Trevor Gibson

Glucosinolate‐accumulating S‐cells in Arabidopsis leaves and flower stalks undergo programmed cell death at early stages of differentiation

The plant secondary metabolites glucosinolates (GSL) have important functions in plant resistance to herbivores and pathogens. We identified all major GSL that accumulated in S-cells in Arabidopsis by MALDI-TOF MS, and estimated by LC-MS that the total GSL concentration in these cells is >130 mM. The precise locations of the S-cells outside phloem bundles in rosette and cauline leaves and in flower stalks were visualised using sulphur mapping by cryo-SEM/energy-dispersive X-ray analysis. S-cells contain up to 40% of the total sulphur in flower stalk tissues. S-cells in emerging flower stalks and developing leaf tissues show typical signs of programmed cell death (PCD) or apoptosis, such as chromatin condensation in the nucleus and blebbing of the membranes. TUNEL staining for DNA double-strand breaks confirmed the occurrence of PCD in S-cells in post-meristematic tissues in the flower stalk as well as in the leaf. Our results indicate that S-cells in post-meristematic tissues show an extreme degree of metabolic specialisation in addition to PCD. Accumulation and maintenance of a high concentration of GSL in these cells are accompanied by degradation of a number of cell organelles. The substantial changes in cell composition during S-cell differentiation indicate the importance of this particular GSL-based phloem defence system. The specific anatomy of the S-cells and the ability to accumulate specialised secondary metabolites is similar to that of the non-articulated laticifer cells in latex plants, suggesting a common evolutionary origin.

Analysis of Phytochemical Composition and Chemoprotective Capacity of Rocket (Eruca sativa and Diplotaxis tenuifolia) Leafy Salad Following Cultivation in Different Environments

Consumption of green leafy vegetables is associated with reduced risk of several types of cancer and cardiovascular disease. These beneficial effects are attributed to a range of phytochemicals including flavonoids and glucosinolates, both of which are found in high levels in Brassicaceous crops. Rocket is the general name attributed to cultivars of Eruca sativa and Diplotaxis tenufolia, known as salad rocket and wild rocket, respectively. We have shown that different light levels during the cultivation period of these crops have a significant impact on the levels of flavonoids present in the crop at harvest, with over 15-fold increase achieved in quercetin, isorhamnetin, and cyanidin in high light conditions. Postharvest storage further affects the levels of both flavonoids and glucosinolates, with cyanidin increasing during shelf life and some glucosinolates, such as glucoiberverin, being reduced over the same storage period. In vitro assays using human colon cell lines demonstrate that glucosinolate-rich extracts of Eruca sativa cv. Sky, but not Diplotaxis tenufolia cv. Voyager, confer significant resistance to oxidative stress on the cells, which is indicative of the chemoprotective properties of the leaves from this species. Our findings indicate that both pre and postharvest environment and genotypic selection, when developing new lines of Brassicaceous vegetables, are important considerations with the goal of improving human nutrition and health.

Dietary quebracho tannins are not absorbed, but increase the antioxidant capacity of liver and plasma in sheep.

A total of sixteen lambs were divided into two groups and fed two different diets. Of these, eight lambs were fed a control diet (C) and eight lambs were fed the C diet supplemented with quebracho tannins (C+T). The objective of the present study was to assess whether dietary quebracho tannins can improve the antioxidant capacity of lamb liver and plasma and if such improvement is due to a direct transfer of phenolic compounds or their metabolites, to the animal tissues. Feed, liver and plasma samples were purified by solid-phase extraction (SPE) and analysed by liquid chromatography-MS for phenolic compounds. Profisitinidin compounds were identified in the C+T diet. However, no phenolic compounds were found in lamb tissues. The liver and the plasma from lambs fed the C+T diet displayed a greater antioxidant capacity than tissues from lambs fed the C diet, but only when samples were not purified with SPE. Profisetinidin tannins from quebracho seem not to be degraded or absorbed in the gastrointestinal tract. However, they induced antioxidant effects in animal tissues.

Platelet-mediated metabolism of the common dietary flavonoid, quercetin.

Background Flavonoid metabolites remain in blood for periods of time potentially long enough to allow interactions with cellular components of this tissue. It is well-established that flavonoids are metabolised within the intestine and liver into methylated, sulphated and glucuronidated counterparts, which inhibit platelet function. Methodology/Principal Findings We demonstrate evidence suggesting platelets which contain metabolic enzymes, as an alternative location for flavonoid metabolism. Quercetin and a plasma metabolite of this compound, 4′-O-methyl quercetin (tamarixetin) were shown to gain access to the cytosolic compartment of platelets, using confocal microscopy. High performance liquid chromatography (HPLC) and mass spectrometry (MS) showed that quercetin was transformed into a compound with a mass identical to tamarixetin, suggesting that the flavonoid was methylated by catechol-O-methyl transferase (COMT) within platelets. Conclusions/Significance Platelets potentially mediate a third phase of flavonoid metabolism, which may impact on the regulation of the function of these cells by metabolites of these dietary compounds.

Novel probiotic-fermented milk with angiotensin I-converting enzyme inhibitory peptides produced by Bifidobacterium bifidum MF 20/5.

In previous research, we have demonstrated that Bifidobacterium bifidum MF 20/5 fermented milk possessed stronger angiotensin converting enzyme (ACE) inhibitory activity than other lactic acid bacteria, including Lactobacillus helveticus DSM 13137, which produces the hypotensive casokinins Ile-Pro-Pro (IPP) and Val-Pro-Pro (VPP). The aim of this study is to investigate the ACE-inhibitory peptides released in B. bifidum MF 20/5 fermented milk. The novel ACE-inhibitory peptide LVYPFP (IC50 = 132 μM) is reported here for the first time. Additionally, other bioactive peptides such as the ACE-inhibitor LPLP (IC50 = 703 μM), and the antioxidant VLPVPQK were identified. Moreover, the peptide and amino acid profiles, the ACE-inhibitory activity (ACEi), pH, and degree of hydrolysis of the fermented milk were determined and compared with those obtained in milk fermented by L. helveticus DSM 13137. The sequences of the major bioactive peptides present in fermented milk of B. bifidum and L. helveticus were identified and quantified. B. bifidum released a larger amount of peptides than L. helveticus but no IPP or VPP were detected in B. bifidum fermented milk. Also the lactotripeptide concentrations and ACEi were higher in L. helveticus fermented milk when the pH was maintained at 4.6. This may represent a technical advantage for B. bifidum that reduces the pH at a slow enough rate to facilitate the peptide generation without the need for pH control. Thus these findings show the potential for the use of this probiotic strain to produce fermented milk with a wider range of health benefits including reduction of blood pressure.

Deciphering the complexity of sainfoin (Onobrychis viciifolia) proanthocyanidins by MALDI-TOF mass spectrometry with a judicious choice of isotope patterns and matrixes.

Use of superdihydroxybenzoic acid as the matrix enabled the analysis of highly complex mixtures of proanthocyanidins from sainfoin (Onobrychis viciifolia) by MALDI-TOF mass spectrometry. Proanthocyanidins contained predominantly B-type homopolymers and heteropolymers up to 12-mers (3400 Da). Use of another matrix, 2,6-dihydroxyacetophenone, revealed the presence of A-type glycosylated dimers. In addition, we report here how a comparison of the isotopic adduct patterns, which resulted from Li and Na salts as MALDI matrix additives, could be used to confirm the presence of A-type linkages in complex proanthocyanidin mixtures. Preliminary evidence suggested the presence of A-type dimers in glycosylated prodelphinidins and in tetrameric procyanidins and prodelphinidins.

Production of angiotensin-I-converting enzyme inhibitory peptides from β-lactoglobulin- and casein-derived peptides: An integrative approach

Angiotensin I‐converting enzyme (ACE) inhibition is one of the mechanisms by which reduction in blood pressure is exerted. Whey proteins are a rich source of ACE inhibitory peptides and have shown a blood pressure reduction effect i.e. antihypertensive activity. The aim of this work was to develop a simplified process using a combination of adsorption and microfiltration steps for the production of hydrolysates from whey with high ACE inhibitory activity and potency; the latter was measured as the IC50, which is the peptide concentration required to reduce ACE activity by half. This process integrates the selective separation of β‐lactoglobulin‐ and casein‐derived peptides (CDP) from rennet whey and their hydrolysis, which results in partially pure, less complex hydrolysates with high bioactive potency. Hydrolysis was carried out with protease N “Amano” in a thermostatically controlled membrane reactor operated in a batch mode. By applying the integrative approach it was possible to produce from the same feedstock two different hydrolysates that exhibited high ACE inhibition. One hydrolysate was mainly composed of casein‐derived peptides with IC50 = 285 μg/mL. In this hydrolysate we identified the well‐known potent ACE‐inhibitor and antihypertensive tripeptide Ile‐Pro‐Pro (IPP) and another novel octapeptide Gln‐Asp‐Lys‐Thr‐Glu‐Ile‐Pro‐Thr (QDKTEIPT). The second hydrolysate was mainly composed of β‐lactoglobulin derived peptides with IC50 =128 μg/mL. This hydrolysate contained a tetrapeptide (Ile‐Ile‐Ala‐Glu) IIAE as one of the two major peptides. A further advantage to this process is that enzyme activity was substantially increased as enzyme product inhibition was reduced.

Chemical characterisation and determination of sensory attributes of hydrolysates produced by enzymatic hydrolysis of whey proteins following a novel integrative process

The overall aim of this work was to characterise the major angiotensin-converting enzyme (ACE) inhibitory peptides produced by enzymatic hydrolysis of whey proteins, through the application of a novel integrative process. This process consisted of the combination of adsorption and microfiltration within a stirred cell unit for the selective immobilisation of β-lactoglobulin and casein-derived peptides (CDP) from whey. The adsorbed proteins were hydrolysed in situ, which resulted in the separation of peptide products from the substrate and fractionation of peptides. Two different hydrolysates were produced: (i) from CDP (IC(50)=287 μg/mL) and (ii) from β-lactoglobulin (IC(50)=128 μg/mL). The well-known antihypertensive peptide IPP and several novel peptides that have structural similarities with reported ACE inhibitory peptides were identified and characterised in both hydrolysates. Furthermore, the hydrolysates were assessed for bitterness. No significant difference was found between the bitterness of the control (milk with no hydrolysate) and hydrolysate samples at different concentrations (at, below and above the IC(50)).

Plant growth-promoting Bacillus suppress Brevicoryne brassicae field infestation and trigger density-dependent and density-independent natural enemy responses

AbstractSoil-dwelling plant growth-promoting (PGP) Bacillus lives in intimate associations with plants; some species offer direct benefits via plant growth promotion while others confer protection against various pathogens. However, the roles of PGP Bacillus as elicitors of plant defences against agricultural pests and as a component of integrated pest management systems remain virtually unexplored. The effects of three major ubiquitous gram-positive rhizobacteria; Bacillus cereus, Bacillus subtilis and Bacillus amyloliquefaciens were studied individually and in admixture on (i) calabrese (sprouting broccoli, Brassica oleracea) vegetative and reproductive growth parameters and (ii) the population dynamics of the specialist cosmopolitan pest, cabbage aphid (Brevicoryne brassicae) infestation, and its important natural enemies; the braconid endoparasitoid (Diaeretiellarapae), ladybird beetle (Coccinella septempunctata) and syrphid fly (all species). We found that all Bacillus treatments efficiently suppressed B. brassicae field populations in varying magnitudes. B. cereus and B. subtilis significantly increased the rates of parasitism by D. rapae, however, none of the other treated plants lured natural enemies, which responded in a density-dependent manner. Although the mixed Bacillus treatment significantly reduced root weight ratio, none of the Bacillus spp. treatments produced significant effects on calabrese growth. Taken together, PGP Bacillus may offer multiple plant benefits through suppressed pest infestation and increased percent parasitism in the field, with potential applications in integrated pest management.

Proportions of A1, A2, B and C β-casein protein variants in retail milk in the UK

The A1 variant protein of the β-casein family has been implicated in various disease states although much evidence is weak or contradictory. The primary objective was to measure, for the first time, the proportions of the key β-casein variant proteins in UK retail milk over the course of one year. In total, 55 samples of semi-skimmed milk were purchased from five supermarkets over the course of a year and the proportions of the A1, A2, B and C casein variant proteins were measured, using high resolution HPLC-MS. The results showed that β-casein in UK retail milk comprises approximately 0.58, 0.31, 0.07 and 0.03 A2, A1, B and C protein variants, respectively. The proportion of A2 is higher than some early studies would predict although the reasons for this and any implications for health are unclear.