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Autism spectrum disorder (ASD) having core characteristics of social interaction problems and repetitive behaviors and interests affects individuals at varying degrees and comorbidities, making it difficult to determine the precise etiology underlying the symptoms. Given its heterogeneity, ASD is difficult to treat and the development of therapeutics is slow due to the scarcity of animal models that are easy to produce and screen with. Based on the theory of excitation/inhibition imbalance in the brain with ASD which involves glutamatergic and/or GABAergic neurotransmission, a pharmacologic agent to modulate these receptors might be a good starting point for modeling. N-methyl-D-aspartic acid (NMDA) is an amino acid derivative acting as a specific agonist at the NMDA receptor and therefore imitates the action of the neurotransmitter glutamate on that receptor. In contrast to glutamate, NMDA selectively binds to and regulates the NMDA receptor, but not other glutamate receptors such as AMPA and kainite receptors. Given this role, we aimed to determine whether NMDA administration could result in autistic-like behavior in adolescent mice. Both male and female mice were treated with saline or NMDA (50 and 75 mg/kg) and were tested on various behavior experiments. Interestingly, acute NMDA-treated mice showed social deficits and repetitive behavior similar to ASD phenotypes. These results support the excitation/inhibition imbalance theory of ASD and that NMDA injection can be used as a pharmacologic model of ASD-like behaviors.

The steelhead trout (Oncorhynchus mykiss) is the species most at risk from selenium (Se) exposure in the San Francisco Bay Delta (SFBD). However, although steelhead trout are usually exposed to environmental Se in the juvenile stage, data to test their sensitivity to excess Se, especially its organic form, in the juvenile stage are scarce. Therefore, the objective of the current study was to assess the sensitivity of juvenile steelhead trout to ecologically relevant forms of Se using integrated sensitive endpoints. Fish (mean weight: 22.3 g) were fed one of five diets containing 1.1 (control), 8.8, 15.4, 30.8, and 61.6 μg Se/g diet dw (Se1.1, Se8.8, Se15.4, Se30.8, and Se61.6, respectively) in the form of selenomethionine for 4 weeks. After 4 weeks, Se significantly accumulated in a dose-dependent manner in all tissues at different rates. The growth rate and plasma cholesterol were significantly depressed in fish fed diets containing Se30.8 and above. Hematological parameters and mortality were significantly elevated in fish fed the Se61.6 diet. Marked histopathological alterations were observed in fish fed the Se8.8 diet (the lowest observed effect concentration, LOEC) and above. The current results suggest that the steelhead trout is more sensitive to excess Se than nonanadromous rainbow trout used in previous studies because of its lower LOEC despite the use of selenomethionine and the shorter experimental duration. Additionally, it should be noted that the current Se levels found in the SFBD are already a threat to the threatened population of steelhead trout on the central California coast.

We investigated the effects of cooking (steaming and microwaving) and processing (freeze-drying and hot-air-drying) methods on the antioxidant activity of broccoli florets. 2,2-diphenyl-1-picrylhydrazyl (DPPH•), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•), and alkyl• free radical scavenging assays were employed to assess anti-oxidant potentials. The cytoprotective effect against oxidative damage induced by H2O2 was studied using hepatocellular carcinoma (HepG2) cells. Anti-proliferative effects were assessed in MCF-7 and MDA-MB-231 breast cancer cells. L-sulforaphane in broccoli extracts was quantified using high-performance liquid chromatography (HPLC). Steam and microwave treatments caused increases in total polyphenol content (TPC), whereas the total flavonoid content (TFC) decreased following steam treatment. A slight increase in TFC was observed in the microwaved samples. Extracts of all broccoli samples showed almost identical radical scavenging and cytoprotective effects. HPLC demonstrated that steamed (3 min)-freeze-dried (F-S3) and microwaved (2 min)-freeze-dried (F-M2) samples exhibited elevated levels of L-sulforaphane. In addition, the F-S3 and F-M2 extracts displayed strong anti-proliferative effects in MCF-7 cells, which correlated with L-sulforaphane content. As we observed no significant decrease in the antioxidant activity of broccoli florets, the cooking and processing methods and conditions studied here are recommended for broccoli.

It is important to evaluate leaching behavior in agricultural soils to prevent the pollution of groundwater by pesticides. We identified the distribution coefficients (Kd) of ten pesticides with different physicochemical properties and compared their leaching characteristics using wick lysimeters from three distinct soil types on Jeju Island. The Kdvalues varied by pesticide and soil, but were within the range of 1.2 to 4231 L kg−1. Based on the European standard (Kd −1), six pesticides (alachlor, ethoprophos, carbofuran, napropamide, tebuconazole, and etridiazole) were mobile in at least one tested soil, and their soil organic carbon affinity was ≤ 5.811. This value differed greatly from the other pesticides (16.533 and higher). The solubility of the six mobile pesticides was ≥ 32 mg L−1, which substantially differed from the other pesticides (≤ 0.71 mg L−1). Thus, we conclude that our mobility assessment, which is based on Kdvalues, can be used to predict the leaching of pesticides in the volcanic ash soils of Jeju Island. The use of pesticides should be strictly controlled to reduce the possibility of groundwater contamination.

The steroid 20-hydroxyecdysone (20E) is a major component of phytoecdysteroid in plants and may play a defensive role against insect pests in higher plants. In spinach, the biosynthesis and accumulation of 20E have been investigated during the vegetative stage; however, these processes have not been clearly studied during the reproductive stage, particularly in male and female individuals. In this study, we analyzed the level and distribution of 20E in individual male and female spinach plants during the reproductive stage via high performance liquid chromatography (HPLC). We found that 20E biosynthesis and accumulation were markedly different between male and female spinach during the late flowering stage. Compared with the male plant, biosynthesis of 20E in the leaves was more active and its accumulation in the floral parts was higher in female plants during the late flowering stage. These results indicate that the female reproductive organs at least in PE-positive plants could be effectively protected against harmful insects via active biosynthesis and accumulation of PE during the late flowering stage to protect floral parts from harmful insects for seed formation and store the available 20E in seeds for the next generation.

A highly coordinated complex known as the microprocessor precisely processes primary transcripts of MIRNA genes into mature miRNAs. In plants, the microprocessor minimally consists of three components: Dicer-like protein 1 (DCL1), HYPONASTIC LEAF 1 (HYL1), and SERRATE (SE). To precisely modulate miRNA maturation, the microprocessor cooperates with at least 12 proteins in plants. In addition, we here show the involvement of a novel gene, HYL1-interacting GIY-YIG-like endonuclease (HIGLE). The encoded protein has a GIY-YIG domain that is generally found within a class of homing endonucleases. HIGLE directly interacts with the microprocessor components HYL1 and SE. Unlike the functions of other GIY-YIG endonucleases, the catalytic core of HIGLE has both DNase and RNase activities that sufficiently processes miRNA precursors into short fragments in vitro.

Algal blooms are one of the greatest aquatic environmental concerns, and the control of algal blooms has become a great challenge in recent years. In this study, we evaluated the effects of Bidens pilosa plant extracts in comparison to those of several widespread plants, including rice (Oryza sativa), Pistia stratiotes, Eichhornia crassipes, and Pteris vittata, on the growth of the bloom-forming blue-green alga Microcystis aeruginosa. Both ethanolic and methanolic extracts of B. pilosa, in contrast to the other plant extracts, exhibited high inhibitory effects on M. aeruginosa growth at a concentration of 500 mg/L (dry weight equivalent, DWE). The inhibition efficiency in terms of the cell density and chlorophyll a concentration significantly reached 84–88% (p

Plant pyruvate decarboxylases (PDC) catalyze the decarboxylation of pyruvate to form acetaldehyde and CO2 and are well known to play a key role in energy supply via fermentative metabolism in oxygen-limiting conditions. In addition to their role in fermentation, plant PDCs have also been hypothesized to be involved in aroma formation although, to date, there is no direct biochemical evidence for this function. We investigated the role of PDCs in fruit volatile biosynthesis, and identified a melon pyruvate decarboxylase, PDC1, that is highly expressed in ripe fruits. In vitro biochemical characterization of the recombinant PDC1 enzyme showed that it could not only decarboxylate pyruvate, but that it also had significant activity toward other straight- and branched-chain α-ketoacids, greatly expanding the range of substrates previously known to be accepted by the plant enzyme. RNAi-mediated transient and stable silencing of PDC1 expression in melon showed that this gene is involved in acetaldehyde, propanal and pentanal production, while it does not contribute to branched-chain amino acid (BCAA)-derived aldehyde biosynthesis in melon fruit. Importantly, our results not only demonstrate additional functions for the PDC enzyme, but also challenge the long standing hypothesis that PDC is involved in BCAA-derived aldehyde formation in fruit.

This study was carried out to investigate residual characteristics of insecticide acetamiprid inasparagus under greenhouse condition from July to August and consequentially to obtain basic data forguideline on safe use of this pesticide in asparagus. Residues of acetamiprid in young stem of asparagusbefore and after removing foliage were analyzed from samples harvested at 0, 1, 3, 5 and 7 days after singleapplication before harvest. As a result, residues of acetamiprid in young stem of asparagus before and afterremoving foliage at 0 day were 0.27 mg/kg and 0.14 mg/kg, respectively, which were higher than tentativelimit (0.1 mg/kg). However, 3 days later residues of acetamiprid were lower than the tentative limit, representing0.08 mg/kg and 0.03 mg/kg in the asparagus before and after removing foliage, respectively. Acetamiprid wasundetectable in both samples at 5 days since the concentrations were less than detection limit (0.02 mg/kg) inthis study. In summary, the half-life of acetamiprid in asparagus regardless of removing foliage was quiteshort under greenhouse condition from July to August, in the range of 1-3 days, and single application ofacetamiprid water dispersible granule in/on asparagus at 7 days before harvest would have no problem onsafety issues about pesticide residue. This result might be basic information to construct guideline for safeuse of acetamiprid in asparagus.Key words Acetamiprid, Asparagus, Pesticide residues

제주에서 생산된 페퍼민트, 스피아민트, 오데코롱민트 3종으로 부터 추출한 에센셜오일의 성분을 기체 크로마토그래피-질량분석기(GC-MS)와 headspace-GC-MS (HS-GC-MS)를 이용하여 분석하였다. GC-MS 분석결과 페퍼민트, 스피아민트, 오데코롱민트 각각에서 13종의 성분이 동정되었다. 전체 이온 크로마토그램의 피크 inte...

In the present study, we investigated the effects of methanol extracts from Alpinia katsumadai Hayata against antiviral potential underlying mechanism in glucosidase inhibition. Syncytium formation in Newcastle disease virus (NDV)-infected baby hamster kidney (BHK) cell originates from the trafficking of viral glycoprotein into cell-surface. Methanol extracts inhibited not only syncytium formation, but also trafficking of glycoprotein, hemagglutinin-neuraminidase (HN), onto cell-surface. A. katsumadai extracts showed the inhibitory activities (IC50 25 μg/mL) against α-glucosidase. These results suggested that blue chanterelle extracts inhibited the cell-surface expression of NDV-HN glycoprotein without significantly affecting HN glycoprotein synthesis in NDV-infected BHK cells.

Trafficking of viral glycoproteins to the cell surface results in syncytium formation in baby hamster kidney (BHK) cells infected with Newcastle disease virus (NDV). An extract from the medicinal Areca catechu L plant inhibited not only syncytium formation, but also trafficking of the hemagglutinin-neuramidase (HN) glycoprotein to the cell-surface. The viral glycoprotein was processed within the endoplasmic reticulum during transit to the cell membrane. Fungal extracts showed inhibitory activities (IC 50 10 µg/mL) against α-glucosidase. These results suggested that A. catechu L. extracts inhibited the cell-surface expression of NDV-HN glycoprotein without significantly affecting HN glycoprotein synthesis in NDV-infected BHK cells.

We developed an efficient Agrobacterium-mediated transformation protocol for spinach (Spinacia oleracea L.) that uses root-derived callus. Evaluation of this protocol was based on the systematic evaluation of factors that influence transformation efficiency. Four of the five factors that were tested significantly affected the transformation efficiency, including spinach cultivar, Agrobacterium tumefaciens strain and density, and the duration of co-cultivation. Transgenic spinach plants were generated based on optimized conditions, consisting of callus explants of the cultivar Gyeowoonae, A. tumefaciens strain EHA105 with OD600 of 0.2, a co-cultivation period of 4 d, and 100 μM acetosyringone supplemented in the inoculation and co-cultivation media. After co-cultivation with A. tumefaciens, explants were cultured in low-selective and then non-selective conditions to enhance the growth of putative transgenic explants. Visualization of the fluorescent marker, enhanced green fluorescent protein (EGFP), was used to select transgenic explants at several stages, including callus, somatic cotyledonary embryo, and plantlet. The best results for fluorescence visualization screening were obtained at the somatic cotyledonary embryo stage. On average, 24.96 ± 6.05% of the initial calli regenerated shoots that exhibited EGFP fluorescence. The putative transgenic plants were subjected to β-glucuronidase (GUS)-staining assay, phosphinothricin acetyltransferase (PAT) strip test, and molecular analyses to assess the transgene incorporation into plant genome and its expression. All EGFP-positive plants tested were confirmed to be transgenic by GUS-staining assay, PAT strip test, and molecular analyses. The transformation system described in this study could be a practical and powerful technique for functional genetic analysis and genetic modification of spinach.

Blue chanterelle (Polyozellus multiplex), known as an edible mushroom, was extracted using methanol to screen on anti-viral agent. Syncytium formation in Newcastle disease virus (NDV)-infected baby hamster kidney (BHK) cell originates from the trafficking of viral glycoprotein into cell-surface. Blue chanterelle inhibited not only syncytium formation, but also trafficking of glycoprotein, hemagglutinin-neuramidase (HN), onto cell-surface. Viral glycoprotein is processed within the endoplasmic reticulum during routing to surface. Blue chanterelle extracts showed the inhibitory activities (IC50 10 μg/mL) against α-glucosidase. These results suggested that blue chanterelle extracts inhibited the cell-surface expression of NDV-HN glycoprotein without significantly affecting HN glycoprotein synthesis in NDV-infected BHK cells.

To better understand 20-hydroxyecdysone (20E) fluctuation and accumulation in perennial plant, 20E concentration in individual organs of Achyranthes japonica during reproductive growth stage were analyzed by high performance liquid chromatography (HPLC). Concentrations of 20E in root and floral part were much higher than those in stem and leaf during reproductive growth stage and rapidly increased from flowering stage in August to seed-setting stage in October, and thereafter decreased at the stage of seed maturing in November. In contrast, the 20E concentrations in stem and leaf gradually decreased during reproductive growth. In the analysis of detailed fluctuation of 20E in floral part, the 20E concentration was highest in the seed at the early stage of seed development, compared to flower, peduncle, seed coat, and/or seed in other growth stages, and decreased during seed maturation. The accumulation of 20E in reproductive organs, especially seed and root, suggests that 20E has a defensive role for protection of especially newly developing organs against phytophagous insects.

STUDY DESIGN Prospective experimental study. PURPOSE To evaluate bacterial contamination during surgery. OVERVIEW OF LITERATURE The participants of surgery and ventilation system have been known as the most significant sources of contamination. METHODS Two pairs of air culture blood agar plate for G(+) bacteria and MacConkey agar plate for G(-) bacteria were placed at 3 different locations in a conventional operation room: in the surgical field, under the airflow of local air conditioner, and pathway to door while performing spine surgeries. One pair of culture plates was retrieved after one hour and the other pair was retrieved after 3 hours. The cultured bacteria were identified and number of colonies was counted. RESULTS There was no G(-) bacteria identified. G(+) bacteria grew on all 90 air culture blood agar plates. The colony count of one hour group was 14.5±5.4 in the surgical field, 11.3±6.6 under the local air conditioner, and 13.1±8.7 at the pathway to the door. There was no difference among the 3 locations. The colony count of 3 hours group was 46.4±19.5, 30.3±12.9, and 39.7±15.2, respectively. It was more at the surgical field than under the air conditioner (p=0.03). The number of colonies of one hour group was 13.0±7.0 and 3 hours group was 38.8±17.1. There was positive correlation between the time and the number of colonies (r=0.76, p=0.000). CONCLUSIONS Conventional operation room was contaminated by G(+) bacteria. The degree of contamination was most high at the surgical field. The number of bacteria increased right proportionally to the time.

The scientific understanding of the Earth's climate system, including the central question of how the climate system is likely to respond to human-induced perturbations, is comprehensively captured in GCMs and Earth System Models(ESM). Diagnosing the simulated climate response, and comparing responses across different models, is crucially dependent on transparent assumptions of how the GCM/ESM has been driven – especially because the implementation can involve subjective decisions and may differ between modelling groups performing the same experiment. This paper outlines the climate forcings and setup of the Met Office Hadley Centre ESM, HadGEM2-ES for the CMIP5 set of centennial experiments. We document the prescribed greenhouse gas concentrations, aerosol precursors, stratospheric and tropospheric ozone assumptions, as well as implementation of land-use change and natural forcings for the HadGEM2-ES historical and future experiments following the Representative Concentration Pathways. In addition, we provide details of how HadGEM2-ES ensemble members were initialised from the control run and how the palaeoclimate and AMIP experiments, as well as the "emission-driven" RCP experiments were performed.

The objective of this study was to develop an efficient system for the regeneration of spinach plants (Spinacia oleracea L.) by investigating the factors influencing callus and shoot induction. All plant growth regulator (PGR) combinations tested induced callus with high frequency (73–100 %), and the combination of 5 μM α-naphthaleneacetic acid (NAA), 10 μM 6-benzyladenine (BA) and 0.1 μM gibberellic acid (GA3) had the most significant effect on callus growth in term of weight (120.98 ± 22.56 mg). A high auxin-containing medium induced competent callus for shoot formation, while high cytokinin-containing media enhanced callus growth and made callus incompetent for shoot regeneration. Longer periods of callus induction in a high auxin-containing medium were required to form competent callus and led to a high regeneration capacity. The PGR combination shift from a high auxin to cytokinin ratio (ACR) to a low ACR resulted in highly efficient regeneration. Among the regeneration systems tested, the combination of 10 μM NAA and 0.3 μM GA3 for callus induction for 6 weeks followed by 2 μM NAA and 5 μM BA resulted in the highest plant regeneration frequency (83.33 ± 6.43 %) and the highest number of plantlets per explant (7.93 ± 1.24). Somatic embryos at cotyledonary stage and plantlets were transferred to PGR-free medium to establish whole plants. Regenerated female plants grew well to maturity in the greenhouse (77.17 ± 9.80 %) and produced seeds (175.21 ± 28.01 firm seeds per plant).

One of the major tasks in transition to a hydrogen economy is to build a cost-effective infrastructure of hydrogen supply. Building an infrastructure of hydrogen supply includes a series of logistical steps from production through delivery and storage to utilization of the hydrogen. This paper attempts to build the cost-effective central hydrogen supply system using a transportation model. As a case study, a hydrogen delivery plan for 2040 was established by dividing the Korean peninsula into two regions, northern and southern regions. Average hydrogen delivery distances for 2040 in the northern and the southern regions are estimated as 69.8 km and 85.2 km. The subsequently derived hydrogen supply network of centrally produced hydrogen in 2040 is expected to be similar to the liquefied natural gas pipeline network in 2005. The derived hydrogen supply network, delivery distances, and delivery costs are expected to provide a set of basic and indispensable information in building a cost-effective infrastructure of hydrogen supply in the Korean peninsula.

With the purpose of improving ginsenoside content in adventitious root cultures of Korean wild ginseng (Panax ginseng Meyer), the roots were treated with different dosages of γ-ray (5, 10, 25, 50, 75, 100, and 200 Gy). The growth of adventitious roots was inhibited at over 100 Gy. The irradiated adventitious roots showed significant variation in the morphological parameters and crude saponin content at 50 to100 Gy. Therefore, four mutant cell lines out of the propagation of 35 cell lines treated with 50 Gy and 100 Gy were selected on the basis of phenotypic morphology and crude saponin contents relative to the wild type control. The contents of 7 major ginsenosides (Rg 1 , Re, Rb 1 , Rb 2 , Rc, Rf, and Rd) were determined for cell lines 1 and 3 from 100 Gy and lines 2 and 4 from 50 Gy treatments. Cell line 2 showed more secondary roots, longer length and superior growth rate than the root controls in flasks and bioreactors. Cell line 1 showed larger average diameter and the growth rate in the bioreactor was comparable with that of the control but greater in the flask cultured roots. Cell lines 1 and 2, especially the former, showed much more ginsenoside contents than the control in flasks and bioreactors. Therefore, we chose cell line 1 for further study of ginsenoside contents. The crude saponin content of line 1 in flask and bioreactor cultures increased by 1.4 and 1.8-fold, respectively, compared to the control. Total contents of 7 ginsenoside types (Rg 1 , Re, Rb 1 , Rb 2 , Rc, Rf, and Rd) increased by 1.8 and 2.3-fold, respectively compared to the control. Crude saponin and ginsenoside contents in the bioreactor culture increased by about 1.4-fold compared to that the flask culture.

Label-free LC-MS/MS-based shot-gun proteomics was used to quantify the differential protein synthesis and metabolite profiling in order to assess metabolic changes during the development of citrus fruits. Our results suggested the occurrence of a metabolic change during citrus fruit maturation, where the organic acid and amino acid accumulation seen during the early stages of development shifted into sugar synthesis during the later stage of citrus fruit development. The expression of invertases remained unchanged, while an invertase inhibitor was up-regulated towards maturation. The increased expression of sucrose-phosphate synthase and sucrose-6-phosphate phosphatase and the rapid sugar accumulation suggest that sucrose is also being synthesized in citrus juice sac cells during the later stage of fruit development.

This study was carried out to investigate a role of the juvenile hormone analog, fenoxycarb (FC) in terminating the reproductive diapause in the black rice bug, Scotinophara lurida (Burmeister) (Heteroptera: Pentatomidae). FC-treated adults collected at different dates developed their ovaries sooner than untreated ones. Topical application of FC accelerated diapause termination of field-collected S. lurida in which FC-treated adults exhibited early oviposition. In FC-treated males, development of their accessory glands and testes accelerated appeared to be prompted. These results suggest that various physiological overwintering traits related to diapause in this species may in part be under gonadotrophic endocrine control.

The mechanisms and strategies for winter survival of the black rice bug Scotinophara lurida are investigated along with the relationship between cold hardiness and diapause. The ability of S. lurida to survive subzero temperatures varies depending on developmental stage, temperature and exposure duration. Mean supercooling point (SCP) varies from –7.6 to –10.7 °C with developmental stage, but is not significantly different between stages examined. The SCP also varies with season, being lowest in January and increasing rapidly in February and remaining almost at the same level (–7.3 to 9.6 °C) until April The osmolality of haemolymph of field-collected S. lurida adults rises dramatically from 53.9 mOsm kg−1 in August to 75.3 mOsm kg−1 in December, and then declines linearly to 57.0 mOsm kg−1 in May. Field-collected S. lurida adults show a peak glucose content in October, glycerol content in November and trehalose content in December. Only trehalose content decreases after the application of the juvenile hormone analogue, fenoxycarb, suggesting that trehalose is a cryoprotectant during diapause. These various physiological and biochemical traits related to cold tolerance in S. lurida may be, at least in part, under the control of juvenile hormone through the reproductive diapause programme.

We established an in vitro plant regeneration system via somatic embryogenesis of Aster scaber, an important source of various biologically active phytochemicals. We examined the callus induction and embryogenic capacities of three explants, including leaves, petioles, and roots, on 25 different media containing different combinations of α-naphthalene acetic acid (NAA) and 6-benzyladenine (BA). The optimum concentrations of NAA and BA for the production of embryogenic calli were 5.0 μM and 0.05 μM, respectively. Media containing higher concentrations of auxin and cytokinin (such as 25 μM NAA and 25 μM BA) were suitable for shoot regeneration, especially for leaf-derived calli, which are the most readily available calli and are highly competent. For root induction from regenerated shoots, supplemental auxin and/or cytokinin did not improve rooting, but instead caused unwanted callus induction or retarded growth of regenerated plants. Therefore, plant growth regulator-free medium was preferable for root induction. Normal plants were successfully obtained from calli under the optimized conditions described above. This is the first report of the complete process of in vitro plant regeneration of A. scaber via somatic embryogenesis.

Acetylcholinesterase (AChE) has been known to be the target of organophosphorous and carbamate insecticides. Only a single AChE, however, existed in insects and was involved in insecticide resistance, recently another AChE is reported in mosquitoes and aphids. We have cloned cDNAs encoding two ace genes, designated as Ha-ace1 and Ha-ace2 by a combined degenerate PCR and RACE strategy from adult heads of the oriental tobacco budworm, Helicoverpa assulta. The Ha-ace1 and Ha-ace2 genes encode 664 and 647 amino acids, respectively and have conserved motifs including a catalytic triad, a choline-binding site and an acyl pocket. Both Ha-AChEs were determined to be secretory proteins based on the existence of a signal peptide. The Ha-ace1 gene, the first reported ace1 in lepidopterans, belongs to the ace1 subfamily whereas the Ha-ace2 gene showed high similarity to those in the ace2 subfamily. Phylogenetic analysis showed that the Ha-ace1 gene was completely diverged from the Ha-ace2, suggesting that the Ha-ace genes are duplicated. Quantitative real time-PCR revealed that expression level of the Ha-ace1 gene was much higher than that of the Ha-ace2 in all body parts examined. The biochemical properties of purified proteins by affinity chromatography showed substrate specificity for acetylthiocholine iodide, and inhibitor specificity for BW284C51 and eserine and their peptide sequences partially identified by a MALDI-TOF mass spectrometer demonstrated that two Ha-AChEs were expressed in vivo.

Pheromone biosynthesis activating neuropeptide (PBAN) produced in the subesophageal ganglion stimulates pheromone production in the pheromone gland. A cDNA isolated from female adult heads of the diamondback moth (Plutella xylostella (L.)) encodes 193 amino acids including PBAN, designated as Plx-PBAN, and four other neuropeptides (NPs): diapause hormone (DH) homologue, alpha-NP, beta-NP and gamma-NP. All of the peptides are amidated in their C-termini and shared a conserved motif, FXPR(or K)L structure, as reported from other PBAN cDNAs. Plx-PBAN consists of 30 amino acids, the shortest PBAN so far reported. Plx-PBAN exhibited below 50% homology, compared with other known PBANs. The Plx-DH homologue is structurally different from DH of Bombyx mori. The length of Plx-beta-NP (16 amino acids) was the shortest and showed relatively low similarity, whereas gamma-NP (10 amino acids in length) was the longest among examined gamma-NPs. When female adults were injected with synthetic Plx-PBAN, pheromone production showed a maximal increase 1h post-injection. RT-PCR screening revealed that Plx-PBAN cDNA was expressed in all examined body parts, with the highest expression level in the head of female adults. Analysis of RT-PCR products indicated the Plx-PBAN sequence was identical in all examined body parts of both sexes. Phylogenetic analysis revealed that the Plx-PBAN gene is distantly related to other PBANs, demonstrated by the relatively low similarity.

This study was carried out to investigate an effect of a juvenile hormone (JH) analog, fenoxycarb (FC), on egg development of the black rice bug, Scotinophara lurida (Burmeister). FC-treated S. lurida females could oviposit significantly earlier and more eggs than the untreated control. This gonadotropic effect of the JH analog followed a dose-dependent manner with a maximum over 200 μg doses per adult. Though developmental period of live eggs laid from the FC-treated females was not significantly different from the control, the FC treatment gave significantly adverse effects on the following embryonic development of S. lurida.

Acetylcholinesterase (AChE) was purified, by affinity chromatography, from adult heads of the housefly (Musca domestica) (GSNU strain) known to have mutated AChE gene. The purified AChE showed a single peak in capillary electrophoresis with an overall yield of 42% representing 957-fold purification and a specific activity of about 290 μ mol/min/mg. It hydrolyzed acetylthiocholine iodide better than S-butyrylthiocholine iodide or propionylthiocholine iodide. AChE-specific inhibitors, BW284C51 and eserine, significantly inhibited the purified AChE, but a butyrylcholinesterase-specific inhibitor, ethopropazine, did not. The housefly AChE was separated into membrane-bound and soluble forms. In vitro inhibition assays with seven organophophates, except for EPN, showed no significant difference between the crude and the purified AChE. Molecular forms of the housefly AChE did not affect the enzyme sensitivity to chlorpyrifos. Inhibition rate (%) of the AChE from the housefly was shown lower than that of the susceptible housefly strain (SRS) to trichlorfon and chlorpyrifos possibly because of the mutated amino acids of the AChE gene.

Acetylcholinesterase (AChE) was purified from adult heads of a Korean housefly, Musca domestica, strain (KNIH) by affinity chromatography on trimethyl (ffj-aminophenyl) ammonium chloride resin. The purified AChE showed the purification factor over 400-fold and the specific activity of about 290 umol/min/mg. The housefly has both the membrane-bound and the soluble forms of AChE. Only the membrane-bound form of the housefly AChE was isolated by Triton X-114 phase partition and revealed on the native gel. The maximum activities of the purified AChE were shown at pH 7.5-8.5 and 40-45°C. Partial cDNA of AChE (795bp) of the KNIH strain showed that its deduced amino acid sequence shared high similarity with that of insecticide-resistant type of insect AChE.

Aphis spiraecola (Patch) virginiparae were reared for 3 generations on the dropwort, Oenathe javanica DC, under low temperature (15°C) and short days (10L:14D) for artificial induction of sexual forms (Hong and Boo, 1998). These oviparae, artificially induced in the third generation in the laboratory, showed a typical behavior of releasing sex pheromone by waving their hind tibiae and abdomen in the air during photophase. The air around these oviparae, individually or in groups, was collected and analyzed by gas chromatography for pheromone components. (+)-(4aS,7S,7aR)-nepetalactone and (-)-(1R,4aS,7S,7aR)- nepetalactol were detected in the ratio of 6:1 ∼ 8:1. For wild oviparae collected from an apple orchard and a spiraea arboretum during Oct. 1995. ∼ Nov. 1996, the ratio between the two components was around 2:1. Oviparae showed a circadian rhythm in release of sex pheromone, with beginning to release nepetalactone at the start of lights-on and stopping with lights-off. This rhythm was also maintained in an all dark condition. The sex pheromone was always detected during the photophase, even when the photoperiod was altered to a longer regime, for example from 10L:14D to 16L:8D.

Sex pheromone composition was analyzed for the oriental fruit moth ( Grapholita molesta ), and field trapping studies were also conducted to develop monitoring system with its sex pheromone in Korea. The calling and mating behavior of G. molesta were seen maximally 2 to 1 hr before lights-off and these behavior ceased after lights-off. Analysis of abdominal tip extracts from virgin females of G. molesta revealed 4 compounds, Z8-dodecenyl acetate ( Z 8-12: Ac), E 8-dodecenyl acetate ( E 8–12: Ac), Z 8-dodecenol ( Z 8–12: OH) and dodecanol (12: OH). In the field trappings, the best capturing activity for G. molesta was observed with the 95:5:1 blend of Z 8–12: Ac, E 8–12: Ac and Z 8–12: OH but 12: OH showed no synergic effect. The most effective amount of sex pheromone blend was 0.5 mg for a lure.

Electroantennograms (EAGs) were recorded from two predatory insect species, the twelve-spotted lady beetle, Coleomegilla maculata and the green lacewing, Chrysoperla carnea in response to semiochemicals emitted from one of their prey species, the pea aphid Acyrthosiphon pisum and their host plant. EAG responses were also recorded from C. maculata in response to extracts from individuals of the opposite sex and to extracts from an herbaceous plant, catnip Nepeta cataria. Extracts of catnip and two sex pheromone components of aphids, (4aS,7S,7aR)-nepetalactone and (1R,4aS,7S,7aR)-nepetalactol, elicited significant EAG responses from the antennae of both predatory species. Of 10 corn volatile compounds tested, C. carnea adults responded most strongly to 2-phenylethanol and (E)-β-farnesene. A significant difference in EAG response to extracts of corn leaf collections was observed between male and female C. carnea. In C. maculata, significant EAG responses were elicited by most of the tested corn volatile compounds, except α-pinene and (E)-2-hexenal. The highest EAG responses were observed in response to (E)-β-farnesene, α-terpineol, 2-phenylethanol, and β-caryophyllene. Sexual differences in EAG responses of C. maculata were only found in response to 1-octen-3-ol. Male antennae of C. maculata produced significant EAG responses to extracts from conspecific females, but not to males, which indicates that some chemicals from females could be involved in sexual communication. A significant EAG response also was recorded in response to the extracts of fluids produced during “reflex bleeding.” Male and female antennae of both species exhibited similar dose–response curves to most of the selected compounds, although female C. maculata antennae exhibited higher thresholds in response to several compounds including α-terpineol, (Z)-3-hexenol, and (4aS,7S,7aR)-nepetalactone. Field tests showed that 2-phenylethanol was highly attractive to both sexes of the two investigated species. Only C. maculata was attracted to traps baited with α-terpineol.

The present study is concerned with cloning and characterizing Has-PBAN cDNA which is 756 nucleotides long, isolated from the brain and suboesophageal ganglion complex (Br–Sg) of Helicoverpa assulta adults. The 194-amino acid sequence deduced from this cDNA possessed the proteolytic endocleavage sites to generate multiple peptides. From the processing of the prepro-hormone, it can be predicted that the cDNA has a PBAN domain with 33 amino acids and four additional peptide domains: 24 amino acid-, 7 amino acid-, 18 amino acid- and 8 amino acid-long sequences, with FXPR(or K)L (X=G, T or S) amidated at their C-termini. The amino acid sequence of all five predicted peptides, including the PBAN, are identical to that of Helicoverpa zea (Raina, A.K., Jaffe, H., Kempe, T.G., Keim, P., Blacher, R.W., Fales, H.M., Riley, C.T., Klun, J.A., Ridgway, R.L., Hayes, D.K., 1989. Identification of a neuropeptide hormone that regulates sex pheromone production in female moths. Science 244, 796–798 and Ma, P.W.K., Knipple, D.C., Roelofs, W.L., 1994. Structural organization of the Helicoverpa zea gene encoding the precursor protein for pheromone biosynthesis-activating neuropeptide and other neuropeptides. Proc. Natl. Acad. Sci., U.S.A. 91, 506–510). A single mRNA species corresponding to the size of Has-PBAN cDNA was detected from the Br–Sg of 1–3-day old female and male adults, and their expression was also at a similar level. Pheromone production was induced upon injection of female or male Br–Sg extracts or synthetic PBAN into the haemocoel of decapitated 1–3-day old female adults during the photophase when they are not supposed to produce pheromone. From these results, H. assulta adult females seem to use their own PBAN for regulating sex pheromone biosynthesis. Functions of the four other peptides ending with FXPR(or K)L in the Has-PBAN cDNA and of the male PBAN remain to be elucidated.

A proteinous antimicrobial substance was purified from the bacteria-challenged larvae of the fall webworm, Hyphantria cunea. It is a cecropin-like antibacterial peptide which exhibits antibacterial activity against Gram-negative and Gram-positive bacteria, and known as Hyphantria cecropin A. The cDNA clones corresponding to this peptide were isolated from a cDNA library constructed from the bacteria-challenged larvae and obtained complete nucleotide sequences. In addition to the Hyphantria cecropin A sequence, we obtained three other cDNAs exhibiting high sequence similarity with Hyphantria cecropin A. We synthesized the C-terminally amidated peptide of 35 residues based on the deduced sequence of the isolated cDNA of Hyphantria cecropin A. The synthetic peptide exhibited strong antibacterial activity against several microbes including medically important bacteria such as Salmonella, Shigella, and fungus such as Candida. A Southern blot experiment using these cloned cDNAs as probes predicted the existence of multiple forms of Hyphantria cecropin genes.

The solvent fractions of rosemary methanol extract were obtained by successive extraction with n-hexane, chloroform, ethyl acetate, butanol, and water. The ethyl acetate fraction (EAF) contained a remarkable amount of polyphenol and flavonoid as well as high levels of alkyl and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging activity. The activity guided fractionation and repeated chromatographic separations over silica gel, RP C18, and Sephadex LH-20 led to isolation of six compounds from the EAF. 1H NMR, 13C NMR, 2D NMR, MS, and IR spectroscopies determined the compounds to be caffeic acid (1), rosmarinic acid (2), rosmarinic acid methyl ester (3), luteolin (4), apigenin (5), and hispidulin (6), and high-performance liquid chromatography quantification was used to determine concentrations in EAF. Among the six isolated compounds, rosmarinic acid methyl ester showed the highest scavenging activities against di(phenyl)-(2,4,6-trinitrophenyl) iminoazanium, alkyl and ABTS radicals. The EAF mixture, but not individual isolated compounds, shielded dermal fibroblast cells from H2O2-induced cytotoxicity at concentrations that encompass the SC50 of alkyl and ABTS radical. Therefore, our findings suggested for the first time that antioxidant capacity of the EAF mixture result in a synergistic effect on the antioxidant action.

Effects of methanol extract of Terminalia chebula on antibacterial activity and the generation of superoxide radical in Bacillus subtilis, as well as on syncytium formation and cytopathic activity in virus-infected baby hamster kidney cells were examined. The extract effectively inhibited syncytium formation in a concentration-dependent manner, and infectious virus production was markedly reduced. However, glycoprotein synthesis was not affected. These results collectively indicate that methanol extract of T. chebula potentially inhibit glycosylation by acting as a suppressor of intracellular glycosylation trafficking.

Strain DS4, a soil microorganism producing an oxygen radical-generating antibacterial substance, bruceanol B, was isolated, and cultural conditions for the antibiotic production were optimized. Bruceanol B was separated by ethyl acetate extraction, silica gel and Sephadex LH-20 gel column chromatographies, and high-performance liquid chromatography (HPLC). The highest antibacterial activity was obtained from medium containing 1.5% soluble starch and 2.0% yeast extract, cultured at 30°C for 3.5 days.

Methanol extracts of Paeonia lactiflora roots (Radix Paeaniae Lactiflora) had antibacterial activity against Bacillus subtilis. These extracts generated superoxide radicals in the B. subtilis lysate. However, the radicals were strongly detoxified by the addition of superoxide dismutase, an indication that superoxide radicals are important in the antibacterial actions of the extracts. The root extracts also showed a pivotal inhibition role in trafficking of viral glycoprotein in virusinfected baby hamster kidney (BHK) cells. The methanol extract effectively inhibited syncytium formation in a concentration-dependent, and it did not affect glycoprotein synthesis. These results suggest that oxygen radical affect the transport process of viral glycoprotein without its synthesis.

There is increasing interest in phytoecdysteroids (PEs) because of their potential role in plant defense against insects. To understand the mechanism regulating their levels in plants, the fluctuation, distribution, and biosynthesis of PE 20-hydroxyecdysone (20E) examined in Achyranthes japonica. The total amount of 20E per individual plant initially remained at a constant level, and increased markedly after the first leaf pair (LP) stage, while the concentration of 20E in a given plant decreased rapidly during vegetative growth. In addition, the incorporation of [2-(14)C]-mevalonic acid into 20E did not differ significantly depending on plant organs and developmental stages, suggesting that biosynthesis of 20E is not restricted to particular organs or growth stages.

Insensitive acetylcholinesterase (AChE) is involved in the resistance of organophosphorous and carbamate insecticides. We cloned a novel full-length AChE cDNA encoding ace1 gene from adult heads of the diamondback moth (DBM, Plutella xylostella). The ace1 gene encoding 679 amino acids has conserved motifs including catalytic triad, choline-binding site and acyl pocket. Northern blot analysis revealed that the ace1 gene was expressed much higher than the ace2 in all examined body parts. The biochemical properties of expressed AChEs showed substrate specificity for acetylthiocholine iodide and inhibitor specificity for BW284C51 and eserine. Three mutations of AChE1 (D229G, A298S, and G324A) were identified in the prothiofos-resistant strain, two of which (A298S and G324A) were expected to be involved in the prothiofos-resistance through three-dimensional modeling. In vitro functional expression of AChEs in Sf9 cells revealed that only resistant AChE1 is less inhibited with paraoxon, suggesting that resistant AChE1 is responsible for prothiofos-resistance.

Production of sex pheromones, Z9-14:OAc and Z11-14:OAc, of the smaller tea tortrix, Adoxophyes sp. was stimulated by injection of the female or male head extracts as well as synthetic pheromone biosynthesis activating neuropeptide (PBAN) into decapitated females. The amount of pheromone produced reached a maximum level 3 h after injection of synthetic PBAN into females. A cDNA isolated from brain-suboesophageal ganglion complex (Br-SEG) of A. sp. females contained an ORF of 576 nucleotides encoding 192 amino acids. Based on endoproteolytic sites, it can be predicted to be cleaved into five putative peptide domains including PBAN and four other neuropeptides. Ado-PBAN consisting of 31-amino acids is the shortest PBAN so far reported. Four other putative PBAN-encoding gene neuropeptides (PGN) are predicted with PGN-24, PGN-7, PGN-20, and PGN-8 amino acids. All of the peptides are amidated in their C-termini with a FXPR(or I, K)L structure, except for PGN-8 (TVKLTPRLamide). PBAN-like immunoreactive material was observed in Br, SEG and ventral nerve cord (VNC) of the female adult. In the brain, 5-7 pairs of neurons containing PBAN-like immunoreactivity were found in each protocerebral hemisphere. Three groups of cell clusters found in the SEG corresponded to the mandibular, maxillary and labial neurons as in other moths. PBAN-like immunoreactive neurons in the VNC were found in thoracic (three pairs) and abdominal ganglia (two pairs). As compared to other moths, a relatively low similarity of peptide sequences deduced from Ado-PBAN gene and a different expression pattern of PBAN-like immunoreactivity could indicate phylogenetical distance from the other species.

The five components, Z9-16:Ald, 16:Ald, Z11-16:Ald, Z9-16:Ac and Z11-16:Ac, of the sex pheromone in Helicoverpa assulta were mostly detected during the scotophase, with their titer peaking at the 4th hour during the scotophase under a 15L/9D regime. They were not detected during the photophase, but were produced during the photophase when decapitated females were injected with extracts of virgin female (FHE), male heads (MHE), homogenates of the brain-suboesophageal ganglion complex (Br-SOG), or synthetic Hez-PBAN. Production of Z9-16:Ald increased during the first 45min after FHE injection and then declined to a very low level after 2h during the photophase. Synthetic Hez-PBAN stimulated the sex pheromone glands for at least 2h and the effect was more or less proportional to the concentration of the peptide. From the present results, we suggest the following: PBAN is released continuously into the haemolymph to stimulate pheromone biosynthesis at least during the first half of the scotophase, PBAN is synthesized and accumulated independent of photoperiod or sex, and the release starts just prior (about 1h) to the beginning of the scotophase.

Sex pheromones of many Lepidopteran species have relatively simple structures consisting of a hydrocarbon chain with a functional group and usually one to several double bonds. The sex pheromones are usually derived from fatty acids through a specific biosynthetic pathway. We investigated the incorporation of deuterium-labeled palmitic and stearic acid precursors into pheromone components of Helicoverpa zea and Helicoverpa assulta. The major pheromone component for H. zea is (Z)11-hexadecenal (Z11-16:Ald) while H. assulta utilizes (Z)9-hexadecenal (Z9-16:Ald). We found that H. zea uses palmitic acid to form Z11-16:Ald via delta 11 desaturation and reduction, but also requires stearic acid to biosynthesize the minor pheromone components Z9-16:Ald and Z7-16:Ald. The Z9-16:Ald is produced by delta 11 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. The Z7-16:Ald is produced by delta 9 desaturation of stearic acid followed by one round of chain-shortening and reduction to the aldehyde. H. assulta uses palmitic acid as a substrate to form Z9-16:Ald, Z11-16:Ald and 16:Ald. The amount of labeling indicated that the delta 9 desaturase is the major desaturase present in the pheromone gland cells of H. assulta; whereas, the delta 11 desaturase is the major desaturase in pheromone glands of H. zea. It also appears that H. assulta lacks chain-shortening enzymes since stearic acid did not label any of the 16-carbon aldehydes.

To study the structural features of genes for luciferase in Luciola lateralis, two different, functional luciferase genes, Luc1 and Luc2, were cloned and their nucleotide sequences were determined. The two genes were nearly identical, but a deletion of 128 bp was found in the 5' upstream region of Luc2, and minor deletions and additions were also found in the 3' downstream region. Seven base substitutions in the coding region produced two amino acid residue differences between the two genes. Southern blot analysis of genomic DNA isolated from 33 individual fireflies revealed that 26 were homozygous for the Luc1 gene and one was homozygous for Luc2. The others were heterozygotes with two, or even three, different alleles for the luciferase gene.