Ultraviolet light-induced oxidative stress: Effects on antioxidant response of Helicoverpa armigera adults

Ultraviolet (UV) light (blacklight), which emits UV in the range of 320-400 nm, has been used worldwide in light trapping of insect pests. In this article, we test the hypothesis that one of the effects of UV light irradiation is to increase oxidative stress on insects. The effects of UV light irradiation on total antioxidant capacity, malondialdehyde (MDA) and protein carbonyl contents and the activities of superoxide dismutase (SOD), catalase (CAT), peroxidases (POX) and glutathione-S-transferase (GST) were investigated in Helicoverpa armigera adults. The adults were exposed to UV light for various time periods (0, 30, 60 and 90 min). We found that exposure to UV light for 30 min resulted in increased total antioxidant capacity, protein carbonyl content and activities of SOD, CAT, POX and GST. When the exposure time lasted for 60 and 90 min, the protein carbonyl content and activities of CAT and GST remained significantly higher than the control. However, the antioxidant capacity and SOD activity returned to control levels, and POX activity decreased at 60 and 90 min. Our results confirm the hypothesis that UV light irradiation increases the level of oxidative stress in H. armigera adults.     

Differential expression of proteins induced by lead in the Dwarf Sunflower Helianthus annuus

The Dwarf Sunflower (Helianthus annuus) is a hyperaccumulator of toxic metals including cadmium (Cd), mercury (Hg), nickel (Ni), and lead (Pb). In order to identify stress response to Pb, plants were exposed to a mixture of 30 mg/l of three ions, Cd, Cr, and Ni, with and without Pb. Soluble proteins were resolved by two-dimensional gel electrophoresis. Four proteins were differentially expressed and very abundant in the leaf samples after plants were exposed to all these four metals. The first protein spot contained two proteins: chitinase and a chloroplast drought-induced stress protein CDSP-34. The second spot contained a thaumatin-like protein. Two proteins in spot 3 were identified as heat-shock cognate 70-1 and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Several peptides were identified in spot 4 but none could be matched to any sequence in the NCBI database.     

Comparative analysis of proteome changes induced by the two spotted spider mite Tetranychus urticae and methyl jasmonate in citrus leaves

Citrus plants are currently facing biotic and abiotic stresses. Therefore, the characterization of molecular traits involved in the response mechanisms to stress could facilitate selection of resistant varieties. Although large cDNA microarray profiling has been generated in citrus tissues, the available protein expression data are scarce. In this study, to identify differentially expressed proteins in Citrus clementina leaves after infestation by the two-spotted spider mite Tetranychus urticae, a proteome comparison was undertaken using two-dimensional gel electrophoresis. The citrus leaf proteome profile was also compared with that of leaves treated over 0-72 h with methyl jasmonate, a compound playing a key role in the defense mechanisms of plants to insect/arthropod attack. Significant variations were observed for 110 protein spots after spider mite infestation and 67 protein spots after MeJA treatments. Of these, 50 proteins were successfully identified by liquid chromatography-mass spectrometry-tandem mass spectrometry. The majority constituted photosynthesis- and metabolism-related proteins. Five were oxidative stress associated enzymes, including phospholipid glutathione peroxidase, a salt stressed associated protein, ascorbate peroxidase and Mn-superoxide dismutase. Seven were defense-related proteins, such as the pathogenesis-related acidic chitinase, the protease inhibitor miraculin-like protein, and a lectin-like protein. This is the first report of differentially regulated proteins after T. urticae attack and exogenous MeJA application in citrus leaves.     

Dormancy of Medicago marina (L.) seed

Medicago marina (L.) is a Mediterranean species whose seeds show strong dormancy that prevents germination. We used an integrated approach of physiological analyses and proteomics to investigate the mechanisms that control M. marina dormancy/germination and that underlie stress tolerance. First, we evaluated the effects on dormancy breaking of the following treatments: mechanical scarification, freezing at −20 °C, storage for 4 months and heating at 100 °C for 1 h. Mechanical scarification and freezing were the most effective treatments in overcoming dormancy. The role of abscisic acid (ABA) in M. marina dormancy was studied by ELISA immuno-enzymatic assay. The ABA content of germinated and non-germinated mature (control) and treated seeds was determined. The level of ABA was higher in treated seeds than in control seeds; the most significant increase occurred in the heated seeds. A comparison of the ABA level in the germinated, control and treated seeds suggests that different mechanisms modulate ABA content in response to different stresses, and that a specific ABA-signalling pathway regulates germination. Proteomic analysis revealed 46 proteins differentially expressed between treated and untreated seeds; 14 of these proteins were subsequently identified by mass spectrometry. Several of the proteins identified are important factors in the stress response, and are involved in such diverse functions as lipid metabolism, protein folding and chromatin protection. Lastly, an analysis of the phosphoproteome maps showed that the function of many proteins in seeds subjected to temperature treatment is modulated through post-translational modifications.     

Proteomic analysis of the compatible interaction between Vitis vinifera and Plasmopara viticola

We analyzed the proteome of grapevine (Vitis vinifera) leaves 24, 48 and 96 h post infection (hpi) with the downy mildew pathogen Plasmopara viticola. Total proteins were separated on 2-DE gels. By MS analysis, we identified 82 unique grapevine proteins differentially expressed after infection. Upregulated proteins were often included in the functional categories of general metabolism and stress response, while proteins related to photosynthesis and energy production were mostly downregulated. As expected, the activation of a defense reaction was observed more often at the late time point, consistent with the establishment of a compatible interaction. Most proteins involved in resistance were isoforms of different PR-10 pathogenesis-related proteins. Although > 50 differentially expressed protein isoforms were observed at 24 and 96 hpi, only 18 were detected at 48 hpi and no defense-related proteins were among this group. This profile suggests a transient breakdown in defense responses accompanying the onset of disease, further supported by gene expression analyses and by a western blot analysis of a PR-10 protein. Our data reveal the complex modulation of plant metabolism and defense responses during compatible interactions, and provide insight into the underlying molecular processes which may eventually yield novel strategies for pathogen control in the field.     

Light-induced and apoptosis-like cell death in the unicellular eukaryote, Blepharisma japonicum

The unicellular eukaryote, Blepharisma japonicum, is a light-sensitive ciliated protozoa. It possesses a photoreceptor pigment called blepharismin that plays critical roles in defensive behavior against predators and step-up photophobic response. In addition, the pigment generates reactive oxygen species such as singlet oxygen and hydroxyl radicals which contribute to photodynamic action. Previous studies reported that intense light (>300 W m⁻²) induced rapid photodynamic killing (necrosis) characterized by cell swelling and plasma efflux, while moderate light (3-30 W m⁻²) only induced pigment extrusion and photooxidation. We have found that moderate light (5 W m⁻²) induced apoptosis-like cell death. Microscopically it was found that >3 h of moderate light irradiation induced macronuclear condensation and plasma efflux without cell swelling. Single cell gel electrophoresis assay showed that DNA fragmentation occurred between 1 and 3 h of irradiation, and the condensed macronuclei contained quite fragmented DNA. Macronuclear DNA extracted from light-irradiated cells contained DNA fragments of 180-200 and 360-400 bp, which were seen as apoptosis ladders.     

Proteomic analysis of hybrid poplar xylem sap

Xylem sap collected from Populus trichocarpa × Populus deltoides using root pressure was estimated to contain more than 100 proteins. Ninety-seven of these proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). These proteins were classified into 10 functional categories including metabolism, signaling, stress response and cell wall functions. The majority of xylem sap proteins were metabolic enzymes involved in processes including translation, proteolysis, and glycolysis. Stress-related proteins were also prevalent. In contrast to xylem sap proteins collected from annual plants, the majority of poplar xylem sap proteins do not appear to be classically secreted since only 33 proteins were predicted to have an N-terminal signal peptide targeting them to the secretory pathway. Of the remaining 64 proteins, 27 were predicted to be secreted non-classically. While a number of proteins identified here have been previously reported in xylem sap proteomes of annual plants, many xylem sap proteins were identified in poplar which may reflect functions specific to perennial plants.     

Characterization of vegetative storage protein (VSP) and low molecular proteins induced by water deficit in stolon of white clover

In stolon of white clover (Trifolium repens L.), the 17.3 kDa protein has been newly identified as a vegetative storage protein (VSP) which has preponderant roles in N accumulation and mobilization to sustain growth when capacity of N uptake is strongly reduced. To characterize the water deficit effect on this protein, the kinetic pattern of soluble protein, SDS–PAGE, Western blotting, and proteomic analysis was studied in the stolon of white clover during 28 days of water-deficit. Water deficit led to decrease protein concentration. SDS–PAGE revealed that two major proteins of 17.3 and 16 kDa were accumulated to high level in response to water stress. These proteins cross-reacted positively with antibodies raised against the 17.3 kDa VSP, a protein which shared biochemical features with stress proteins implied in dehydration tolerance. Using two-dimensional electrophoresis (2-DE) gel and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS) analysis, it was demonstrated that 19.5 and 17.3 kDa protein spots were up-regulated by water stress, and both spots were identical to nucleoside diphosphate kinase (NDPK) and lipid transfer proteins (LTPs), respectively. These results suggest that low molecular proteins induced by water-deficit in the stolon of white clover act as an alternative N reserves or play significant roles in plant protection against water-deficit stress.     

Cell disruption using a different methodology for proteomics analysis of Trypanosoma cruzi strains

We have developed a cell disruption method to produce a protein extract using Trypanosoma cruzi cells based on a straightforward hypoosmotic lysis protocol. The procedure consists of three steps: incubation of the cells in a hypoosmotic lysis buffer, sonication in a water bath, and centrifugation. The final protein extract was designated TcS12. The stages of cell disruption at different incubation times were monitored by differential interference contrast microscopy. After 30 min of incubation in lysis buffer at 4 °C, the T. cruzi epimastigote forms changed from slender to round-shaped parasites. Nevertheless, cell disruption took place following sonication of the sample for 30 min. The efficiency of the methodology was also validated by flow cytometry, which resulted in 72% of propidium iodide (PI)-labeled cells. To estimate the protein extraction yield and the differential protein expression, the proteomics profile of four T. cruzi strains (CL-Brener, Dm28c, Y, and 4167) were analyzed by liquid chromatography tandem mass spectrometry (LCMS/MS) on a SYNAPT HDMS system using the label-free MS^E approach. ProteinLynx Global Server (version 2.5) with Expression^E analysis identified a total of 1153 proteins and revealed 428 differentially expressed proteins among the strains. Gene ontology analysis showed that not only cytosolic proteins but also nuclear and organellar ones were present in the extract.     

Synergistic anti-bacterial and proteomic effects of epigallocatechin gallate on clinical isolates of imipenem-resistant Klebsiella pneumoniae

Imipenem-resistant Klebsiella pneumoniae (IRKP) were used to explore the synergistic anti-bacterial and proteomic effects of imipenem alone or in combination with epigallocatechin gallate (EGCg). The minimal inhibitory concentrations (MICs) of EGCG for 12 clinically isolated IRKP strains ranged from 300 to 650 μg/ml. Each of the 12 IRKP strains experienced a 4- to 64-fold reduction in the MIC of imipenem upon co-incubation with 0.25 × MIC level of EGCg. The time-kill method was used on the 12 IRKP clinical isolates to evaluate the bactericidal activities of imipenem alone or with EGCg. Compared to imipenem alone, EGCg with imipenem demonstrated enhanced bactericidal activity. Two-dimensional polyacrylamide gel electrophoresis identified eight down-regulated and four up-regulated proteins in the IRKP strain upon exposure to 1 × MIC of EGCg. Analysis of the outer membrane protein profiles of IRKP cultures treated with EGCg revealed unique changes in outer membrane proteins. In addition, scanning electron microscopic analysis demonstrated the presence of cells with wrinkled surfaces containing perforations and irregular rod-shaped forms after treatment with EGCg or imipenem. These studies demonstrate that EGCg can synergize the bacterial activity of imipenem and differentially stimulate the expression of various proteins in IRKP.     

Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle

Bacillus thuringiensis (Bt) Cry8D insecticidal proteins are unique among Cry8 family proteins in terms of its insecticidal activity against adult Scarab beetles, such as Japanese beetle (Popillia japonica Newman). From the sequence homology with other Bt Cry proteins especially those active against beetles, such as Cry3Aa whose 3D structure is available, the structure of the Cry8D protein has been predicted to be a typical three-domain Cry protein type. In addition, the activation process of Cry8D in gut juice of susceptible insects is presumed to be similar to that of Cry3A (Yamaguchi et al., 2008). In this study, the activation process of Cry8Da in insect gut juice was closely examined. Japanese beetle gut juice proteases digested the 130 kDa Cry8Da protein to produce a 64 kDa protein. This 64 kDa protein was active against both adult and larval Japanese beetle and considered to be an activated toxin. N-terminal sequencing of this 64 kDa protein revealed that the Cry8Da leader sequence consisting of 63 amino acid residues from M¹ to F⁶³ was removed. As in the case of Cry3Aa, the proteases further digested the 64 kDa protein to two 8 kDa and 54 kDa fragments. N-terminal amino acid analysis of these smaller fragments indicated that the proteases digested the loop between Alpha Helix (Alpha for short) 3 and Alpha 4. This means that the 8 kDa fragment consists of Alpha 1-3 of Domain I and that the 54 kDa fragment contains the remaining Domain I and full Domain II and Domain III. Size exclusion chromatography and anion exchange chromatography could not separate these 64, 54 and 8 kDa proteins suggesting that the 54 kDa and 8 kDa fragments are still forming the toxin complex equivalent to the 64 kDa protein by size and ionic charge. The sequencing and chromatography results suggest that the gut juice proteases merely nicked the loop between Alpha 3 and Alpha 4. This nicking process appeared to be essential for receptor binding of the Cry8Da toxin. BBMV binding assay revealed that the Cry8Da toxin bound to BBMV preparations from both adult and larval Japanese beetle only after the loop was nicked. Only the 54 kDa fragment bound to the BBMV preparations but not the 64 kDa protein. Ligand blot showed that the protease activated Cry8Da toxin, presumably the 54 kDa fragment, bound to specific BBMV proteins, one or more of those would be receptor(s). The sizes and binding affinities of these Cry8Da-bound proteins of Japanese beetle BBMV differed between larvae and adults.     

Selection of improved Beauveria bassiana (Bals.) Vuill. strains based on 2-deoxy-d-glucose resistance and physiological analysis

A series of 2-deoxy-d-glucose resistant mutants was obtained from wild type Beauveria bassiana 88 (Bb 88) by UV irradiation. Five mutants were characterized on Sabouraud Dextrose Agar and Chitin Agar for both radial extension rate (V_r) and specific growth rate (μ). These values were obtained after adjusting morphometric data to a mathematical model used for filamentous fungi. Additionally, the protease and lipase potency index, conidial size, viability, and production levels were analyzed. The highest values for those physiological measurements were obtained by mutant 882.5 which, relative to Bb 88, showed a 30% reduction in half-life (LT₅₀) on Sphenarium purpurascens, 70% on Acheta domesticus, and 71% on Tenebrio molitor larvae and adults. The half lethal concentration (LC₅₀) on T. molitor larvae was 2.8 × 10⁵ conidia/mL (con/mL) and 1.5 × 10⁶ con/mL, respectively, for mutant 882.5 and Bb 88. This demonstrates that mutant 882.5 is more virulent, with up to an 80% reduction in LC₅₀. This work provides a convenient method for improving strains to be used in biocontrol as a suitable alternative to transgenic constructs.     

Up-regulation of annexin A2 in cholangiocarcinoma caused by Opisthorchis viverrini and its implication as a prognostic marker

Cholangiocarcinoma (CCA), or cancer of the bile ducts, is primarily associated with infection with the liver fluke Opisthorchis viverrini in northeast Thailand. The disease is associated with late presentation, poses challenges for diagnosis and has a high mortality rate - features that highlight the need for tumor markers. At present, there are no specific tumor markers that can indicate the early stages and status of CCA. Proteomic analysis of the proteins expressed on the surface of tumor cells is particularly difficult since proteome-wide analysis of surface membrane proteins has thus far been hampered by the lack of effective strategies to profile hydrophobic membrane proteins. In this study, a sequential protein extraction was utilized to overcome this problem. Membrane protein was extracted from four CCA cell lines with different tumor forming capabilities. The non-tumor H69 biliary cell line was used as a control. Two-dimensional-PAGE followed by MALDI-TOF-MS was used to identify differentially expressed proteins. Among 20 up-regulated membrane proteins identified in the CCA cell lines was ANXA2, a participant in tumor invasion and metastasis in other cancers. Accordingly, ANXA2 was verified in human subjects by probing, using a commercial anti-mouse monoclonal antibody and a tissue microarray of CCA (301 diagnosed cases), where it was found to associate with one of several tumor progression stages as reflected by lymphatic invasion (P = 0.014) and metastasis (P = 0.026). Patients with high expression of ANXA2 had a significantly shorter survival time (P = 0.011). ANXA2 expression in tumors may be useful for predicting the poor outcome of CCA patients.     

The influence of the fungal pathogen Mycocentrospora acerina on the proteome and polyacetylenes and 6-methoxymellein in organic and conventionally cultivated carrots (Daucus carota) during post harvest storage

Many carrots are discarded during post harvest cold storage due to development of fungal infections, caused by, e.g., Mycocentrospora acerina (liquorice rot). We compared the susceptibility of carrots grown under conventional and organic agricultural practices. In one year, organically cultivated carrots showed 3 × to 7 × more symptoms than conventionally cultivated, when studying naturally occurring disease at 4 and 6 months, respectively. On the other hand, we have developed a bioassay for infection studies of M. acerina on carrots and observed that organic roots were more susceptible after one month of storage than conventional ones, but no differences were apparent after four or six months storage. Levels of polyacetylenes (falcarinol, falcarindiol and falcarindiol-3-acetate) did not change, whereas the isocoumarin phytoalexin (6-methoxymellein) accumulated in infected tissue as well as in healthy tissue opposite the infection. The proteomes of carrot and M. acerina were characterized, the intensity of 33 plant protein spots was significantly changed in infected roots including up regulation of defence and stress response proteins but also a decrease of proteins involved in energy metabolism. This combined metabolic and proteomic study indicates that roots respond to fungal infection through altered metabolism: simultaneous induction of 6-methoxymellein and synthesis of defence related proteins.