Cell walls of developing wheat starchy endosperm: comparison of composition and RNA-Seq transcriptome

The transcriptome of the developing starchy endosperm of hexaploid wheat (Triticum aestivum) was determined using RNA-Seq isolated at five stages during grain fill. This resource represents an excellent way to identify candidate genes responsible for the starchy endosperm cell wall, which is dominated by arabinoxylan (AX), accounting for 70% of the cell wall polysaccharides, with 20% (1,3; 1,4)-beta-D-glucan, 7% glucomannan, and 4% cellulose. A complete inventory of transcripts of 124 glycosyltransferase (GT) and 72 glycosylhydrolase (GH) genes associated with cell walls is presented. The most highly expressed GT transcript (excluding those known to be involved in starch synthesis) was a GT47 family transcript similar to Arabidopsis (Arabidopsis thaliana) IRX10 involved in xylan extension, and the second most abundant was a GT61. Profiles for GT43 IRX9 and IRX14 putative orthologs were consistent with roles in AX synthesis. Low abundances were found for transcripts from genes in the acyl-coA transferase BAHD family, for which a role in AX feruloylation has been postulated. The relative expression of these was much greater in whole grain compared with starchy endosperm, correlating with the levels of bound ferulate. Transcripts associated with callose (GSL), cellulose (CESA), pectin (GAUT), and glucomannan (CSLA) synthesis were also abundant in starchy endosperm, while the corresponding cell wall polysaccharides were confirmed as low abundance (glucomannan and callose) or undetectable (pectin) in these samples. Abundant transcripts from GH families associated with the hydrolysis of these polysaccharides were also present, suggesting that they may be rapidly turned over. Abundant transcripts in the GT31 family may be responsible for the addition of Gal residues to arabinogalactan peptide.

The potential to intensify sulforaphane formation in cooked broccoli (Brassica oleracea var. italica) using mustard seeds (Sinapis alba)

Sulforaphane, a naturally occurring cancer chemopreventive, is the hydrolysis product of glucoraphanin, the main glucosinolate in broccoli. The hydrolysis requires myrosinase isoenzyme to be present in sufficient activity; however processing leads to its denaturation and hence reduced hydrolysis. In this study, the effect of adding mustard seeds, which has a more resilient isoform of myrosinase, to processed broccoli was investigated with a view to intensify the formation of sulforaphane. Thermal inactivation of myrosinase from both broccoli and mustard seeds was studied. Thermal degradation of broccoli glucoraphanin was investigated in addition to the effects of thermal processing on the formation of sulforaphane and sulforaphane nitrile. Limited thermal degradation of glucoraphanin (less than 12 %) was observed when broccoli was placed in vacuum sealed bag (sous vide) and cooked in a water bath at 100 ºC for 8 and 12 min. Boiling broccoli in water prevented the formation of any significant levels of sulforaphane due to inactivated myrosinase. However, addition of powdered mustard seeds to the heat processed broccoli significantly increased the formation of sulforaphane.

Roles of proteolysis in regulation of G protein-coupled receptor function

The enzymatic activity of peptidases must be tightly regulated to prevent uncontrolled hydrolysis of peptide bonds, which could have devastating effects in biological systems. Peptidases are often generated as inactive propeptidases, secreted with endogenous inhibitors or they are compartmentalized. Propeptidases become active after proteolytic removal of N-terminal activation peptides by other peptidases. Some peptidases only become active towards substrates only at certain pHs, thus confining activity to specific compartments or conditions. This review discusses the different roles proteolysis plays in regulating G protein-coupled receptors (GPCRs). At the cell-surface, certain GPCRs are regulated by the hydrolytic inactivation of bioactive peptides by membrane-anchored peptidases, which prevents signaling. Conversely, cell-surface peptidases can also generate bioactive peptides that directly activate GPCRs. Alternatively, cell-surface peptidases activated by GPCRs, can generate bioactive peptides to cause transactivation of receptor tyrosine kinases, thereby promoting signaling. Certain peptidases can signals directly to cells, by cleaving GPCR to initiate intracellular signaling cascades. Intracellular peptidases also regulate GPCRs; lysosomal peptidases destroy GPCRs in lysosomes to permanently terminate signaling and mediate downregulation; endosomal peptidases cleave internalized peptide agonists to regulate GPCR recycling, resensitization and signaling; and soluble intracellular peptidases also participate in GPCR function by regulating the ubiquitination state of GPCRs, thereby altering GPCR signaling and fate. Although the use of peptidase inhibitors has already brought success in the treatment of diseases such as hypertension, the discovery of new regulatory mechanisms involving proteolysis that control GPCRs may provide additional targets to modulate dysregulated GPCR signaling in disease.

Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese(II)-dependent enzyme

The mammalian bradykinin-degrading enzyme aminopeptidase P (AP-P; E. C. 3.4.11.9) is a metal-dependent enzyme and is a member of the peptidase clan MG. AP-P exists as membrane-bound and cytosolic forms, which represent distinct gene products. A partially truncated clone encoding the cytosolic form was obtained from a human pancreatic cDNA library and the 5' region containing the initiating Met was obtained by 5' rapid accumulation of cDNA ends (RACE). The open reading frame encodes a protein of 623 amino acids with a calculated molecular mass of 69,886 Da. The full-length cDNA with a C-terminal hexahistidine tag was expressed in Escherichia coli and COS-1 cells and migrated on SDS-PAGE with a molecular mass of 71 kDa. The expressed cytosolic AP-P hydrolyzed the X-Pro bond of bradykinin and substance P but did not hydrolyze Gly-Pro-hydroxyPro. Hydrolysis of bradykinin was inhibited by 1,10-phenanthroline and by the specific inhibitor of the membrane-bound form of mammalian AP-P, apstatin. Inductively coupled plasma atomic emission spectroscopy of AP-P expressed in E. coli revealed the presence of 1 mol of manganese/mol of protein and insignificant amounts of cobalt, iron, and zinc. The enzymatic activity of AP-P was promoted in the presence of Mn(II), and this activation was increased further by the addition of glutathione. The only other metal ion to cause slight activation of the enzyme was Co(II), with Ca(II), Cu(II), Mg(II), Ni(II), and Zn(II) all being inhibitory. Removal of the metal ion from the protein was achieved by treatment with 1,10-phenanthroline. The metal-free enzyme was reactivated by the addition of Mn(II) and, partially, by Fe(II). Neither Co(II) nor Zn(II) reactivated the metal-free enzyme. On the basis of these data we propose that human cytosolic AP-P is a single metal ion-dependent enzyme and that manganese is most likely the metal ion used in vivo.

Peroxidative bromination and oxygenation of organic compounds: synthesis, X-ray crystal structure and catalytic implications of mononuclear and binuclear oxovanadium(V) complexes containing Schiffbase ligands

Treatment of of (R,R)-N,N-salicylidene cyclohexane 1,2-diamine(H(2)L(1)) in methanol with aqueous NH(4)VO(3) solution in perchloric acid medium affords the mononuclear oxovanadium(V) complex [VOL(1)(MeOH)]-ClO(4) (1) as deep blue solid while the treatment of same solution of (R,R)-N,N-salicylidene cyclohexane 1,2-diamine(H(2)L(1)) with aqueous solution of VOSO(4) leads to the formation of di-(mu-oxo) bridged vanadium(V) complex [VO(2)L(2)](2) (2) as green solid where HL(2) = (R,R)-N-salicylidene cyclohexane 1,2-diamine. The ligand HL(2) is generated in situ by the hydrolysis of one of the imine bonds of HL(1) ligand during the course of formation of complex [VO(2)L(2)](2) (2). Both the compounds have been characterized by single crystal X-ray diffraction as well as spectroscopic methods. Compounds 1 and 2 are to act as catalyst for the catalytic bromide oxidation and C-H bond oxidation in presence of hydrogen peroxide. The representative substrates 2,4-dimethoxy benzoic acid and para-hydroxy benzoic acids are brominated in presence of H(2)O(2) and KBr in acid medium using the above compounds as catalyst. The complexes are also used as catalyst for C-H bond activation of the representative hydrocarbons toluene, ethylbenzene and cyclohexane where hydrogen peroxide acts as terminal oxidant. The yield percentage and turnover number are also quite good for the above catalytic reaction. The oxidized products of hydrocarbons have been characterized by GC Analysis while the brominated products have been characterized by (1)H NMR spectroscopic studies.

One-pot three component reaction involving cyclohexyl isocyanide for the synthesis of furo[3,4-b]chromenes

On stirring an equimolar mixture of 4-oxo-4H-chromene-3-carbaldehyde, ninhydrin and cyclohexyl isocyanide in CH(2)Cl(2)-MeOH (7: 1) at room temperature produces 3-cyclohexylimino-1-(2-hydroxy-1,3-dioxo-2,3-dihydro-1H-inden-2-yl)-1,3-dihydro-9H-furo[3,4-b]chromen-9-one which on hydrolysis produces 1-(2-hydroxy-1,3-dioxo-2,3-dihydro-1H-inden-2-yl)-1H-furo[3,4-b]chromene-3,9-dione. The structure of the latter compound was confirmed by single crystal X-ray diffraction

Production of novel ACE inhibitory peptides from β-lactoglobulin using Protease N Amano

Potent angiotensin I-converting enzyme (ACE) inhibitory peptide mixtures were obtained from the hydrolysis of β-lactoglobulin (βLg) using Protease N Amano, a food-grade commercial proteolytic preparation. Hydrolysis experiments were carried out for 8 h at two different temperatures and neutral pH. Based on their ACE inhibitory activity, samples of 6 h of digestion were chosen for further analysis. The temperature used for the hydrolysis had a marked influence on the type of peptides produced and their concentration in the hydrolysate. Protease N Amano was found to produce very complex peptide mixtures; however, the partially fractionated hydrolysates had already very potent ACE inhibitory activity. The novel heptapeptide SAPLRVY was isolated and characterised. It corresponded to βLg f(36–42) and had an IC50 value of 8 μm, which is considerably lower than the most potent ACE inhibitory peptides derived from bovine βLg reported so far.

Variation in root-associated phosphatase activities in wheat contributes to the utilization of organic P substrates in vitro, but does not explain differences in the P-nutrition of plants when grown in soils

To understand whether genotypic variation in root-associated phosphatase activities in wheat impacts on its ability to acquire phosphorus (P), various phosphatase activities of roots were measured in relation to the utilization of organic P substrates in agar, and the P-nutrition of plants was investigated in a range of soils. Root-associated phosphatase activities of plants grown in hydroponics were measured against different organic P substrates. Representative genotypes were then grown in both agar culture and in soils with differing organic P contents and plant biomass and P uptake were determined. Differences in the activities of both root-associated and exuded phosphodiesterase and phosphomonoesterase were observed, and were related to the P content of plants supplied with either ribonucleic acid or glucose 6-phosphate, respectively, as the sole form of P. When the cereal lines were grown in different soils, however, there was little relationship between any root-associated phosphatase activity and plant P uptake. This indicates that despite differences in phosphatase activities of cereal roots, such variability appears to play no significant role in the P-nutrition of the plant grown in soil, and that any benefit derived from the hydrolysis of soil organic P is common to all genotypes.

Association of lipoprotein lipase gene polymorphisms with obesity and type 2 diabetes in an Asian Indian population

AIMS: Lipoprotein lipase (LPL), a pivotal enzyme in lipoprotein metabolism, catalyzes the hydrolysis of triglycerides of very low-density lipoproteins and chylomicrons. Assuming that the variants in the promoter of the LPL gene may be associated with changes in lipid metabolism leading to obesity and type 2 diabetes, we examined the role of promoter variants (-T93G and -G53C) in the LPL gene in an urban South Indian population. METHODS: The study subjects (619 type 2 diabetic and 731 normal glucose-tolerant (NGT) subjects) were chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The polymorphisms were genotyped using polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP). Linkage disequilibrium (LD) was estimated from the estimates of haplotypic frequencies. RESULTS: The two polymorphisms studied were not in LD. The -T93G was not associated with type 2 diabetes but was associated with obesity. 11.5% of the obese subjects (62/541) had the XG(TG+GG) genotype compared with 6.4% of the nonobese subjects (52/809; P=0.001). The odds ratio for obesity for the XG genotype was 1.766 (95% CI: 1.19-2.63, P=0.005). Subjects with XG genotype also had higher body mass index and waist circumference compared with those with TT genotype. With respect to G53C, subjects with the XC(GC+CC) genotype had 0.527 and 0.531 times lower risk for developing type 2 diabetes and obesity, respectively. CONCLUSIONS: Among Asian Indians, the -T93G SNP of the LPL gene is associated with obesity but not type 2 diabetes, whereas the -G53C SNP appears to be protective against both obesity and type 2 diabetes.

Endocannabinoids in neuroendopsychology: multiphasic control of mitochondrial function

The endocannabinoid system (ECS) is a construct based on the discovery of receptors that are modulated by the plant compound tetrahydrocannabinol and the subsequent identification of a family of nascent ligands, the 'endocannabinoids'. The function of the ECS is thus defined by modulation of these receptors-in particular, by two of the best-described ligands (2-arachidonyl glycerol and anandamide), and by their metabolic pathways. Endocannabinoids are released by cell stress, and promote both cell survival and death according to concentration. The ECS appears to shift the immune system towards a type 2 response, while maintaining a positive energy balance and reducing anxiety. It may therefore be important in resolution of injury and inflammation. Data suggest that the ECS could potentially modulate mitochondrial function by several different pathways; this may help explain its actions in the central nervous system. Dose-related control of mitochondrial function could therefore provide an insight into its role in health and disease, and why it might have its own pathology, and possibly, new therapeutic directions.

Intra-crystalline protein diagenesis (IcPD) in Patella vulgata. Part II: Breakdown and temperature sensitivity

Artificial diagenesis of the intra-crystalline proteins isolated from Patella vulgata was induced by isothermal heating at 140 °C, 110 °C and 80 °C. Protein breakdown was quantified for multiple amino acids, measuring the extent of peptide bond hydrolysis, amino acid racemisation and decomposition. The patterns of diagenesis are complex; therefore the kinetic parameters of the main reactions were estimated by two different methods: 1) a well-established approach based on fitting mathematical expressions to the experimental data, e.g. first-order rate equations for hydrolysis and power-transformed first-order rate equations for racemisation; and 2) an alternative model-free approach, which was developed by estimating a “scaling” factor for the independent variable (time) which produces the best alignment of the experimental data. This method allows the calculation of the relative reaction rates for the different temperatures of isothermal heating. High-temperature data were compared with the extent of degradation detected in sub-fossil Patella specimens of known age, and we evaluated the ability of kinetic experiments to mimic diagenesis at burial temperature. The results highlighted a difference between patterns of degradation at low and high temperature and therefore we recommend caution for the extrapolation of protein breakdown rates to low burial temperatures for geochronological purposes when relying solely on kinetic data.

Consumer acceptability and sensory profile of cooked broccoli with mustard seeds added to improve chemoprotective properties

Broccoli, a rich source of glucosinolates, is a commonly consumed vegetable of the Brassica family. Hydrolysis products of glucosinolates, isothiocyanates, have been associated with health benefits and contribute to the flavour of Brassica. However, boiling broccoli causes the myrosinase enzyme needed for hydrolysis to denature. In order to ensure hydrolysis, broccoli must either be mildly cooked or active sources of myrosinase, such as mustard seed powder, can be added post-cooking. In this study, samples of broccoli were prepared in six different ways; standard boiling with and without mustard seeds, sous-vide cooking at low temperature (70 °C) and sous-vide cooking at higher temperature (100 ºC) without mustard and with mustard at two different concentrations. The majority of consumers disliked the mildly cooked broccoli samples (70 ºC, 12 min, sous-vide) which had a hard and stringy texture. The highest mean consumer liking was for standard boiled samples (100 ºC, 7 min). Addition of 1% mustard seed powder developed sensory attributes such as pungency, burning sensation, mustard odour and flavour. One cluster of consumers (32%) found mustard seeds to be a good complement to cooked broccoli, however, the majority disliked the mustard-derived sensory attributes. Where the mustard seeds were partially processed, doubling the addition to 2% led to only the same level of mustard flavour and pungency as 1% unprocessed seeds, and mean consumer liking remained unaltered. This suggests that optimisation of the addition level of partially processed mustard seeds may be a route to enhance bioactivity of cooked broccoli without compromising consumer acceptability.

Olfactory selection of Plantago lanceolata by snails declines with seedling age

Background and Aims Despite recent recognition that (1) plant–herbivore interactions during the establishment phase, (2) ontogenetic shifts in resource allocation and (3) herbivore response to plant volatile release are each pivotal to a comprehensive understanding of plant defence, no study has examined how herbivore olfactory response varies during seedling ontogeny. Methods Using a Y-tube olfactometer we examined snail (Helix aspersa) olfactory response to pellets derived from macerated Plantago lanceolata plants harvested at 1, 2, 3, 4, 5, 6 and 8 weeks of age to test the hypothesis that olfactory selection of plants by a generalist herbivore varies with plant age. Plant volatiles were collected for 10 min using solid-phase microextraction technique on 1- and 8-week-old P. lanceolata pellets and analysed by gas chromatography coupled with a mass spectrometer. Key Results Selection of P. lanceolata was strongly negatively correlated with increasing age; pellets derived from 1-week-old seedlings were three times more likely to be selected as those from 8-week-old plants. Comparison of plant selection experiments with plant volatile profiles from GC/MS suggests that patterns of olfactory selection may be linked to ontogenetic shifts in concentrations of green leaf volatiles and ethanol (and its hydrolysis derivatives). Conclusions Although confirmatory of predictions made by contemporary plant defence theory, this is the first study to elucidate a link between seedling age and olfactory selection by herbivores. As a consequence, this study provides a new perspective on the ontogenetic expression of seedling defence, and the role of seedling herbivores, particularly terrestrial molluscs, as selective agents in temperate plant communities.

Thermal and pressure stability of myrosinase enzymes from black mustard (Brassica nigra L. W.D.J Koch. var. nigra), brown mustard (Brassica juncea L. Czern. var. juncea) and yellow mustard (Sinapsis alba L. Subsp Maire) seeds

This study investigates the effects of temperature and pressure on inactivation of myrosinase extracted from black, brown and yellow mustard seeds. Brown mustard had higher myrosinase activity (2.75 un/mL) than black (1.50 un/mL) and yellow mustard (0.63 un/mL). The extent of enzyme inactivation increased with pressure (600-800 MPa) and temperature (30-70 °C) for all the mustard seeds. However, at combinations of lower pressures (200-400 MPa) and high temperatures (60-80 °C), there was less inactivation. For example, application of 300 MPa and 70 °C for 10 minutes retained 20%, 80% and 65% activity in yellow, black and brown mustard, respectively, whereas the corresponding activity retentions when applying only heat (70 °C, 10min) were 0%, 59% and 35%. Thus, application of moderate pressures (200-400 MPa) can potentially be used to retain myrosinase activity needed for subsequent glucosinolate hydrolysis.

Endothelin-1 and fibroblast growth factors stimulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes. The potential role of the cascade in the integration of two signaling pathways leading to myocyte hypertrophy.

Maximally effective concentrations of endothelin-1 (ET-1), acidic FGF (aFGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA) activated mitogen-activated protein kinase (MAPK) by 3-4-fold in crude extracts of myocytes cultured from neonatal rat heart ventricles. Maximal activation was achieved after 5 min. Thereafter, MAPK activity stimulated by ET-1 or aFGF declined to control values within 1-2 h, whereas activation by TPA was more sustained. Two peaks of MAPK activity (a 42- and a 44-kDa MAPK) were resolved in cells exposed to ET-1 or aFGF by fast protein liquid chromatography on a Mono Q column. One major and one minor peak of MAPK kinase (MAPKK) was stimulated by ET-1 or aFGF. Cardiac myocytes expressed protein kinase C (PKC)-alpha, -delta, -epsilon and -zeta as shown immunoblotting. Exposure to 1 microM TPA for 24 h down-regulated PKC-alpha, -delta, and -epsilon, but not PKC-zeta. This maneuver wholly abolished the activation of MAPK on re-exposure to TPA but did not affect the response to aFGF. The effect of ET-1 was partially down-regulated. ET-1 stimulated phospho[3H]inositide hydrolysis 18-fold, whereas aFGF stimulated by only 30%. Agonists which initially utilize dissimilar signaling pathways may therefore converge at the level of MAPKK/MAPK and this may be relevant to the hypertrophic response of the heart.