In order to confirm the relationship between glutathione-peroxidase (GPx1) and biological significance on steroidogenesis, we have studied the immunocytochemical localization of GPx1 in the rat adrenal cortical cells. GPx1 was observed not only in cytoplasm (cytosol GPx1) but in mitochondria (mitochondrial GPx1). The staining intensity was altered by the functional state of the adrenal cortical cells. Furthermore, cytosol- and mitochondrial-GPx1 was modified by lipoperoxidative damage in the adrenal cortical cells. Therefore, we proposed that the pattern of GPx1 staining should be a more sensitive and specific indicator of oxidative damage in cells. Thus, the staining pattern of GPx1 is thought to be a useful marker for lipid peroxidation in the adrenal cortical cells.
After publication of reports describing the presence of stem/progenitor cells among non-hormone-producing cells in the pituitary, the mechanism responsible for proliferation and differentiation generated considerable interest. Several studies have suggested that Notch signaling is involved. In the present study, we examined the histochemical relationship between Notch signaling molecules and the transcription factor SOX2 in rat pituitary. Combined in situ hybridization and immunohistochemistry showed that Notch2 mRNA and SOX2 were co-expressed at embryonic day 14.5 in most cells in the adenohypophyseal primordium. In adult rat pituitary, double immunohistochemistry showed that SOX2 and either Notch2 or the Notch signaling target HES1 were co-localized within cells with large oval nuclei in both the marginal cell layer and cell aggregates in the main part of the anterior lobe, which are believed to be stem cell niches. Furthermore, when the Notch signaling inhibitor DAPT was added to a primary culture of adult rat anterior pituitary cells, the proportion of SOX2-expressing cells within Notch2-positive cells was approximately 30% lower. These findings suggest that Notch signaling has a role in maintaining the stemness of precursor cells in the adult rat pituitary gland.
We used suncus (Suncus murinus; house musk shrew) to generate partner cells for cell fusion to produce suncus monoclonal antibodies. Suncus are insectivores that are genetically distant to rodents, and recognize antigens and epitopes that are not immunogenic in mice and rats, which are the animals most commonly used in basic life science research and from which monoclonal antibodies are usually produced. To date, monoclonal antibodies from suncus have not been generated due to the lack of a plasmacytoma fusion partner. To obtain suncus plasmacytoma cell lines suitable as a cell fusion partner, we injected suncus at both sides of the tail base with antigen emulsion, collected the lymph nodes and spleens, and cultured the cells to obtain immortalized lymphoid cell lines visually resembling mouse SP2/0-Ag14 myeloma cells. Three suncus immunized with the antigen provided 4 cell lines of suncus plasmacytoma, but they did not secrete immunoglobulins. Antibody-producing hybrid cells were generated from these cell lines using a cell fusion technique. Using one of the cell lines as a fusion partner, we obtained six lines of immunoglobulin-producing hybrid cells which secreted an unidentified monoclonal IgG. When these 6 lines were used as new fusion partners, we obtained several hybrid cell lines which secreted immunogen-specific monoclonal antibodies. These hybrid cells can be cloned and cryopreserved. We also obtained another good fusion partner which initially secreted antibody but later stopped doing so. These suncus-suncus hybrid cell lines will be useful for the production of suncus monoclonal antibodies.
The estrogen receptor (ER) functions as a dimer and is involved in several different biological functions. However ER dimeric proteins have not been identified by in situ methodologies. Structured illumination microscopy (SIM) has been recently developed, which enabled the localization of protein and protein interaction. Therefore, in this study, we firstly demonstrated that ERs formed both homodimers and heterodimers in breast carcinoma cell lines using Nikon’s SIM (N-SIM). ERα/α homodimers were detected in the nuclei of both ERα-positive MCF-7 and T-47D cells; 23.0% and 13.4% of ERα proteins formed ERα/α homodimers, respectively. ERα/β heterodimers were also detected in MCF-7 and T-47D. Approximately 6.6% of both ERα and ERβ1 proteins formed ERα/β1 heterodimers in MCF-7. In addition, 18.1% and 22.4% of ERα and ERβ proteins formed ERα/β2 heterodimers and ERα/β5 heterodimers in MCF-7, respectively. In addition, by using proximity ligation assay (PLA) in MCF-7, estradiol-induced ERα/α homodimers and ERα/β1 heterodimers were both detected after 15 to 45 min of treatment and at 15 min, respectively. The percentage of total ER proteins could also be determined using N-SIM. By using both methods, it has become possible to evaluate precise localization and ratio of ER dimers among different cell types.
Both prokineticin receptor 2 (pkr2) and prokineticin 2 (pk2) gene-deficient mice have hypoplasia of the main olfactory bulb (MOB). This hypoplasia has been attributed to disruption of the glomerulus that is caused by loss of afferent projection from olfactory sensory neurons (OSN), and to the impaired migration of granule cells, a type of interneuron. In the present study, we examined whether migration of the second type of interneuron, periglomerular cells (PGC), is dependent on the pkr2 expression by observing the localization of distinct subpopulations of PGC: calretinin (CR)-, calbindin (CB)- and tyrosine hydroxylase (TH)-expressing neurons. In the Pkr2−/− mice, the construction of the layered structure of the MOB was partially preserved, with the exception of the internal plexiform layer (IPL) and the glomerular layer (GL). In the outermost layer of the MOB, abundant CR- and CB-immunopositive neurons were observed in the hypoplastic olfactory bulb. In addition, although markedly decreased, TH-immunopositive neurons were also observed in the outermost cell-dense region in the Pkr2−/−. The findings suggest that the migration of PGC to the MOB, as well as the migration from the core to the surface region of the MOB, is not driven by the PK2-PKR2 system.
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