Two-dimensional HPLC on-line analysis of phosphopeptides using titania and monolithic columns

Conventional and comprehensive two-dimensional (2D) HPLC systems using the combination of titania and monolithic columns were established for the on-line analysis of phosphopeptides. Compared with immobilized metal affinity chromatography of a general method for the analysis of phosphopeptides, the use of titania columns in the analysis permits the specific isolation of phosphopeptides in a higher yield. Using the current 2D HPLC systems, phosphopeptides were specifically isolated from nonphosphorylated peptides by the first-dimension titania column, followed by the high-speed separation of the phosphopeptides by the second-dimension monolithic column. Proteolytic digests of beta-casein were analyzed within 30 min using the comprehensive 2D HPLC system; all phosphopeptides from beta-casein could be efficiently isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The comprehensive 2D HPLC system coupled with mass spectrometry will be useful for high-throughput and on-line phosphoproteome analyses.     

Distinct contributions of vacuolar Qabc- and R-SNARE proteins to membrane fusion specificity

In eukaryotic endomembrane systems, Qabc-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) on one membrane and R-SNARE on the opposing membrane assemble into a trans-QabcR-SNARE complex to drive membrane fusion. However, it remains ambiguous whether pairing of Qabc- and R-SNAREs mediates membrane fusion specificity. Here, we explored the fusion specificity of reconstituted proteoliposomes bearing purified SNAREs in yeast vacuoles and other organelles. We found that not only vacuolar R-SNARE Nyv1p but also the non-cognate R-SNAREs, endosomal Snc2p, and endoplasmic reticulum-Golgi Sec22p caused efficient fusion with vacuolar Qabc-SNAREs. In contrast, their fusion is blocked completely by replacing vacuolar Qc-SNARE Vam7p with the non-cognate endosomal Tlg1p and Syn8p, although these endosomal Qc-SNAREs fully retained the ability to form cis-SNARE complexes with vacuolar SNAREs in solution and on membranes. Thus, our current study establishes that an appropriate assembly of Qabc-SNAREs is crucial for regulating fusion specificity, whereas R-SNARE itself has little contribution to specificity.     

Characterization of 1-(2-[18F] fluoro-3-pyridyl)-4-(2-isopropyl-1-oxo- isoindoline-5-yl)-5-methyl-1H-1,2,3-triazole, a PET ligand for imaging the metabotropic glutamate receptor type 1 in rat and monkey brains

Neurovascular degeneration contributes to the pathogenesis of Alzheimer's disease (AD). Because erythropoietin (EPO) promotes endothelial regeneration, we investigated the therapeutic effects of EPO in animal models of AD. In aged Tg2576 mice, EPO receptors (EPORs) were expressed in the cortex and hippocampus. Tg2576 mice were treated with daily injection of EPO (5000 IU/kg/day) for 5 days. At 14 days, EPO improved contextual memory as measured by fear-conditioning test. EPO enhanced endothelial proliferation and the level of synaptophysin expression in the brain. EPO also increased capillary density, and decreased the level of the receptor for advanced glycation endproducts (RAGE) in the brain, while decreasing in the amount of amyloid plaque and amyloid-β (Aβ). In cultured human endothelial cells, EPO enhanced angiogenesis and suppressed the expression of the RAGE. These results show that EPO improves memory and ameliorates endothelial degeneration induced by Aβ in AD models. This pre-clinical evidence suggests that EPO may be useful for the treatment of AD.     

Method of testing insecticide susceptibility of Stenotus rubrovittatus (Hemiptera: Miridae) by topical application

Appropriate droplet size and the preferred sex and age of insects for insecticides used in topical application were investigated to standardize the method of testing the susceptibility of Stenotus rubrovittatus (Matsumura). The overflow of acetone droplets applied on the notum of the thorax of S. rubrovittatus adults occurred when the droplet size exceeded 0.20 μl per insect, and the overflow occurred more frequently in males than in females. No effect of acetone solution on mortality was observed at less than 0.20 μl droplet size per insect within 72 h after treatment. The LD₅₀ values and relative potencies of fenitrothion 24 and 48 h after treatment were almost the same in each sex. The fenitrothion susceptibility of males was twice higher than that of females. The fenitrothion susceptibility increased with age after emergence. However, there were small differences in LD₅₀ values and relative potencies between 4–7 and 8–11 days after emergence because the females 4–11 days after emergence had a small difference in body weight. These results led us to conclude that the recommended size of acetone droplets per insect is less than 0.20 μl and that it is suitable to use S. rubrovittatus females 4–11 days after emergence for topical application.     

Exploring warm-season cover crops as carbon sources for anaerobic soil disinfestation (ASD)

Background and aims

Anaerobic soil disinfestation (ASD) has been shown to be an effective strategy for controlling soilborne plant pathogens and plant-parasitic nematodes in vegetable and other specialty crop production systems. Anaerobic soil disinfestation is based upon supplying labile carbon (C) to stimulate microbially-driven anaerobic soil conditions in moist soils covered with polyethylene mulch. To test the effectiveness of warm-season cover crops as C sources for ASD, a greenhouse study was conducted using a sandy field soil in which several warm-season legumes and grasses were grown and incorporated and compared to molasses-amended and no C source controls.

Methods

Greenhouse pots were irrigated to fill soil porosity and covered with a transparent polyethylene mulch to initiate a 3-week ASD treatment prior to planting tomatoes. Soilborne plant pathogen inoculum packets, yellow nutsedge (Cyperus esculentus L.) tubers, and Southern root-knot nematode (Meloidogyne incognita (Kofoid & White) Chitwood; M.i.) eggs and juveniles were introduced at cover crop incorporation.

Results

In nearly all cases, ASD treatment utilizing cover crops as a C source resulted in soil anaerobicity values that were equal to the molasses-amended fallow control and greater than the no C source fallow control. In trial 1, Fusarium oxysporum Schlechtend.:Fr. (F.o.) survival was reduced by more than 97% in all C source treatments compared to the no C source control but there was no effect of C source in Trial 2. Carbon source treatments were inconsistent in their effects on survival of Sclerotium rolfsii Sacc. (S.r). In general, the number of M.i. extracted from tomato root tissue and root gall ratings were low in all treatments with cover crop C source, molasses C source, or composted poultry litter. Germination of yellow nutsedge tubers was highest in the no C source control (76%), lowest in the molasses control (31%), and intermediate from cover crop treatments (49% to 61%).

Conclusions

Warm-season cover crops have potential to serve as a C source for ASD in vegetable and other crop production systems, but more work is needed to improve consistency and further elucidate mechanisms of control of soilborne plant pathogens and weeds during ASD treatment utilizing cover crops.     

Labeling and enrichment of Arabidopsis thaliana matrix metalloproteases using an active-site directed, marimastat-based photoreactive probe

Matrix metalloproteases (MMPs) are secreted or membrane-bound zinc-containing proteases that play diverse roles in development and immunity in plants and in tissue remodeling in animals. We developed a photoreactive probe based on the MMP inhibitor marimastat, conjugated to a 4-azidotetrafluorobenzoyl moiety as photoreactive group and biotin as detection or sorting function. The probe labels At2-MMP, At4-MMP, At5-MMP, and likely other plant MMPs in leaf extracts, as shown by transient At-MMP expression in Nicotiana benthamiana, protein blot, and LC-MS/MS analysis. This MMP probe is a valuable tool to study the post-translational status of MMPs during plant immunity and other MMP-regulated processes.     

Protection of bioreactor from microbial contamination by high dissolved oxygen tension

Summary Repeated batch hydrolysis of casein using immobilized protease were carried out at various do tensions and pH. At any of the pH tested, microbial contamination could be satisfactorily suppressed by maintaining high do. In continuous reaction, contamination could be suppressed throughout 40 days under the do of 190 ppm, whereas contamination was observed after 24 hours with the do of 2.5 ppm.     

Improved immobilization of DNA to microwell plates for DNA-DNA hybridization.

An improved and simplified protocol for DNA immobilization was developed to enhance DNA-DNA hybridization on microwell plates. Target DNA was immobilized by simple dry-adsorption. Efficiencies of DNA immobilization and retention were enhanced 1.4-6.5 times and 4.2-19.6 times, respectively, compared with a conventional method. The overall hybridization efficiency was increased 3.1-5.2 times. This simple new protocol can reduce the consumption of scarce DNA samples.     

Sampling efficiency of the pale grass blue butterfly Zizeeria maha (Lepidoptera: Lycaenidae): A versatile indicator species for environmental risk assessment in Japan

The pale grass blue butterfly, Zizeeria maha (Lepidoptera, Lycaenidae), has successfully been employed as an indicator species to evaluate acute biological changes in polluted human-living environments after the Fukushima nuclear accident. Here, we quantitatively examined the field sampling efficiency of the butterfly and its associated sexual sampling bias under different conditions, which may provide important information for environmental risk assessment. Sampling data were obtained in 2014 and 2015 from 87 localities across Japan. Across the localities, the mean capture rate was 17 individuals per hour per person, and males represented approximately 80% of the collected butterflies. No significant difference was detected in the capture rate under different weather conditions. Among the four habitats, the riverside showed a significantly higher capture rate than the city park. A shorter sampling time tended to yield a higher capture rate. Comparisons among prefectures and districts of collection localities revealed that the Kanto district had significantly higher percentages of collected males than 4 other districts. Fukushima prefecture also had high percentage, although not significant. A generalized linear mixed model indicated that the capture rate was significantly negatively affected by the agricultural village, the city park, the rainy weather, and latitude and positively by longitude. Together, the present study showed high sampling efficiency and versatility of this butterfly as an indicator for environmental risk assessment but simultaneously suggested preferable conditions for a field sampling design. This study also suggested a possibility that the Fukushima nuclear accident or other factors might have affected behavioral or population dynamics of the butterfly.