Establishment of import tolerance for the insecticide thiacloprid in strawberry.

To promote exports, import tolerance (IT) of thiacloprid in strawberry was proposed using the Organization for Economic Cooperation and Development (OECD) maximum residue limit (MRL) calculator after conducting three different field trials. The pre-harvest interval of residual pattern and degradation dynamics of thiacloprid in strawberry were determined using ultra-performance liquid chromatography-tandem mass spectrometry. Samples were extracted with acetonitrile and a mixture of salts and dilution was performed for purification. A six-point matrix-matched calibration curve was constructed which provided excellent linearity with coefficient of determination (R2 ) of 0.9998 or more. Detection and quantification limits were 0.003 and 0.01 mg/kg, respectively. The method was validated in quintuplicate at three different concentrations, which resulted in acceptable recovery ranging from 80.86% to 101.71% with relative standard deviation of 6.50 or less among the three field sites. The developed method was applied to the field-treated sample harvested at different intervals. In the pre-harvest interval trial, the amount of thiacloprid residues ranged from 0.24 to 0.70 mg/kg in field site 1 (Nonsan), 0.16 to 0.50 mg/kg in field site 2 (Sunchang), and 0.36 to 0.50 mg/kg in field site 3 (Sacheon). By contrast, in the degradation trial, the observed residues were 0.03-0.81 mg/kg in field site 1 and 0.02-0.48 mg/kg in field site 2. Consequently, the IT of thiacloprid in strawberry using the OECD MRL calculator was proposed as 2 mg/kg, which is exactly the same as the MRL established by the Republic of Korea. In conclusion, the residue study proposes 2.0 mg/kg as the MRL of thiacloprid in strawberries.

Characterization and quantification of γ-oryzanol in grains of 16 Korean rice varieties.

γ-Oryzanol, a mixture of ferulic acid esters of triterpene alcohols and sterols, is a nutritionally important group of rice secondary metabolites. A library of 27 γ-oryzanol was assembled from existing data and used to assist identification and quantification of γ-oryzanol isolated from 16 Korean rice varieties (11 white and 5 pigmented). γ-Oryzanol was analyzed with liquid chromatography with diode array detection and electrospray ionization mass spectrometry. Nineteen different γ-oryzanol were observed and identified as stigmasterol, campesterol and sitosterol or common and hydroxylated triterpene alcohols. In the 16 varieties, the total γ-oryzanol content averaged 43.8 mg/100 g (range, 26.7-61.6 mg/100 g), which Josaengheugchal exhibited the highest level (61.6 mg/100 g). The Korean rice varieties were classified based on qualitative and quantitative γ-oryzanol data by multivariate statistical analysis. Clusters of specialty rice varieties exhibited higher γ-oryzanol levels than those of common rice varieties.

Dietary lufenuron reduces egg hatch and influences protein expression in the fruit fly Bactrocera latifrons (Hendel).

Lufenuron (LFN), a chitin synthase inhibitor, impacts the fertility of Ceratitis capitata, Bactrocera dorsalis, B. cucurbitae, and B. latifrons. We posed the hypothesis that LFN curtails egg hatch in the solanaceous fruit fly, B. latifrons. In this study, newly emerged virgin adults were sexed and fed for 12 days with varying concentrations of LFN-laced agar diets until sexual maturation. Eggs were collected from 12-d-old adults and the egg hatch was assessed. Egg hatch decreased in adults reared on LFN-treated diets. LFN-treated media did not influence fertility after one gender was reared on experimental and the other on control media before mating. Exposure to LFN-treated medium after mating led to reduced egg hatch. We infer that LFN is not a permanent sterilant, and reduced egg hatch depends on continuous exposure to dietary LFN after mating. Proteomic analysis identified two differentially expressed proteins, a pheromone binding protein and a chitin binding protein, between adults maintained on LFN-treated and control diets. Expression of two genes encoding chitin synthase 2, and chitin binding protein, was altered in adults exposed to dietary LFN. LFN treatments also led to increased expression of two odorant binding proteins one in females and one in males. We surmise these data support our hypothesis and provide insight into LFN actions.

The Bdtf1 gene in Alternaria brassicicola is important in detoxifying brassinin and maintaining virulence on Brassica species.

Brassinin is an antifungal compound induced in Brassica plants after microbial infection. Molecular evidence is incomplete, however, in supporting the importance of brassinin in plant resistance to pathogens. To test the importance of brassinin in plant defense, we studied the functions of the gene Bdtf1 in the necrotrophic fungus Alternaria brassicicola. Several strains of mutants of this gene were weakly virulent on Brassica species, causing lesions 70% smaller in diameter than the wild type on three Brassica species. These mutants, however, were as virulent as the wild type on Arabidopsis thaliana. They were similar to the wild type in spore germination, colony morphology, and mycelial growth in nutrient-rich media, both with and without stress-inducing chemicals. Unlike wild-type A. brassicicola, however, the mutants failed to germinate and their hyphal growth was arrested in the presence of 200 µM brassinin. When grown in a medium containing 100 µM brassinin, wild-type mycelium entirely converted the brassinin into a nontoxic derivative, of which the precise chemical nature was not established. Mutants of the Bdtf1 gene were unable to perform this conversion. Our results support the hypothesis that the ability of A. brassicicola to detoxify brassinin is necessary for successful infection of Brassica species.

Phn and Nag-like dioxygenases metabolize polycyclic aromatic hydrocarbons in Burkholderia sp. C3.

Burkholderia sp. C3 can transform polycyclic aromatic hydrocarbons (PAHs), a class of ubiquitous pollutants, through multiple pathways, indicating existence of multiple dioxygenases (Seo et al., in Biodegradation 18:123-131, 2006a). Both phn and nag-like genes in C3 were cloned and identified with the DNA sequence alignment and the gene organization in the clusters. When cloned and expressed in Escherichia coli, either the alpha- and beta-subunits of dioxygenase of the phn genes or the ferredoxin-, alpha- and beta-subunits of the nag-like genes transformed naphthalene, phenanthrene and dibenzothiophene but at different rates. The E. coli transformant containing the phn genes transformed phenanthrene faster than that containing the nag-like genes, which was consistent with higher transcription of the phnAc gene than the nagAc-like gene in C3 in response to phenanthrene. 1-Hydroxy-2-naphthanoic acid (1H2NA) and 2-hydroxy-1-naphthanoic acid (2H1NA) (3,4- and 1,2-dioxygenation metabolites of phenanthrene, respectively) were detected in the culture medium of the phn genes transformed E. coli. The concentration of 1H2NA was 262-fold higher than 2H1NA in the medium of the phn genes transformed E. coli. The results suggested that the phn genes play a major role in 1,2-/3,4-dioxygenation and 3,4-dioxygenation dominates. Twenty-eight PAH degradation-associated enzymes including those encoded by the nag-like and phn genes in phenanthrene-grown C3 cells were identified via alignment of amino acid sequences of the detected polypeptides with those in protein databases. The polypeptides were determined with nano liquid chromatography-ion trap mass spectrometry after tryptic in-gel digestion of the enzymes on 1D SDS-PAGE.

Identification of protein related to dietary vitamin B3 deficiency in Mediterranean fruit fly larvae.

Mediterranean fruit fly (medfly), Ceratitis capitata, is among the most destructive agricultural pest. The sterile insect technique (SIT) can effectively control medfly populations. To rear healthy medflies for the purpose of SIT, it is essential to supplement B vitamins in the diet. However, the function of the dietary B vitamins in C. capitata larvae is not known. With the microscopic analysis, several organs in the head were examined and the spiracle formation and sensory organs were normally formed between the niacin-supplied and niacin-absent groups. However, formation of the ocular depression was differently developed between the two groups, although the hypostomal sclerite was formed properly. These results signify that niacin deficiency maybe interrupt development of medfly larvae ocular depression. Proteomic analyses using LC MS/MS detected a total of 1845 proteins in two flies. A total of 607 of the 1845 proteins were overexpressed and one third (598 proteins) were downexpressed in the niacin-deficient larvae, while about one third were similarly expressed. Overexpressed proteins in the niacin-deficient larvae included ryanodine receptor 44 F, intergrin-PS, spalt-major homeotic protein, and chiffon protein. One of important overexpressed proteins was optomotor-blind protein in relation to wing development in the niacin-deficient medfly larvae.

Insecticidal activity of basil oil, trans-anethole, estragole, and linalool to adult fruit flies of Ceratitis capitata, Bactrocera dorsalis, and Bactrocera cucurbitae.

Basil oil and its three major active constituents (trans-anethole, estragole, and linalool) obtained from basil (Oscimum basilicum L.) were tested on three tephritid fruit fly species [Ceratitis capitata (Wiedemann), Bactrocera dorsalis (Hendel), and Bactrocera cucurbitae (Coquillett)] for insecticidal activity. All test chemicals acted fast and showed a steep dose-response relationship. The lethal times for 90% mortality/knockdown (LT90) of the three fly species to 10% of the test chemicals were between 8 and 38 min. The toxic action of basil oil in C. capitata occurred significantly faster than in B. cucurbitae but slightly faster than in B. dorsalis. Estragole acted faster in B. dorsalis than in C. capitata and B. cucurbitae. Linalool action was faster in B. dorsalis and C. capitata than in B. cucurbitae. trans-Anethole action was similar to all three species. Methyl eugenol acted faster in C. capitata and B. cucurbitae than in B. dorsalis. When linalool was mixed with cuelure (attractant to B. cucurbitae male), its potency to the three fly species decreased as the concentration of cuelure increased. This was due to linalool hydrolysis catalyzed by acetic acid from cuelure degradation, which was confirmed by chemical analysis. When methyl eugenol (B. dorsalis male attractant) was mixed with basil oil, trans-anethole, estragole, or linalool, it did not affect the toxicity of basil oil and linalool to B. dorsalis, but it did significantly decrease the toxicity of trans-anethole and estragole. Structural similarity between methyl eugenol and trans-anethole and estragole suggests that methyl eugenol might act at a site similar to that of trans-anethole and estragole and serve as an antagonist if an action site exists. Methyl eugenol also may play a physiological role on the toxicity reduction.

Application of multibounce attenuated total reflectance fourier transform infrared spectroscopy and chemometrics for determination of aspartame in soft drinks.

Aspartame is a low-calorie sweetener commonly used in soft drinks; however, the maximum usage dose is limited by the U.S. Food and Drug Administration. Fourier transform infrared (FTIR) spectroscopy with attenuated total reflectance sampling accessory and partial least-squares regression (PLS) was used for rapid determination of aspartame in soft drinks. On the basis of spectral characterization, the highest R2 value, and lowest PRESS value, the spectral region between 1600 and 1900 cm(-1) was selected for quantitative estimation of aspartame. The potential of FTIR spectroscopy for aspartame quantification was examined and validated by the conventional HPLC method. Using the FTIR method, aspartame contents in four selected carbonated diet soft drinks were found to average from 0.43 to 0.50 mg/mL with prediction errors ranging from 2.4 to 5.7% when compared with HPLC measurements. The developed method also showed a high degree of accuracy because real samples were used for calibration, thus minimizing potential interference errors. The FTIR method developed can be suitably used for routine quality control analysis of aspartame in the beverage-manufacturing sector.

Cytotoxic Effects of 24-Methylenecyloartanyl Ferulate on A549 Nonsmall Cell Lung Cancer Cells through MYBBP1A Up-Regulation and AKT and Aurora B Kinase Inhibition.

Lung cancer is the second most prevalent cancer. Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer. The low efficacy in current chemotherapies impels us to find new alternatives to prevent or treat NSCLC. Rice bran oil is cytotoxic to A549 cells, a NSCLC cell line. Here, we identified 24-methylenecyloartanyl ferulate (24-mCAF) as the main component responsible for the cytotoxicity in A549 cells. An iTRAQ-based quantitative proteomics analysis revealed that 24-mCAF inhibits cell proliferation and activates cell death and apoptosis. 24-mCAF induces up-regulation of Myb binding protein 1A (MYBBP1A), a tumor suppressor that halts cancer progression. 24-mCAF inhibits the activity of AKT and Aurora B kinase, two Ser/Thr kinases involved in MYBBP1A regulation and that represent important targets in NSCLC. This study provides the first insight of the effect of 24-mCAF, the main component of rice bran oil, on A459 cells at the cellular and molecular levels.

Application of an enzyme-linked immunosorbent assay for the analysis of imidacloprid in wiliwili tree, Erythrina sandwicensis O. Deg, for control of the wasp Quadrastichus erythrinae.

A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for the neonicotinoid insecticide imidacloprid was evaluated for its reproducibility, accuracy, and comparability to results from a conventional high-performance liquid chromatography (HPLC) for the analysis of imidacloprid in the endemic wiliwili tree (Erythrina sandwicensis O. Deg) found in dryland forests and landscapes in Hawaii. Imidacloprid was applied to these wiliwili trees in an attempt to control the newly introduced erythrina gall wasp, Quadrastichus erythrinae Kim. Leaf samples were freeze-dried and extracted with acidic aqueous methanol followed by methylene chloride partitioning. After solvent removal, the extract residue was reconstituted in 1 mL of water/methanol (1:1, v/v) for ELISA; no significant matrix interference was observed at 10-fold or more dilution. The average recoveries of imidacloprid from fortified samples ranged from 78% to 100% by ELISA. The correlation between the ELISA and HPLC results was excellent (r2 = 0.98). Imidacloprid was detected with the ELISA in all treated samples and its level varied in the samples among different treatments and in those from different parts of the trees. The infestation severity rating of leaf samples was inversely related to the concentration of imidacloprid. It is clear that imidacloprid effectively controls the wasps. The ELISA is a suitable method for quantitative and reliable determination of imidacloprid in wiliwili trees and the application provides information to understand how to control the wasps.

Nicotinamide in relation to dietary nicotinic acid and nine other vitamins and larval development of Ceratitis capitata (Diptera: Tephritidae).

This study was undertaken to understand why Ceratitis capitata larvae reared on a diet fortified with nine vitamins except nicotinic acid had 100% mortality, while those reared on a 10-vitamin-free diet had 66% survival (Chang, C. L.; Li, Q. X. Ann. Entomol. Soc. Am. 2004, 97, 536-540). Our results showed that nicotinamide was detected at a level of 0.07 microg/g in second-instar larvae reared on the 10-vitamin-free diet and 0.30 microg/g in the corresponding spent diet, while it was not detected in either the larvae reared on the diet fortified with 707 microg/g of nine vitamins (nicotinic acid absent) or the corresponding spent diet. Nicotinamide was detected at concentrations of 0.13 and 0.15 microg/g in the larvae fed the diets that were fortified with 707 microg/g of nine other vitamins and 2 and 20 microg/g of nicotinic acid, respectively, but it was not found in the larvae fed the 0.2 microg/g of nicotinic acid diet. Nicotinamide was detected at concentrations of 0.44, 0.52, and 0.55 microg/g in the spent diets that were fortified with the nine vitamins (707 microg/g) and 0.2, 2, and 20 microg/g of nicotinic acid, respectively. Nicotinamide adenine dinucleotide (NAD) was in the live larvae, but not in the dead larvae. These findings indicate that dietary nicotinic acid is converted to nicotinamide, which, in turn, is used to synthesize NAD, and suggest a positive relationship between C. capitata larvae survival rates and concentrations of dietary nicotinic acid and nitcotinamide in the larvae as well as in the spent diets. The result shows that nicotinamide derived from supplemental nicotinic acid is essential for the development and survival of C. capitata larvae. Nicotinamide may be a biomarker for larval survival and development.

Pulmonary Proteome and Protein Networks in Response to the Herbicide Paraquat in Rats.

Paraquat (PQ) has been one of the most widely used herbicides in the world. PQ, when ingested, is toxic to humans and may cause acute respiratory distress syndrome. To investigate molecular perturbation in lung tissues caused by PQ, Sprague Dawley male rats were fed with PQ at a dose of 25 mg/kg body weight for 20 times in four weeks. The effects of PQ on cellular processes and biological pathways were investigated by analyzing proteome in the lung tissues in comparison with the control. Among the detected proteins, 321 and 254 proteins were over-represented and under-represented, respectively, in the PQ-exposed rat lung tissues in comparison with the no PQ control. All over- and under-represented proteins were subjected to Ingenuity Pathway Analysis to create 25 biological networks and 38 pathways of interacting protein clusters. Over-represented proteins were involved in the C-jun-amino-terminal kinase pathway, caveolae-mediated endocytosis signaling, cardiovascular-cancer-respiratory pathway, regulation of clathrin-mediated endocytosis, non-small cell lung cancer signaling, pulmonary hypertension, glutamate receptor, immune response and angiogenesis. Under-represented proteins occurred in the p53 signaling pathway, mitogen-activated protein kinase signaling pathway, cartilage development and angiogenesis inhibition in the PQ-treated lungs. The results suggest that PQ may generate reactive oxygen species, impair the MAPK/p53 signaling pathway, activate angiogenesis and depress apoptosis in the lungs.

Identification and Classification of Rhizobia by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry.

Mass spectrometry (MS) has been widely used for specific, sensitive and rapid analysis of proteins and has shown a high potential for bacterial identification and characterization. Type strains of four species of rhizobia and Escherichia coli DH5α were employed as reference bacteria to optimize various parameters for identification and classification of species of rhizobia by matrix-assisted laser desorption/ionization time-of-flight MS (MALDI TOF MS). The parameters optimized included culture medium states (liquid or solid), bacterial growth phases, colony storage temperature and duration, and protein data processing to enhance the bacterial identification resolution, accuracy and reliability. The medium state had little effects on the mass spectra of protein profiles. A suitable sampling time was between the exponential phase and the stationary phase. Consistent protein mass spectral profiles were observed for E. coli colonies pre-grown for 14 days and rhizobia for 21 days at 4°C or 21°C. A dendrogram of 75 rhizobial strains of 4 genera was constructed based on MALDI TOF mass spectra and the topological patterns agreed well with those in the 16S rDNA phylogenetic tree. The potential of developing a mass spectral database for all rhizobia species was assessed with blind samples. The entire process from sample preparation to accurate identification and classification of species required approximately one hour.

Large-scale identification of membrane proteins with properties favorable for crystallization.

Membrane protein crystallography is notoriously difficult due to challenges in protein expression and issues of degradation and structural stability. We have developed a novel method for large-scale screening of native sources for integral membrane proteins that have intrinsic biochemical properties favorable for crystallization. Highly expressed membrane proteins that are thermally stable and nonaggregating in detergent solutions were identified by mass spectrometry from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebrum. Many of the membrane proteins identified had been crystallized previously, supporting the promise of the approach. Most identified proteins have known functions and include high-value targets such as transporters and ATPases. To validate the method, we recombinantly expressed and purified the yeast protein, Yop1, which is responsible for endoplasmic reticulum curvature. We demonstrate that Yop1 can be purified with the detergent dodecylmaltoside without aggregating.

Diet-induced over-expression of flightless-I protein and its relation to flightlessness in Mediterranean fruit fly, Ceratitis capitata.

The Mediterranean fruit fly (medfly), Ceratitis capitata is among the most economically important pests worldwide. Understanding nutritional requirement helps rearing healthy medfly for biocontrol of its population in fields. Flight ability is a high priority criterion. Two groups of medfly larvae were reared with two identical component diets except one with fatty acids (diet A) and another without it (diet B). Adults from larvae reared on diet B demonstrated 20±8% of normal flight ability, whereas those from larvae reared on diet A displayed full flight ability of 97±1%. Proteomes were profiled to compare two groups of medfly pupae using shotgun proteomics to study dietary effects on flight ability. When proteins detected in pupae A were compared with those in pupae B, 233 and 239 proteins were, respectively, under- and over-expressed in pupae B, while 167 proteins were overlapped in both pupae A and B. Differential protein profiles indicate that nutritional deficiency induced over-expression of flightless-I protein (fli-I) in medfly. All proteins were subjected to Ingenuity Pathway Analysis (IPA) to create 13 biological networks and 17 pathways of interacting protein clusters in human ortholog. Fli-I, leucine-rich repeat (LRR)-containing G protein-coupled receptor 2, LRR protein soc-2 and protein wings apart-like were over-expressed in pupae B. Inositol-1,4,5-trisphosphate receptor, protocadherin-like wing polarity protein stan and several Wnt pathway proteins were under-expressed in pupae B. These results suggest down-regulation of the Wnt/wingless signaling pathway, which consequently may result in flightlessness in pupae B. The fli-I gene is known to be located within the Smith-Magenis syndrome (SMS) region on chromosome 17, and thus, we speculate that nutritional deficiency might induce over-expression of fli-I (or fli-I gene) and be associated with human SMS. However, more evidence would be needed to confirm our speculation.

Suitability of a magnetic particle immunoassay for the analysis of PBDEs in Hawaiian euryhaline fish and crabs in comparison with gas chromatography/electron capture detection-ion trap mass spectrometry.

A gas chromatograph/electron capture detector-ion trap mass spectrometer (GC/ECD-ITMS) was used for the determination of polybrominated diphenyl ethers (PBDEs) in euryhaline fish and crabs. GC/ECD-ITMS results showed that average recoveries from the spiked fish samples are in a range of 58-123% with relative standard deviations (RSDs) of 5-19%. PBDE concentrations obtained from GC/ECD-ITMS ranged from 28 ng/g to 1845 ng/g lipid weight (lw) in all aquatic species collected from Hawaiian brackish waters. The general BDE congener concentration profile observed in this study is BDE-47>BDE-100>BDE-154>BDE-99>BDE-153>BDE-28>BDE-183. The ELISA results expressed as BDE-47 equivalents correlated well with those of GC/ECD-ITMS, with a correlation coefficient (R(2)=0.68) and regression coefficient (slope=0.82). Comparison of ELISA with GC/ECD-ITMS results demonstrated that ELISA provides a timely and cost-effective method to screen PBDEs in fish and crab samples.