Same but different - pseudo-pectin in the charophytic alga Chlorokybus atmophyticus.

All land-plant cell walls possess hemicelluloses, cellulose and anionic pectin. The walls of their cousins, the charophytic algae, exhibit some similarities to land plants' but also major differences. Charophyte 'pectins' are extractable by conventional land-plant methods, although they differ significantly in composition. Here, we explore 'pectins' of an early-diverging charophyte, Chlorokybus atmophyticus, characterising the anionic polysaccharides that may be comparable to 'pectins' in other streptophytes. Chlorokybus 'pectin' was anionic and upon acid hydrolysis gave GlcA, GalA and sulphate, plus neutral sugars (Ara≈Glc>Gal>Xyl); Rha was undetectable. Most Gal was the l-enantiomer. A relatively acid-resistant disaccharide was characterised as ß-d-GlcA-(1→4)-l-Gal. Two Chlorokybus 'pectin' fractions, separable by anion-exchange chromatography, had similar sugar compositions but different sulphate-ester contents. No sugars were released from Chlorokybus 'pectin' by several endo-hydrolases [(1,5)-α-l-arabinanase, (1,4)-ß-d-galactanase, (1,4)-ß-d-xylanase, endo-polygalacturonase] and exo-hydrolases [α- and ß-d-galactosidases, α-(1,6)-d-xylosidase]. 'Driselase', which hydrolyses most land-plant cell wall polysaccharides to mono- and disaccharides, released no sugars except traces of starch-derived Glc. Thus, the Ara, Gal, Xyl and GalA of Chlorokybus 'pectin' were not non-reducing termini with configurations familiar from land-plant polysaccharides (α-l-Araf, α- and ß-d-Galp, α- and ß-d-Xylp and α-d-GalpA), nor mid-chain residues of α-(1→5)-l-arabinan, ß-(1→4)-d-galactan, ß-(1→4)-d-xylan or α-(1→4)-d-galacturonan. In conclusion, Chlorokybus possesses anionic 'pectic' polysaccharides, possibly fulfilling pectic roles but differing fundamentally from land-plant pectin. Thus, the evolution of land-plant pectin since the last common ancestor of Chlorokybus and land plants is a long and meandering path involving loss of sulphate, most l-Gal and most d-GlcA; re-configuration of Ara, Xyl and GalA; and gain of Rha.

Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls.

BACKGROUND AND AIMS: The programmed softening occurring during fruit development requires scission of cell wall polysaccharides, especially pectin. Proposed mechanisms include the action of wall enzymes or hydroxyl radicals. Enzyme activities found in fruit extracts include pectate lyase (PL) and endo-polygalacturonase (EPG), which, in vitro, cleave de-esterified homogalacturonan in mid-chain by ß-elimination and hydrolysis, respectively. However, the important biological question of whether PL exhibits action in vivo had not been tested. METHODS: We developed a method for specifically and sensitively detecting in-vivo PL products, based on Driselase digestion of cell wall polysaccharides and detection of the characteristic unsaturated product of PL action. KEY RESULTS: In model in-vitro experiments, pectic homogalacturonan that had been partially cleaved by commercial PL was digested to completion with Driselase, releasing an unsaturated disaccharide ('ΔUA-GalA'), taken as diagnostic of PL action. ΔUA-GalA was separated from saturated oligogalacturonides (EPG products) by electrophoresis, then subjected to thin-layer chromatography (TLC), resolving ΔUA-GalA from higher homologues. The ΔUA-GalA was confirmed as 4-deoxy-ß-l-threo-hex-4-enopyranuronosyl-(1→4)-d-galacturonic acid by NMR spectroscopy. Driselase digestion of cell walls from ripe fruits of date (Phoenix dactylifera), pear (Pyrus communis), rowan (Sorbus aucuparia) and apple (Malus pumila) yielded ΔUA-GalA, demonstrating that PL had been acting in vivo in these fruits prior to harvest. Date-derived ΔUA-GalA was verified by negative-mode mass spectrometry, including collision-induced dissociation (CID) fragmentation. The ΔUA-GalA:GalA ratio from ripe dates was roughly 1:20 (mol mol-1), indicating that approx. 5 % of the bonds in endogenous homogalacturonan had been cleaved by in-vivo PL action. CONCLUSIONS: The results provide the first demonstration that PL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.

Direct monitoring of biocatalytic deacetylation of amino acid substrates by 1H NMR reveals fine details of substrate specificity.

Amino acids are key synthetic building blocks that can be prepared in an enantiopure form by biocatalytic methods. We show that the l-selective ornithine deacetylase ArgE catalyses hydrolysis of a wide-range of N-acyl-amino acid substrates. This activity was revealed by 1H NMR spectroscopy that monitored the appearance of the well resolved signal of the acetate product. Furthermore, the assay was used to probe the subtle structural selectivity of the biocatalyst using a substrate that could adopt different rotameric conformations.

Oral cysteamine as an adjunct treatment in cystic fibrosis pulmonary exacerbations: An exploratory randomized clinical trial.

BACKGROUND: Emerging data suggests a possible role for cysteamine as an adjunct treatment for pulmonary exacerbations of cystic fibrosis (CF) that continue to be a major clinical challenge. There are no studies investigating the use of cysteamine in pulmonary exacerbations of CF. This exploratory randomized clinical trial was conducted to answer the question: In future pivotal trials of cysteamine as an adjunct treatment in pulmonary exacerbations of CF, which candidate cysteamine dosing regimens should be tested and which are the most appropriate, clinically meaningful outcome measures to employ as endpoints? METHODS AND FINDINGS: Multicentre double-blind randomized clinical trial. Adults experiencing a pulmonary exacerbation of CF being treated with standard care that included aminoglycoside therapy were randomized equally to a concomitant 14-day course of placebo, or one of 5 dosing regimens of cysteamine. Outcomes were recorded on days 0, 7, 14 and 21 and included sputum bacterial load and the patient reported outcome measures (PROMs): Chronic Respiratory Infection Symptom Score (CRISS), the Cystic Fibrosis Questionnaire-Revised (CFQ-R); FEV1, blood leukocyte count, and inflammatory markers. Eighty nine participants in fifteen US and EU centres were randomized, 78 completed the 14-day treatment period. Cysteamine had no significant effect on sputum bacterial load, however technical difficulties limited interpretation. The most consistent findings were for cysteamine 450mg twice daily that had effects additional to that observed with placebo, with improved symptoms, CRISS additional 9.85 points (95% CI 0.02, 19.7) p = 0.05, reduced blood leukocyte count by 2.46x109 /l (95% CI 0.11, 4.80), p = 0.041 and reduced CRP by geometric mean 2.57 nmol/l (95% CI 0.15, 0.99), p = 0.049. CONCLUSION: In this exploratory study cysteamine appeared to be safe and well-tolerated. Future pivotal trials investigating the utility of cysteamine in pulmonary exacerbations of CF need to include the cysteamine 450mg doses and CRISS and blood leukocyte count as outcome measures. CLINICAL TRIAL REGISTRATION: NCT03000348; www.clinicaltrials.gov.

Characterisation of the non-oxidative degradation pathway of dehydroascorbic acid in slightly acidic aqueous solution.

Although l-ascorbate (vitamin C) is an important biological antioxidant, its degradation pathways in vivo remain incompletely characterised. Ascorbate is oxidised to dehydroascorbic acid, which can be either hydrolysed to diketogulonate (DKG) or further oxidised. DKG can be further degraded, oxidatively or non-oxidatively. Here we characterise DKG products formed non-enzymically and non-oxidatively at 20 °C and at a slightly acidic pH typical of the plant apoplast. High-voltage electrophoresis revealed at least five products, including two novel CPLs (epimers of 2-carboxy-l-threo-pentonolactone), which slowly interconverted with CPA (2-carboxy-l-threo-pentonate). One of the two CPLs has an exceptionally low pKa. The CPL structures were supported by MS [(C6H7O7)-] and by 1H and 13C NMR spectroscopy. Xylonate and its lactone also appeared. Experiments with [1-14C]DKG showed that all five products (including the 5-carbon xylonate and its lactone) retained DKG's carbon-1; therefore, most xylonate arose by decarboxylation of CPLs or CPA, one of whose -COOH groups originates from C-2 or C-3 of DKG after a 'benzilic acid rearrangement'. Since CPLs appeared before CPA, a DKG lactone is probably the main species undergoing this rearrangement. CPA and CPL also form non-enzymically in vivo, where they may be useful to researchers as 'fingerprints', or to organisms as 'signals', indicating a non-oxidative, slightly acidic biological compartment.

Montbresides A-D: antibacterial p-coumaroyl esters of a new sucrose-based tetrasaccharide from Crocosmia × crocosmiiflora (montbretia) flowers.

Crocosmia × crocosmiiflora (montbretia) flowers yielded four esters (montbresides A-D) of a new sucrose-based tetrasaccharide, 3-O-ß-d-glucopyranosyl-4´-O-α-d-rhamnopyranosyl-sucrose [ß-d-Glc-(1 → 3)-α-d-Glc-(1↔2)-ß-d-Fru-(4 ← 1)-α-d-Rha]. All four possess O-p-coumaroyl residues on C-3 of fructose and C-4 of α-glucose, plus O-acetyl residues on C-2 and C-3 of rhamnose and C-6 of fructose. Montbresides A and B are additionally O-acetylated on C-1 of fructose. The p-coumaroyls are trans- in montbresides A and C and cis- in B and D. Elemental compositions were determined from MS data, and structures from 1D and 2D NMR spectra. Monosaccharide residues were identified from selective 1D TOCSY spectra and TLC, and acylation sites from 2D HMBC spectra. Enantiomers were distinguished by enzymic digestion. Montbretia flower extracts were cytotoxic against six human cancerous cell-lines, but purified montbresides lacked cytotoxicity. Each montbreside displayed antibacterial activity against Staphylococcus aureus (minimal inhibitory concentration ~6 µg/ml). Montbretia is a potential source of new cytotoxins and antibacterial agents.

Scottish consensus statement on the role of FeNO in adult asthma.

Fractional exhaled nitric oxide (FeNO) is the only available point of care test to assess type-2 inflammation in asthma. In making a diagnosis of asthma, FeNO should be used together with blood eosinophils and spirometry, alongside a history. Raised FeNO in conjunction with blood eosinophilia are treatable traits of type 2 inflammation in asthma, which in turn may guide personalised management. A FeNO suppression test can be used to assess adherence and device use with ICS therapy. Furthermore FeNO may be used to provide feedback to patients in response to ICS, especially when spirometry is normal. FeNO may facilitate appropriate referral to secondary care for more definitive specialist investigations. In summary, FeNO is cost effective in the diagnosis and management of asthma and should be incorporated into primary and secondary care as part of routine clinical practice.

Further Studies on the Pyrolytic Domino Cyclization of Stabilized Phosphonium Ylides Bearing an Ortho-Aminophenyl Group.

Four new, stabilized phosphonium ylides containing a 2-(benzyl(methyl)amino)phenyl group have been prepared and characterized and are found, upon pyrolysis under gas-phase flow conditions, to lose Ph3PO and benzyl radicals to afford new heterocyclic products resulting from domino cyclization of both C- and N-centered radicals. Most products arise from processes of the former type and have quinoline, phenanthridine, or ring-fused phenanthridine structures, while in one case, a process of the latter type leads to a benzocarbazole product. The X-ray structure of a 2-(methyl(tosyl)amino)phenyl ylide is also reported.

The pectic disaccharides lepidimoic acid and ß-d-xylopyranosyl-(1→3)-d-galacturonic acid occur in cress-seed exudate but lack allelochemical activity.

BACKGROUND AND AIMS: Cress-seed (Lepidium sativum) exudate exerts an allelochemical effect, promoting excessive hypocotyl elongation and inhibiting root growth in neighbouring Amaranthus caudatus seedlings. We investigated acidic disaccharides present in cress-seed exudate, testing the proposal that the allelochemical is an oligosaccharin-lepidimoic acid (LMA; 4-deoxy-ß-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnose). METHODS: Cress-seed exudate was variously treated [heating, ethanolic precipitation, solvent partitioning, high-voltage paper electrophoresis and gel-permeation chromatography (GPC)], and the products were bioassayed for effects on dark-grown Amaranthus seedlings. Two acidic disaccharides, including LMA, were isolated and characterized by electrophoresis, thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) spectroscopy, and then bioassayed. KEY RESULTS: Cress-seed exudate contained low-Mr, hydrophilic, heat-stable material that strongly promoted Amaranthus hypocotyl elongation and inhibited root growth, but that separated from LMA on electrophoresis and GPC. Cress-seed exudate contained ∼250 µmLMA, whose TLC and electrophoretic mobilities, susceptibility to mild acid hydrolysis and NMR spectra are reported. A second acidic disaccharide, present at ∼120 µm, was similarly characterized, and shown to be ß-d-xylopyranosyl-(1→3)-d-galacturonic acid (Xyl→GalA), a repeat unit of xylogalacturonan. Purified LMA and Xyl→GalA when applied at 360 and 740 µm, respectively, only slightly promoted Amaranthus hypocotyl growth, but equally promoted root growth and thus had no effect on the hypocotyl:root ratio, unlike total cress-seed exudate. CONCLUSIONS: LMA is present in cress seeds, probably formed by rhamnogalacturonan lyase action on rhamnogalacturonan-I during seed development. Our results contradict the hypothesis that LMA is a cress allelochemical that appreciably perturbs the growth of potentially competing seedlings. Since LMA and Xyl→GalA slightly promoted both hypocotyl and root elongation, their effect could be nutritional. We conclude that rhamnogalacturonan-I and xylogalacturonan (pectin domains) are not sources of oligosaccharins with allelochemical activity, and the biological roles (if any) of the disaccharides derived from them are unknown. The main allelochemical principle in cress-seed exudate remains to be identified.

Sugar composition of the pectic polysaccharides of charophytes, the closest algal relatives of land-plants: presence of 3-O-methyl-D-galactose residues.

BACKGROUND AND AIMS: During evolution, plants have acquired and/or lost diverse sugar residues as cell-wall constituents. Of particular interest are primordial cell-wall features that existed, and in some cases abruptly changed, during the momentous step whereby land-plants arose from charophytic algal ancestors. METHODS: Polysaccharides were extracted from four charophyte orders [Chlorokybales (Chlorokybus atmophyticus), Klebsormidiales (Klebsormidium fluitans, K. subtile), Charales (Chara vulgaris, Nitella flexilis), Coleochaetales (Coleochaete scutata)] and an early-diverging land-plant (Anthoceros agrestis). 'Pectins' and 'hemicelluloses', operationally defined as extractable in oxalate (100 °C) and 6 m NaOH (37 °C), respectively, were acid- or Driselase-hydrolysed, and the monosaccharides analysed chromatographically. One unusual monosaccharide, 'U', was characterized by (1)H/(13)C-nuclear magnetic resonance spectroscopy and also enzymically. KEY RESULTS: 'U' was identified as 3-O-methyl-D-galactose (3-MeGal). All pectins, except in Klebsormidium, contained acid- and Driselase-releasable galacturonate, suggesting homogalacturonan. All pectins, without exception, released rhamnose and galactose on acid hydrolysis; however, only in 'higher' charophytes (Charales, Coleochaetales) and Anthoceros were these sugars also efficiently released by Driselase, suggesting rhamnogalacturonan-I. Pectins of 'higher' charophytes, especially Chara, contained little arabinose, instead possessing 3-MeGal. Anthoceros hemicelluloses were rich in glucose, xylose, galactose and arabinose (suggesting xyloglucan and arabinoxylan), none of which was consistently present in charophyte hemicelluloses. CONCLUSIONS: Homogalacturonan is an ancient streptophyte feature, albeit secondarily lost in Klebsormidium. When conquering the land, the first embryophytes already possessed rhamnogalacturonan-I. In contrast, charophyte and land-plant hemicelluloses differ substantially, indicating major changes during terrestrialization. The presence of 3-MeGal in charophytes and lycophytes but not in the 'intervening' bryophytes confirms that cell-wall chemistry changed drastically between major phylogenetic grades.

NMR methodology for complex mixture 'separation'.

Mixture 'separation' by NMR is demonstrated through the development of a pseudo 4D NMR experiment, 3D IPAP INEPT-INADEQUATE-HSQC, designed for the structural elucidation of (13)C tagged compounds.

An unexpectedly lichenase-stable hexasaccharide from cereal, horsetail and lichen mixed-linkage ß-glucans (MLGs): implications for MLG subunit distribution.

Mixed-linkage (1→3),(1→4)-ß-d-glucan (MLG) is a biologically and technologically important hemicellulose, known to occur in three widely separated lineages: the Poales (including grasses and cereals), Equisetum (fern-allies), and some lichens e.g. Iceland moss (Cetraria islandica). Lichenase (E.C. 3.2.1.73) is widely assumed to hydrolyse all (1→4) bonds that immediately follow (1→3) bonds in MLG, generating predominantly the tetrasaccharide ß-d-Glcp-(1→4)-ß-d-Glcp-(1→4)-ß-d-Glcp-(1→3)-d-Glc (G4G4G3G; MLG4), the corresponding trisaccharide (G4G3G; MLG3), and sometimes also laminaribiose (G3G; MLG2). The ratio of the oligosaccharides produced characterises each polysaccharide. We report here that digestion of MLG from barley (Hordeum vulgare), Equisetum arvense and C. islandica by Bacillus subtilis lichenase also yields the unexpectedly stable hexasaccharide, ß-d-Glcp-(1→3)-ß-d-Glcp-(1→4)-ß-d-Glcp-(1→4)-ß-d-Glcp-(1→4)-ß-d-Glcp-(1→3)-d-Glc (G3G4G4G4G3G, i.e. MLG2-MLG4), identified by thin-layer chromatography, gel-permeation chromatography, HPLC (HPAEC), ß-glucosidase digestion, (1)H/(13)C-NMR spectroscopy and mass spectrometry. On HPLC, G3G4G4G4G3G is the major constituent of a peak previously ascribed solely to the nonasaccharide G4G4G4G4G4G4G4G3G. Because it was widely presumed that lichenase would cleave G3G4G4G4G3G to MLG2+MLG4, our data both redefine the substrate specificity of Bacillus lichenase and show previous attempts to characterise MLGs by HPLC of lichenase-digests to be flawed. MLG2 subunits are particularly underestimated; often reported as negligible, they are here shown to be an appreciable constituent of MLGs from all three lineages. We also show that there is no appreciable yield of water-soluble lichenase products with DP>9; potential identities of products previously labelled DP>9 are suggested. Finally, this discovery also provides a opportunity to investigate the spatial distribution of subunits along the MLG chain. We show that MLG2 subunits in barley and Cetraria MLG are not randomly distributed, but predominantly found at the non-reducing end of MLG4 subunits.

New gas-phase cascade reactions of stabilized phosphorus ylides leading to ring-fused indoles and to quinolines.

Gas-phase cyclization processes of stabilized phosphorus ylides bearing a suitably substituted 2-aminophenyl group lead efficiently either to 3-substituted quinolines or benzo[c]carbazole and heterocyclic-fused analogues depending on the substituents present.