Starfish saponins. XII. Sulfated steroid glycosides from the starfish Hacelia attenuata

Three minor sulfated steroidal xylosides were isolated from the Mediterranean starfish H. attenuata. The major one, attenuatoside S-I, contains the aglycon 24-ethyl-5α-cholestane-3β,6β,8,15α,16β,29-hexol; the xylopyranosyl unit is β-glycosidically attached to C(29); the sulfate is at C(15α). The 2nd one, attenuatoside S-II, is the Δ27 analog. Their structures were deduced by spectroscopic methods and confirmed by transformation of desulfated attenuatoside S-II to desulfated attenuatoside S-I. The 3rd compd., attenuatoside S-III, present only in trace amt., is isomeric with attenuatoside S-I in which the primary hydroxyl is apparently at C(26) rather than at C(29). Another asterosaponin, attenuatoside D, contains the novel aglycon 24-northornasterol A [3β,6α,20-trihydroxy-24-nor-5α-cholest-9(11)-en-23-one], 5 sugar units attached by a glycosidic linkage to C(6) of the aglycon and a sulfate group at C(3)

Shall I compare thee to a GM potato?

A fundamental issue in the safety assessment of genetically modified crops is the question of whether unintentional changes have occurred in the crop plant as a consequence of the genetic modification. This question was addressed recently by using a powerful metabolite fingerprinting and metabolite profiling method to assess whether genetically modified potatoes are substantially similar to their corresponding conventional cultivars

Stability of the major allergen Brazil nut 2S albumin (Ber e 1) to physiologically-relevant in vitro gastrointestinal digestion

The major 2S albumin allergen from Brazil nuts, Ber e 1, was subjected to gastrointestinal digestion using a physiologically relevant in vitro model system either before or after heating (100 °C for 20 min). Whilst the albumin was cleaved into peptides, these were held together in a much larger structure even when digested by using a simulated phase 1 (gastric) followed by a phase 2 (duodenal) digestion system. Neither prior heating of Ber e 1 nor the presence of the physiological surfactant phosphatidylcholine affected the pattern of proteolysis. After 2 h of gastric digestion, ≈ 25% of the allergen remained intact, ≈ 50% corresponded to a large fragment of Mr 6400, and the remainder comprised smaller peptides. During duodenal digestion, residual intact 2S albumin disappeared quickly, but a modified form of the ‘large fragment’ remained, even after 2 h of digestion, with a mass of ≈ 5000 Da. The ‘large fragment’ comprised several smaller peptides that were identified, by using different MS techniques, as deriving from the large subunit. In particular, sequences corresponding to the hypervariable region (Q37–M47) and to another peptide (P42–P69), spanning the main immunoglobulin E epitope region of 2S albumin allergens, were found to be largely intact following phase 1 (gastric) digestion. They also contained previously identified putative T-cell epitopes. These findings indicate that the characteristic conserved skeleton of cysteine residues of 2S albumin family and, particularly, the intrachain disulphide bond pattern of the large subunit, play a critical role in holding the core protein structure together even after extensive proteolysis, and the resulting structures still contain potentially active B- and T-cell epitopes.This research was supported by a Marie Curie Fellowship of the European Community programme ‘Qualityof Life and Management of Living Resources’ under contract number QLK1-CT-2001–51997 and the BBSRC competitive strategic grant to IFR. MALDI-TOF and Q-TOF mass spectrometry was carried outin the Joint IFR-JIC Proteomics Facility which is funded in part by BBSRC (JREI grant numbers: JRE10832, JE412701, JE412631) and by grants from Syngenta and Unilever.Peer Reviewe

Isolation, identification and stability of acylated derivatives of apigenin 7-O-glucoside from chamomile (Chamomilla recutita [L.] Rauschert)

The major flavonoids in the white florets of chamomile (Chamomilla recutita [L.] Rauschert) were rapidly purified using a combination of polyamide solid-phase extraction and preparative HPLC. From the combined LC/MS, LC/MS/MS, and NMR data the apigenin glucosides were identified as apigenin 7-O-glucoside (Ap-7-Glc), Ap-7-(6''-malonyl-Glc), Ap-7-(6''-acetyl-Glc), Ap-7-(6''- caffeoyl-Glc), Ap-7-(4-''acetyl-Glc), Ap-7-(4''-acetyl,6-''malonyl-Glc), and a partially characterised apigenin-7-(mono-acetyl/monomalonylglucoside) isomer. Malonyl and caffeoyl derivatives of Ap-7-Glc have not previously been identified in chamomile. The two mono-acetyl/mono-malonyl flavonoids have not previously been reported in any plant species. These acylated glucosides are unstable and degrade to form acetylated compounds or Ap-7-Glc. The degradation products formed are dependent on the extraction and storage conditions, i.e. temperature, pH and solvent

Mass spectrometry and structural characterization of 2S albumin isoforms from Brazil nuts (Bertholletia excelsa)

Proteomic approaches have been used to characterise the main 2S albumin isoforms from Brazil nuts (Bertholletia excelsa). Whilst most isoforms (∼10 discrete protein species) exhibited molecular masses of around 12 kDa with a high amino acid sequence homology, important charge heterogeneity was found, with pIs varying between 4.6 and 6.6, with one ≥7.0. Proteomic analysis showed that these corresponded to a total of six National Center for Biotechnology Information (NCBI) accessions and that three isoforms had been purified to homogeneity corresponding to gi/384327, 112754 and 99609. The latter sequence corresponds to an isoform, previously only identified at the nucleotide sequence level, had a slightly higher molecular weight (13.4 kDa), and with noticeable differences in the primary structure. Proteins corresponding to six different NCBI accessions were identified, the heterogeneity of which had been increased by posttranslational processing. Evidence was found of cyclization of the N-terminal glutamine residue in two isoforms, together with ragged C-termini, indicative of carboxypeptidase activity within the vacuole following posttranslational processing. No evidence of glycosylation was found. Circular dichroism (CD) and Fourier transform-infrared (FT-IR) spectroscopy indicated all the studied isoforms were predominantly α-helical in nature, but that the Mr 13 400 species was structurally distinct, with a higher proportion of α-helical structure.This research has been supported by a Marie Curie Fellowship of the European Community programme “Quality of Life and Management of Living Resources” under contract number QLK1-CT-2001-51997 and the BBSRC competitive strategic grant for IFR.Peer Reviewe

Rerouting the Plant Phenylpropanoid Pathway by Expression of a Novel Bacterial Enoyl-CoA Hydratase/Lyase Enzyme Function

The gene for a bacterial enoyl-CoA hydratase (crotonase) homolog (HCHL) previously shown to convert 4-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA to the corresponding hydroxybenzaldehydes in vitro provided an opportunity to subvert the plant phenylpropanoid pathway and channel carbon flux through 4-hydroxybenzaldehyde and the important flavor compound 4-hydroxy-3-methoxybenzaldehyde (vanillin). Expression of the Pseudomonas fluorescens AN103 HCHL gene in two generations of tobacco plants caused the development of phenotypic abnormalities, including stunting, interveinal chlorosis and senescence, curled leaf margins, low pollen production, and male sterility. In second generation progeny, the phenotype segregated with the transgene and transgenic siblings exhibited orange/red coloration of the vascular ring, distorted cells in the xylem and phloem bundles, and lignin modification/reduction. There was depletion of the principal phenolics concomitant with massive accumulation of novel metabolites, including the glucosides and glucose esters of 4-hydroxybenzoic acid and vanillic acid and the glucosides of 4-hydroxybenzyl alcohol and vanillyl alcohol. HCHL plants exhibited increased accumulation of transcripts for phenylalanine ammonia-lyase, cinnamate-4-hydroxylase, and 4-coumarate:CoA ligase, whereas β-1,3-glucanase was suppressed. This study, exploiting the ability of a bacterial gene to divert plant secondary metabolism, provides insight into how plants modify inappropriately accumulated metabolites and reveals the consequences of depleting the major phenolic pools