Induction of apoptotic change in the rat hippocampus caused by ferric nitrilotriacetate

Iron, a source of oxidative stress, plays a major role in the pathology of neurodegenerative disease. In Alzheimer's disease, the hippocampus is vulnerable to oxidative stress, leading to impairment in memory formation. In our previous study, a brain oxidative reaction was induced after intraperitoneal injection of ferric nitrilotriacetate (Fe-NTA). However, since only a small amount of iron reached the brain in the previous study, Fe-NTA was administered into the hippocampus using an osmotic pump in this study. After continuous injection of Fe-NTA for 2 weeks, a high level of apoptotic change was induced in the hippocampus, in accordance with the iron localization. After injection for 4 weeks, the hippocampus was totally destroyed. A small amount of iron infiltrated into the cerebral cortex and the striatum, and deposition was observed at the choroid plexus and ependymal cells. However, no apoptotic reaction or clear tissue injury was observed in these areas. In addition, muscarinic acetylcholine receptors (M1, M2, and M4) were decreased in both the cortex and hippocampus while it increased in the striatum. Thus, the hippocampus is likely vulnerable to oxidative stress from Fe-NTA, and the oxidative stress is considered to bring the disturbance in the muscarinic acetylcholine receptors.     

Differentiation of embryonic stem cells into fibroblast-like cells in three-dimensional type I collagen gel cultures

Fibroblasts are heterogeneous mesenchymal cells that play important roles in the production and maintenance of extracellular matrix. Although their heterogeneity is recognized, progenitor progeny relationships among fibroblasts and the factors that control fibroblast differentiation are poorly defined. The current study was designed to develop a reliable method that would permit in vitro differentiation of fibroblast-like cells from human and murine embryonic stem cells (ESCs). Undifferentiated ESCs were differentiated into embryoid bodies (EBs) with differentiation media. EBs were then cast into type I collagen gels and cultured for 21?d with basal media. The spindle-shaped cells that subsequently grew from the EBs were released from the gels and subsequently cultured as monolayers in basal media supplemented with serum. Differentiated cells showed a characteristic spindle-shaped morphology and had ultrastructural features consistent with fibroblasts. Immunocytochemistry showed positive staining for vimentin and alpha-smooth muscle actin but was negative for stage-specific embryonic antigens and cytokeratins. Assays of fibroblast function, including proliferation, chemotaxis, and contraction of collagen gels demonstrated that the differentiated cells, derived from both human and murine ESCs, responded to transforming growth factor-β1 and prostaglandin E(2) as would be expected of fibroblasts, functions not expected of endothelial or epithelial cells. The current study demonstrates that cells with the morphologic and functional features of fibroblasts can be reliably derived from human and murine ESCs. This methodology provides a means to investigate and define the mechanisms that regulate fibroblast differentiation.     

Antigen-Specific IgG ameliorates allergic airway inflammation via Fcγ receptor IIB on dendritic cells

Background

There have been few reports on the role of Fc receptors (FcRs) and immunoglobulin G (IgG) in asthma. The purpose of this study is to clarify the role of inhibitory FcRs and antigen presenting cells (APCs) in pathogenesis of asthma and to evaluate antigen-transporting and presenting capacity by APCs in the tracheobronchial mucosa.

Methods

In FcγRIIB deficient (KO) and C57BL/6 (WT) mice, the effects of intratracheal instillation of antigen-specific IgG were analysed using the model with sensitization and airborne challenge with ovalbumin (OVA). Thoracic lymph nodes instilled with fluorescein-conjugated OVA were analysed by fluorescence microscopy. Moreover, we analysed the CD11c⁺ MHC class II⁺ cells which intaken fluorescein-conjugated OVA in thoracic lymph nodes by flow cytometry. Also, lung-derived CD11c⁺ APCs were analysed by flow cytometry. Effects of anti-OVA IgG1 on bone marrow dendritic cells (BMDCs) in vitro were also analysed. Moreover, in FcγRIIB KO mice intravenously transplanted dendritic cells (DCs) differentiated from BMDCs of WT mice, the effects of intratracheal instillation of anti-OVA IgG were evaluated by bronchoalveolar lavage (BAL).

Results

In WT mice, total cells and eosinophils in BAL fluid reduced after instillation with anti-OVA IgG1. Anti-OVA IgG1 suppressed airway inflammation in hyperresponsiveness and histology. In addition, the number of the fluorescein-conjugated OVA in CD11c⁺ MHC class II⁺ cells of thoracic lymph nodes with anti-OVA IgG1 instillation decreased compared with PBS. Also, MHC class II expression on lung-derived CD11c⁺ APCs with anti-OVA IgG1 instillation reduced. Moreover, in vitro, we showed that BMDCs with anti-OVA IgG1 significantly decreased the T cell proliferation. Finally, we demonstrated that the lacking effects of anti-OVA IgG1 on airway inflammation on FcγRIIB KO mice were restored with WT-derived BMDCs transplanted intravenously.

Conclusion

Antigen-specific IgG ameliorates allergic airway inflammation via FcγRIIB on DCs.     

Microenvironmental geometry guides platelet adhesion and spreading: a quantitative analysis at the single cell level

To activate clot formation and maintain hemostasis, platelets adhere and spread onto sites of vascular injury. Although this process is well-characterized biochemically, how the physical and spatial cues in the microenvironment affect platelet adhesion and spreading remain unclear. In this study, we applied deep UV photolithography and protein micro/nanostamping to quantitatively investigate and characterize the spatial guidance of platelet spreading at the single cell level and with nanoscale resolution. Platelets adhered to and spread only onto micropatterned collagen or fibrinogen surfaces and followed the microenvironmental geometry with high fidelity and with single micron precision. Using micropatterned lines of different widths, we determined that platelets are able to conform to micropatterned stripes as thin as 0.6 μm and adopt a maximum aspect ratio of 19 on those protein patterns. Interestingly, platelets were also able to span and spread over non-patterned regions of up to 5 μm, a length consistent with that of maximally extended filopodia. This process appears to be mediated by platelet filopodia that are sensitive to spatial cues. Finally, we observed that microenvironmental geometry directly affects platelet biology, such as the spatial organization and distribution of the platelet actin cytoskeleton. Our data demonstrate that platelet spreading is a finely-tuned and spatially-guided process in which spatial cues directly influence the biological aspects of how clot formation is regulated.     

Post-translational regulation of CND41 protease activity in senescent tobacco leaves

The degradation of chloroplast proteins is an important occurrence in the mobilization of nutrients from senescing leaves to reproductive organs during senescence. Recently, we proved that tobacco CND41 protease is involved in Rubisco degradation and the translocation of nitrogen during senescence. In this study, we show the post-translational regulation of CND41 protease. Using very specific antibodies that were prepared against CND41-specific peptide (anti-Val 186 to Ser 206), immunoblot analysis clearly indicated a change in the accumulation and processing of CND41 during the maturation of leaves in whole plants. The developmental modification of CND41 was also observed in transgenic tobacco with constitutive expression of CND41 under cauliflower mosaic virus 35S promoter. Further studies of seedlings under senescence induced by combined treatment with nitrogen-starvation and high sucrose confirmed that the processing of CND41 was important for protease activity and senescence. A possible mechanism for the regulation of CND41 activity is discussed.     

Carbon cycling and budget in a forested basin of southwestern Hokkaido,northern Japan

Quantification of annual carbon sequestration is very important in order to assess the function of forest ecosystems in combatting global climate change and the ecosystem responses to those changes. Annual cycling and budget of carbon in a forested basin was investigated to quantify the carbon sequestration of a cool-temperate deciduous forest ecosystem in the Horonai stream basin, Tomakomai Experimental Forest, northern Japan. Net ecosystem exchange, soil respiration, biomass increment, litterfall, soil-solution chemistry, and stream export were observed in the basin from 1999–2001 as a part of IGBP-TEMA project. We found that 258 g C m^–2 year^–1 was sequestered annually as net ecosystem exchange (NEE) in the forested basin. Discharge of carbon to the stream was 4 g C m^–2 year^–1 (about 2% of NEE) and consisted mainly of dissolved inorganic carbon (DIC). About 43% of net ecosystem productivity (NEP) was retained in the vegetation, while about 57% of NEP was sequestered in soil, suggesting that the movement of sequestered carbon from aboveground to belowground vegetation was an important process for net carbon accumulation in soil. The derived organic carbon from aboveground vegetation that moved to the soil mainly accumulated in the solid phase of the soil, with the result that the export of dissolved organic carbon to the stream was smaller than that of dissolved inorganic carbon. Our results indicated that the aboveground and belowground interaction of carbon fluxes was an important process for determining the rate and retention time of the carbon sequestration in a cool-temperate deciduous forest ecosystem in the southwestern part of Hokkaido, northern Japan.     

Sericin enhances attachment of cultured human skin fibroblasts

Human skin fibroblasts were cultured on sericin prepared from cocoon shells. The living cell number after 72 h was enhanced to 250% of the no-sericin control. The increase was due to the acceleration of the initial attachment of the cells. It was found that sericin M, the main component of about 400 kDa, and its serine-rich repetitive domain were the active principles.