharide donor 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroimidate, while benzoylation of 3 followed by selective removal of acetyl groups yielded the disaccharide acceptor allyl alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside. Coupling of 5 with 6 gave the tetrasaccharide allyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside, which were converted into the tetrasaccharide donor 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroimdate and the tetrasaccharide acceptor allyl alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside, respectively, by the same strategies as used for conversion of 3 into 5 and 6. Condensation of 5 with 13 gave the hexasaccharide 14, while condensation of 12 with 13 gave the octasaccharide 17. Dodecasaccharide 21 was obtained by the coupling of 12 with the octasaccharide acceptor 20. Similar strategies were used for the syntheses of beta-(1-->6)-linked glucose di-, tri-, tetra-, hexa-, and octamers. Deprotection of the oligosaccharides in ammonia-saturated methanol yielded the free alpha-(1-->6)-linked mannosyl and beta-(1-->6)-linked glucosyl oligomers.     

Detection of Candida sp. mannan antigen by indirect ELISA-inhibition

Summary We have obtained mannans from four Candida species: C. albicans A, C. albicans B and C. tropicalis; antimannan sera against C. albicans A, C. albicans B and C. tropicalis were obtained by immunizing rabbits sub-cutaneously with the respective yeast extract. The efficacy of these sera in reacting with mannans obtained from three Candida sp. has been proven by indirect ELISA-inhibition.Any of three immune sera can be used to detect mannan antigen from the three Candida sp. tested. This confirms the existence of crossed reactivity and the possibility of detecting mannan antigen in serum from patients infected by different Candida sp., although we had only one immune serum and one Candida mannan.     

Different effects of native Candida albicans mannan and mannan-derived oligosaccharides on antigen-stimulated lymphoproliferation in vitro

Yeast cell wall mannan polysaccharide has been proposed to contribute to immune dysfunction associated with chronic infections involving Candida albicans. This influence of mannan has been suggested based partially upon studies of the in vitro immunoinhibitory effects of mannans prepared from Saccharomyces cerevisiae or C. albicans by precipitation with Fehling's reagent, which provides a structurally modified product contaminated with copper. We have therefore evaluated the immunoinhibitory influence of a more native C. albicans mannan prepared by complexation with cetyltrimethylammonium bromide (CTAB). CTAB mannan was a potent stimulator of lymphoproliferation when added to human PMBC from donors responsive to Candida; it has no inhibitory influence on lympho-proliferation induced by Candida or other Ag. In contrast, members of a family of mannose oligosaccharides (disaccharide through hexasaccharide) derived from the CTAB mannan by weak alkaline degradation did not stimulate lymphoproliferation, but were potent inhibitors of lymphoproliferation stimulated by Candida and other Ag. Fifty percent inhibitory doses were 260 to 34 microM, respectively. Compositional analyses of these immunoinhibitory oligomers showed them to be composed of mannose and free of contaminating protein. We propose that mannan-derived oligosaccharides produced by catabolism of mannan in vivo are immunoinhibitory and contribute to the deficit in cell-mediated immune function associated with chronic candidiasis.     

Immunochemical study on the mannans of Candida albicans NIH A-207, NIH B-792, and J-1012 strains prepared by fractional precipitation with cetyltrimethylammonium bromide

The mannans of Candida albicans NIH A-207 (A strain, serotype A), C. albicans NIH B-792 (B strain, serotype B), and C. albicans J-1012 (J strain, serotype C) prepared by fractional precipitation with cetyltrimethylammonium bromide (Cetavlon) were investigated for their immunochemical properties. Upon treatment with 10 mM HCl at 100 degrees C for 60 min, the mannans of A and B strains each released a mixture of manno-oligosaccharides ranging from hexaose to mannose together with (for each one) an acid-modified mannan, while J-strain mannan released lower oligosaccharides, tetraose to mannose. The acid-modified mannan of B strain did not show antibody-precipitating activity against homologous antiserum, whereas acid-modified A- and J-strain mannans retained most of this activity. The acid-released oligosaccharides were assumed to consist of beta-1,2-linked D-mannopyranosyl residues from the results of specific rotation and proton magnetic resonance studies.     

Antigenic relationship between Candida parapsilosis and Candida albicans serotype B

We examined the antigenic relationship between Candida parapsilosis and C. albicans serotype B with respect to antigenic factors 13 and 13b, specific for the former species and common to both species, respectively. Acetolysis of C. albicans serotype B cell-wall mannan gave six oligosaccharides. Their chemical structure was determined by 1H-nuclear magnetic resonance (NMR) spectroscopy, methylation analysis, and partial acid hydrolysis. The structure of the hexasaccharide derived from C. albicans serotype B mannan was alpha-D-Manp-(1-2)-alpha-D-Manp-(1-3)-alpha-D-Manp-(1- 2)-alpha-D-Manp-(1-2)- alpha-D-Manp-(1-2)-D-Man (M6) which is identical to that from C. parapsilosis mannan. Inhibition of two precipitin reaction systems (anti-C. albicans serotype B serum and anti-C. parapsilosis serum to the respective homologous mannan), by oligosaccharides from homologous and heterologous mannans indicated that M6 from either C. albicans serotype B or C. parapsilosis was the most effective inhibitor. Moreover inhibition of the agglutination reaction between factor serum containing anti-factors 13 and 13b and C. albicans serotype B or C. parapsilosis cells by oligosaccharides from both mannans also indicated that the M6s were the most effective inhibitors. These results suggest that the M6s derived from the two species are identical in their chemical structure, although the structures of the whole mannans of the two species are not identical as demonstrated by gel diffusion precipitation patterns, and that M6s may be involved in the specificities of antigenic factors 13 and 13b. The amount of M6 is larger in C. parapsilosis cell-wall mannan, suggesting that high repeating frequency of M6 fragment may induce the antibody specific for C. parapsilosis.     

An efficient and concise synthesis of a beta-(1-->6)-linked D-galactofuranosyl hexasaccharide

A beta-(1-->6)-linked D-galactofuranosyl hexasaccharide was synthesized efficiently in a block construction manner by the well-known Schmidt glycosylation method using 6-O-acetyl-2,3,5-tri-O-benzoyl-beta-D-galactofuranosyl trichloroacetimidate (1) and allyl 2,3,5-tri-O-benzoyl-beta-D-galactofuranoside (3) as the key synthons. Coupling of 3 with 1 gave beta-(1-->6)-linked disaccharide 4. Subsequent selective deacetylation of 4 afforded the disaccharide acceptor 5, while deallylation of 4 followed by trichloroacetimidate formation produced the disaccharide donor 6. Condensation of 5 with 6 gave the tetrasaccharide 7, and subsequent deacetylation afforded the tetrasaccharide acceptor 8. Finally, coupling of 8 with 6 followed by deacylation yielded the target beta-(1-->6)-linked galactofuranose hexasaccharide 10. All of the reactions in the synthesis were carried out smoothly and in high yield.     

Multimeric bivalent immunogens from recombinant tetanus toxin H<Subscript>C</Subscript> fragment,synthetic hexasaccharides,and a glycopeptide adjuvant

Using recombinant tetanus toxin H_C fragment (rTT-H_C) as carrier, we prepared multimeric bivalent immunogens featuring the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Ogawa, in combination with either the synthetic hexasaccharide fragment of O-PS of Vibrio cholerae O:1, serotype Inaba, or a synthetic disaccharide tetrapeptide peptidoglycan fragment as adjuvant. The conjugation reaction was effected by squaric acid chemistry and monitored in virtually real time by SELDI-TOF MS. In this way, we could prepare well-defined immunogens with predictable carbohydrate–carrier ratio, whose molecular mass and the amount of each saccharide attached could be independently determined. The ability to prepare such neoglycoconjugates opens unprecedented possibilities for preparation of conjugate vaccines for bacterial diseases from synthetic carbohydrates.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

The structure of the O-glycosylically-linked oligosaccharide chains of LPG-I,a glycoprotein present in articular lubricating fraction of bovine synovial fluid

Periodate oxidation of LPG-1 established that N-acetylneuraminic acid residues are linked preponderantly α-(2→3) to D-galactose residues. The resistance of 2-acetamido-2-deoxyD-galactose residues to periodate oxidation suggests that they are linked at either O-3 or O-4 to D-galactose residues. After treatment of LPG-I with alkaline sulfite, ≈80% of 2-acetamido-2-deoxygalactose was recovered as the sulfonic acid derivative. The Gal→GalNAc disaccharide released from sialic-acid-free LPG-I by digestion with endo-2-acetamido-2-deoxy-α-D-galactosidase (which suggests an α-D-GalNAc→-L-Ser or -L-Thr linkage) gave a high color-yield in the Morgan—Elson reaction, indicating that 2-acetamido-2-deoxy-D-galactose residues are linked at C-3 to D-galactose residues. The migration of the released Gal-GalNAc disaccharide was the same as that of a standard sample of O-β-D-galactosyl-(1→3)-2-acetamido-2-deoxy-D-galactose. Treatment of sialic acid-free LPG-I with Streptococcus pneumoniae β-D-galactosidase, which hydrolyzes only galactosides linked β-D-(1→4) gave no free D-galactose, whereas treatment of LPG-I with bovine testes β-D-galactosidase released > 90% of D-galactose. These results provide evidence for β-D-Galp-(1→3)-α-D-GalNAcp-(1→3)-L-Ser or -L-Thr and α-NeuAc-(2→3)-β-D-Galp-(1→3)-α-D- GalNAcp-(1→3)-L-Ser or -L-Thr structures. The sensitivity of the methods used and the recovery of constituents following treatment of LPG-I do not rule out the occurrence of small amounts of other tri- or tetra-saccharide chains.     

Biochemical and Immunochemical Studies of Fungi

Crude mannans extracted from Candida albicans and Saccharomyces cerevisiae by autoclaving yeast cells in citrate buffer (pH 7.0) according to Peat's method, were fractionated repeatedly by column chromatography on DEAE-Sephadex, acetate form, yielding neutral and acidic mannans. The former fraction showed a single peak by boundary electrophoresis and ultracentrifugal analysis, while the latter contained small amounts of phosphorus and protein. Using purified mannans as controls, various serological experiments were carried out with mannan antigens extracted from C. albicans with 45% phenol water and with 3% NaOH. No remarkable differences were observed in the antigenic activity of 4 mannan antigens from C. albicans, and the purified mannan exhibited very high antigenic activity. It was found that the mannan of S. cerevisiae was antigenically less specific than that of C. albicans mannan. The difference in serological specificity between mannans of both species may reflect not only differences in mannopyranose linkages but differences in the structure of the macromolecules.