Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: lipid peroxidation,sugar hydrolysis,Maillard reactions and their relationship to glass state transition

Two primary biochemical reactions in seed ageing (lipid peroxidation and non-enzymatic protein glycosylation with reducing sugars) have been studied under different seed water contents and storage temperatures, and the role of the glassy state in retarding biochemical deterioration examined. The viability loss of Vigna radiata seeds during storage is associated with Maillard reactions; however, the contribution of primary biochemical reactions varies under different storage conditions. Biochemical deterioration and viability loss are greatly retarded in seeds stored below a high critical temperature (approximately 40 degrees C above glass transition temperature). This high critical temperature corresponds to the cross-over temperature (T(c)) of glass transition where molecular dynamics changes from a solid-like system to a normal liquid system. The data show that seed ageing slows down significantly, even before seed tissue enters into the glassy state.     

Maillard reaction-like lysine modification by a lipid peroxidation product: immunochemical detection of protein-bound 2-hydroxyheptanal in vivo

2-Hydroxyheptanal (2-HH) is one of the reactive aldehyde species generated during the peroxidation of n-6 polyunsaturated fatty acids, such as linoleic and arachidonic acids. Analogous to the Maillard reaction of reducing sugars, 2-HH readily reacts with lysine epsilon-amino groups. In the present study, to define the occurrence of the Maillard reaction-like lysine modification by 2-HH in vivo, we raised a monoclonal antibody directed to a trihydropyridinone (THPO) structure, 1-alkyl-4-butyl-5-pentyl-1,2,6-trihydropyridin-3-one, formed from 2-HH and lysine, and examined the presence of the antigenic structure in the human atherosclerotic aorta. Mice were immunized with the 2-HH-modified keyhole limpet hemocyanin (KLH) as the immunogen. Using a THPO-carrier protein conjugate, we screened the hybridomas and finally obtained a clone that produced the monoclonal antibody 3C8 (mAb3C8). The antibody strongly recognized bovine serum albumin (BSA) treated with 2-HH, but showed no cross-reactivity with BSAs modified with other related aldehydes. By using this antibody, it was revealed that the antigenic structure was indeed present in atherosclerotic lesions of the human aorta.     

Aldehyde adducts generated during lipid peroxidation modification of proteins

Abstract

Various lines of evidence indicate that an important part in the pathogenesis of atherosclerosis is the modification of the plasma low-density lipoproteins (LDLs). A large number of pro-inflammatory and pro-atherogenic properties have been ascribed to the oxidatively modified LDLs and their components. There is considerable evidence to support the role of lipid peroxidation products, reactive aldehydes in particular, originating from the oxidized LDL as important signaling molecules in the context of the atherosclerotic lesion. These aldehydes generated during the peroxidation of LDL exhibit a facile reactivity with proteins, generating a variety of intra- and intermolecular covalent adducts on the apolipoprotein B-100 particle in LDL. Characterization of the aldehyde adducts generated in the protein is therefore critical in understanding the nature of the oxidized LDL. However, the majority of adducts generated during the oxidative modification of LDL have not yet been chemically characterized. In this review, the current status of aldehyde adducts quantitatively analyzed in the Cu²⁺-oxidized LDL is reviewed.     

The multiple Maillard reactions of ribose and deoxyribose sugars and sugar phosphates

Ribose 5-phosphate (R5P) undergoes the Maillard reaction with amines at significantly higher rates than most other sugars and sugar phosphates. The presence of an intramolecular phosphate group, which catalyzes the early stages of the Maillard reaction, provides the opportunity for the R5P molecule to undergo novel reaction paths creating unique Maillard products. The initial set of reactions leading to an Amadori product (phosphorylated) and to an alpha-dicarbonyl phosphate compound follows a typical Maillard reaction sequence, but an observed phosphate hydrolysis accompanying the reaction adds to the complexity of the products formed. The reaction rate for the loss of R5P is partially dependent on the pK(a) of the amine but also is correlated to the protonation of an early intermediate of the reaction sequence. In the presence of oxygen, a carboxymethyl group conjugated to the amine is a major product of the reaction of R5P with N-acetyllysine while little of this product is generated in the absence of oxygen. Despite lacking a critical hydroxyl group necessary for the Maillard reaction, 2-deoxyribose 5-phosphate (dR5P) still generates an Amadori-like product (with a carbonyl on the C-3 carbon) and undergoes phosphate cleavage. Two highly UV-absorbing products of dR5P were amine derivatives of 5-methylene-2-pyrrolone and 2-formylpyrrole. The reaction of dR5P with certain amines generates a set of products that exhibit an interesting absorbance at 340nm and a high fluorescence.     

"Enzymatic" lipid peroxidation: reactions of mammalian lipoxygenases

Lipoxygenase is a dioxygenase which incorporates one molecule of oxygen at a certain position of unsaturated fatty acids such as arachidonic and linolenic acids. The enzymatic oxygenation of unsaturated fatty acids is stereospecific concomitant with a stereoselective abstraction of hydrogen atom. Fatty acid cyclooxygenase is an atypical lipoxygenase incorporating two molecules of oxygen, and initiates the biosynthesis of prostaglandins and thromboxanes. Arachidonate 5-lipoxygenase is responsible for the leukotriene synthesis. No such bioactive compound has been found as a metabolite of the 12- and 15-lipoxygenase pathways, and their physiological roles are still unclarified. These enzymes have been purified, and their molecular and catalytic properties have been investigated. Their cDNA clones have been isolated, and their nucleotide sequences have been determined deducing the primary structures of the enzymes.     

Vitamin E is essential for seed longevity and for preventing lipid peroxidation during germination

Tocopherols (vitamin E) are lipophilic antioxidants synthesized by all plants and are particularly abundant in seeds. Despite cloning of the complete suite of tocopherol biosynthetic enzymes and successful engineering of the tocopherol content and composition of Arabidopsis thaliana leaves and seeds, the functions of tocopherols in plants have remained elusive. To address this issue, we have isolated and characterized two VITAMIN E loci (VTE1 and VTE2) in Arabidopsis that when mutated result in tocopherol deficiency in all tissues. vte1 disrupts tocopherol cyclase activity and accumulates a redox-active biosynthetic intermediate, whereas vte2 disrupts homogentisate phytyl transferase activity and does not accumulate pathway intermediates. Mutations at either locus cause significantly reduced seed longevity compared with the wild type, indicating a critical role for tocopherols in maintaining viability during quiescence. However, only vte2 mutants exhibited severe seedling growth defects during germination and contained levels of lipid hydroperoxides and hydroxy fatty acids elevated up to 4- and 100-fold, respectively, relative to the wild type. These data demonstrate that a primary function of tocopherols in plants is to limit nonenzymatic lipid oxidation during seed storage, germination, and early seedling development. The vte mutant phenotypes also explain the strong selection for retention of tocopherol biosynthesis during the evolution of seed-bearing plants.     

The 21-aminosteroid inhibitors of lipid peroxidation: reactions with lipid peroxyl and phenoxy radicals

The 21-aminosteroids U74006F and U74500A have been examined for their ability to scavenge the lipid peroxyl (LOO.) and phenoxy (PhO.) radicals. Lipid peroxidation was followed by measuring the formation of linoleic acid hydroperoxide (LOOH; 18:200H) from linoleic acid during incubations in methanol at 37 degrees C. Initiation of lipid peroxidation was by the radical generator 2,2'-azobis(2,4-dimethylvaleronitrile; AMVN), which under the conditions employed, initiated LOOH formation at a constant rate of 22 microM/h with a kinetic chain length of 21. Alpha-tocopherol (alpha TC) nearly completely blocked the chain reaction by scavenging LOO., reducing its formation to that essentially attributable to initiation alone. The average inhibition rate constant kinh for alpha TC at 37 degrees C was calculated as 4.9 x 10(5) M-1 sec-1. U74006F or U74500A also inhibited LOOH formation, reducing its rate to a constant fraction of control in a concentration dependent manner. U74500A was a more potent scavenger of LOO. than U74006F; however, both compounds were considerably less potent than alpha TC based upon their respective kinh's at 37 degrees C. Similarly, alpha TC, U74006F and U74500A scavenged PhO.. As seen with LOO. scavenging, alpha TC was orders of magnitude more reactive toward PhO. than either 21-aminosteroid as judged by their respective second order rate constants (k2). Both U74006F and U74500A were degraded during their reaction with LOO. or PhO. to as yet uncharacterized product(s). The data indicate that while the 21-aminosteroids can scavenge lipid radicals, their activity in this regard is less than expected based upon their ability to inhibit iron dependent lipid peroxidation.     

Oxidative modification of low-density lipoprotein: lipid peroxidation by myeloperoxidase in the presence of nitrite

Oxidative modification of low-density lipoprotein (LDL) is a pivotal process in early atherogenesis and can be brought about by myeloperoxidase (MPO), which is capable of reacting with nitrite, a NO metabolite. We studied MPO-mediated formation of conjugated dienes in isolated human LDL in dependence on the concentrations of nitrite and chloride. This reaction was strongly stimulated by low concentrations (5-50 microM) of nitrite which corresponds to the reported concentration in the arterial vessel wall. Under these conditions no protein tyrosine nitration occurred; this reaction required much higher nitrite concentrations (100 microM-1 mM). Chloride neither supported lipid peroxidation alone nor was its presence mandatory for the effect of nitrite. We propose a prominent role of lipid peroxidation for the proatherogenic action of the MPO/nitrite system, whereas peroxynitrite may be competent for protein tyrosine nitration of LDL. Monomeric and oligomeric flavan-3-ols present in cocoa products effectively counteracted, at micromolar concentrations, the MPO/nitrite-mediated lipid peroxidation of LDL. Flavan-3-ols also suppressed protein tyrosine nitration induced by MPO/nitrite or peroxynitrite as well as Cu2+-mediated lipid peroxidation of LDL. This multi-site protection by (-)-epicatechin or other flavan-3-ols against proatherogenic modification of LDL may contribute to the purported beneficial effects of dietary flavan-3-ols for the cardiovascular system.     

Oxidative stress, phospholipid loss and lipid hydrolysis during drying and storage of intermediate seeds

The biochemical and physiological basis of intermediate seed storage behaviour was examined by investigating the effects of equilibrium drying under relative humidities (RHs) of 9–81% and of storage at 20 or 5°C on coffee seed viability and antioxidant, lipid and sugar status. Slow drying induced a significant decrease in the concentrations of the pools of two major antioxidants, glutathione and ascorbate, and an increase in the free fatty acid (FFA) content of seeds, independent of the RH employed. Seeds stored at 81% RH and 20°C lost their viability very rapidly and showed an extensive loss and oxidation of antioxidants, an accumulation of FFA and a selective loss of phospholipids, in particular phosphatidylethanolamine (PE). Interestingly, the changes in PE content were not due to fatty acid de-esterification and the increase in FFA levels resulted from neutral lipid hydrolysis. Decreasing the storage temperature to 5°C considerably slowed both the loss of seed viability and the level of oxidative stress as well as the rates of lipid hydrolysis. No decline in seed viability was observed under storage conditions of 45% RH/20°C. After 1 year under 45% RH/5°C, the loss of seed viability was found to be due to imbibitional damage and could be circumvented by pre-humidifying or pre-heating seeds before sowing.     

The interrelationship of the processes of lipid peroxidation and lipid phospholipase hydrolysis in the synaptosomes

The influence of Fe2+, alpha-tocopherol, phospholipase A2 and mepacrine on the activity of lipid peroxidation (LPO) and phospholipid hydrolysis (PLH) was studied in synaptosomes. It was established that there is the tight direct interconnection between LPO and PLH in synaptosomes. It is assumed that activation of endogenous phospholipases in neurons is one of the causes of uncompensated LPO-activation during epileptogenesis.     

Different roles for nitrogen monoxide and peroxynitrite in lipid peroxidation induced by activated neutrophils

We studied the roles of nitrogen monoxide (NO&z.rad;) and peroxynitrite produced by the polymorphonuclear leukocytes (PMNs) isolated from an inflammatory exudate. PMNs were incubated either in a medium with a submicromolar concentration of iron or in a diethylenetriaminepenta-acetic acid (DTPA)-containing medium, and stimulated with phorbol 12-myristate 13-acetate (PMA) to generate free radicals. In both conditions superoxide anion (O(2)(*)(-)), NO&z.rad; and peroxynitrite were produced. In the presence of arachidonic acid, malondialdehyde (MDA) was generated. This MDA was generated in one of two way; the peroxynitrite iron-independent mechanism (40%) and the Fenton reaction, caused by free iron (60%). We also observed that the addition of L-arginine was followed by a 42% reduction in MDA, which can be explained by the antioxidant effect of NO&z.rad;. These results indicate that lipid peroxidation can occur in the absence of iron, through a peroxynitrite-mediated mechanism, and that NO&z.rad; may act as an antioxidant when it is produced in large amounts.     

Maillard reactions by alpha-oxoaldehydes: detection of glyoxal-modified proteins

Proteins can be chemically modified by sugars by glycation, or the Maillard reaction. The Maillard reaction produces irreversible adducts on proteins that are collectively known as advanced glycation end products, or AGEs. Recent studies indicate that several alpha-dicarbonyl compounds, including glyoxal (GXL), are precursors of AGEs in vivo. We developed antibodies against a GXL-modified protein (GXL-AGE) and purified a mixture of GXL-AGE-specific antibodies by chromatography on GXL-modified bovine serum albumin (BSA-GXL) coupled to EAH-Sepharose. This preparation was then processed on a human serum albumin-carboxymethyllysine (HSA-CML)-NHS-Sepharose to remove CML-specific antibodies. We used the resulting purified antibody in a competitive ELISA to probe GXL-AGEs in vitro and in vivo. We found increasingly greater antibody binding with increasing concentrations of GXL-modified BSA, but the antibody failed to react with either free CML or protein-bound CML. Incubation experiments with BSA revealed that glyceraldehyde, ribose and threose could be precursors of GXL-AGEs as well. Experiments in which GXL was incubated with N-alpha-acetyl amino acids showed that the antibody reacts mostly with lysine modifications. The GXL-derived lysine-lysine crosslinking structure, GOLD was found to be one of the antigenic epitopes for the antibody. Analysis of human plasma proteins revealed significantly higher levels of GXL-AGE antigens in type II diabetic subjects compared with normal controls (P<0.0001). We also found GXL-AGEs in human lens proteins. Bovine aortic endothelial cells cultured for 7 days with 30 mM glucose did not accumulate intracellular GXL-AGEs. These studies underscore the importance of GXL for extracellular AGE formation (except in lens where it is likely to be formed intracellularly) and suggest that changes associated with age and diabetes might be prevented by alteration of GXL-AGE formation.     

A role for caleosin in degradation of oil-body storage lipid during seed germination

Caleosin is a Ca(2+)-binding oil-body surface protein. To assess its role in the degradation of oil-bodies, two independent insertion mutants lacking caleosin were studied. Both mutants demonstrated significant delay of breakdown of the 20:1 storage lipid at 48 and 60 h of germination. Additionally, although germination rates for seeds were not affected by the mutations, mutant seedlings grew more slowly than wild type when measured at 48 h of germination, a defect that was corrected with continued growth for 72 and 96 h in the light. After 48 h of germination, wild-type central vacuoles had smooth contours and demonstrated internalization of oil bodies and of membrane containing alpha- and delta-tonoplast intrinsic proteins (TIPs), markers for protein storage vacuoles. In contrast, mutant central vacuoles had distorted limiting membranes displaying domains with clumps of the two TIPs, and they contained fewer oil bodies. Thus, during germination caleosin plays a role in the degradation of storage lipid in oil bodies. Its role involves both the normal modification of storage vacuole membrane and the interaction of oil bodies with vacuoles. The results indicate that interaction of oil bodies with vacuoles is one mechanism that contributes to the degradation of storage lipid.     

Iron-induced lipid peroxidation and protein modification in endoplasmic reticulum membranes. Protection by stobadine

Treatment with FeSO(4)/EDTA (0.2 micromol Fe(II) per mg of protein) was used to study the effect of oxidative stress on lipid peroxidation and structural properties of endoplasmic reticulum (ER) membranes isolated from rabbit brain. Oxidative stress resulted in conjugated diene formation and a decrease of 1-anilino-8-naphthalenesulfonate (ANS) fluorescence in a time-dependent manner. In contrast, fluorescence anisotropy of 1, 6-diphenyl-1,3,5-hexatriene was increased early after the initiation of lipid peroxidation and no further increase was observed after 1, 2 and 3 h of peroxidation. FeSO(4)/EDTA treatment was accompanied by formation of conjugates of lipid peroxidation products with membrane proteins, as detected by the increase in fluorescence excitation (350-360 nm) and emission (440-450 nm) maximum. Oxidative stress also induced a marked decrease of the intrinsic fluorescence of aromatic amino acids, suggesting modification or changes in the environment of these amino acid residue(s). The lipid antioxidant, stobadine, completely prevented the changes of ANS fluorescence and production of peroxidized lipid-protein conjugates whereas tryptophan fluorescence was only partially protected. These results suggest that Fe(II) induces both lipid-mediated- and lipid peroxidation independent-modification of ER membrane proteins. The study also demonstrates that stobadine is a potent inhibitor of Fe(II)-induced protein modification.     

Maillard reactions and increased enzyme inactivation during oligosaccharide synthesis by a hyperthermophilic glycosidase

The thermostable Pyrococcus furiosus beta-glycosidase was used for oligosaccharide production from lactose in a kinetically controlled reaction. Our experiments showed that higher temperatures are beneficial for the absolute as well as relative oligosaccharide yield. However, at reaction temperatures of 80 degrees C and higher, the inactivation rate of the enzyme in the presence of sugars was increased by a factor of 2 compared to the inactivation rate in the absence of sugars. This increased enzyme inactivation was caused by the occurrence of Maillard reactions between the sugar and the enzyme. The browning of our reaction mixture due to Maillard reactions was modeled by a cascade of a zeroth- and first-order reaction and related to enzyme inactivation. From these results we conclude that modification of only a small number of amino groups already gives complete inactivation of the enzyme.     

Protein modification by oxidized phospholipids and hydrolytically released lipid electrophiles: Investigating cellular responses

Oxygen is essential for the growth and function of mammalian cells. However, imbalances in oxygen or abnormalities in the ability of a cell to respond to oxygen levels can result in oxidative stress. Oxidative stress plays an important role in a number of diseases including atherosclerosis, rheumatoid arthritis, cancer, neurodegenerative diseases and asthma. When membrane lipids are exposed to high levels of oxygen or derived oxidants, they undergo lipid peroxidation to generate oxidized phospholipids (oxPL). Continual exposure to oxidants and decomposition of oxPL results in the formation of reactive electrophiles, such as 4-hydroxy-2-nonenal (HNE). Reactive lipid electrophiles have been shown to covalently modify DNA and proteins. Furthermore, exposure of cells to lipid electrophiles results in the activation of cytoprotective signaling pathways in order to promote cell survival and recovery from oxidant stress. However, if not properly managed by cellular detoxification mechanisms, the continual exposure of cells to electrophiles results in cytotoxicity. The following perspective will discuss the biological importance of lipid electrophile protein adducts including current strategies employed to identify and isolate protein adducts of lipid electrophiles as well as approaches to define cellular signaling mechanisms altered upon exposure to electrophiles. This article is part of a Special Issue entitled: Oxidized phospholipids-their properties and interactions with proteins.     

Oxidation of vitamin E during iron-catalyzed lipid peroxidation: evidence for electron-transfer reactions of the tocopheroxyl radical.

Incubation of phosphatidylcholine liposomes containing the biological antioxidant alpha-tocopherol (alpha-TH) with xanthine, xanthine oxidase, and FeCl2 caused alpha-TH oxidation to alpha-tocopherol quinone (alpha-TQ) and 8a-hydroperoxytocopherone (2). In addition, 4a,5-epoxy-8a-hydroperoxytocopherone (3), 7,8-epoxy-8a-hydroperoxytocopherone (4), and their respective hydrolysis products 2,3-epoxy-alpha-tocopherol quinone (6) and 5,6-epoxy-alpha-tocopherol quinone (7) also were formed. alpha-TQ was the major product at less than 20% alpha-TH oxidation, whereas epoxides were the predominant products when alpha-TH was more extensively oxidized. 8a-(Alkyldioxy)tocopherones 1, which are formed when peroxyl radicals oxidize alpha-TH in other systems and which are precursors to alpha-TQ, were not found. 8a-Hydroxytocopherone (5), rather than 8a-(alkyldioxy)tocopherones 1, appeared to be the precursor to alpha-TQ. Approximately 30% of the alpha-TH consumed was regenerated by treatment of samples with ascorbic acid or nordehydroguaiaretic acid (NDGA) at pH 3, but not at pH 7. The stability of the ascorbic acid- and NDGA-reducible species and pH dependence for regeneration matched those of 8a-hydroxytocopherone (5) and contrasted with the properties of the tocopheroxyl radical (alpha-T.). Incubation of liposomes containing alpha-TH with the diphenylpicrylhydrazyl (DPPH) radical, which oxidizes alpha-TH to alpha-T. in high yield, formed an ascorbic acid-reducible species with properties identical to those of compound 5. The results indicate that phospholipid peroxyl radicals oxidize alpha-T. to epoxides, 8a-hydroperoxytocopherone (2), and the tocopherone cation (alpha-T+), which hydrolyzes to 5, the immediate precursor to alpha-TQ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Drought stress during soybean seed filling affects storage compounds through regulation of lipid and protein metabolism

Soybean seeds have high lipid and protein contents. Adverse environmental conditions restrict seed yield and quality. We examined the changes in storage compounds caused by drought stress from R5 stage (beginning seed growth stage). Under drought stress, contents of lipid in seed were remarkably low compared to control at 24 and 29 days after treatment. Protein contents in seed were immediately decreased after water deficit treatment. On the other hand, soluble sugar contents in seed were increased by drought stress. Drought stress decreased the expression of genes involved in lipid biosynthesis (PK, BCCP2, and KAS1) and increased the genes expression involved in lipid degradation (ACX2, MS, and PEPCK). These results suggest that the increasing of sugar content in seed under drought stress was complemented by degradation of lipids. The expressions of genes encoding storage protein (Gy4 and β-conglycinin) were also decreased by drought stress. This study showed how drought stress during seed filling affects seed quality, especially lipid and protein contents, that may facilitate further research on seed storage compounds metabolism under environmental stresses.     

Inhibition of phospholipid hydrolysis by phospholipase A2 in microsomal and mitochondrial membranes subjected to lipid peroxidation

The rate of phospholipid hydrolysis in rat liver microsomal and mitochondrial membranes catalyzed by phospholipase A2 was shown to decrease after ascorbate + Fe2+-induced lipid peroxidation. The degree of inhibition was linearly dependent on the amount of lipid peroxidation products (malonyl dialdehyde) accumulated in the membrane. The decreased phospholipid hydrolysis rate in membranes after lipid peroxidation was registered using phospholipases A2 from two sources: porcine pancreas and bee venom. It was established that the inhibitory action of phospholipid peroxidation products was not linked with a direct effect on the enzyme and was not caused by depletion of phospholipase reaction substrates (as a result of lipid peroxidation). A possible role of lateral separation of oxidized and non-oxidized lipid phases in the mechanisms of inhibition of phospholipid hydrolysis by phospholipase A2 is discussed.