Chemical structures of Streptococcus pneumoniae capsular polysaccharide type 39 (CPS39), CPS47F, and CPS34 characterized by nuclear magnetic resonance spectroscopy and their relation to CPS10A.

Structural characterization of Streptococcus pneumoniae capsular polysaccharides (CPS) is a prerequisite for unraveling both antigenic and genetic relationships that exist between different serotypes. In the current study, comparative structural studies of S. pneumoniae CPS serogroup 10 (CPS10) were extended to include genetically related S. pneumoniae CPS34, CPS39, and CPS47F. High-resolution heteronuclear nuclear magnetic resonance (NMR) spectroscopy confirmed the published structure of CPS34 and, in conjunction with glycosyl composition analyses, revealed the following repeat unit structures of the other serotypes, which have not been previously characterized: [structure: see text] Common and unique structural features of these polysaccharides, including different positions of O-acetylation, were unambiguously associated with specific genes in each corresponding cps locus. The only exception involved the gene designated wcrC, which is associated with the α1-2 transfer of Gal pyranoside (Galp) to ribitol-5-phosphate in the synthesis of CPS10A, CPS47F, and CPS34 but with α1-1 transfer of Gal to ribitol-5-phosphate in the synthesis of CPS39. The corresponding gene in the cps39 locus, although related to wcrC, more closely resembled a previously identified gene (i.e., wefM) of Streptococcus oralis that is associated with α1-1 transfer of Galp to ribitol-5-phosphate. These and other recent findings identify linkages from α-Galp to ribitol-5-phosphate and from this residue to adjacent Gal furanoside (Galf) as important sites of CPS structural and genetic diversity.

Cutin deficiency in the tomato fruit cuticle consistently affects resistance to microbial infection and biomechanical properties, but not transpirational water loss.

Plant cuticles are broadly composed of two major components: polymeric cutin and a mixture of waxes, which infiltrate the cutin matrix and also accumulate on the surface, forming an epicuticular layer. Although cuticles are thought to play a number of important physiological roles, with the most important being to restrict water loss from aerial plant organs, the relative contributions of cutin and waxes to cuticle function are still not well understood. Tomato (Solanum lycopersicum) fruits provide an attractive experimental system to address this question as, unlike other model plants such as Arabidopsis, they have a relatively thick astomatous cuticle, providing a poreless uniform material that is easy to isolate and handle. We identified three tomato mutants, cutin deficient 1 (cd1), cd2 and cd3, the fruit cuticles of which have a dramatic (95-98%) reduction in cutin content and substantially altered, but distinctly different, architectures. This cutin deficiency resulted in an increase in cuticle surface stiffness, and in the proportions of both hydrophilic and multiply bonded polymeric constituents. Furthermore, our data suggested that there is no correlation between the amount of cutin and the permeability of the cuticle to water, but that cutin plays an important role in protecting tissues from microbial infection. The three cd mutations were mapped to different loci, and the cloning of CD2 revealed it to encode a homeodomain protein, which we propose acts as a key regulator of cutin biosynthesis in tomato fruit.

Alkaloids from Menispermum dauricum.

The alkaloids, dechloroacutumidine and 1-epidechloroacutumine, together with three known alkaloids, acutumidine, acutumine, and dechloroacutumine, were isolated from the rhizomes of Menispermum dauricum and their structures established by spectral and chemical methods. The cytotoxicity of each compound against the growth of human cell lines was studied, and acutumine selectively inhibited T-cell growth.

NMR of glycans: shedding new light on old problems.

The diversity in molecular arrangements and dynamics displayed by glycans renders traditional NMR strategies, employed for proteins and nucleic acids, insufficient. Because of the unique properties of glycans, structural studies often require the adoption of a different repertoire of tailor-made experiments and protocols. We present an account of recent developments in NMR techniques that will deepen our understanding of structure-function relations in glycans. We open with a survey and comparison of methods utilized to determine the structure of proteins, nucleic acids and carbohydrates. Next, we discuss the structural information obtained from traditional NMR techniques like chemical shifts, NOEs/ROEs, and coupling-constants, along with the limitations imposed by the unique intrinsic characteristics of glycan structure on these approaches: flexibility, range of conformers, signal overlap, and non-first-order scalar (strong) coupling. Novel experiments taking advantage of isotopic labeling are presented as an option for overcoming spectral overlap and raising sensitivity. Computational tools used to explore conformational averaging in conjunction with NMR parameters are described. In addition, recent developments in hydroxyl detection and hydrogen bond detection in protonated solvents, in contrast to traditional sample preparations in D2O for carbohydrates, further increase the tools available for both structure information and chemical shift assignments. We also include previously unpublished data in this context. Accurate determination of couplings in carbohydrates has been historically challenging due to the common presence of strong-couplings. We present new strategies proposed for dealing with their influence on NMR signals. We close with a discussion of residual dipolar couplings (RDCs) and the advantages of using (13)C isotope labeling that allows gathering one-bond (13)C-(13)C couplings with a recently improved constant-time COSY technique, in addition to the commonly measured (1)H-(13)C RDCs.

Constant time INEPT CT-HSQC (CTi-CT-HSQC) - A new NMR method to measure accurate one-bond J and RDCs with strong 1H-1H couplings in natural abundance.

Strong (1)H-(1)H coupling can significantly reduce the accuracy of (1)J(CH) measured from frequency differences in coupled HSQC spectra. Although accurate (1)J(CH) values can be extracted from spectral simulation, it would be more convenient if the same accurate (1)J(CH) values can be obtained experimentally. Furthermore, simulations reach their limit for residual dipolar coupling (RDC) measurement, as many significant, but immeasurable RDCs are introduced into the spin system when a molecule is weakly aligned, thus it is impossible to have a model spin system that truly represents the real spin system. Here we report a new J modulated method, constant-time INEPT CT-HSQC (CTi-CT-HSQC), to accurately measure one-bond scalar coupling constant and RDCs without strong coupling interference. In this method, changing the spacing between the two 180° pulses during a constant time INEPT period selectively modulates heteronuclear coupling in quantitative J fashion. Since the INEPT delays for measuring one-bond carbon-proton spectra are short compared to (3)J(HH), evolution due to (strong) (1)H-(1)H coupling is marginal. The resulting curve shape is practically independent of (1)H-(1)H coupling and only correlated to the heteronuclear coupling evolution. Consequently, an accurate (1)J(CH) can be measured even in the presence of strong coupling. We tested this method on N-acetyl-glucosamine and mannose whose apparent isotropic (1)J(CH) values are significantly affected by strong coupling with other methods. Agreement to within 0.5Hz or better is found between (1)J(CH) measured by this method and previously published simulation data. We further examined the strong coupling effects on RDC measurements and observed an error up to 100% for one bond RDCs using coupled HSQC in carbohydrates. We demonstrate that RDCs can be obtained with higher accuracy by CTi-CT-HSQC, which compensates the limitation of simulation method.

More accurate 1J(CH) coupling measurement in the presence of 3J(HH) strong coupling in natural abundance.

J couplings are essential for measuring RDCs (residual dipolar couplings), now routinely used to deduce molecular structure and dynamics of glycans and proteins. Accurate measurement of (1)J(CH) is critical for RDCs to reflect the true structure and dynamics in the molecule of interest. We report noticeable discrepancies between (1)J(CH) values measured with HSQC type pulse sequences in the (1)H dimension from those measured in the (13)C dimension for 17 sugars and show that these discrepancies arise from strong scalar coupling. In order to determine how to minimize errors in measuring (1)J(CH), we analyze the strong coupling effects in detail using the product operator-formalism and spectral simulations based on the solution of the Liouville equation (not considering relaxation effects) in the presence of strong coupling. We report that the apparent (1)J(CH) measured with 2D HSQC-based sequences in either dimension can be in error by up to 4 Hz and that the values measured in the (1)H dimension can disagree with those in the (13)C dimension by up to 7 Hz. We demonstrate that spectral simulations can reproduce the errors induced by strong coupling and that these can be used to extract true (1)J(CH) values. We find that the (1)J(CH) values measured using a modified Z-filtered coupled HSQC are still affected by strong coupling. We conclude that spectral simulation yields accurate (1)J(CH) with errors as low as 1% in the presence of strong coupling.

Isolation and identification of oligomers from partial degradation of lime fruit cutin.

Complementary degradative treatments with low-temperature hydrofluoric acid and methanolic potassium hydroxide have been used to investigate the protective biopolymer cutin from Citrus aurantifolia (lime) fruits, augmenting prior enzymatic and chemical strategies to yield a more comprehensive view of its molecular architecture. Analysis of the resulting soluble oligomeric fragments with one- and two-dimensional NMR and MS methods identified a new dimer and three trimeric esters of primary alcohols based on 10,16-dihydroxyhexadecanoic acid and 10-oxo-16-hydroxyhexadecanoic acid units. Whereas only 10-oxo-16-hydroxyhexadecanoic acid units were found in the oligomers from hydrofluoric acid treatments, the dimer and trimer products isolated to date using diverse degradative methods included six of the seven possible stoichiometric ratios of monomer units. A novel glucoside-linked hydroxyfatty acid tetramer was also identified provisionally, suggesting that the cutin biopolymer can be bound covalently to the plant cell wall. Although the current findings suggest that the predominant molecular architecture of this protective polymer in lime fruits involves esters of primary and secondary alcohols based on long-chain hydroxyfatty acids, the possibility of additional cross-linking to enhance structural integrity is underscored by these and related findings of nonstandard cutin molecular architectures.

Magic-angle spinning NMR studies of cell wall bound aromatic-aliphatic biopolyesters associated with strengthening of intercellular adhesion in potato (Solanum tuberosum L.) tuber parenchyma.

Intercellular adhesion strengthening, a phenomenon that compromises the texture and the edible quality of potatoes (Solanum tuberosum L.), has been induced reproducibly by exposure to low-pH acetic acid solutions under tissue culture conditions. The resulting parenchyma tissues have been examined by solid-state nuclear magnetic resonance (NMR) in order to characterize the biopolymer(s) thought to be associated with this syndrome. Cross polarization-magic angle spinning (CPMAS) (13)C NMR has been used to establish the presence of a polyphenol-suberin-like aromatic-aliphatic polyester within an abundant cell wall polysaccharide matrix in potato tubers that exhibit hardening due to strengthened intercellular adhesion. Dipolar dephasing and CP chemical shift anisotropy experiments suggest that the aromatic domain is composed primarily of guaiacyl and sinapyl groups. Two-dimensional wide-line separation experiments show that the biopolymer associated with parenchyma hardening contains rigid polysaccharide cell walls and mobile aliphatic long-chain fatty acids; (1)H spin diffusion experiments show that these flexible aliphatic chains are proximal to both the phenolics and a subpopulation of the cell wall polysaccharides. Finally, high-resolution MAS NMR of parenchyma samples swelled in DMSO in conjunction with two-dimensional through-bond and through-space NMR spectroscopy provides evidence for covalent linkages among the polysaccharide, phenolic, and aliphatic domains of the intercellular adhesion-strengthening biopolymer in potato parenchyma tissue.

Deoxysarpagine hydroxylase--a novel enzyme closing a short side pathway of alkaloid biosynthesis in Rauvolfia.

Microsomal preparations from cell suspension cultures of the Indian plant Rauvolfia serpentina catalyze the hydroxylation of deoxysarpagine under formation of sarpagine. The newly discovered enzyme is dependent on NADPH and oxygen. It can be inhibited by typical cytochrome P450 inhibitors such as cytochrome c, ketoconazole, metyrapone, tetcyclacis and carbon monoxide. The CO-effect is reversible with light (450 nm). The data indicate that deoxysarpagine hydroxylase is a novel cytochrome P450-dependent monooxygenase. A pH optimum of 8.0 and a temperature optimum of 35 degrees C were determined. K(m) values were 25 microM for NADPH and 7.4 microM for deoxysarpagine. Deoxysarpagine hydroxylase activity was stable in presence of 20% sucrose at -25 degrees C for >3 months. The analysis of presence of the hydroxylase in nine cell cultures of seven different families indicates a very limited taxonomic distribution of this enzyme.

Alkaloids from Stemona collinsae.

A new alkaloid, isostenine (1), together with two known ones neotuberostemonine (2) and bisdehydroneotuberostemonine (3) were isolated from the root of Stemona collinsae. Their structures were determined by various spectral methods.

Nitrotyrasacutuminine from Menispermum dauricum.

Nitrotyrasacutuminine, an unusual nitrated morphine-type alkaloid was isolated from the roots of Menispermum dauricum. Its structure was determined by various 2D spectra and chemical methods.