Effect of phenolic-rich plant materials on protein and lipid oxidation reactions

The antioxidant activity of natural plant materials rich in phenolic compounds is being widely investigated for protection of food products sensitive to oxidative reactions. In this thesis plant materials rich in phenolic compounds were studied as possible antioxidants to prevent protein and lipid oxidation reactions in different food matrixes such as pork meat patties and corn oil-in water emulsions. Loss of anthocyanins was also measured during oxidation in corn oil-in-water emulsions. In addition, the impact of plant phenolics on amino acid level was studied using tryptophan as a model compound to elucidate their role in preventing the formation of tryptophan oxidation products. A high-performance liquid chromatography (HPLC) method with ultraviolet and fluorescence detection (UV-FL) was developed that enabled fast investigation of formation of tryptophan derived oxidation products. Byproducts of oilseed processes such as rapeseed (Brassica rapa L.), camelina (Camelina sativa) and soy meal (Glycine max L.) as well as Scots pine bark (Pinus sylvestris) and several reference compounds were shown to act as antioxidants toward both protein and lipid oxidation in cooked pork meat patties. In meat, the antioxidant activity of camelina, rapeseed and soy meal were more pronounced when used in combination with a commercial rosemary extract (Rosmarinus officinalis). Berry phenolics such as black currant (Ribes nigrum) anthocyanins and raspberry (Rubus idaeus) ellagitannins showed potent antioxidant activity in corn oil-in-water emulsions toward lipid oxidation with and without β-lactoglobulin. The antioxidant effect was more pronounced in the presence of β-lactoglobulin. The berry phenolics also inhibited the oxidation of tryptophan and cysteine side chains of β-lactoglobulin. The results show that the amino acid side chains were oxidized prior the propagation of lipid oxidation, thereby inhibiting fatty acid scission. In addition, the concentration and color of black currant anthocyanins decreased during the oxidation. Oxidation of tryptophan was investigated in two different oxidation models with hydrogen peroxide (H2O2) and hexanal/FeCl2. Oxidation of tryptophan in both models resulted in oxidation products such as 3a-hydroxypyrroloindole-2-carboxylic acid, dioxindolylalanine, 5-hydroxy-tryptophan, kynurenine, N-formylkynurenine and β-oxindolylalanine. However, formation of tryptamine was only observed in tryptophan oxidized in the presence of H2O2. Pine bark phenolics, black currant anthocyanins, camelina meal phenolics as well as cranberry proanthocyanidins (Vaccinium oxycoccus) provided the best antioxidant effect toward tryptophan and its oxidation products when oxidized with H2O2. The tryptophan modifications formed upon hexanal/FeCl2 treatment were efficiently inhibited by camelina meal followed by rapeseed and soy meal. In contrast, phenolics from raspberry, black currant, and rowanberry (Sorbus aucuparia) acted as weak prooxidants. This thesis contributes to elucidating the effects of natural phenolic compounds as potential antioxidants in order to control and prevent protein and lipid oxidation reactions. Understanding the relationship between phenolic compounds and proteins as well as lipids could lead to the development of new, effective, and multifunctional antioxidant strategies that could be used in food, cosmetic and pharmaceutical applications.

Differential recognition by tick-resistant cattle of the recombinantly expressed Rhipicephalus microplus serine protease inhibitor-3 (RMS-3)

Rhipicephalus micro plus is an important bovine ectoparasite, widely distributed in tropical and subtropical regions of the world causing large economic losses to the cattle industry. Its success as an ectoparasite is associated with its capacity to disarm the antihemostatic and anti-inflammatory reactions of the host. Serpins are protease inhibitors with an important role in the modulation of host-parasite interactions. The cDNA that encodes for a R. microplus serpin was isolated by RACE and subsequently cloned into the pPICZ alpha A vector. Sequence analysis of the cDNA and predicted amino acid showed that this cDNA has a conserved serpin domain. B- and T-cell epitopes were predicted using bioinformatics tools. The recombinant R. microplus serpin (rRMS-3) was secreted into the culture media of Pichia pastoris after methanol induction at 0.2 mg l(-1) qRT-PCR expression analysis of tissues and life cycle stages demonstrated that RMS-3 was mainly expressed in the salivary glands of female adult ticks. Immunological recognition of the rRMS-3 and predicted B-cell epitopes was tested using tick-resistant and susceptible cattle sera. Only sera from tick-resistant bovines recognized the B-cell epitope AHYNPPPPIEFT (Seq7). The recombinant RMS-3 was expressed in P. pastoris, and ELISA screening also showed higher recognition by tick-resistant bovine sera. The results obtained suggest that RMS-3 is highly and specifically secreted into the bite site of R. microplus feeding on tick-resistant bovines. Capillary feeding of semi-engorged ticks with anti-AHYNPPPPIEFT sheep sera led to an 81.16% reduction in the reproduction capacity of R. microplus. Therefore, it is possible to conclude that R. microplus serpin (RMS-3) has an important role in the host-parasite interaction to overcome the immune responses in resistant cattle. (C) 2012 Elsevier GmbH. All rights reserved.

Studies on the fractionation of total transfer ribonucleic acid and purification of an alanine transfer ribonucleic acid from chick embryo

Chick embryo tRNA, prepared by a simple large-scale method, was fractionated on three different ion-exchange columns. In all cases simple chromatographic patterns for various tRNA species were observed, indicating the presence of only a few major species of tRNA for each amino acid. By repeated chromatography one species of alanine tRNA was purified to approx. 80% purity. T1 ribonuclease digest of this purified tRNA gave a simple chromatographic pattern. Because of the simplicity of the method of preparation of tRNA from this readily available source and the presence of only a few species of tRNA for each amino acid, chick embryo is suited for the study of tRNA and its various functions in higher systems.

Induced Variations in Brassinosteroid Genes Define Barley Height and Sturdiness, and Expand the Green Revolution Genetic Toolkit

Reduced plant height and culm robustness are quantitative characteristics important for assuring cereal crop yield and quality under adverse weather conditions. A very limited number of short-culm mutant alleles were introduced into commercial crop cultivars during the Green Revolution. We identified phenotypic traits, including sturdy culm, specific for deficiencies in brassinosteroid biosynthesis and signaling in semidwarf mutants of barley (Hordeum vulgare). This set of characteristic traits was explored to perform a phenotypic screen of near-isogenic short-culm mutant lines from the brachytic, breviaristatum, dense spike, erectoides, semibrachytic, semidwarf, and slender dwarf mutant groups. In silico mapping of brassinosteroid-related genes in the barley genome in combination with sequencing of barley mutant lines assigned more than 20 historic mutants to three brassinosteroid-biosynthesis genes (BRASSINOSTEROID-6-OXIDASE, CONSTITUTIVE PHOTOMORPHOGENIC DWARF, and DIMINUTO) and one brassinosteroid-signaling gene (BRASSINOSTEROID-INSENSITIVE1 [HvBRI1]). Analyses of F2 and M2 populations, allelic crosses, and modeling of nonsynonymous amino acid exchanges in protein crystal structures gave a further understanding of the control of barley plant architecture and sturdiness by brassinosteroid-related genes. Alternatives to the widely used but highly temperature-sensitive uzu1.a allele of HvBRI1 represent potential genetic building blocks for breeding strategies with sturdy and climate-tolerant barley cultivars.

Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

Summary We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3′-N-P-P3-M-G-P6-L-5′. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3′ leader and 5′ trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses.

Effect of some "strong" excitants of central neurones on the uptake of L-glutamate and L-aspartate by synaptosomes

"Strong" excitants of central neurones such as β N-oxalyl L α,β-diaminopropionic acid (ODAP), N-methyl-D-aspartic acid (NMDA) and kainic acid (KA) were found to inhibit the high affinity uptake of glutamate and aspartate in synaptosomes isolated from young rat brain. The potency of these "strong" excitants as convulsants appear to parallel their ability to inhibit glutamate uptake by synaptosomes. The data suggest the possibility that the convulsive effect of these "strong" excitants could be mediated by glutamate/aspartate.

Synthetic polypeptide with antifreeze activity

MUCH information has been gathered in recent years on the so-called 'antifreeze' proteins which lower the freezing point of the serum of certain marine fishes living in sub-zero water temperatures1−4. The proteins from the Antarctic fish Trematomus borchgrevinki are glycoproteins with a repeating alanyl-alanyl-threonyl tripeptide sequence, the threonyl residue being linked to a disaccharide1,2. In contrast, the antifreeze protein from the winter flounder Pseudopleuronectus americanus in the North American Atlantic coastal region is made up of eight ammo acids with no apparent repeating sequence of the residues and no sugar moiety (ref. 4 and unpublished work of C. L. Hew, C. C. Yip & G. Fletcher). The antifreeze activity of these proteins is not compatible with the known colligative properties of solutes in solution and the mechanism of their action is not yet fully understood. But a common feature of both types of the antifreeze proteins is the preponderance of alanine which accounts for over 60% of the total amino residues. This fact, together with the absence of the carbohydrate in the protein from the winter flounder, prompted us to attempt the synthesis of polypeptide analogues having comparable proportions of alanine in them along with suitable other amino acids. As a first step, we made use of the lack of any obvious periodicity in the distribution of the alanyl residues in the flounder's protein and attempted the synthesis of a random copolypeptide containing about 65 mol % of alanine and 35 mol % of aspartic acid. The choice of aspartic acid was made on the basis of its being the next major amino acid in the flounder's protein3,4 and on the expectation that its polar character will help the water-solubility of the alanine-rich copolypeptide, as in other studies on alanine-containing random copolymers. In addition, Duman and DeVries4 have earlier indicated the involvement of carboxyl groups on the antifreeze activity by chemical modification studies. We report here the synthesis of this polypeptide and show that it possesses antifreeze activity.

Pentitol phosphate dehydrogenases: Discovery, characterization and use in D-arabitol and xylitol production by metabolically engineered Bacillus subtilis

The ultimate goal of this study has been to construct metabolically engineered microbial strains capable of fermenting glucose into pentitols D-arabitol and, especially, xylitol. The path that was chosen to achieve this goal required discovery, isolation and sequencing of at least two pentitol phosphate dehydrogenases of different specificity, followed by cloning and expression of their genes and characterization of recombinant arabitol and xylitol phosphate dehydrogenases. An enzyme of a previously unknown specificity, D-arabitol phosphate dehydrogenase (APDH), was discovered in Enterococcus avium. The enzyme was purified to homogenity from E. avium strain ATCC 33665. SDS/PAGE revealed that the enzyme has a molecular mass of 41 ± 2 kDa, whereas a molecular mass of 160 ± 5 kDa was observed under non-denaturing conditions implying that the APDH may exist as a tetramer with identical subunits. Purified APDH was found to have narrow substrate specificity, converting only D-arabitol 1-phosphate and D-arabitol 5-phosphate into D-xylulose 5-phosphate and D-ribulose 5-phosphate, respectively, in the oxidative reaction. Both NAD+ and NADP+ were accepted as co-factors. Based on the partial protein sequences, the gene encoding APDH was cloned. Homology comparisons place APDH within the medium chain dehydrogenase family. Unlike most members of this family, APDH requires Mn2+ but no Zn2+ for enzymatic activity. The DNA sequence surrounding the gene suggests that it belongs to an operon that also contains several components of phosphotransferase system (PTS). The apparent role of the enzyme is to participate in arabitol catabolism via the arabitol phosphate route similar to the ribitol and xylitol catabolic routes described previously. Xylitol phosphate dehydrogenase (XPDH) was isolated from Lactobacillus rhamnosus strain ATCC 15820. The enzyme was partially sequenced. Amino acid sequences were used to isolate the gene encoding the enzyme. The homology comparisons of the deduced amino acid sequence of L. rhamnosus XPDH revealed several similar enzymes in genomes of various species of Gram-positive bacteria. Two enzymes of Clostridium difficile and an enzyme of Bacillus halodurans were cloned and their substrate specificities together with the substrate specificity of L. rhamnosus XPDH were compared. It was found that one of the XPDH enzymes of C. difficile and the XPDH of L. rhamnosus had the highest selectivity towards D-xylulose 5-phosphate. A known transketolase-deficient and D-ribose-producing mutant of Bacillus subtilis (ATCC 31094) was further modified by disrupting its rpi (D-ribose phosphate isomerase) gene to create D-ribulose- and D-xylulose-producing strain. Expression of APDH of E. avium and XPDH of L. rhamnosus and C. difficile in D-ribulose- and D-xylulose-producing strain of B. subtilis resulted in strains capable of converting D-glucose into D-arabitol and xylitol, respectively. The D-arabitol yield on D-glucose was 38 % (w/w). Xylitol production was accompanied by co-production of ribitol limiting xylitol yield to 23 %.

Electron and proton transfer in NADH:ubiquinone oxidoreductase (Complex I) from Escherichia coli

Cells of every living organism on our planet − bacterium, plant or animal − are organized in such a way that despite differences in structure and function they utilize the same metabolic energy represented by electrochemical proton gradient across a membrane. This gradient of protons is generated by the series of membrane bound multisubunit proteins, Complex I, II, III and IV, organized in so-called respiratory or electron transport chain. In the eukaryotic cell it locates in the inner mitochondrial membrane while in the bacterial cell it locates in the cytoplasmic membrane. The function of the respiratory chain is to accept electrons from NADH and ubiquinol and transfer them to oxygen resulting in the formation of water. The free energy released upon these redox reactions is converted by respiratory enzymes into an electrochemical proton gradient, which is used for synthesis of ATP as well as for many other energy dependent processes. This thesis is focused on studies of the first member of the respiratory chain − NADH:ubiquinone oxidoreductase or Complex I. This enzyme has a boot-shape structure with hydrophilic and hydrophobic domains, the former of which has all redox groups of the protein, the flavin and eight to nine iron-sulfur clusters. Complex I serves as a proton pump coupling transfer of two electrons from NADH to ubiquinone to the translocation of four protons across the membrane. So far the mechanism of energy transduction by Complex I is unknown. In the present study we applied a set of different methods to study the electron and proton transfer reactions in Complex I from Escherichia coli. The main achievement was the experiment that showed that the electron transfer through the hydrophilic domain of Complex I is unlikely to be coupled to proton transfer directly or to conformational changes in the protein. In this work for the first time properties of all redox centers of Complex I were characterized in the intact purified bacterial enzyme. We also probed the role of several conserved amino acid residues in the electron transfer of Complex I. Finally, we found that highly conserved amino acid residues in several membrane subunits form a common pattern with a very prominent feature – the presence of a few lysines within the membrane. Based on the experimental data, we suggested a tentative principle which may govern the redox-coupled proton pumping in Complex I.

Platelet endothelial cell adhesion molecule 1 (PECAM-1) and its interactions with glycosaminoglycans: 1. Molecular modeling studies

Platelet endothelial cell adhesion molecule 1 (PECAM-1) has many functions, including its roles in leukocyte extravasation as part of the inflammatory response and in the maintenance of vascular integrity through its contribution to endothelial cell−cell adhesion. PECAM-1 has been shown to mediate cell−cell adhesion through homophilic binding events that involve interactions between domain 1 of PECAM-1 molecules on adjacent cells. However, various heterophilic ligands of PECAM-1 have also been proposed. The possible interaction of PECAM-1 with glycosaminoglycans (GAGs) is the focus of this study. The three-dimensional structure of the extracellular immunoglobulin (Ig) domains of PECAM-1 were constructed using homology modeling and threading methods. Potential heparin/heparan sulfate-binding sites were predicted on the basis of their amino acid consensus sequences and a comparison with known structures of sulfate-binding proteins. Heparin and other GAG fragments have been docked to investigate the structural determinants of their protein-binding specificity and selectivity. The modeling has predicted two regions in PECAM-1 that appear to bind heparin oligosaccharides. A high-affinity binding site was located in Ig domains 2 and 3, and evidence for a low-affinity site in Ig domains 5 and 6 was obtained. These GAG-binding regions were distinct from regions involved in PECAM-1 homophilic interactions.

Prediction of heparin binding sites in bone morphogenetic proteins (BMPs)

Heparin is a glycosaminoglycan known to bind bone morphogenetic proteins (BMPs) and the growth and differentiation factors (GDFs) and has strong and variable effects on BMP osteogenic activity. In this paper we report our predictions of the likely heparin binding sites for BMP-2 and 14. The N-terminal sequences upstream of TGF-β-type cysteine-knot domains in BMP-2, 7 and 14 contain the basic residues arginine and lysine, which are key components of the heparin/HS-binding sites, with these residues being highly non-conserved. Importantly, evolutionary conserved surfaces on the beta sheets are required for interactions with receptors and antagonists. Furthermore, BMP-2 has electropositive surfaces on two sides compared to BMP-7 and BMP-14. Molecular docking simulations suggest the presence of high and low affinity binding sites in dimeric BMP-2. Histidines were found to play a role in the interactions of BMP-2 with heparin; however, a pKa analysis suggests that histidines are likely not protonated. This is indicative that interactions of BMP-2 with heparin do not require acidic pH. Taken together, non-conserved amino acid residues in the N-terminus and residues protruding from the beta sheet (not overlapping with the receptor binding sites and the dimeric interface) and not C-terminal are found to be important for heparin–BMP interactions.

Characterization of sequence and structural features of the Candida krusei Enolase

The incidence of human infections by the fungal pathogen Candida species has been increasing in recent years. Enolase is an essential protein in fungal metabolism. Sequence data is available for human and a number of medically important fungal species. An understanding of the structural and functional features of fungal enolases may provide the structural basis for their use as a target for the development of new anti-fungal drugs. We have obtained the sequence of the enolase of Candida krusei (C. krusei), as it is a significant medically important fungal pathogen. We have then used multiple sequence alignments with various enolase isoforms in order to identify C. krusei specific amino acid residues. The phylogenetic tree of enolases shows that the C. krusei enolase assembles on the tree with the fungal genes. Importantly, C. krusei lacks four amino acids in the active site compared to human enolase, as revealed by multiple sequence alignments. These differences in the substrate binding site may be exploited for the design of new anti-fungal drugs to selectively block this enzyme. The lack of the important amino acids in the active site also indicates that C. krusei enolase might have evolved as a member of a mechanistically diverse enolase superfamily catalying somewhat different reactions.

A comparative structural bioinformatics analysis of the insulin receptor family ectodomain based on phylogenetic information

The insulin receptor (IR), the insulin-like growth factor 1 receptor (IGF1R) and the insulin receptor-related receptor (IRR) are covalently-linked homodimers made up of several structural domains. The molecular mechanism of ligand binding to the ectodomain of these receptors and the resulting activation of their tyrosine kinase domain is still not well understood. We have carried out an amino acid residue conservation analysis in order to reconstruct the phylogeny of the IR Family. We have confirmed the location of ligand binding site 1 of the IGF1R and IR. Importantly, we have also predicted the likely location of the insulin binding site 2 on the surface of the fibronectin type III domains of the IR. An evolutionary conserved surface on the second leucine-rich domain that may interact with the ligand could not be detected. We suggest a possible mechanical trigger of the activation of the IR that involves a slight ‘twist’ rotation of the last two fibronectin type III domains in order to face the likely location of insulin. Finally, a strong selective pressure was found amongst the IRR orthologous sequences, suggesting that this orphan receptor has a yet unknown physiological role which may be conserved from amphibians to mammals.

Assembly of physalis mottle virus capsid protein in Escherichia coli and the role of amino and carboxy termini in the formation of the icosahedral particles

The coat protein gene of physalis mottle tymovirus (PhMV) was over expressed in Escherichia coli using pET-3d vector. The recombinant protein was found to self assemble into capsids in vivo. The purified recombinant capsids had an apparent s value of 56.5 S and a diameter of 29(±2) nm. In order to establish the role of amino and carboxy-terminal regions in capsid assembly, two amino-terminal deletions clones lacking the first 11 and 26 amino acid residues and two carboxy-terminal deletions lacking the last five and ten amino acid residues were constructed and overexpressed. The proteins lacking N-terminal 11 (PhCPN1) and 26 (PhCPN2) amino acid residues self assembled into T = 3 capsids in vivo, as evident from electron microscopy, ultracentrifugation and agarose gel electrophoresis. The recombinant, PhCPN1 and PhCPN2 capsids were as stable as the empty capsids formed in vivo and encapsidated a small amount of mRNA. The monoclonal antibody PA3B2, which recognizes the epitope within region 22 to 36, failed to react with PhCPN2 capsids while it recognized the recombinant and PhCPN1 capsids. Disassembly of the capsids upon treatment with urea showed that PhCPN2 capsids were most stable. These results demonstrate that the N-terminal 26 amino acid residues are not essential for T = 3 capsid assembly in PhMV. In contrast, both the proteins lacking the C-terminal five and ten amino acid residues were present only in the insoluble fraction and could not assemble into capsids, suggesting that these residues are crucial for folding and assembly of the particles.

Stereochemistry of peptides and polypeptides containing omega amino acids

The omega amino acids have a larger degree of conformational variability than the alpha amino acids, leading to a greater diversity of backbone structures in peptides and polypeptides. The synthetic accessibility of chiral beta-amino acids and the recent observation of novel helical folds in oligomers of cyclic beta-amino acids has led to renewed interest in the stereochemistry of omega-amino acid containing peptides. This review focuses on the conformational characteristics of the polymethylene chain in omega-amino acid segments and surveys structural features in peptides established by X-ray diffraction. The literature on polymers of achiral omega-amino acids (nylon derivatives) and chiral, substituted derivatives derived from trifunctional alpha-amino acids, reveals that while sheet-like, intermolecular hydrogen bonded structures are formed by the former, folded helices appear favoured by the latter. omega-Amino acids promise to expand the repertoire of peptide folds.