Structure and physicochemical properties of barley non-starch polysaccharides — I. Water-extractable β-glucans and arabinoxylans

Water-soluble non-starch polysaccharides were extracted from a Canadian malting barley (cv. Harrington) by sequential treatment with water at 40 °C (WE40) and 65 °C (WE65). The yields were 1.4 and 1.3% (w/w), respectively, of the dry barley grist. The WE40 extract was composed of 82.5% glucose, 8.9% xylose, and 7.0% arabiose residues, whereas WE65 contained 93.3% Glc, 3.3% Xyl, and 2.5% Ara. Only minute amounts of mannose and galactose residues were found in either fraction. Both extracts were further fractionated by stepwise (NH₄)₂SO₄ precipitation into several polysaccharide populations. Subfractions from both extracts, obtained up to 45% saturation with (NH₄)₂SO₄, contained mostly β-glucans, whereas subfractions precipitated at increasing saturation levels of (NH₄)₂SO₄ (45–100%) contained progressively more arabinoxylans and less β-glucans. Compared to WE40, the WE65 extract was enriched in β-glucan populations with higher molecular size, higher limiting viscosity values, and higher content of β-(1 → 4) linkages. The ratio of tri-/ tetrasaccharide oligomers was also higher in β-glucans extracted at 65 °C than those extracted at 40 °C. Arabinoxylans in both extracts, WE40 and WE65, were highly substituted and contained large proportions of doubly substituted xylose residues.     

Amphiphilic properties of polysaccharides: dextrin chains as example.

Polysaccharide chains are usually considered to be highly hydrophilic, since they contain no obvious apolar moieties. However, it is possible for even these chains to display hydrophobic character, arising out of stereochemical constraints in the chain. We had earlier shown that linear dextrin chains display amphiphilic properties, since all the hydroxyl groups are disposed on one side or face of the chain and the hydrogens disposed on the other. We provide further evidence here for this conclusion that dextrins are amphiphilic chains. In contrast, dextrans and cellulosic chains do not display amphiphilicity. Oligosaccharides that can adopt incipient helical structures might display amphiphilicity. This property might be relevant to intermolecular recognition on cell surfaces, lectin-sugar binding, antigen-antibody interactions and the like, and might be manifested more in a heteromolecular recognition process than as homomolecular self-aggregation.     

Thermal properties of polysaccharides at low moisture: 1—An endothermic melting process and water-carbohydrate interactions

The thermal properties of a broad range of polysaccharides containing 5–25% w/w water have been studied by differential scanning calorimetry and dynamic mechanical thermal analysis (DMTA). Following room temperature conditioning, an endothermic event accompanied by material softening is observed at 45–80°C for all samples except those above their glass transition temperature. The temperature of the event is determined by thermal history and is apparently independent of polymer type or moisture content. The associated enthalpy increases with water content. Variable frequency DMTA analysis suggests a structural melting event rather than a relaxation process. The endothermic event is recovered over the days timescale after heating, and can be annealed to higher temperatures with increasing holding temperature.

Results are interpreted in terms of a dynamic hydration model in which specific energetic water-carbohydrate interactions occur but with a lifetime defined by their local effective microviscosity. The observation of the endotherm below glass transition temperatures suggests that in aqueous polysaccharide glasses, enthalpic structures involving the solvent can be made and broken.     

Cortisol metabolism in vitro—III. Inhibition of microsomal 6β—hydroxylase and cytosolic 4-ene-reductase

The in vitro metabolism of cortisol in human liver fractions is highly complex and variable. Cytosolic metabolism proceeds predominantly via A-ring reduction (to give 3,5β-tetrahydrocortisol; 3,5β-THF), while microsomal incubations generate upto 7 metabolites, including 6β-hydroxycortisol (6β-OHF), and 6β-hydroxycortisone (6β-OHE), products of the cytochrome P450 (CYP) 3A subfamily. The aim of the present study was, therefore, to examine two of the main enzymes involved in cortisol metabolism, namely, microsomal 6β-hydroxylase and cytosolic 4-ene-reductase. In particular, we wished to assess the substrate specificity of these enzymes and identify compounds with inhibitory potential. Incubations for 30 min containing [³H]cortisol, potential inhibitors, microsomal or cytosolic protein (3 mg), and co-factors were followed by radiometric HPLC analysis. The K_m value for 6β-OHF and 6β-OHE formation was 15.2 ± 2.1 μM (mean ± SD; n = 4) and the V_max value 6.43 ± 0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6β-hydroxylase was ketoconazole (K_i = 0.9 ± 0.4 μM; N = 4), followed by gestodene (K_i = 5.6 ± 0.6 μM) and cyclosporine (K_i = 6.8 ± 1.4 μM). Both betamethasone and dexamethasone produced some inhibition (K_i = 31.3 and 54.5 μ, respectively). However, substrates for CYP2C (tolbutamide), CYP2D (quinidine), and CYP1A (theophylline) were essentially non-inhibitory. The K_m value for cortisol 4-ene-reductase was 26.5 ± 11.2 μM (n = 4) and the V_max value 107.7 ± 46.0 pmol/min/mg cytosolic protein. The most potent inhibitors were androstendione (K_i = 17.8 ± 3.3 μM) and gestodene (K_i = 23.8 ± 3.8 μM). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive.     

Scaling properties of cell and organelle size

How size is controlled is a fundamental question in biology. In this review, we discuss the use of scaling relationships—for example, power laws of the form y ∝ xα—to provide a framework for comparison and interpretation of size measurements. Such analysis can illustrate the biological and physical principles underlying observed trends, as has been proposed for the allometric dependence of metabolic rate or limb structure on organism mass. Techniques for measuring size at smaller length-scales continue to improve, leading to more data on the control of size in cells and organelles. Size scaling of these structures is expected to influence growth patterns, functional capacity, and intracellular transport. Furthermore, organelles such as the nucleus, mitochondria, and endoplasmic reticulum show widely varying morphologies which affect their scaling properties. We provide brief summaries of these issues for individual organelles, and conclude with a discussion on how to apply this concept to better understand the mechanisms of size control in the cellular environment.     

Configurational statistics of polysaccharide chains. Part III. Linear β-(1 → 4′) xylan and mannan

The characteristic ratio C_N = 〈r²〉₀/Nl_v² of the β-D (1 → 4′)-linked polysaccharides xylan and mannan has been computed as a function of the angle τ at the bridge oxygen atom and the degree of polymerization N. The calculated values of the characteristic ratio C_N are very high relative to their free rotational dimensions. The characteristic ratio of these polysaecharides converges to the asymptotic value at low degree of polymerization at higher τ values. The low values of the calculated characteristic ratio of xylan compared to cellulose and mannan for the same τ value indicate that the former is more flexible and assumes a compact configuration. A pronounced difference in the values of the characteristic ratio C_N of cellulose and mannan has also been observed lower τ angles (<120°). On the other hand, nearly the same values of C_N have been obtained at higher τ angles (120°–125°), which suggests that, cellulose and mannan may have similar configuralons in certain solvents.     

Cell wall carbohydrates from fruit pulp of Argania spinosa: structural analysis of pectin and xyloglucan polysaccharides

Isolated cell walls of Argania spinosa fruit pulp were fractionated into their polysaccharide constituents and the resulting fractions were analysed for monosaccharide composition and chemical structure. The data reveal the presence of homogalacturonan, rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) in the pectic fraction. RG-I is abundant and contains high amounts of Ara and Gal, indicative of an important branching in this polysaccharide. RG-II is less abundant than RG-I and exists as a dimer. Structural characterisation of xyloglucan using enzymatic hydrolysis, gas chromatography, MALDI-TOF-MS and methylation analysis shows that XXGG, XXXG, XXLG and XLLG are the major subunit oligosaccharides in the ratio of 0.6:1:1.2:1.6. This finding demonstrates that the major neutral hemicellulosic polysaccharide is a galacto-xyloglucan. In addition, Argania fruit xyloglucan has no XUFG, a novel xyloglucan motif recently discovered in Argania leaf cell walls. Finally, the isolation and analysis of arabinogalactan-proteins showed that Argania fruit pulp is rich in these proteoglycans.     

Molecular discrimination of structurally equivalent Lys 63‐linked and linear polyubiquitin chains

At least eight types of ubiquitin chain exist, and individual linkages affect distinct cellular processes. The only distinguishing feature of differently linked ubiquitin chains is their structure, as polymers of the same unit are chemically identical. Here, we have crystallized Lys 63‐linked and linear ubiquitin dimers, revealing that both adopt equivalent open conformations, forming no contacts between ubiquitin molecules and thereby differing significantly from Lys 48‐linked ubiquitin chains. We also examined the specificity of various deubiquitinases (DUBs) and ubiquitin‐binding domains (UBDs). All analysed DUBs, except CYLD, cleave linear chains less efficiently compared with other chain types, or not at all. Likewise, UBDs can show chain specificity, and are able to select distinct linkages from a ubiquitin chain mixture. We found that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO (NF‐κB essential modifier) binds to linear chains exclusively, whereas the NZF (Npl4 zinc finger) domain of TAB2 (TAK1 binding protein 2) is Lys 63 specific. Our results highlight remarkable specificity determinants within the ubiquitin system.     

Solution conformations of pectin polysaccharides: Determination of chain characteristics by small angle neutron scattering,viscometry, and molecular modeling

The solution behavior of pectin polysaccharides has been investigated by small angle neutron scattering (SANS), viscosimetric, and molecular modeling studies. The samples used in the experimental study were obtained from apple and citrus and had degrees of methylation ranging from 28 to 73%, with a rhamnose content lying between 0.6 and 2.2%. Persistence lengths, derived from intrinsic viscosity measurements, ranged from 59 to 126 Å, whereas those derived by SANS were between 45 and 75 Å. These values correspond to 10–17 monomer units. The modeling simulations were performed for both homogalacturonan itself and homogalacturonan carrying various degrees of rhamnose inserts (rhamnogalacturonan). This required the evaluation of the accessible conformational space for the eight disaccharides that represent the constituent repeating segments of the homogalacturonan and rhamnogalacturonan polysaccharides. For each dimer, complete conformational analysis was accomplished using the flexible residue method of the MM3 molecular mechanics procedure and the results used to access the configurational statistics of representative pectic polysaccharide chains. For homogalacturonan, an extended chain conformation having a persistence length of 135 Å (corresponding to 30 monomers) was predicted. The inclusion of varying amounts of rhamnose units (5–25%) in the model in strict alternating sequence with galacturonate residues (equivalent to the rhamnogalacturonan “hairy region” chains) only slightly reduced the calculated persistence length. The extended overall chain conformation remained relatively unchanged as a consequence of the self-cancellation of the kinking effects of successive paired rhamnose units. © 1996 John Wiley & Sons, Inc.