Development of SSR markers by next-generation sequencing of Korean landraces of chamoe (Cucumis melo var. makuwa)

The oriental melon (Cucumis melo var. makuwa), called ‘chamoe’ in Korean, is a popular fruit crop cultivated mainly in Asia and a high-market value crop in Korea. To provide molecular breeding resources for chamoe, we developed and characterized genomic SSR markers from the preliminary Illumina read assemblies of Gotgam chamoe (one of the major landraces; KM) and SW3 (the breeding parent). Mononucleotide motifs were the most abundant type of markers, followed by di-, tri-, tetra-, and pentanucleotide motifs. The most abundant dinucleotide was AT, followed by AG and AC, and AAT was the most abundant trinucleotide motif in both assemblies. Following our SSR-marker development strategy, we designed a total of 370 primer sets. Of these, 236 primer sets were tested, exhibiting 93 % polymorphism between KM and SW3. Those polymorphic SSRs were successfully amplified in the netted and Kirkagac melons, which respectively exhibited 81 and 76 % polymorphism relative to KM, and 32 and 38 % polymorphism relative to SW3. Seven selected SSR markers with a total of 17 alleles (2–3 alleles per locus) were used to distinguish between KM, SW3, and four chamoe cultivars. Our results represent the first attempt to provide genomic resources for Korean landraces for the purposes of chamoe breeding, as well as to discover a set of SSR markers capable of discriminating chamoe varieties from Korea and the rest of Asia, which possess little genetic diversity. This study establishes a highly efficient strategy for developing SSR markers from preliminary Illumina assemblies of AT-rich genomes.     

The structure of a shellfish specific GST class glutathione S‐transferase from antarctic bivalve Laternula elliptica reveals novel active site architecture

Glutathione‐S‐transferases have been identified in all the living species examined so far, yet little is known about their function in marine organisms. In a previous report, the recently identified GST from Antarctic bivalve Laternula elliptica (LeGST) was classified into the rho class GST, but there are several unique features of LeGST that may justify reclassification, which could represent specific shellfish GSTs. Here, we determined the crystal structure of LeGST, which is a shellfish specific class of GST. The structural analysis showed that the relatively open and wide hydrophobic H‐site of the LeGST allows this GST to accommodate various substrates. These results suggest that the H‐site of LeGST may be the result of adaptation to their environments as sedentary organisms. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Engineering flavonoid glycosyltransferases for enhanced catalytic efficiency and extended sugar-donor selectivity

Flavonoids are predominantly found as glycosides in plants. The glycosylation of flavonoids is mediated by uridine diphosphate-dependent glycosyltransferases (UGT). UGTs attach various sugars, including arabinose, glucose, galactose, xylose, and glucuronic acid, to flavonoid aglycones. Two UGTs isolated from Arabidopsis thaliana, AtUGT78D2 and AtUGT78D3, showed 89 % amino acid sequence similarity (75 % amino acid sequence identity) and both attached a sugar to the 3-hydroxyl group of flavonols using a UDP-sugar. The two enzymes used UDP-glucose and UDP-arabinose, respectively, and AtUGT78D2 was approximately 90-fold more efficient than AtUGT78D3 when judged by the k _cat/K _m value. Domain exchanges between AtUGT78D2 and AtUGT78D3 were carried out to find UGTs with better catalytic efficiency for UDP-arabinose and exhibiting dual sugar selectivity. Among 19 fusion proteins examined, three showed dual sugar selectivity, and one fusion protein had better catalytic efficiency for UDP-arabinose compared with AtUGT78D3. Using molecular modeling, the changes in enzymatic properties in the chimeric proteins were elucidated. To the best of our knowledge, this is the first report on the construction of fusion proteins with expanded sugar-donor range and enhanced catalytic efficiencies for sugar donors.     

The inhibitory effect of naringenin on atopic dermatitis induced by DNFB in NC/Nga mice

Aims

Atopic dermatitis (AD) is a chronic and relapsing inflammatory dermatitis characterized by pruritic and eczematous skin lesions. Here, we investigated the therapeutic effect of the fruit flavonoid naringenin on DNFB induced atopic dermatitis mice model.

Main methods

AD-like skin lesion was induced by repetitive skin contact with DNFB in NC/Nga mice and the effects of the fruit flavonoid naringenin were evaluated on the basis of histopathological findings of skin, ear swelling and cytokine production of CD4⁺T cells.

Key findings

Intraperitoneal injection of naringenin for one week after DNFB challenge significantly lowered ear swelling and improved back skin lesions. In addition, naringenin significantly suppressed production of interferon-gamma (IFN-γ) by activated CD4⁺ T cells and serum IgE level. Furthermore, naringenin reduced DNFB-induced infiltration of eosinophils, mast cells, CD4⁺ T cells, and CD8⁺ T cells in skin lesions.

Significance

Naringenin may suppress the development of AD-like skin lesions in DNFB-treated NC/Nga mice by reducing IFN-γ production of activated CD4⁺ T cells, serum IgE levels and infiltration of immune cells to skin lesion.     

First Record of Bourgelatia diducta (Nematoda: Chabertiidae) from Wild Boars in the Republic of Korea

This study describes the first record of Bourgelatia diducta (Nematoda: Chabertiidae) from wild boars in the Republic of Korea (=South Korea). Gastrointestinal tracts of 87 Korean wild boars (Sus scrofa coreanus) hunted in mountains in the south-western part of South Korea between 2009 and 2012 were examined for their visceral helminths. B. diducta, as identified by morphological characteristics of the head and tail, were recovered from the large intestine of 47 (54%) wild boars. The average length of adult female worms was 11.3±0.87 mm and the thickest part of the body measured 0.54±0.04 mm in maximum width, while those of males were 9.8±0.72 and 0.45±0.03 mm, respectively. The characteristic J-shaped type II ovejector was observed in females, and the type II dorsal ray with 2 rami on each side of the median fissure was uniquely seen in males. The buccal capsule was small, relatively thin-walled, cylindrical, very short, and ring-shaped. The externodorsal ray arose from a common stem with the dorsal ray. The cervical groove was absent. The anterior extremity was equipped with 20-22 external corona radiata, 4 cephalic papillae and 2 lateral amphids around the mouth. The eggs were 66.0×38.9 µm in average size. By the present study, B. diducta (Nematoda: Chabertiidae) is recorded for the first time in South Korea. Additionally, morphological characteristics and identification keys provided in the present study will be helpful in the faunistic or taxonomic studies for strongylid nematodes related.     

Proliferation of Toxoplasma gondii Suppresses Host Cell Autophagy

Autophagy is a process of cytoplasmic degradation of endogenous proteins and organelles. Although its primary role is protective, it can also contribute to cell death. Recently, autophagy was found to play a role in the activation of host defense against intracellular pathogens. The aims of our study was to investigate whether host cell autophagy influences Toxoplasma gondii proliferation and whether autophagy inhibitors modulate cell survival. HeLa cells were infected with T. gondii with and without rapamycin treatment to induce autophagy. Lactate dehydrogenase assays showed that cell death was extensive at 36-48 hr after infection in cells treated with T. gondii with or without rapamycin. The autophagic markers, LC3 II and Beclin 1, were strongly expressed at 18-24 hr after exposure as shown by Western blotting and RT-PCR. However, the subsequent T. gondii proliferation suppressed autophagy at 36 hr post-infection. Pre-treatment with the autophagy inhibitor, 3-methyladenine (3-MA), down-regulated LC3 II and Beclin 1. The latter was also down-regulated by calpeptin, a calpain inhibitor. Monodansyl cadaverine (MDC) staining detected numerous autophagic vacuoles (AVs) at 18 hr post-infection. Ultrastructural observations showed T. gondii proliferation in parasitophorous vacuoles (PVs) coinciding with a decline in the numbers of AVs by 18 hr. FACS analysis failed to confirm the presence of cell apoptosis after exposure to T. gondii and rapamycin. We concluded that T. gondii proliferation may inhibit host cell autophagy and has an impact on cell survival.     

Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Cav3.2 T-type channel

Voltage-activated Ca²⁺ channels are membrane protein machinery performing selective permeation of external calcium ions. The main Ca²⁺ selective filters of all high-voltage-activated Ca²⁺ channel isoforms are commonly composed of four Glu residues (EEEE), while those of low-voltage-activated T-type Ca²⁺ channel isoforms are made up of two Glu and two Asp residues (EEDD). We here investigate how the Asp residues at the pore loops of domains III and IV affect biophysical properties of the Ca_v3.2 channel. Electrophysiological characterization of the pore mutant channels in which the pore Asp residue(s) were replaced with Glu, showed that both Asp residues critically control the biophysical properties of Ca_v3.2, including relative permeability between Ba²⁺ and Ca²⁺, anomalous mole fraction effect (AMFE), voltage dependency of channel activation, Cd²⁺ blocking sensitivity, and pH effects, in distinctive ways.     

Molecular Basis of Cannabinoid CB1 Receptor Coupling to the G Protein Heterotrimer Gαiβγ: IDENTIFICATION OF KEY CB1 CONTACTS WITH THE C-TERMINAL HELIX α5 OF Gαi*

The cannabinoid (CB1) receptor is a member of the rhodopsin-like G protein-coupled receptor superfamily. The human CB1 receptor, which is among the most expressed receptors in the brain, has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. Different classes of CB1 agonists evoke signaling pathways through the activation of specific subtypes of G proteins. The molecular basis of CB1 receptor coupling to its cognate G protein is unknown. As a first step toward understanding CB1 receptor-mediated G protein signaling, we have constructed a ternary complex structural model of the CB1 receptor and G_i heterotrimer (CB1-G_i), guided by the x-ray structure of β₂-adrenergic receptor (β₂AR) in complex with G_s (β₂AR-G_s), through 824-ns duration molecular dynamics simulations in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer environment. We identified a group of residues at the juxtamembrane regions of the intracellular loops 2 and 3 (IC2 and IC3) of the CB1 receptor, including Ile-218^3.54, Tyr-224^IC2, Asp-338^6.30, Arg-340^6.32, Leu-341^6.33, and Thr-344^6.36, as potential key contacts with the extreme C-terminal helix α₅ of Gα_i. Ala mutations of these residues at the receptor-G_i interface resulted in little G protein coupling activity, consistent with the present model of the CB1-G_i complex, which suggests tight interactions between CB1 and the extreme C-terminal helix α₅ of Gα_i. The model also suggests that unique conformational changes in the extreme C-terminal helix α₅ of Gα play a crucial role in the receptor-mediated G protein activation.