Stability and biological activity of wild blueberry (Vaccinium angustifolium) polyphenols during simulated in vitro gastrointestinal digestion.

Wild blueberries are rich in polyphenols and have several potential health benefits. Understanding the factors that affect the bioaccessibility and bioavailability of polyphenols is important for evaluating their biological significance and efficacy as functional food ingredients. Since the bioavailability of polyphenols such as anthocyanins is generally low, it has been proposed that metabolites resulting during colonic fermentation may be the components that exert health benefits. In this study, an in vitro gastrointestinal model comprising sequential chemostat fermentation steps that simulate digestive conditions in the stomach, small intestine and colon was used to investigate the breakdown of blueberry polyphenols. The catabolic products were isolated and biological effects tested using a normal human colonic epithelial cell line (CRL 1790) and a human colorectal cancer cell line (HT 29). The results showed a high stability of total polyphenols and anthocyanins during simulated gastric digestion step with approximately 93% and 99% of recovery, respectively. Intestinal digestion decreased polyphenol- and anthocyanin- contents by 49% and 15%, respectively, by comparison to the non-digested samples. During chemostat fermentation that simulates colonic digestion, the complex polyphenol mixture was degraded to a limited number of phenolic compounds such as syringic, cinnamic, caffeic, and protocatechuic acids. Only acetylated anthocyanins were detected in low amounts after chemostat fermentation. The catabolites showed lowered antioxidant activity and cell growth inhibition potential. Results suggest that colonic fermentation may alter the biological activity of blueberry polyphenols.

PpERF3b, a transcriptional repressor from peach, contributes to disease susceptibility and side branching in EAR-dependent and -independent fashions.

KEY MESSAGE: Peach ERF3b is a potent transcriptional repressor for defense-related genes even in the presence of similar levels of transcriptional activators and can interfere with plant development through pathways independent of the EAR motif. Ethylene response factors (ERFs) are a major group of plant transcription factors with either activation or repression capabilities on gene transcription. Repressor-type ERFs are characterised by an intrinsic motif, namely the ERF-associated amphiphilic repression motif (EAR). Here we report the identification of three genes from peach (Prunus persica), PpERF12, PpERF3a and PpERF3b, encoding for ERF repressors. The transcription kinetics of these genes was investigated by qRT-PCR after inoculation of peach leaves with Xanthomonas campestris pv. pruni. All three genes showed higher induction in the susceptible 'BabyGold 5', than in the resistant 'Venture' peach varieties suggesting a negative role for these genes in disease resistance. The functional potency of PpERF3b has been confirmed in vivo by its ability to repress the expression of GUS-reporter gene. To better understand the functional role of PpERF3b, the full-length and the EAR-truncated (PpERF3b∆EAR) genes were overexpressed in tobacco (Nicotiana tabacum). Both transgenic plants (PpERF3b and PpERF3b∆EAR) uniformly exhibited precocious side branching, which suggests the interference of PpERF3b with auxin-mediated dormancy of lateral shoots. Consistent with that the expression of auxin-response factors (Nt-ARF1, Nt-ARF6 and Nt-ARF8) was significantly downregulated in transgenic plants compared to the wild type (WT). Although side branching was independent of EAR motif, the response of transgenic plants to inoculation by Pseudomonas syringae pv. tabaci was EAR dependent. Transgenic plants overexpressing PpERF3b∆EAR showed less disease symptoms than those overexpressing the full-length gene or WT plants. Resistance of PpERF3b∆EAR plants was associated with enhanced induction of pathogenesis-related (PR) genes. Our results indicate that repressor-type ERFs might act through pathways that are dependent or independent of the EAR motif.

Differential expression of peach ERF transcriptional activators in response to signaling molecules and inoculation with Xanthomonas campestris pv. pruni.

Ethylene response factors (ERFs) are a large family of transcription factors (TFs) that have diverse functions in plant development and immunity. However, very little is known about the molecular regulation of these TFs in stone fruits during disease incidence. In the present study, we describe the identification of five peach ERFs (Pp-ERFs), aiming to elucidate their potential roles in defense against Xanthomonas campestris pv. pruni (Xcp), the causal agent of bacterial spot disease. The phylogenetic analysis along with sequence comparisons indicated that all Pp-ERFs are transcriptional activators belonging to groups IX and IIV ERFs. The transactivation capacity of these proteins was verified in vivo where they all induced the expression of the GUS reporter gene and in a GCC-dependent manner. The nuclear localization was also confirmed for two of these proteins, Pp-ERF2.b and Pp-ERF2.c, after their transient expression in onion epidermal cells. The induction kinetics of Pp-ERFs after inoculation with Xcp was determined by qRT-PCR. Except for Pp-ERF2.b, transcript levels of Pp-ERFs increased strongly and rapidly in the resistant 'Venture' compared to the susceptible 'BabyGold 5' cultivar after infection with Xcp. In contrast, the expression of Pp-ERF2.b was several-fold higher in the susceptible cultivar after bacterial infection. The expression of Pp-ERFs was also monitored after treating with signaling compounds; salicylic acid (SA) (1 mM), ethephon (1 mM) and methyl jasmonate (MeJA) (50 µM). Although the results generally emphasize the role of ethylene/jasmonic acid (ET/JA) signaling pathways in regulating the expression of Pp-ERFs, there was a coordination of the timing of ET/JA responses, suggesting compensatory rather than synergistic interactions between these pathways during defense against Xcp.

Consumption of guar gum and retrograded high-amylose corn resistant starch increases IL-10 abundance without affecting pro-inflammatory cytokines in the colon of pigs fed a high-fat diet.

Increases in dietary intake of viscous and nonviscous soluble fiber are reported to improve bowel health. However, related biological mechanisms are not very clear. This study was conducted to examine if colonic inflammation would occur in a typical Western diet model and determine if consumption of soluble fiber components would attenuate potential detrimental effects by differentially affecting colonic abundances of anti-inflammatory cytokine IL-10 and 2 pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) and IL-6 in pigs fed a high-fat basal diet supplemented, respectively, with 15% viscous soluble fiber guar gum (GG) and 15% nonviscous soluble fiber, that is, retrograded high-amylose corn (Zea mays) resistant starch (RS). A total of 24 Yorkshire growing barrows were assigned into a standard corn and soybean (Glycine max) meal (SBM)-based grower diet as a positive control (PC), an animal protein-based high-fat basal diet as the negative control (NC), and 2 NC basal diets supplemented with 15% GG and 15% RS, respectively, according to a completely randomized block design for 4 wk. Abundance of these cytokines in homogenized and extracted colonic tissue supernatant samples was measured by ELISA. Although colonic IL-10 abundance was lower (P < 0.05) in the corn and SBM-based PC group than that in the high-fat basal NC group, there were no differences (P > 0.05) in colonic abundances of TNF-α and IL-6 between NC and PC groups and among all of the treatment groups. Compared with the NC group, consumption of GG and RS at 15% increased (P < 0.05) colonic IL-10 abundance. Moreover, there was no difference (P > 0.05) in colonic IL-10 abundance between the 15% GG and the 15% RS groups. Thus, consumption of a typical high-fat Western diet did not induce colonic inflammation. Diets supplemented with 15% GG or 15% RS may protect the colon from developing inflammation by enhancing IL-10 abundance.

Molecular characterization of peach PR genes and their induction kinetics in response to bacterial infection and signaling molecules.

'Venture' and 'BabyGold 5' are two peach cultivars with a demonstrated resistance and susceptibility, respectively, to bacterial spot disease caused by Xanthomonas campestris pv. pruni (Xcp). To explore the differences between these cultivars at the molecular level, two PR1 (Pp-PR1a, Pp-PR1b) and three PR5 (Pp-TLP1, Pp-TLP2 and Pp-TLP3) genes were isolated from peach (Prunus persica L.) and investigated by in silico and in situ approaches. The analysis of gene expression by qRT-PCR indicated that all PR genes, except Pp-PR1a, were induced to a significantly higher degree in the resistant cultivar. In response to signaling molecules, Pp-PR1a was induced chiefly by SA treatment, while other PR genes were induced mainly by ethephon or MeJA treatments. The induction of the same set of PR genes in response to bacterial infection, MeJA or ethephon suggests the involvement of jasmonic acid (JA)/ethylene (ET)-signaling pathways in mediating resistance against Xcp, which is consistent with the potential hemibiotrophic nature of this bacterium. The identification of binding sites for ERF and MYC2 transcription factors in the promoter of Pp-TLP1 and Pp-TLP2 genes further supported the role of JA/ET pathways in the transcription regulation of these genes. The role of stomata in defense against Xcp was also investigated by measuring stomatal apertures in both 'Venture' and 'BabyGold 5' leaves after 1 and 3 HPI. While most stomata closed in both cultivars within 1 HPI, stomata reopened again at 3 HPI with a higher percentage recorded for 'BabyGold 5', suggesting a potential role of stomata in the susceptibility of this cultivar.

Effect of processing conditions on phospholipase D activity of corn kernel subcellular fractions.

The influence of physicochemical conditions on the phospholipase D (PLD) activity of subcellular preparations of sweet corn (Zea mays L. cv. Peaches and Cream) kernels has been studied. The microsomal, mitochondrial, and cytosolic preparations of corn kernels possessed PLD activity albeit at varying proportions. The microsomal and cytosolic PLD activities were stimulated 2-fold between 5 and 15 degrees C. Ethanol had varying modulatory effects on PLD activity. By contrast, acetaldehyde was a potent inhibitor of PLD. As well, a naturally occurring C(6) aldehyde such as hexanal and an alcohol such as hexanol inhibited PLD activity efficiently. Divalent cations such as calcium chloride and magnesium chloride stimulated PLD activity at micromolar levels. Monovalent cations such as KCl and NaCl did not appear to affect PLD activity. Partial purification of PLD from the microsomal, mitochondrial, and cytosolic fractions separately revealed four major isoforms with relative molecular masses of 200, 140-150, 102-108, and 60-66 kDa. The importance of PLD in the maintenance of processed food quality is discussed.

Influence of salicylic acid on H2O2 production, oxidative stress, and H2O2-metabolizing enzymes. Salicylic acid-mediated oxidative damage requires H2O2.

We investigated how salicylic acid (SA) enhances H2O2 and the relative significance of SA-enhanced H2O2 in Arabidopsis thaliana. SA treatments enhanced H2O2 production, lipid peroxidation, and oxidative damage to proteins, and resulted in the formation of chlorophyll and carotene isomers. SA-enhanced H2O2 levels were related to increased activities of Cu,Zn-superoxide dismutase and were independent of changes in catalase and ascorbate peroxidase activities. Prolonging SA treatments inactivated catalase and ascorbate peroxidase and resulted in phytotoxic symptoms, suggesting that inactivation of H2O2-degrading enzymes serves as an indicator of hypersensitive cell death. Treatment of leaves with H2O2 alone failed to invoke SA-mediated events. Although leaves treated with H2O2 accumulated in vivo H2O2 by 2-fold compared with leaves treated with SA, the damage to membranes and proteins was significantly less, indicating that SA can cause greater damage than H2O2. However, pretreatment of leaves with dimethylthiourea, a trap for H2O2, reduced SA-induced lipid peroxidation, indicating that SA requires H2O2 to initiate oxidative damage. The relative significance of the interaction among SA, H2O2, and H2O2-metabolizing enzymes with oxidative damage and cell death is discussed.

Changes in Activities of Antioxidant Enzymes and Their Relationship to Genetic and Paclobutrazol-Induced Chilling Tolerance of Maize Seedlings.

The potential role of antioxidant enzymes in protecting maize (Zea mays L.) seedlings from chilling injury was examined by analyzing enzyme activities and isozyme profiles of chilling-susceptible (CO 316) and chilling-tolerant (CO 328) inbreds. Leaf superoxide dismutase (SOD) activity in CO 316 was nearly one-half that of CO 328, in which the high activity was maintained during the chilling and postchilling periods. Activity of glutathione reductase (GR) was much higher in roots than in leaves. CO 328 also possessed a new GR isozyme that was absent in roots of CO 316. Ascorbate peroxidase (APX) activity was considerably lower in leaves of CO 328 than in CO 316, and nearly similar in roots. Paclobutrazol treatment of CO 316 induced several changes in the antioxidant enzyme profiles and enhanced their activities, especially those of SOD and APX, along with the induction of chilling tolerance. These results suggest that increased activities of SOD in leaves and GR in roots of CO 328, as well as SOD and APX in leaves and roots of paclobutrazol-treated CO 316, contribute to their enhanced chilling tolerance.

Ultraviolet-B- and ozone-induced biochemical changes in antioxidant enzymes of Arabidopsis thaliana.

Earlier studies with Arabidopsis thaliana exposed to ultraviolet B (UV-B) and ozone (O3) have indicated the differential responses of superoxide dismutase and glutathione reductase. In this study, we have investigated whether A. thaliana genotype Landsberg erecta and its flavonoid-deficient mutant transparent testa (tt5) is capable of metabolizing UV-B- and O3-induced activated oxygen species by invoking similar antioxidant enzymes. UV-B exposure preferentially enhanced guaiacol-peroxidases, ascorbate peroxidase, and peroxidases specific to coniferyl alcohol and modified the substrate affinity of ascorbate peroxidase. O3 exposure enhanced superoxide dismutase, peroxidases, glutathione reductase, and ascorbate peroxidase to a similar degree and modified the substrate affinity of both glutathione reductase and ascorbate peroxidase. Both UV-B and O3 exposure enhanced similar Cu,Zn-superoxide dismutase isoforms. New isoforms of peroxidases and ascorbate peroxidase were synthesized in tt5 plants irradiated with UV-B. UV-B radiation, in contrast to O3, enhanced the activated oxygen species by increasing membrane-localized NADPH-oxidase activity and decreasing catalase activities. These results collectively suggest that (a) UV-B exposure preferentially induces peroxidase-related enzymes, whereas O3 exposure invokes the enzymes of superoxide dismutase/ascorbate-glutathione cycle, and (b) in contrast to O3, UV-B exposure generated activated oxygen species by increasing NADPH-oxidase activity.

Isolation of nonsedimentable lipid-protein particles from insect intestine.

Nonsedimentable lipid-protein particles have been isolated from intestinal tissue of the American cockroach, Periplaneta americana. Most of the particles were within the range 30-50 nm in diameter and appear to originate from larger structures. Lipid analysis of the particles showed them to be enriched in neutral lipid components relative to microsomal membranes. Specifically, there is a decline in the amounts of phosphatidylcholine and phosphatidylethanolamine in the nonsedimentable particles compared with the microsomal membranes. Also, in contrast to microsomal membranes, the particles have a higher content of phosphatidic acid along with 1,2- and 1,3-diacyglycerols, free fatty acids and an unidentified lipid that co-migrates with sterol ester, wax ester and hydrocarbon standards in thin layer chromatograms. The cytosol, separated from the particles by ultrafiltration, contained phosphatidic acid, free fatty acids and the unidentified lipid. By contrast, the composition of neutral lipids in the cytosol resembles that of the particles. SDS-PAGE analysis of microsomal membranes, the particles and particle free cytosol shows an enrichment of low molecular weight proteins in the particles and cytosol. The particles and cytosol appear to possess proteolytic activity that is distinguishable from that of corresponding microsomal membranes since the incubation of these components with BSA resulted in the formation of distinct polypeptides. Many characteristics of these particles resemble those of the deteriosomes that have been isolated from plant tissue.

Nonsedimentable microvesicles from senescing bean cotyledons contain gel phase-forming phospholipid degradation products.

A mixture of liquid-crystalline and gel-phase lipid domains is detectable by wide angle x-ray diffraction in smooth microsomal membranes isolated from senescent 7-day-old cotyledons, whereas corresponding membranes from young 2-day-old cotyledons are exclusively liquid-crystalline. The gel-phase domains in the senescent membranes comprise phospholipid degradation products including diacylglycerols, free fatty acids, long-chain aldehydes, and long-chain hydrocarbons. The same complement of phospholipid degradation products is also present in nonsedimentable microvesicles isolated from senescent 7-day-old cotyledons by filtration of a 250,000g, 12-hour supernatant through a 300,000 dalton cut-off filter. The phospholipid degradation products in the microvesicles form gel-phase lipid domains when reconstituted into phospholipid liposomes. Nonsedimentable microvesicles of a similar size, which are again enriched in the same gel-phase-forming phospholipid degradation products, are also generated in vitro from smooth microsomal membranes isolated from 2-day-old cotyledons when Ca(2+) is added to activate membrane-associated lipolytic enzymes. The Ca(2+)-treated membranes do not contain detectable gel-phase domains, suggesting that the phospholipid degradation products are completely removed by microvesiculation. The observations collectively indicate that these nonsedimentable microvesicles serve as a vehicle for moving phospholipid degradation products out of membrane bilayers into the cytosol. As noted previously (Yao K, Paliyath G, Humphrey RW, Hallett FR, Thompson JE [1991] Proc Natl Acad Sci USA 88: 2269-2273), the term "deteriosome" connotes this putative function and would serve to distinguish these microvesicles from other cytoplasmic microvesicles unrelated to deterioration.

Identification and characterization of nonsedimentable lipid-protein microvesicles.

Previously uncharacterized lipid-protein microvesicles have been isolated from young and senescing bean cotyledon tissue. The microvesicles are nonsedimentable and enriched in phospholipid degradation products (free fatty acids, long-chain aldehydes, and long-chain hydrocarbons). They range from 70 to 170 nm (radius) with a mean radius of 132 nm, and it is clear from freeze-fracture electron micrographs that they are bilayered in nature. Nonsedimentable lipid-protein microvesicles containing the same products of phospholipid degradation but smaller were also formed in vitro when smooth microsomal membranes from young cotyledon tissue were treated with Ca2+ to stimulate enzymatic degradation of phospholipids. The data suggest that these microvesicles comprise an intermediate stage of membrane lipid deterioration. They appear to serve as a vehicle for moving phospholipid degradation products out of membranes into the cytosol during senescence and perhaps also during normal membrane lipid turnover.

Characteristics of a membrane-associated lipoxygenase in tomato fruit.

Microsomal membranes isolated from the pericarp of maturegreen tomato (Lycopersicon esculentum) fruit rapidly metabolize exogenous radiolabeled linoleic acid into fatty acid oxidation products at 22 degrees C. The reaction is strongly inhibited by n-propyl gallate, an inhibitor of lipoxygenase. The membranes also rapidly metabolize 16:0/18:2(*) phosphatidylcholine into radiolabeled oxidation products that comigrate on TLC plates with those formed from free linoleic acid. At 30 degrees C, the formation of fatty acid oxidation products from 16:0/18:2(*) phosphatidylcholine is slower, and there is an initial accumulation of radiolabeled linoleic acid that is not evident at 22 degrees C, which can be attributed to the action of lipolytic acyl hydrolase. Radiolabeled phosphatidic acid and diacylglycerol are also formed during metabolism of 16:0/18:2(*) phosphatidylcholine by the microsomal membranes, and there is no breakdown of either linoleic acid or phosphatidylcholine by heat-denatured membranes. When Triton X-100 treated membranes were used, the same patterns of metabolite formation from radiolabeled linoleic acid and 16:0/18:2(*) phosphatidylcholine were observed. Thus, the enzymes mediating the breakdown of these radiolabeled compounds appear to be tightly associated with the membranes. Collectively, the data indicate that there is a lipoxygenase associated with microsomal membranes from tomato fruit that utilizes free fatty acid substrate released from phospholipids. The microsomal lipoxygenase is strongly active over a pH range of 4.5 to 8.0, comprises approximately 38% of the total (microsomal plus soluble) lipoxygenase activity in the tissue, has an apparent K(m) of 0.52 millimolar and an apparent V(max) of 0.186 millimoles per minute per milligram of protein. The membranous enzyme also cross-reacts with polyclonal antibodies raised against soybean lipoxygenase-1 and has an apparent molecular mass of 100 kilodaltons.

Senescence-Related Changes in ATP-Dependent Uptake of Calcium into Microsomal Vesicles from Carnation Petals.

Microsomal membrane vesicles isolated from the petals of young carnation (Dianthus caryophyllus L. cv White Sim) flowers accumulate Ca(2+) in the presence of ATP. The specific activity of ATP-dependent uptake is approximately 20 nanomoles per milligram of protein per 30 minutes. The membranes also hydrolyze ATP, but Ca(2+) stimulation of ATP hydrolysis was not discernible above the high background of Ca(2+)-insensitive ATPase activity. The initial velocity of uptake showed a sigmoidal rise with increasing Ca(2+) concentration, suggesting that Ca(2+) serves both as substrate and activator for the enzyme complex mediating its uptake. The concentration of Ca(2+) at half maximal velocity of uptake (S(0.5)) was 12.5 micromolar and the Hill coefficient (n(H)) was 2.5. The addition of calmodulin to membrane preparations that had been isolated in the presence of chelators did not promote ATP-dependent accumulation of Ca(2+), although this may reflect the fact that the treatment with chelators did not fully remove endogenous calmodulin. Transport of Ca(2+) into membrane vesicles was unaffected by 50 micromolar ruthenium red and 5 micromolar sodium azide, indicating that uptake is primarily into vesicles of non-mitochondrial origin. By subfractionating the microsomes on a linear sucrose gradient, it was established that the ATP-dependent Ca(2+) transport activity comigrates with endoplasmic reticulum and plasma membrane. During post-harvest development of cut flowers, ATP-dependent uptake of Ca(2+) into microsomal vesicles declined by approximately 70%. This occurred before the appearance of petal-inrolling and the climacteric-like rise in ethylene production, parameters that denote the onset of senescence. There were no significant changes during this period in S(0.5) or n(H), but V(max) for ATP-dependent Ca(2+) uptake decreased by approximately 40%. A similar decline in ATP-dependent uptake of Ca(2+) into microsomal vesicles was induced by treating young flowers with physiological levels of exogenous ethylene.

Promotion of beta-glucan synthase activity in corn microsomal membranes by calcium and protein phosphorylation.

Regulation of the activity of beta-glucan synthase was studied using microsomal preparations from corn coleoptiles. The specific activity as measured by the incorporation of glucose from uridine diphospho-D-[U-14C]glucose varied between 5 to 15 pmol (mg protein)-1 min-1. Calcium promoted beta-glucan synthase activity and the promotion was observed at free calcium concentrations as low as 1 micromole. Kinetic analysis of substrate-velocity curve showed an apparent Km of 1.92 x 10(-4) M for UDPG. Calcium increased the Vmax from 5.88 x 10(-7) mol liter-1 min-1 in the absence of calcium to 9.52 x 10(-7) mol liter-1 min-1 and 1.66 x 10(-6) mol liter-1 min-1 in the presence of 0.5 mM and 1 mM calcium, respectively. The Km values remained the same under these conditions. Addition of ATP further increased the activity above the calcium-promoted level. Sodium fluoride, a phosphoprotein phosphatase inhibitor, promoted glucan synthase activity indicating that phosphorylation and dephosphorylation are involved in the regulation of the enzyme activity. Increasing the concentration of sodium fluoride from 0.25 mM to 10 mM increased glucan synthase activity five-fold over the + calcium + ATP control. Phosphorylation of membrane proteins also showed a similar increase under these conditions. Calmodulin, in the presence of calcium and ATP stimulated glucan synthase activity substantially, indicating that calmodulin could be involved in the calcium-dependent phosphorylation and promotion of beta-glucan synthase activity. The role of calcium in mediating auxin action is discussed.

Calcium- and calmodulin-regulated breakdown of phospholipid by microsomal membranes from bean cotyledons.

Evidence for the involvement of Ca(2+) and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U-(14)C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U-(14)C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca(2+), whereas lipolytic acyl hydrolase proved to be insensitive to Ca(2+). Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca(2+). Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC(50) values ranging from 10 to 15 micromolar. Thus the Ca(2+)-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca(2+) on phospholipase D is independent of calmodulin. The role of Ca(2+) as a second messenger in the initiation of membrane lipid degradation is discussed.

Identification of naturally occurring calmodulin inhibitors in plants and their effects on calcium- and calmodulin-promoted protein phosphorylation.

While studying the calmodulin activity in post-climacteric apples, a heat stable, dialyzable component that inhibited calmodulin-promoted phosphodiesterase activity was detected. The compound(s) that inhibited calmodulin activity did not bind to Dowex-50, H+ or Dowex-2, Cl- and was exclusively present in the neutral fraction. The inhibitors irreversibly bound to polyvinylpolypyrrolidone indicating their phenolic nature. Fractionation of the neutral fraction on a C18-microbondapak column and analysis for the inhibition of calmodulin-promoted phosphodiesterase activity showed significant inhibitory activity associated with fractions eluted 5 min, 15 min and 18 min after injection. Perdeuteriomethylation and combined gas chromatographic-mass spectrometric analysis of the inhibitors showed them to be flavonoids. (+)-Catechin was identified in the fraction eluted 5 min after injection that also showed maximum inhibition. Other flavonoids such as epicatechin, quercetin and naringenin also inhibited calmodulin-promoted phosphodiesterase activity. Among the phenolic compounds commonly encountered in plant tissue only caffeic acid inhibited calmodulin-promoted phosphodiesterase activity. Inhibition by catechin and caffeic acid could be reversed by increasing the calmodulin concentration in the assay mixture. Both catechin and caffeic acid inhibited Ca- and calmodulin-promoted phosphorylation of soluble proteins from corn coleoptiles. The physiological properties of flavonoids are discussed in light of this evidence.

Biosynthesis of alkanes by particulate and solubilized enzyme preparations from pea leaves (Pisum sativum).

Enzymatic activity responsible for the conversion of fatty acids to alkanes catalyzed by pea leaf homogenate was found to be mainly in the microsomal fraction. This particulate preparation catalyzed alkane formation from n-C18, n-C22, and n-C24 acids at rates comparable to that observed with n-C32 acid with O2 and ascorbate as required cofactors. In each case the major alkane contained two carbon atoms less than the precursor acid. Since the preparation also catalyzed alpha-oxidation, it was suspected that some alpha-oxidation intermediate, with one less carbon atom than the substrate acid, might lose another carbon to generate the alkane. Thin-layer and radio-gas-liquid chromatographic analysis of the products generated from [U-14C]stearic acid by the particulate preparation after different periods of incubation showed that, at all time periods, alpha-hydroxy C18 acid, C17 aldehyde, and C17 acid were the major products. Since C16 alkane was the major product even after short periods of reaction, the C17 aldehyde might have been the immediate precursor of the alkane. Exogenous labeled C18 and C24 aldehyde were converted to alkanes. The alkane-synthesizing activity was solubilized from the microsomal preparation using Triton X-100. The solubilized preparation was retarded in a Sepharose 6-B column, but the hydrocarbon-forming activity was not resolved from alpha-oxidation. The solubilized preparation produced alkane with two carbon atoms less than the parent acid in a time- and protein-dependent manner. The soluble preparation also required O2 and ascorbate and, like the microsomal preparation, was inhibited by dithioerythritol and metal ion chelating agents.

Calmodulin inhibitor in senescing apples and its physiological and pharmacological significance.

While assaying calmodulin activity in senesced apple extracts by using its property of promoting the activity of activator-deficient 3',5'-cyclic AMP 5'-nucleotidohydrolase (phosphodiesterase, EC 3.1.4.17) from bovine heart, we detected a heat-stable, dialyzable, low molecular weight component that inhibited calmodulin activity. Specific activity of calmodulin as calculated from the linearly increasing portion of the activity curve was in the range of 150 to 160 units/mg of protein in crude extracts from apples stored at 2 degrees C for a period of 6 months with or without calcium treatment. Apple extract that was passed through a phenothiazine-Sepharose affinity column did not promote phosphodiesterase activity, whereas the EGTA eluate of the column promoted phosphodiesterase activity similar to the original extract. The inhibition of calmodulin activity appeared to be lower in extracts from apples stored at 2 degrees C after calcium treatment. The inhibition was found to increase after storage of apples at room temperature for 30 days. Activity of purified bovine brain calmodulin was also inhibited by the inhibitor present in apple extracts, which indicated that the inhibition was not specific to plant calmodulin alone and could have wide applications. The importance of the inhibitor in relation to senescence/aging and its possible pharmacological applications are discussed.