Food Allergy,Oral Tolerance and Immunomodulation—Their Molecular and Cellular Mechanisms

This paper describes the molecular and cellular mechanisms of food allergy and oral tolerance including immunomodulation. Food allergy is triggered by an aberrant immune response elicited by oral administration of dietary antigens. Oral tolerance is a state of immunological unresponsiveness induced by oral administration of dietary antigens and is thought to serve to suppress food allergy. This review first describes the characteristic properties of T and B cells relating to milk allergy and also the location of binding sites to T and B cells on allergen molecules. The immunogenicity of allergens is shown to be reduced by the modulations of the T cell binding site, using sophisticated methods such as site-specific mutagenesis. Furthermore, this review focuses on oral tolerance with special reference to the identification of lymphocyte compartment subsets and the immunological mechanism relating to oral tolerance. Finally, the application of oral tolerance for the treatment of allergy is speculated on.     

Molecular cloning,expression and characterization of Pekin duck interferon-λ

Interferons (IFNs) are the first line of defense against viral infections in vertebrates. Type III interferon (IFN-λ) is recognized for its key role in innate immunity of tissues of epithelial origin. Here we describe the identification of the Pekin duck IFN-λ ortholog (duIFN-λ). The predicted duIFN-λ protein has an amino acid identity of 63%, 38%, 37% and 33% with chicken IFN-λ and human IFN-λ3, IFN-λ2 and IFN-λ1, respectively. The duck genome contains a single IFN-λ gene that is comprised of five exons and four introns. Recombinant duIFN-λ up-regulated OASL and Mx-1 mRNA in primary duck hepatocytes. Our observations suggest evolutionary conservation of genomic organization and structural features implicated in receptor binding and antiviral activity. The identification and expression of duIFN-λ will facilitate further study of the role of type III IFN in antiviral defense and inflammatory responses of the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health.     

Monosialogangliosides and Their Interaction with Cholera Toxin—Investigation by Molecular Modeling and Molecular Mechanics

Abstract

Molecular mechanics and molecular dynamics studies are performed to investigate the conformational preference of cell surface monosialogangliosides (GM3, GM2 and GM1) in aqueous environment. Water mediated hydrogen bonding network plays a significant role in the structural stabilization of GM3, GM2 and GM1. The spatial flexibility of NeuNAc of gangliosides at the binding site of cholera toxin reveals a limited allowed eulerian space of 2.4% with a much less allowed eulerian space (1.4%) for external galactose of GM1. The molecular mechanics of monosialoganglioside-cholera toxin complex reveals that cholera toxin can accommodate the monosialogangliosides in three different modes. Direct and water mediated hydrogen bonding interactions stabilize these binding modes and play an essential role in defining the order of specificity for different monosialogangliosides towards cholera toxin. This study identifies the NeuNAc binding site as a site for design of inhibitors that can restrict the pathogenic activity of cholera toxin.     

Molecular Basis for the Recognition and Cleavages of IGF-II, TGF-α, and Amylin by Human Insulin-Degrading Enzyme

Insulin-degrading enzyme (IDE) is involved in the clearance of many bioactive peptide substrates, including insulin and amyloid-β, peptides vital to the development of diabetes and Alzheimer's disease, respectively. IDE can also rapidly degrade hormones that are held together by intramolecular disulfide bond(s) without their reduction. Furthermore, IDE exhibits a remarkable ability to preferentially degrade structurally similar peptides such as the selective degradation of insulin-like growth factor (IGF)-II and transforming growth factor-α (TGF-α) over IGF-I and epidermal growth factor, respectively. Here, we used high-accuracy mass spectrometry to identify the cleavage sites of human IGF-II, TGF-α, amylin, reduced amylin, and amyloid-β by human IDE. We also determined the structures of human IDE-IGF-II and IDE-TGF-α at 2.3 Å and IDE-amylin at 2.9 Å. We found that IDE cleaves its substrates at multiple sites in a biased stochastic manner. Furthermore, the presence of a disulfide bond in amylin allows IDE to cut at an additional site in the middle of the peptide (amino acids 18-19). Our amylin-bound IDE structure offers insight into how the structural constraint from a disulfide bond in amylin can alter IDE cleavage sites. Together with NMR structures of amylin and the IGF and epidermal growth factor families, our work also reveals the structural basis of how the high dipole moment of substrates complements the charge distribution of the IDE catalytic chamber for the substrate selectivity. In addition, we show how the ability of substrates to properly anchor their N-terminus to the exosite of IDE and undergo a conformational switch upon binding to the catalytic chamber of IDE can also contribute to the selective degradation of structurally related growth factors.     

Molecular Divergence of Lysozymes and α-Lactalbumin

Abstract

The vast number of proteins that sustain the currently living organisms have been generated from a relatively small number of ancestral genes that has involved a variety of processes. Lysozyme is an ancient protein whose origin goes back an estimated 400 to 600 million years. This protein was originally a bacteriolytic defensive agent and has been adapted to serve a digestive function on at least two occasions, separated by nearly 40 million years. The origins of the related goose type and T4 phage lysozyme that are distinct from the more common C type are obscure. They share no discernable amino acid sequence identity and yet they possess common secondary and tertiary structures. Lysozyme C gene also gave rise, after gene duplication 300 to 400 million years ago, to a gene that currently codes for α-lactalbumin, a protein expressed only in the lactating mammary gland of all but a few species of mammals. It is required for the synthesis of lactose, the sugar secreted in milk. α-Lactalbumin shares only 40% identity in amino acid sequence with lysozyme C, but it has a closer spatial structure and gene organization. Although structurally similar, functionally they are quite distinct. Specific amino acid substitutions in α-lactalbumin account for the loss of the enzyme activity of lysozyme and the acquisition of the features necessary for its role in lactose synthesis. Evolutionary implications are as yet unclear but are being unraveled in many laboratories.     

Synthesis,Antifungal Activity,and Molecular Simulation Study of L–Carvone-Derived 1,3,4-Oxadiazole-Thioether Compounds

To discover potent antifungal molecules with new and distinctive structures, 20 novel L-carvone-derived 1,3,4-oxadiazole-thioether compounds 5 a – 5 t were synthesized through multi-step reaction of L-carvone, and their structures were confirmed by FT-IR, ¹H-NMR, ¹³C-NMR, and HR-MS. The antifungal activities of compounds 5 a – 5 t were preliminarily tested by in vitro method, and the results indicated that all of the title compounds displayed certain antifungal activities against the eight tested plant fungi, especially for P. piricola. Among them, compound 5 i (R=p-F) with the most significant antifungal activity deserved further study for discovering and developing novel natural product-based antifungal agents. Moreover, two molecular simulation technologies were employed for the investigation of their structure–activity relationships (SARs). Firstly, a reasonable and effective 3D-QSAR model was established by the comparative molecular field (CoMFA) method, and the relationship of the substituents linked with the benzene rings and the inhibitory activities of the title compounds against P. piricola was elucidated. Then, the binding mode of compound 5 i (R=p-F) and its potential biological target (CYP51) was simulated by molecular docking, and it was found that compound 5 i could readily bind with CYP51 in the active site, and the ligand-receptor interactions involved three hydrogen bonds and several hydrophobic effects.     

Molecular Mechanics of the α-Actinin Rod Domain: Bending,Torsional, and Extensional Behavior

α-Actinin is an actin crosslinking molecule that can serve as a scaffold and maintain dynamic actin filament networks. As a crosslinker in the stressed cytoskeleton, α-actinin can retain conformation, function, and strength. α-Actinin has an actin binding domain and a calmodulin homology domain separated by a long rod domain. Using molecular dynamics and normal mode analysis, we suggest that the α-actinin rod domain has flexible terminal regions which can twist and extend under mechanical stress, yet has a highly rigid interior region stabilized by aromatic packing within each spectrin repeat, by electrostatic interactions between the spectrin repeats, and by strong salt bridges between its two anti-parallel monomers. By exploring the natural vibrations of the α-actinin rod domain and by conducting bending molecular dynamics simulations we also predict that bending of the rod domain is possible with minimal force. We introduce computational methods for analyzing the torsional strain of molecules using rotating constraints. Molecular dynamics extension of the α-actinin rod is also performed, demonstrating transduction of the unfolding forces across salt bridges to the associated monomer of the α-actinin rod domain.     

Molecular aspects of pancreatic β-cell dysfunction: Oxidative stress,microRNA, and long noncoding RNA

Metabolic syndrome is known as a frequent precursor of type 2 diabetes mellitus (T2D). This disease could affect 8% of the people worldwide. Given that pancreatic β-cell dysfunction and loss have central roles in the initiation and progression of the disease, the understanding of cellular and molecular pathways associated with pancreatic β-cell dysfunction can provide more information about the underlying pathways involved in T2D. Multiple lines evidence indicated that oxidative stress, microRNA, and long noncoding RNA play significant roles in various steps of diseases. Oxidative stress is one of the important factors involved in T2D pathogenesis. This could affect the function and survival of the β cell via activation or inhibition of several processes and targets, such as receptor-signal transduction, enzyme activity, gene expression, ion channel transport, and apoptosis. Besides oxidative stress, microRNAs and noncoding RNAs have emerged as epigenetic regulators that could affect pancreatic β-cell dysfunction. These molecules exert their effects via targeting a variety of cellular and molecular pathways involved in T2D pathogenesis. Here, we summarized the molecular aspects of pancreatic β-cell dysfunction. Moreover, we highlighted the roles of oxidative stress, microRNAs, and noncoding RNAs in pancreatic β-cell dysfunction.     

Left–Right Determination: Involvement of Molecular Motor KIF3, Cilia,and Nodal Flow

Mammalian left–right determination is a good example for how multiple cell biological processes coordinate in the formation of a basic body plan. The leftward movement of fluid at the ventral node, called nodal flow, is the central process in symmetry breaking on the left–right axis. Nodal flow is autonomously generated by the rotation of posteriorly tilted cilia that are built by transport via KIF3 motor on cells of the ventral node. How nodal flow is interpreted to create left–right asymmetry has been a matter of debate. Recent evidence suggests that the leftward movement of sheathed lipidic particles, called nodal vesicular parcels (NVPs), may result in the activation of the noncanonical hedgehog signaling pathway, an asymmetric elevation in intracellular Ca²⁺ and changes in gene expression.Although the human body is apparently bilaterally symmetrical on the surface, the visceral organs are arranged asymmetrically in a stereotyped manner. The heart, spleen, and pancreas reside on the left side of the body, whereas the gall bladder and most of the liver are on the right side (Fig. 1A). Because the human body is formed from a spherically symmetrical egg (oocyte), symmetry breakdown is one of the fundamental processes of development.Open in a separate windowFigure 1.(A) Left–right asymmetric arrangements of internal organs in the human body. (Left) Normal arrangement (situs solitus). Most humans (>99%) have the heart on the left side and the liver on the right side. (Right) Mirrored arrangement (situs inversus). Half of patients with Kartagener''s syndrome have this arrangement, whereas the remaining patients are normal. Therefore, the left–right bilateral symmetry is randomly broken in this disease. (B–E) Scanning electron micrographs of wild-type (B, D) and Kif3b^−/− (C, E) mouse embryos. (B, C) Full-length images. Wild-type embryos at this stage have already turned with a right-sided tail (B), whereas Kif3b^−/− embryos remain unturned (C). In panel C, the dilated pericardial sac has been removed, and the heart loop is inverted (arrow). (D, E) Higher-magnification images and schematic representations of the heart loops showing a normal loop in the wild-type embryo (D) and an inverted loop in the mutant embryo (E). (F–I) Scanning electron micrographs of a mouse node. (F) Low-magnification view of a mouse embryo at 7.5 days postcoitum. Reichert''s membrane is removed, and the embryo is observed from the ventral side. The node is indicated by a black rectangle. The orientation is indicated in the panel as anterior (A), posterior (P), left (L), and right (R). Scale bar = 100 µm. (G) Higher-magnification view of the mouse node. The orientation is the same as in panel A. Scale bar = 20 µm. (H) Higher-magnification view of the nodal cilia (arrows) and nodal pit cells. Scale bar = 5 µm. (I) Nodal pit cells of Kif3b^−/− embryos. Nodal cilia are absent in these genetically manipulated embryos. (J) Intraflagellar transport. Protein components in the cilia and flagella are transported by KIF3A/B complexes (light and dark blue) along the doublet microtubules of the axoneme. (Panels A and J were reproduced with permission from JT Biohistory Research Hall/TokyoCinema. B–I were modified from Nonaka et al. 1998, Okada et al. 2005, and Hirokawa et al. 2006, with permission.)In many lower vertebrates and invertebrates, eggs are asymmetrical even before fertilization (as in Drosophila [Gilbert 2003]). In some organisms, such as fish and frog, the dorsoventral (DV) and anteroposterior (AP) axes are determined at fertilization by the distribution of the yolk and the entry position of the sperm (Gilbert 2003). In human, mouse, and other mammals, the embryo is initially cylindrically symmetrical when it implants itself into the wall of the uterus. The DV axis is first to be specified as the proximal–distal axis from the implantation site. Subsequently, the AP axis is arbitrarily determined in the plane perpendicular to the DV axis (Alarcon and Marikawa 2003; Beddington and Robertson 1999). In either case, L/R is thus the last axis to be determined, and needs to be consistent with the preceding DV and AP axes. Because the chirality of the body is predetermined by chiral molecules, such as amino acids and nucleic acids, the laterality or orientation of the L/R axis is established theoretically or potentially once the AP and DV axes are determined. The problem here is how this potentially established laterality is materialized through developmental events. This mechanism is still totally unknown for the invertebrates. However, recent studies of mouse embryo clarified the L/R determination mechanism in mammalian embryos (Hirokawa et al. 2006).Until recently, little was known about the mechanism for breaking L/R symmetry. The initial clue to tackling this question was a human genetic disease called Kartagener''s syndrome. Approximately half of these patients have their organs in the reversed orientation (situs inversus). Thus, the L/R determination is randomized in this syndrome. Patients with Kartagener''s syndrome also suffer from sinusitis and bronchiectasis (Kartagener 1933). A number of male patients with Kartagener''s syndrome are sterile. Affected individuals have immotile sperm and defective cilia in their airway (Afzelius 1976). Airway cilia and sperm from these patients have abnormal ultrastructures. Specifically, the axonemes, the interior supramolecular complexes that produce the movement of cilia and flagella, lack dynein arms, the molecular motors required for cilia and flagella motility (Afzelius 1976). However, the relationships between these phenotypes and the causes of situs inversus remained unknown for more than 20 years.Approximately 10 years ago, molecular biological studies identified several genes that are asymmetrically expressed in the L/R orientation before L/R asymmetric morphogenesis of the embryo. Morphological L/R asymmetry first becomes apparent with the orientation of the heart-tube loop (Fig. 1B,D) (Kaufman 1992), but initial L/R asymmetric gene expression precedes the morphological changes. In early embryos (7.5 days for mice) at the stage of somatogenesis, genes such as Lefty-2 (Ebaf), Nodal, and Pitx2 are expressed in the left lateral plate mesoderm, a structure located on the side of the embryos (Capdevila et al. 2000; Hamada 2002; Harvey 1998; Levin 2005; Yost 1999). However, the upstream phenomena that cause asymmetrical expression of these genes remain enigmatic.Many studies have suggested that the so-called node, a concave triangular region transiently formed during gastrulation at the ventral midline surface of early embryos, is important for L/R determination (Harvey 1998). When viewed from above the ventral side, the node of mouse embryos appears as a roughly triangular depression with the apex pointed toward the anterior (Fig. 1F,G), and it is 50–100 µm in width and 10–20 µm in depth. This nodal pit is covered by Reichert''s membrane, and the cavity is filled with extraembryonic fluid. The ventral embryonic surface of the nodal pit consists of an epithelial sheet of a few hundred monociliated cells (nodal pit cells).Nodal pit cells have one or sometimes two cilia that appear as rodlike protrusions approximately 5 µm in length and 0.3 µm in diameter (Fig. 1G,H). Because Kartagener''s syndrome suggests a potential link between cilia motility and L/R determination, the cilia in the node have been postulated to be motile and responsible for L/R determination.However, the ultrastructure of these nodal cilia is similar to that of immotile primary cilia. In motile cilia and flagella, nine pairs of doublet microtubules are arranged longitudinally along the axes (Fig. 2A) (Satir and Christensen 2007). Adjacent pairs of doublet microtubules are connected by dynein arms, which generate the motility of cilia and flagella. In addition, there are two microtubules in the center of cilia and flagella, referred to as the central pair, which define the direction of the beating plane. This essential central pair of microtubules is missing in primary cilia. Similar to other immotile primary cilia, the monocilia of nodal pit cells lack the central pair of microtubules and thus have a 9 + 0 microtubule arrangement. Therefore, based on their ultrastructure and initial video-microscopic observations, nodal monocilia were originally considered immotile (Bellomo et al. 1996).Open in a separate windowFigure 2.(A) Ultrastructures of normal cilia and primary cilia. (Left) Normal cilia and flagella have nine pairs of doublet microtubules (yellow) and two central microtubules (yellow). Adjacent doublet microtubules are connected with dynein motors (blue and green). The orientation of the central pair of microtubules is considered to determine the beating plane (purple). (Right) The central pair of microtubules is missing in immotile primary cilia and nodal cilia. In nodal cilia, the dynein motors remain in a chiral arrangement and produce a rotation-like movement (purple). (B–E) Rotation of nodal cilia and leftward nodal flow. The images are views from the ventral side. The orientation is indicated in the panels as anterior (A), posterior (P), left (L), and right (R). (B) Trajectory of a fluorescent bead attached to the tip of a nodal cilium (Movies 1 and 2). Three consecutive video frames with 33-ms exposures at 16-ms intervals (interlaced scan) are shown. The moving bead produces arc-shaped images (traced by green arrows). The beads rotate clockwise when viewed above the node. (C) Trajectories of the tips of nodal cilia traced from a high-speed video sequence (500 frames/s) (Movie 3). The red circles show the positions of the ends of the cilia at 10-ms time intervals, and the yellow circles show the positions of the roots of the cilia. The white ellipses show the trajectories of the tips. Scale bar = 5 µm. (D) The positions of beads that entered the node from the right edge traced for 4 seconds at 0.33-second intervals. Different symbols indicate different beads. Most beads go straight to the left edge of the node. Scale bar = 20 µm. (E) The trajectories of four beads selected to illustrate the streamline of the nodal flow. The flow is mostly laminar and straight in the middle of the node, but often makes small vortices near the left edge (arrowheads). (Panel A was reproduced with permission from JT Biohistory Research Hall/TokyoCinema. B–E were modified from Okada et al. [1999, 2005] with permission.)

Movie 1

Download video file.^{(3.2M, mov)}Leftward nodal flow. Nodal flow was visualized by adding fluorescent beads to the medium surrounding the ventral node of a mouse embryo at the early somite stage. 4× time lapse.

Movie 2

Open in a separate windowClick here to view.^{(104K, gif)}Rotatory movement of nodal cilia.

Movie 3

Download video file.^{(39M, mov)}Posteriorly-tilted rotation of nodal cilia. Nodal cilia were observed by high-speed video microscopy (500 frames/s). The focus was adjusted to approximately 3 µm above the surface of the ventral node. The images of the cilia thus blur when they are near the floor and become clear when they come up to the focal plane. Note that the particle (highlighted by a red circle) does not go down to the surface but eventually goes toward the left side of the node due to the movement of the cilia. Reproduced from Okada et al. (2005) with permission.Through studies of the flow of materials within cells, we serendipitously found that nodal cilia are actually motile and vigorously rotating. This rotation generates the leftward flow of extraembryonic fluid in the nodal pit. The directionality of this flow, termed nodal flow, determines laterality. Thus, quite unexpectedly, a physical process, fluid flow, was identified as the initial L/R symmetry-breaking event. In this review, we first summarize the discovery of nodal flow and then discuss how this leftward linear flow is generated in a fluid dynamic manner by the rotational movement of cilia. We further discuss the mechanisms by which L/R asymmetry is determined by this nodal flow.     

Molecular activation of NF-κB, pro-inflammatory mediators, and signal pathways in γ-irradiated mice

The effects of γ-irradiation on inflammatory gene expression, including NF-κB activation, in the kidney of C57/BL6 mice exposed to 1–9 Gy doses of ⁶⁰Co γ-irradiation. Radiation enhanced the NF-κB activation and oxidative stress caused a dose-dependent disruption in the redox balance. The significance of this study is the new molecular information gained on γ-irradiation effects through the activation of pro-inflammatory genes by NF-κB via the MAPK signaling pathway. Considering the exquisite sensitivity of NF-κB and other pro-inflammatory mediators to the redox status, we conclude that the activation of inflammatory processes by irradiation is likely initiated by increased oxidative stress.     

Molecular cloning and characterization of a gibberellin-inducible, putative α-glucosidase gene from barley

A putative -glucosidase clone has been isolated from a cDNA library constructed from mRNA of barley aleurones treated with gibberellin A₃ (GA). The clone is 2752 bp in length and has an uninterrupted open reading frame encoding a polypeptide of 877 amino acids. A 680 amino acid region is 43% identical to human lysosomal -glucosidase and other glycosyl hydrolases. In isolated aleurones, the levels of the corresponding mRNA increase strongly after the application of GA, similar to the pattern exhibited by low-pI -amylase mRNA. High levels are also observed in the aleurone and scutellum after germination, while low levels are found in developing seeds. The genome contains a single form of this -glucosidase gene and two additional sequences that may be related genes or pseudogenes.Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Molecular systematics of Brassica and allied genera (Subtribe Brassicinae,Brassiceae) —chloroplast genome and cytodeme congruence

Summary Chloroplast DNA restriction sites for 20 endonucleases were mapped using cpDNA probes from Brassica juncea and site variation was surveyed in 33 diploid taxa of the Subtribe Brassicinae. A total of 419 mutations was observed, including both site (i.e., gain/ loss) and fragment length (i.e., insertions or deletions); 221 (53%) mutations showed variation at the interspecific level. Phylogenetic analysis indicated a clear division of the subtribe into two ancient evolutionary lineages. These were (I) the Nigra lineage: Brassica nigra, B. fruticulosa, B. tournefortii, Sinapis pubescens, S. alba, S. flexuosa, S. arvensis, Coincya cheiranthos, Erucastrum canariense, and Hirschfeldia incana, and (II) the Rapa/ Oleracea lineage: Brassica rapa, B. oleracea ssp. oleracea and ssp. alboglabra, B. rupestris-villosa complex (B. rupestris, B. drepanensis, B. macrocarpa, B. villosa), B. barrelieri, B. deflexa, B. oxyrrhina, B. gravinae, Diplotaxis erucoides, D. tenuifolia, Eruca sativa, Raphanus raphanistrum, R. sativus, and Sinapis aucheri. In the Nigra lineage, Brassica nigra was most closely related to the annual Sinapis species, S. arvensis and S. alba. In the Rapa/Oleracea lineage, the Brassica rapa and B. oleracea genomes formed a distinct group whose closest relatives were the wild species of the B. oleracea (n=9) complex (i.e., B. rupestris-villosa complex). Species with n=7 chromosomes exist in both lineages. Hirschfeldia incana (n=7), in the Nigra lineage, was most closely related to Sinapis pubescens. In the Rapa/Oleracea lineage three taxa with n=7 — B. deflexa, D. erucoides, and S. aucheri — were closely related, advanced in the lineage, and were the closest apparent relatives (particularly D. erucoides) to B. rapa, B. oleracea, and its wild relatives. Levels of genetic divergence suggested by the cpDNA data were consistent with cytodeme recognition in the subtribe, but provided evidence for inconsistencies in the current generic delimitations based on morphology. Very low levels of genetic divergence were evident among taxa/accessions within a cytodeme. Raphanus was closely related to the Brassica rapa and B. oleracea genomes and clearly belongs in Subtribe Brassicinae. Several cytoplasmic genetic markers of potential use in plant breeding programs were identified for each of the cytodemes.