Detection of endogenous and food-derived collagen dipeptide prolylhydroxyproline (Pro-Hyp) in allergic contact dermatitis-affected mouse ear

Generation of collagen dipeptides and deposition of orally administered prolylhydroxyproline (Pro-Hyp) in local inflammatory sites were examined in mice with hapten (2,4-dinitrofluorobenzene)-induced dermatitis in the ear. Pro-Hyp content in the hapten-treated ear was significantly higher in the chronic phase of contact dermatitis than the vehicle control. In contrast, hydroxyprolylglycine contents remained at lower levels in all cases compared to Pro-Hyp. Four hours after the ingestion of [¹³C₅,¹⁵N]Pro and [¹³C₅,¹⁵N]Pro-Hyp, labeled-Pro-Hyp and Pro, respectively, appeared only in the ear with dermatitis. Thus, Pro-Hyp is generated and degraded as part of the rapid synthesis and degradation of collagen in the ear with dermatitis. In addition to the endogenously generated Pro-Hyp, the orally administered Pro-Hyp was deposited in the ears.     

13C n.m.r. studies of the carbohydrate portion of glucoamylase from Aspergillus oryzae

Proton-decoupled, natural abundance ¹³C n.m.r. spectroscopy was used to investigate the carbohydrate structure and content of glucoamylase from Aspergillus oryzae. We found α-d-mannopyranose was the dominant sugar present (⋍91 residues). The Elson-Morgan assay showed that hexosamine was also present as a minor component (2.6% of the total carbohydrate). The intermannose linkages appear to be random. Integration data suggest that 41 α-d-mannopyranose residues are O-2 and O-3 glycosylated and 17 α-d-mannopyranose residues are involved in O-4 glycosylation. Treatment of glucoamylase with α-mannosidase appeared to remove all the carbohydrate residues present.     

Chemical Taxonomy of Economically Important Tyrophagus Mites (Acariformes,Acaridae)

Rosefuran and perillene were isolated from the opisthonotal glands secretion of the Tyrophagus neiswanderi mite. A facile synthesis of the former was accomplished by the lithiation of 3-methyl- furan with butyllithium-tetramethylethylenediamine, and a subsequent reaction with 3-methyl-2- butenylbromide. A mixture of rosefuran, 4-methyl-2-(3-methyl-2-butenyl)-furan and 3-methyl-2,5-bis- (3-methyl-2-butenyl)-furan was obtained in a high yield. The occurrence of these and other recently identified monoterpenoids, namely, α- and β-acaridial and 2,3-epoxyneral, is discussed relative to the CP-Sil 19 CB column profiles as worthy for the identification of economically important mites of the genus Tyrophagus, which is a difficult but essential task for the successful application of the species-specific alarm pheromones in the management of these pests.     

Bacillus thuringiensis Cry4Aa insecticidal protein: Functional importance of the intrinsic stability of the unique α4–α5 loop comprising the Pro-rich sequence

The long loop connecting transmembrane α4 and α5 of the Bacillus thuringiensis Cry4Aa toxin possesses a unique feature with Pro-rich sequence (Pro¹⁹³Pro¹⁹⁴_Pro¹⁹⁶) which was shown to be crucial for toxicity. Here, the structural role in the intrinsic stability of the Pro-rich sequence toward toxin activity was investigated. Three Val-substituted mutants (P193V, P194V and P196V) and one Phe-substituted mutant (P193F) were generated and over-expressed in Escherichia coli as inclusions at levels equal to the wild-type. Bioassays demonstrated that all mutants, particularly P193V and P193F whose inclusions were hardly soluble in carbonate buffer (pH 9.0), exhibited reduced toxicity, suggesting an essential role in toxin function by the specific cyclic structure of individual Pro residues. Analysis of the 65-kDa Cry4Aa structure from 10-ns molecular dynamics (MD) simulations revealed that the α4–α5 loop is substantially stable as it showed low structural fluctuation with a 1.2-Å RMSF value. When the flexibility of the α4–α5 loop was increased through P193G, P194G and P196G substitutions, decreased toxicity was also observed for all mutants, mostly for the P193G mutant with low alkali-solubility, suggesting a functional importance of loop-rigidity attributed by individual Pro-cyclic side-chains, particularly Pro¹⁹³. Further MD simulations revealed that the most critical residue−Pro¹⁹³ for which mutations vastly affect toxin solubility and larval toxicity is in close contact with several surrounding residues, thus playing an additional role in the structural arrangement of the Cry4Aa toxin molecule. Altogether, our data signify that the intrinsic stability of the unique Cry4Aa α4–α5 loop structure comprising the Pro-rich sequence plays an important role in toxin activity.     

Lipopolysaccharide of Pseudomonas solanacearum

A novel monoterpene, 2(E)-(4-methyl-3-pentenyl)butenedial (α-acaridial (1) for the trivial name), was isolated from the secretion of the acarid mite Tyrophagus perniciosus. The structure was clarified in the light of spectral data, and the geometry of the double bond in the butenedial moiety was assigned based on the γ-deshielding effect on a methylene and on an aldehyde group. Coupling the Grignard reagent (CH₃)₂C =CHCH₂CH₂MgBr to THP-OCH₂(C = O)CH₂CH₂O-THP, and dehydration and deprotection of the OH groups gave α-(E)- and α-(Z)-acaridiol, which were fully assigned byMS and NMR. Matching the spectral data of the synthetic alcohols with those of the alcohol derived from the natural product, or of natural acaridial with those of synthetic α-(E)- and α-(Z)-acaridial, corroborated beyond all doubt the structure of this new monoterpene dial.     

His180 in the pore-lining α4 of the Bacillus thuringiensis Cry4Aa δ-endotoxin is crucial for structural arrangements of the α4-α5 transmembrane hairpin and hence biotoxicity

One proposed toxic mechanism of Bacillus thuringiensis Cry δ-endotoxins involves pore formation in target membranes by the α4-α5 transmembrane hairpin constituting their pore-forming domain. Here, nine selected charged and uncharged polar residues in the pore-lining α4 of the Cry4Aa mosquito-active toxin were substituted with Ala. All mutant toxins, i.e., D169A, R171A, Q173A, H178A, Y179A, H180A, Q182A, N183A and E187A, were over-expressed in Escherichia coli as 130-kDa protoxin inclusions at levels comparable to the wild-type toxin. Bioassays against Aedes aegypti larvae revealed that only H178A and H180A mutants displayed a drastic reduction in biotoxicity, albeit almost complete insolubility observed for H178A, but not for H180A inclusions. Further mutagenic analysis showed that replacements of His¹⁸⁰ with charged (Arg, Lys, Asp, Glu), small uncharged polar (Ser, Cys) or small non-polar (Gly, Val) residues severely impaired the biotoxicity, unlike substitutions with relatively large uncharged (Asn, Gln, Leu) or aromatic (Phe, Tyr, Trp) residues. Similar to the trypsin-activated wild-type toxin, both bio-active and -inactive H180 mutants were still capable of releasing entrapped calcein from lipid vesicles and producing cation-selective channels with ~130-pS maximum conductance. Analysis of the Cry4Aa structure revealed the existence of a hydrophobic cavity near the critical His¹⁸⁰ side-chain. Analysis of simulated structures revealed that His¹⁸⁰-to-smaller residue conversions create a gap disrupting such cavity's hydrophobicity and hence structural arrangements of the α4-α5 hairpin. Altogether, our data disclose a critical involvement in Cry4Aa-biotoxicity of His¹⁸⁰ exclusively present in the lumen-facing α4 for providing proper environment for the α4-α5 hairpin prior to membrane-inserted pore formation.     

The inhibitory effect of naringenin on atopic dermatitis induced by DNFB in NC/Nga mice

Aims

Atopic dermatitis (AD) is a chronic and relapsing inflammatory dermatitis characterized by pruritic and eczematous skin lesions. Here, we investigated the therapeutic effect of the fruit flavonoid naringenin on DNFB induced atopic dermatitis mice model.

Main methods

AD-like skin lesion was induced by repetitive skin contact with DNFB in NC/Nga mice and the effects of the fruit flavonoid naringenin were evaluated on the basis of histopathological findings of skin, ear swelling and cytokine production of CD4⁺T cells.

Key findings

Intraperitoneal injection of naringenin for one week after DNFB challenge significantly lowered ear swelling and improved back skin lesions. In addition, naringenin significantly suppressed production of interferon-gamma (IFN-γ) by activated CD4⁺ T cells and serum IgE level. Furthermore, naringenin reduced DNFB-induced infiltration of eosinophils, mast cells, CD4⁺ T cells, and CD8⁺ T cells in skin lesions.

Significance

Naringenin may suppress the development of AD-like skin lesions in DNFB-treated NC/Nga mice by reducing IFN-γ production of activated CD4⁺ T cells, serum IgE levels and infiltration of immune cells to skin lesion.     

Molecular structure of the alpha-lytic protease from Myxobacter 495 at 2.8 Angstroms resolution.

The α-lytic protease was isolated from an extracellular filtrate of the soil microorganism Myxobacter 495. Trigonal crystals (space group, P3₂21) of this serine enzyme were grown from 1·3 m-Li₂SO₄ at pH 7·2. X-ray reflections from crystals of the native enzyme, comprising the 2·8 Å limiting sphere, were phased by the multiple isomorphous replacement technique. Five heavy-atom derivatives were used and the overall mean figure of merit 〈m?〉 is 0·83. The resulting native electron density map of α-lytic protease has been interpreted in conjunction with the published sequence (Olson et al., 1970) of 198 amino-acid residues.α-Lytic protease has a structural core similar to that of the pancreatic serine proteases (108 α-carbon atom positions are topologically equivalent (within 2·0 Å) to residues of porcine elastase) and its tertiary structure is even more closely related to the two other bacterial serine protease structures previously determined (James et al., 1978; Brayer et al., 1978b; Delbaere et al., 1979a). α-Lytic protease has the following distinctive features in common with the bacterial serine enzymes, Streptomyces griseus proteases A and B: an amino terminus that is exposed to solvent on the enzyme surface, a considerably shortened uranyl loop (residues 65 to 84), a major segment of polypeptide chain from the autolysis loop deleted (residues 144 to 155), a buried guanidinium group of Arg138 in an ion-pair bond with Asp194, and an altered conformation of the methionine loop (residues 168 to 182) relative to the pancreatic enzymes.At the present resolution, the members of the catalytic quartet (Ser214, Asp102, His57 and Ser195) adopt the conformation found in all members of the Gly-Asp-Ser-Gly-Gly serine protease family. The carboxylate of Asp102 is in a highly polar environment, as it is the recipient of four hydrogen bonds. The interaction between the N^ε2 atom of the imidazole ring in His57 and O^γ atom of Ser195 is very weak (3·3 Å) and supports the concept that there is little, if any, enhanced nucleophilicity of the side-chain of Ser195 in the native enzyme.The molecular basis for the observed substrate specificity of α-lytic protease is clear from the distribution of amino acid side-chains in the neighborhood of the active site. An insertion of five residues at position 217, and the conformation of the side-chain of Met192 account for the fact that the specificity pocket can bind only small residues, such as Ala, Ser or Val.     

Ear Mite Removal in the Santa Catalina Island Fox (Urocyon littoralis catalinae): Controlling Risk Factors for Cancer Development

Ear mites (Otodectes cynotis) and ear canal tumors are highly prevalent among federally endangered Island foxes (Urocyon littoralis catalinae) living on Santa Catalina Island off the coast of Southern California. Since studies began in the 1990s, nearly all foxes examined were found to be infected with ear mites, and ceruminous gland tumors (carcinomas and adenomas) were detected in approximately half of all foxes ≥ 4 years of age. We hypothesized that reduction of ear mite infection would reduce otitis externa and ceruminous gland hyperplasia, a risk factor for tumor development. In this study, we conducted a randomized field trial to assess the impact of acaricide treatment on ear mite prevalence and intensity of infection, otitis externa, ceruminous gland hyperplasia, and mite-specific IgG and IgE antibody levels. Treatment was highly effective at eliminating mites and reducing otitis externa and ceruminous gland hyperplasia, and mite-specific IgG antibody levels were significantly lower among uninfected foxes. Ceruminous gland hyperplasia increased in the chronically infected, untreated foxes during the six month study. Our results provide compelling evidence that acaricide treatment is an effective means of reducing ear mites, and that mite removal in turn reduces ear lesions and mite-specific IgG antibody levels in Santa Catalina Island foxes. This study has advanced our understanding of the underlying pathogenesis which results in ceruminous gland tumors, and has helped inform management decisions that impact species conservation.     

Dynamics of sialyl Lewis(a) in aqueous solution and prediction of the structure of the sialyl Lewis(a)-SelectinE complex

In this article we investigate all possible three-dimensional structures for sialyl Lewis^a (SLe^a) in aqueous solution and we predict without a priori experimental information its conformation when bound to SelectinE by using a combination of long molecular dynamics (MD) simulations. Based on 10 ns MD studies, three structures differing in glycosidic conformations are proposed for SLe^a in aqueous solution. Based on a 4 ns MD study of the SLe^a-SelectinE complex with initial structures derived from our prediction tools, we find that, fucose and N-acetyl neuraminic acid are in close contact with SelectinE and therefore expect interactions of the protein with these two sugar rings to be significantly more important than in the case of galactose and N-acetyl glucosamine. Our predictions indicate that the N-acetyl glucosamine of SLe^a is positioned primarily in the aqueous phase. In order to be able to interact with SLe^a the side chains of amino acid residues Lys99 and Lys111 in SelectinE appear to undergo large conformational changes when contrasted with the positions of these residues in the X-ray crystal structure. Furthermore, amino acid residues Arg97, Glu98 and Lys99 are acting as a holding arm to position the NeuNAc of SLe^a in the binding pocket.     

Highly sulphated galactan from Halymenia durvillei (Halymeniales,Rhodophyta), a red seaweed of Madagascar marine coasts

Halymenia durvillei is a red seaweed with a great potential as sulphated galactan producer collected in the coastal waters of small island of Madagascar (Nosy-be in Indian Ocean). To elucidate the structure of its polysaccharide, NMR (¹H and ¹³C), FTIR, HPAEC and different colorimetric methods were carried out. It has been shown that this polysaccharide, consisted mainly of galactose, was branched by xylose and galactose in minor amounts. Arabinose and fucose were also detected. This galactan was found highly sulphated (42%, w/w) and pyruvylated (1.8%, w/w). Analysis of glycosidic linkages by CPG-MS and ¹³C NMR indicated that the polysaccharide has the defining linear backbone of alternating 3-β-d-galactopyranosyl units and 4-linked α-l/d-galactopyranosyl residues. 3,6-Anhydrogalactose units have been also detected in minor quantity. This λ-carrageenan like polysaccharide has shown original sulphatation patterns with 2-O (26%) or 2/6-O (58%) sulphated 3-linked β-d-galactopyranosyl units and 6-O (19%) or 2/6-O (47%) 4-linked α-l/d-galactopyranosyl residues.     

In vivo assessment of tumor-induced nonspecific suppression of contact sensitivity: I. Correlation with in vitro studies

Fibrosarcoma-bearing BALB/c mice were assessed for 2,4-dinitroflurobenzene (DNFB)-induced contact sensitivity by a quantitative radioisotopic ear assay. Measurement of contact sensitivity was based on the localization of intraperitoneally injected iodinated-human serum albumin ([¹²⁵I]HSA) in the challenged ear. Normal and tumor-bearing mice (TBM) had optimal localization 4 days after sensitization, as determined by challenging with DNFB ear application and measuring increased vascular permeability of the challenged ear over the unchallenged ear. However, at all times TBM responsiveness to challenge was significantly lower than that of the normal population. Kinetic experiments indicated that the most dramatic decrease in TBM primary and secondary cell-mediated immune response to the contact sensitizing agent occurred 15 to 19 days post tumor transplant, flattening out to a consistently low level during the fourth and fifth week of tumor growth. Results from these in vivo experiments strongly corroborate our previous in vitro inhibition data from tumor-induced nonspecific suppressor cells.     

Specific interaction of concanavalin a with glycolipid monolayers

The effect of ¹³¹I-labelled concanavalin A on the surface pressure and surface radioactivity of monolayers formed from phospholipids and from natural and synthetic glycolipids has been studied. The lectin binds to and penetrates dipalmitoyl phosphatidylcholine monolayers at a surface pressure of 15 dynes/cm and this interaction is inhibited by the presence of α-methyl mannose int he subphase. At surface pressures of 25 dynes/cm or higher, concanavalin A will interact with monoglucosyl diglyceride or diglucosyl diglyceride from Acholeplasma laidlawii and with synthetic glycolipids containing $\text{2}\text{or}\text{3 α1 → 4-}\text{linked}$ D-glucose residues in the headgroup, but not with phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, or with the ganglioside II³NeuAc-GgOse₄-Cer. The binding to the glycolipid sugar group and penetration of the hydrocarbon region seem to occur simultaneously, as the time courses for the development of surface pressure and surface radioactivity coincide.     

Modification of hERG1 channel gating by Cd2+

Each of the four subunits in a voltage-gated potassium channel has a voltage sensor domain (VSD) that is formed by four transmembrane helical segments (S1–S4). In response to changes in membrane potential, intramembrane displacement of basic residues in S4 produces a gating current. As S4 moves through the membrane, its basic residues also form sequential electrostatic interactions with acidic residues in immobile regions of the S2 and S3 segments. Transition metal cations interact with these same acidic residues and modify channel gating. In human ether-á-go-go–related gene type 1 (hERG1) channels, Cd²⁺ coordinated by D456 and D460 in S2 and D509 in S3 induces a positive shift in the voltage dependence of activation of ionic currents. Here, we characterize the effects of Cd²⁺ on hERG1 gating currents in Xenopus oocytes using the cut-open Vaseline gap technique. Cd²⁺ shifted the half-point (V_1/2) for the voltage dependence of the OFF gating charge–voltage (Q_OFF-V) relationship with an EC₅₀ of 171 µM; at 0.3 mM, V_1/2 was shifted by +50 mV. Cd²⁺ also induced an as of yet unrecognized small outward current (I_Cd-out) upon repolarization in a concentration- and voltage-dependent manner. We propose that Cd²⁺ and Arg residues in the S4 segment compete for interaction with acidic residues in S2 and S3 segments, and that the initial inward movement of S4 associated with membrane repolarization displaces Cd²⁺ in an outward direction to produce I_Cd-out. Co²⁺, Zn²⁺, and La³⁺ at concentrations that caused ∼+35-mV shifts in the Q_OFF-V relationship did not induce a current similar to I_Cd-out, suggesting that the binding site for these cations or their competition with basic residues in S4 differs from Cd²⁺. New Markov models of hERG1 channels were developed that describe gating currents as a noncooperative two-phase process of the VSD and can account for changes in these currents caused by extracellular Cd²⁺.     

Statistical mechanical treatment of α-helices and extended structures in proteins with inclusion of short- and medium-range interactions

A statistical mechanical model of protein conformation with medium-range interactions between theith and (i+k)^th residues (k<-4) is presented. Two two-state models, an α-helix-coil and an extended-structure-coil model, are formulated using the same form of the partition function, but the two models are applied independently to predict the locations of α-helical, extended, and coil segments; in the relatively few cases (<2%) where the predictions from the two models are in conflict, the prediction is scored as an incorrect one. Two independent sets of statistical weights (one set for each model) are derived to describe the interactions between the 20 amino acid residues for each range of interactionk; they are evaluated by minimizing an objective function so that the probability profiles for the α-helix or extended structure, respectively, in proteins computed from these statistical weights correlate optimally with the experimentally observed native conformations of these proteins. Examination of the resulting statistical weights shows that those for the interactions between hydrophobic residues and between a hydrophobic and a hydrophilic residue have reasonable magnitudes compared to what would be expected from the spatial arrangements of the side chains in the α-helix and the extended structure, and that those for the α-helix-coil model correlate well with experimentally determined values of the Zimm-Bragg parameterss and σ of the helix-coil transition theory. From the point of view of a method to predict the conformational states (i.e., α-helix, extended structure, and coil) of each residue, the statistical weights (as inall empirical prediction schemes) depend very much on the proteins used for the data base, since the presently available set of proteins of known structure is still too small for very high predictability; as a result, the correctness of the prediction is not very good for proteins not included in the data base. However, the correctness of the prediction, at least for the 37 proteins utilized as the data base in this study, is 91% and 87% for the α-helix-coil and the extended-structure-coil models, respectively; further, 79% of all the residues are predicted correctly when both the α-helix-coil and extended-structure-coil models are applied independently.     

First Feline Case of Otodectosis in the Republic of Korea and Successful Treatment with Imidacloprid/Moxidectin Topical Solution

In April 2010, pruritic symptoms were recognized in 3 privately-owned Siamese cats raised in Gwangju, Korea. Examination of ear canals revealed dark brown, ceruminous otic exudates that contain numerous live mites at various developmental stages. Based on morphological characteristics of adult mites in which caruncles were present on legs 1 and 2 in adult females and on legs 1, 2, 3, and 4 in adult males while the tarsus of leg 3 in both sexes was equipped with 2 long setae, the mite was identified as Otodectes cynotis. Ten ear mite-free domestic shorthaired cats were experimentally infected with O. cynotis to evaluate the efficacy of 10% imidacloprid/1% moxidectin spot-on. Live mites were recovered from 1 of 10 treated cats on day 9 post-treatment (PT) while no live mites were observed from the ear canals of treated cats on days 16 and 30 PT. The efficacy of 10% imidacloprid/1% moxidectin spot-on on O. cynotis in cats was, therefore, 90% on day 9 and 100% on days 16 and 30 PT. This is the first report of otodectosis in 3 cats naturally infested with O. cynotis in Gwang-ju, Korea. Both natural and experimental infestations were successfully treated with 10% imidacloprid/1% moxidectin spot-on.     

Altered secretary efficiency of Streptomyces hygroscopicus transglutaminase in Escherichia coli by the pro-peptide modification

Streptomyces transglutaminase (TGase) is naturally synthesized as a zymogen (pro-TGase), which is then processed to produce the active enzyme through removal of its pro-peptide. In this study, we investigated the effect of the pro-peptide on the secretion of Streptomyces hygroscopicus TGase in Escherichia coli by modifying its pro-peptide. Four N-terminal amino acid residues (Tyr12, Asn27, Asn30, and Arg32) in the pro-peptide predicted to interact with TGase region through hydrogen bonds. When the four amino acid residues were mutated into Ala, the secretion of TGase was partially or completely inhibited. Furthermore, deletion of the C-terminal α-helix^37–42 in the pro-peptide concomitantly decreased the secretion of TGase. However, deletion of the C-terminal loop^43–52 of the pro-peptide, resulted in increased secretion of TGase by approximately 70% as compared with the control native pro-peptide. These findings indicate that modification of the pro-peptide has a significant impact on the secretory efficiency of TGase in E. coli.     

Characterization of a water-soluble glucan from angelica acutiloba

A water-soluble glucan, AR-Glucan, from the roots of Angelica acutiloba was obtained homogeneous as determined by ultracentrifugal analysis, electrophoresis, and gel filtration. AR-Glucan was composed Of d-glucose, and its MW was estimated to be 13 500. Methylation analysis indicated that AR-Glucan contained 4-O- and 4,6-di-O-substituted glucosyl residues. ¹H and ¹³C NMR data accorded with the results of methylation analysis, and the glycosidic linkages in AR-Glucan were shown to have the α-configuration. The results of β-amylase, α-amylase, and pullulanase treatments of AR-Glucan showed that it contained (1 → 4) linked α-d-glucosyl side chains of long chain length such as amylopectin. Thus, AR-Glucan is a (1 → 4) linked α-d-glucan to which are attached glucosyl side chains at O-6 of the glucosyl residues of the main chain.     

Structural analysis of leuconostoc dextrans containing 3-O-α-d-glucosylated α-d-glucosyl residues in both linear-chain and branch-point positions,or only in branch-point positions,by methylation and by 13C-N.M.R. spectroscopy

It had been established by methylation-structural analysis that dextran fraction S from Leuconostoc mesenteroides NRRL B-1355 has two types of α-d-glucopyranosyl residues that are linked through O-3, i.e., 35% of the residues carry a (1→3)-bond, and ～10% carry a (1→6)-bond in addition to a (1→3)-bond. Two similarly constituted dextrans have now been identified by methylation-structural analysis, namely, the S-type fractions from L. mesenteroides strains NRRL B-1498 and B-1501. The S-type fractions from L. mesenteroides strains B-1355, B-1498, and B-1501 are structurally differentiated from the α-d-glucans (characteristically insoluble) of certain cariogenic Streptococci which also contain both 3-O- and 3,6-di-O-substituted α-d-glucopyranosyl residues. ¹³C-N.m.r. spectra have been recorded at 90° for both the S- and L-type fractions of strains B-1355, b-1498, and B-1501. The L-type fractions have a low degree of branching through 3,6-di-O-substituted αd-glucopyranosyl residues, but no 3-mono-O-substituted residues. (Dextran fraction S of Streptococcus 5000 g.l.c. instrument equipped with hydrogen-flame detectors. On-column injection of glass columns (2 mm i.d. x 1.23 m) was employed for all such chromatography.The ¹³C-n.m.r. conditions and methods for preparation of dextran samples have been described(su4). In general, a Varian XL-100-15 spectrometer equipped with a Nicolet TT-100 system was employed in the Fourier-transform mode. Chemical shifts are expressed in p.p.m. relative to external tetramethylsilane, but were actually calculated by reference to the lock signal.