Analysis of internal motions of interleukin-13 variant associated with severe bronchial asthma using (15)N NMR relaxation measurements

The single nucleotide polymorphism interleukin-13 (IL-13) R110Q is associated with severe bronchial asthma because its lower affinity leads to the augmentation of local IL-13 concentration, resulting in an increase in the signal transduction via IL-13R. Since the mutation site does not directly bind to IL-13Ralpha2, we carried out NMR relaxation analyses of the wild-type IL-13 and IL-13-R110Q in order to examine whether the R110Q mutation affects the internal motions in IL-13 molecules. The results showed that the internal motion in the micro- to millisecond time scale on helix D, which is suggested to be important for the interaction between IL-13 and IL-13Ralpha2, is increased in IL-13-R110Q compared with that in the wild-type IL-13. It therefore appears that the difference in the internal motions on helix D between the wild-type IL-13 and IL-13-R110Q may be involved in their affinity differences with IL-13Ralpha2.     

Role of Glutathione Peroxidase 4 in Glutamate-Induced Oxytosis in the Retina

Purpose

The purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in glutamate-induced oxytosis in the retina.

Methods

For in vitro studies, an immortalized rat retinal precursor cell line R28 was used. Cells were transfected with siRNA specifically silencing GPx4 or with scrambled control siRNA. Lipid peroxidation was evaluated by 4-hydroxy-2-nonenal (4-HNE) immunostaining. Cytotoxicity and cell death were evaluated using an LDH activity assay and annexin V staining, respectively. Cells transfected with GPx4 siRNA or control siRNA were treated with glutamate (1 or 2 mM), and the cytotoxicity was evaluated using the LDH activity assay. For in vivo studies, retinal ganglion cell damage was induced by intravitreal injection of 25-mM N-methyl-D-aspartate (NMDA, 2 μL/eye) in GPx4^+/+ and GPx4^+/− mice. The evaluation of lipid peroxidation (4-HNE immunostaining), apoptosis (TUNEL staining), and cell density in the ganglion cell layer (GCL) were performed at 12 h, 1 day, and 7 days after the NMDA injection.

Results

GPx4 knockdown significantly increased LDH activity by 13.9-fold (P < 0.01) and increased peroxidized lipid levels by 3.2-fold in R28 cells (P < 0.01). In cells transfected with scrambled control siRNA, treatment with glutamate at 1 or 2 mM did not increase LDH activity; whereas, in cells transfected with GPx4 siRNA, glutamate treatment significantly increased LDH activity (1.52-fold, P < 0.01). GPx4^+/− mice exhibited higher levels of lipid peroxidation in retinas treated with NMDA than GPx4^+/+ mice (1.26-fold, P < 0.05). GPx4^+/− mice had more TUNEL-positive cells induced by NMDA in GCL (1.45-fold, P < 0.05). In addition, the cell density in GCL of GPx4^+/− mice was 19% lower than that in GPx4^+/+ mice after treatment with NMDA (P < 0.05).

Conclusion

These results suggest that defective GPx4 expression is associated with enhanced cytotoxicity by glutamate-induced oxytosis in the retina.     

Characteristics of Serum Endocan Levels in Infection

Objectives

Endocan is a newly recognized biomarker of sepsis. However, there have been no studies of the trends in endocan levels during infection and their associations with other clinical factors. The aim of this study was to assess the time course of endocan levels and the associations of endocan with clinical factors during infection by comparison with other biomarkers.

Methods

Serum samples and blood cultures were obtained from patients who were diagnosed with infection from June 2013 to March 2014. Serum endocan, C-reactive protein (CRP), and procalcitonin (PCT) levels during four periods during infection were measured (day 0, day 1-2, day 3-5, and day 6-10).

Results

A total of 78 patients were enrolled in this study. The median endocan level decreased by only 23% during infection, whereas both serum CRP and PCT levels decreased by more than 80%. Endocan levels were correlated to neither CRP levels nor PCT levels in each period. Endocan levels at day 0 in patients with bacteremia were higher than those without bacteremia (1.09 ng/mL vs 0.82 ng/mL, P=0.002), but neither CRP levels nor PCT levels at day 0 were different between the two groups. Areas under the receiver operator characteristic (ROC) curves of endocan, CRP, and PCT at day 0 were 0.662, 0.343, and 0.563, respectively. Positive blood cultures tended to be related to high endocan levels, but not significantly (odds ratio: 4.24, 95% CI: 0.99-10.34, P=0.05).

Conclusions

In bacteremic cases, serum endocan levels in bacteremia tended to be higher than in non-bacteremic cases. Although endocan level was not identified as a prognostic factor of bacteremia, further prospective study concerning the relationship between serum endocan level and bacteremia would be needed.     

Expression of human IL-13 receptor alpha2 extracellular domain in Pichia pastoris

Interleukin-13 receptor alpha2 (IL-13Ralpha2) binds IL-13 with high affinity and plays an important role in IL-13 signaling as a decoy receptor. We expressed the extracellular domain of human IL-13Ralpha2 (1-313) in methylotrophic yeast Pichia pastoris. SDS-PAGE analysis by PAS staining and Western blot analysis detected the product of the extracellular domain of human IL-13Ralpha2 as glycoprotein from P. pastoris. The yield of purified extracellular domain of human IL-13Ralpha2 was 2mg from 1L of culture. From CD analysis, the 2D structure of the purified IL-13Ralpha2 showed the typical beta-sheet. ELISA of the purified IL-13Ralpha2 detected the binding activity for human IL-13. Thus, it was found that the active extracellular domain of human IL-13Ralpha2 was expressed from P. pastoris.     

FUNCTIONAL REDUCTION OF THE SOUTHERN MINKE WHALE (BALAENOPTERA ACUTOROSTRATA) TESTIS DURING THE FEEDING SEASON

Seasonal variation in reproductive activity in the male southern minke whales was investigated. The animals were killed and collected during the feeding season (December-March) in the Antarctic Ocean in the eastern part of Area III (35°E-70°E, south of 60°S) and Area IV (70°E-130°E, south of 60°S). Blood samples, testes, epididymides, and vasa deferentia were collected from 62 males, which had testes weighing over 400 g each. Changes in testicular morphology and plasma testosterone, estradiol-17β and LH concentrations measured by enzyme immunoassay were investigated. Reduction of testicular function associated with different capture periods was found in the summer season. From December to March, body length and body weight remained steady, but decreases in testicular weight, epididymal weight, and testicular volume were found in February. During the same period, plasma testosterone concentration also declined. A significant decrease in the number of germ cells continued during the period of feeding season. An increase in area of seminiferous tubule tended to proceed in a number of germ cells. These changes reflected the percentage of spermatozoa in the vasa deferentia ; in particular, motile spermatozoa were observed in December. Based on morphological observation, spermatogenesis had also declined. These results indicate regulation of testicular function by testosterone, as in terrestrial mammals. A gradual decrease of testicular function occurred during December-January.     

Glypican-3, overexpressed specifically in human hepatocellular carcinoma,is a novel tumor marker

With the global pandemic of hepatitis B and C infections, the incidence of Hepatocellular carcinoma (HCC) is rapidly increasing world wide. We identified glypican-3 (GPC3), a novel oncofetal gene over-expressed specifically in human HCC, as based on data of cDNA microarrays. As GPC3 is a GPI-anchored membrane protein and could be secreted, we attempted to detect secreted GPC3 protein in sera from HCC patients using Western blotting and ELISA. GPC3 protein was positive in sera of 40.0% (16/40) of HCC patients, and negative in sera from subjects with liver cirrhosis (LC) (0/13), chronic hepatitis (CH) (0/34), and healthy donors (0/60). All subjects were Japanese. Although 12 of 40 HCC patients were negative for both alpha-fetoprotein (AFP) and PIVKA-II well known tumor markers of HCC, four of these were GPC3-positive in the sera. We also observed vanishing GPC3 protein in the sera of three patients after the surgical treatment for HCC. On the other hand, immunohistochemical analysis revealed that HCC expressed GPC3 protein in all 14 HCC patients tested. In conclusion, GPC3, as defined in this study was shown to be a useful tumor marker for cancer-diagnosis for large numbers of patients with HCC.     

Astrocytic contributions to blood-brain barrier (BBB) formation by endothelial cells: A possible use of aortic endothelial cell for In vitro BBB model

Astrocytic contribution of endothelial cell monolayer permeability was examined in two blood-brain barrier (BBB) models, using the coculture in a double chamber system: rat astrocytes and bovine aortic endothelial cells (BAECs) or bovine brain endothelial cells (BBECs). In system 1, where astrocytes were separated from endothelial cells, a 40% reduction in -glucose permeability of the BBEC monolayer, but not the BAEC monolayer, was observed by cocultivation with astrocytes. Although several passages of BBEC in culture elicited morphological transformation from spindle-shapes to cobblestone-like features, the passaged BBECs remained responsive to astrocytes in coculture in system 1 (37% reduction of the -glucose permeability). By contrast, in system 2, where respective endothelial cells and astrocytes layered on the upper and lower surfaces of a membrane, the permeability of both BAEC and BBEC monolayers was reduced by cocultivation with astrocytes (75% reduction for BAEC and 40% reduction for BBEC). BAECs in this contiguous coculture (system 2) with astrocytes showed numerous tight junction-like structures characteristic of the BBB in vivo. These results suggest that primary cultured BBECs, which had been primed by astrocytes in vivo, retain a higher sensitivity to astrocytes possibly through an astrocytic soluble factor (s) to exhibit BBB-specific phenotypes, and that even BAEC from extra-neural tissues, when cultured with astrocytes in close proximity in vitro, may acquire the similar phenotypes and serve for an extensive use of BBB model in vitro.