Involvement of p38alpha in kainate-induced seizure and neuronal cell damage

We investigated how p38alpha mitogen-activated protein kinase (p38) is related to kainate-induced epilepsy and neuronal damages, by using the mice with a single copy disruption of the p38 alpha gene (p38alpha(+/-)). Mortality rate and seizure score of p38alpha(+/-) mice administered with kainate were significantly reduced compared with the case of wild-type (WT) mice. This was clearly supported by the electroencephalography data in which kainate-induced seizure duration and frequency in the brain of p38alpha(+/-) mice were significantly suppressed compared to those of WT mice. As a consequence of seizure, kainate induced delayed neuronal damages in parallel with astrocytic growth in the hippocampus and ectopic innervation of the mossy fibers into the stratum oriens in the CA3 region of hippocampus in WT mice, whose changes were moderate in p38alpha(+/-) mice. Likewise, kainate-induced phosphorylation of calcium/calmodulin-dependent kinase II in the hippocampus of p38alpha (+/-) mice was significantly decreased compared to that of WT mice. These results suggest that p38alpha signaling pathway plays an important role in epileptic seizure and excitotoxicity.     

Cytoskeleton and cell wall function in penetration resistance

Plants successfully repel the vast majority of potential pathogens that arrive on their surface, with most microorganisms failing to breach the outer epidermal wall. Resistance to penetration at the epidermis is a key component of basal defence against disease and critically depends on fortification of the cell wall at the site of attempted penetration through the development of specialised cell wall appositions rich in antimicrobial compounds. Formation of cell wall appositions is achieved by rapid reorganisation of actin microfilaments, actin-dependent transport of secretory products to the infection site and local activation of callose synthesis. Plants are finely tuned to detect the presence of pathogens on their surface, perceiving both chemical and physical signals of pathogen origin. In the on-going evolution of interaction strategies, plants must continually monitor and out manoeuvre pathogen avoidance or suppression of plant defences in order to preserve the effectiveness of penetration resistance.     

Comparative profiling of serum glycoproteome by sequential purification of glycoproteins and 2-nitrobenzensulfenyl (NBS) stable isotope labeling: a new approach for the novel biomarker discovery for cancer

The recent progress in various proteomic technologies allows us to screen serum biomarker including carbohydrate antigens. However, only a limited number of proteins could be detected by current conventional methods such as shotgun proteomics, primarily because of the enormous concentration distribution of serum proteins and peptides. To circumvent this difficulty and isolate potential cancer-specific biomarkers for diagnosis and treatment, we established a new screening system consisting of the sequential steps of (1) immunodepletion of 6 high-abundance proteins, (2) targeted enrichment of glycoproteins by lectin column chromatography, and (3) the quantitative proteome analysis using 12C6- or 13C6-NBS (2-nitrobenzenesulfenyl) stable isotope labeling followed by MALDI-QIT-TOF mass spectrometric analysis. Through this systematic analysis for five serum samples derived from patients with lung adenocarcinoma, we identified as candidate biomarkers 34 serum glycoproteins that revealed significant difference in alpha1,6-fucosylation level between lung cancer and healthy control, clearly demonstrating that the carbohydrate-focused proteomics could allow for the detection of serum components with cancer-specific features. In addition, we developed a more simplified and practical technique, mass spectrometry-based glycan structure analysis and lectin blotting, in order to validate glycan structure of candidate biomarkers that could be applicable in clinical use. Our new glycoproteomic strategy will provide highly sensitive and quantitative profiling of specific glycan structures on multiple proteins, which should be useful for serum biomarker discovery.     

Effects of cyclic irrigation on water and nitrogen mass balances in a paddy field

Cyclic irrigation is considered an effective water management practice for reducing pollutant loads from a paddy-field district. The objective of this study was to clarify the effects of cyclic irrigation on mass balance in paddy plots. At the study site, cyclic irrigation with a high cyclic irrigation ratio (% reused water in irrigation water) was conducted from late April to late June. We found a complementary relationship between the volume of irrigation water and rainfall, which together totaled about 1400-1600 mm during the irrigation period each year. We concluded that a cyclic irrigation system that enables the paddy-field district to use a high cyclic irrigation ratio may lead to more efficient use of rainfall for crop irrigation. Nitrogen concentrations in both irrigation water and ponded water tended to be higher during the cyclic irrigation period than during the lake water irrigation period. Nitrogen input from irrigated water accounted for about 8-16% of the total input of nitrogen. It is suggested that fertilizer application of nitrogen can be reduced by its return through cyclic irrigation.     

Dissecting electrostatic interactions in Bacillus circulans xylanase through NMR-monitored pH titrations

NMR-monitored pH titration curves of proteins provide a rich source of structural and electrostatic information. Although relatively straightforward to measure, interpreting pH-dependent chemical shift changes to obtain site-specific acid dissociation constants (pK (A) values) is challenging. In order to analyze the biphasic titrations exhibited by the side chain (13)C(γ) nuclei of the nucleophilic Glu78 and general acid/base Glu172 in Bacillus circulans xylanase, we have revisited the formalism for the ionization equilibria of two coupled acidic residues. In general, fitting NMR-monitored pH titration curves for such a system will only yield the two macroscopic pK (A) values that reflect the combined effects of both deprotonation reactions. However, through the use of mutations complemented with ionic strength-dependent measurements, we are able to extract the four microscopic pK (Ai) values governing the branched acid/base equilibria of Glu78 and Glu172 in BcX. These data, confirmed through theoretical calculations, help explain the pH-dependent mechanism of this model GH11 xylanase by demonstrating that the kinetically determined pK (A) values and hence catalytic roles of these two residues result from their electrostatic coupling.     

Long-term monitoring of fecal steroid hormones in female snow leopards (Panthera uncia) during pregnancy or pseudopregnancy

Knowledge of the basic reproductive physiology of snow leopards is required urgently in order to develop a suitable management conditions under captivity. In this study, the long-term monitoring of concentrations of three steroid hormones in fecal matter of three female snow leopards was performed using enzyme immunoassays: (1) estradiol-17β, (2) progesterone and (3) cortisol metabolite. Two of the female animals were housed with a male during the winter breeding season, and copulated around the day the estradiol-17β metabolite peaked subsequently becoming pregnant. The other female was treated in two different ways: (1) first housed with a male in all year round and then (2) in the winter season only. She did not mate with him on the first occasion, but did so latter around when estradiol-17β metabolite peaked, and became pseudopregnant. During pregnancy, progesterone metabolite concentrations increased for 92 or 94 days, with this period being approximately twice as long as in the pseudopregnant case (31, 42, 49 and 53 days). The levels of cortisol metabolite in the pseudopregnant female (1.35 μg/g) were significantly higher than in the pregnant females (0.33 and 0.24 μg/g) (P<0.05). Similarly, during the breeding season, the levels of estradiol-17β metabolite in the pseudopregnant female (2.18 μg/g) were significantly higher than those in the pregnant females (0.81 and 0.85 μg/g) (P<0.05). Unlike cortisol the average levels of estradiol-17β during the breeding season were independent of reproductive success.The hormone levels may also be related to housing conditions and the resulting reproductive success in female leopards. The female housed with a male during the non-breeding season had high levels of cortisol metabolites and low levels of estradiol-17β in the breeding season, and failed to become pregnant. This indicates that housing conditions in snow leopards may be an important factor for normal endocrine secretion and resulting breeding success.     

Increase in Alphaproteobacteria in association with a polychaete,Capitella sp. I,in the organically enriched sediment

We conducted bioremediation experiments on the organically enriched sediment on the sea floor just below a fish farm, introducing artificially mass-cultured colonies of deposit-feeding polychaete, Capitella sp. I. To clarify the association between the Capitella and bacteria on the efficient decomposition of the organic matter in the sediment in the experiments, we tried to identify the bacteria that increased in the microbial community in the sediment with dense patches of the Capitella. The relationship between TOC and quinone content of the sediment as an indicator of the bacterial abundance was not clear, while a significant positive correlation was found between Capitella biomass and quinone content of the sediment. In particular, ubiquinone-10, which is present in members of the class Alphaproteobacteria, increased in the sediment with dense patches of the Capitella. We performed denaturing gradient gel electrophoresis (DGGE) analyses to identify the alphaproteobacterial species in the sediment with dense patches of the worm, using two DGGE fragments obtained from the sediment samples and one fragment from the worm body. The sequences of these DGGE fragments were closely related to the specific members of the Roseobacter clade. In the associated system with the Capitella and the bacteria in the organically enriched sediment, the decomposition of the organic matter may proceed rapidly. It is very likely that the Capitella works as a promoter of bacteria in the organically enriched sediment, and feeds the increased bacteria as one of the main foods, while the bacteria decompose the organic matter in the sediment with the assistance of the Capitella.     

Antibody responses to NY-ESO-1 in primary breast cancer identify a subtype target for immunotherapy

The highly immunogenic human tumor antigen NY-ESO-1 (ESO) is a target of choice for anti-cancer immune therapy. In this study, we assessed spontaneous antibody (Ab) responses to ESO in a large cohort of patients with primary breast cancer (BC) and addressed the correlation between the presence of anti-ESO Ab, the expression of ESO in the tumors and their characteristics. We found detectable Ab responses to ESO in 1% of the patients. Tumors from patients with circulating Ab to ESO exhibited common characteristics, being mainly hormone receptor (HR)⁻ invasive ductal carcinomas of high grade, including both HER2⁻ and HER2⁺ tumors. In line with these results, we detected ESO expression in 20% of primary HR⁻ BC, including both ESO Ab⁺ and Ab⁻ patients, but not in HR⁺ BC. Interestingly, whereas expression levels in ESO⁺ BC were not significantly different between ESO Ab⁺ and Ab⁻ patients, the former had, in average, significantly higher numbers of tumor-infiltrated lymph nodes, indicating that lymph node invasion may be required for the development of spontaneous anti-tumor immune responses. Thus, the presence of ESO Ab identifies a tumor subtype of HR⁻ (HER2⁻ or HER2⁺) primary BC with frequent ESO expression and, together with the assessment of antigen expression in the tumor, may be instrumental for the selection of patients for whom ESO-based immunotherapy may complement standard therapy.