A noninvasive transfer system for polarized renal tubule epithelial cell sheets using temperature-responsive culture dishes

We used temperature-responsive culture dishes onto which the temperature-responsive polymer, poly(Nisopropylacrylamide), was covalently grafted for tissue engineering. Confluent cells harvested as intact sheets from these surfaces by simple temperature reduction can be transferred to various surfaces including additional culture dishes, other cell sheets, and tissues. In order to examine the maintenance of cell polarity, Madin-Darby canine kidney cells and human primary renal proximal tubule epithelial cells which had developed apical-basal cell polarity in culture, were subjected to cell sheet transfer. This functional and structural cell polarity, which is susceptible to treatment with trypsin, was examined by immunohistochemistry and transmission electron microscopy. Using our cell-sheet method, the noninvasive transfer of these cell sheets retaining typical distributions of Na+/K+-ATPase, GLUT-1, SGLT-1, aquaporin-1, neutral endopeptidase and dipeptidylendopeptidase IV, could be achieved. The transferred cell sheets also developed numerous microvilli and tight junctions at the apical and lateral membranes, respectively. For biochemical analysis, immunoblotting of occludin, a transmembrane protein that composes tight junctions, was conducted and results confirmed that occludin remained intact after cell sheet transfer. This two-dimensional cell sheet manipulation method promises to be useful for tissue engineering as well as in the investigation of epithelial cell polarity

The Legume–Rhizobia Symbiosis

The symbiotic nitrogen fixation (SNF) with legumes is the primary source of biologically fixed nitrogen for agricultural system. It is performed by a group of bacteria commonly called rhizobia. It is characterized by a host preference, and the differences among symbioses between rhizobial strains and legume genotypes are related to infection, nodule development and effectiveness in N2 fixation. The interaction between a rhizobia and the legume is mediated by a lipochitin oligosaccharide secreted by the rhizobia, and called “Nod factor”. It is recognized by transmembrane receptors on the root-hair cells of the legume. It can regulate the nodule organogenesis by inducing changes in the cytokinin balance of the root, during nodule initiation. N2 fixation in legume nodules is catalyzed by the nitrogenase enzyme depending upon the photosynthate supply, the O2 concentration, and the fixed-N export. Among environmental factors that influence the SNF, the temperature is essential for nodule formation; the salinity and drought decrease the nodule permeability to O2 and the photosynthate supply to the nodule, the phosphorus deficiency inhibits the nodule development and the total N2 fixation. Rhizobia strains differ in their efficiency in N2 fixation with host legume. There is evidence of genotypic variability for SNF at different levels of available P which show a possibility of selecting cultivars able to support biological N2 fixation under low P soils