Although the hypothalamus is classified into more than 10 compartments, it still contains uncharacterized areas. In this study, we identified a new triangular-shaped area between the paraventricular hypothalamic nucleus (PVN) and the fornix area in the mouse anterior hypothalamus, which is enriched in chondroitin sulfate proteoglycans (CSPGs). We designated this region as the perifornical area of the anterior hypothalamus (PeFAH) based on its anatomical location. As evidenced by Nissl staining, the PeFAH was distinguishable as an area of relatively low density. Immunohistochemical and DNA microarray analyses indicated that PeFAH contains sparsely distributed calretinin-positive neurons and densely clustered enkephalin-positive neurons. Furthermore, the PeFAH was shown to have bidirectional neural connections with the lateral septum. Indeed, we confirmed enkephalinergic projections from PeFAH neurons to the lateral septum, and inversely, calbindin-positive lateral septum neurons as afferents to the PeFAH. Finally, c-Fos expression analysis revealed that the activity of certain PeFAH neuronal populations tended to be increased by psychological stressors, but not that of enkephalinergic neurons. We proposed PeFAH as a new region in the AH.
Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets.
Estrogen affects mitochondrial function in various tissues, but the precise mechanism remains unclear. We, therefore investigated the effect on estrogen-regulated mitochondrial morphology by dynamin-related protein 1 (Drp1) and its Ser616-phosphorylated derivative (pDrp1Ser616) are involved in mitochondrial fission. MCF7 human breast cancer cells were treated with 17β-estradiol (E2), an estrogen receptor (ER) α and β antagonist (ICI 182, 780), an ERα antagonist (MPP), and an ERβ antagonist (PHTPP) for 24 hr. The expression of Drp1 and pDrp1Ser616 was analyzed by western blotting and immunohistochemistry. Mitochondrial morphology was analyzed by transmission electron microscopy (TEM). In control cells, Drp1 was detected in the cytoplasm of all cells while pDrp1 was observed in the cytoplasm of 3.4 ± 1.0% of the total population. After E2 treatment, pDrp1Ser616-positive cells comprised 30.6 ± 5.6% of the total population, 10.5 ± 1.7% after E2 + ICI treatment, 12.4 ± 4.2% after E2 + MPP treatment, and 24.0 ± 2.2% after E2 + PHTPP treatment. In ERα knockdown MCF7 cells, pDrp1 expression was decreased after E2 treatment compared to E2-treated wild type cells. Tubular pattern mitochondria were found in the control cells but the number of short and small pattern mitochondria (< 0.5 μm2) was significantly increased after E2 treatment (as observed by TEM). We, therefore concluded that the phosphorylation of Drp1 is important for E2-dependent mitochondrial morphological changes through ERα.
Inflammatory bowel disease (IBD) is an inflammatory disorder of the gastrointestinal tract that is caused by multiple factors, including dysfunction of the immune system and genetic and epigenetic alterations. Aberrant epigenetic regulation, especially histone acetylation, was found in biopsies from IBD patients and mouse models of colitis, suggesting that an epigenetic treatment approach may be useful for IBD therapy. Therefore, we investigated the effects of the histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA), in a mouse model of dextran sulfate sodium (DSS)-induced colitis. C57BL/6 mice were treated with 1.5% DSS for 5 days and/or SAHA (25 mg/kg BW/day) for 26 days. Levels of mRNA for the pro-inflammatory cytokines, interleukin (IL)-6 and tumor necrosis factor (TNF)-α, and the chemokines, Ccl2, were examined by qRT-PCR. CD11b, a marker of dendritic cells, macrophages, and monocytes, as well as Ccl2 expression, were examined by immunohistochemistry. IL-6, TNF-α, and Ccl2 gene expression peaked on day 5 in DSS-treated mouse colon, whereas SAHA treatment significantly decreased pro-inflammatory gene expression. Ccl2 protein expression resembled Ccl2 gene expression results. Moreover, localization of CD11b showed that migratory inflammatory cells were dramatically decreased by SAHA treatment compared to DSS-treated mouse colon. Thus, we conclude that the HDAC inhibitor, SAHA, attenuates inflammatory changes in DSS-induced colitis by suppressing local secretion of pro-inflammatory cytokines and chemokines and also by suppressing mobilization and accumulation of inflammatory cells.
This study investigates the significance of the expression and dynamics of podoplanin in mechanostress and mineralization in cultured murine osteoblasts. Podoplanin increased in osteoblasts subjected to straining in non-mineralization medium, suggesting that the mechanostress alone is a podoplanin induction factor. In osteoblasts subjected to vertical elongation straining in the mineralization medium, the mRNA amounts of podoplanin, osteopontin, and osteocalcin were significantly larger than those in cells not subjected to straining, suggesting that mechanostress is the cause of a synergistic effect in the expression of these proteins. In osteoblasts in the mineralization medium, significant increases in osteocalcin mRNA occurred earlier in cells subjected to straining than in the cells not subjected to straining, suggesting that the mechanostress is a critical factor to enhance the expression of osteocalcin. Western blot and ELISA analysis showed increased podoplanin production in osteoblasts with longer durations of straining. There was significantly less mineralization product in osteoblasts with antibodies for podoplanin, osteopontin, and osteocalcin. There was also less osteopontin and osteocalcin produced in osteoblasts with anti-podoplanin. These findings suggest that mechanostress induces the production of podoplanin in osteoblasts and that podoplanin may play a role in mineralization in cooperation with bone-associated proteins.
Guidance molecules, such as Netrin-1, and their receptors have important roles in controlling axon pathfinding, modulate biological activities of various cancer cells, and may be a useful target for cancer therapy. Dorsal repulsive axon guidance protein (Draxin) is a novel guidance molecule that binds not only common guidance molecule receptors with Netrin-1, but also directly binds the EGF domain of Netrin-1 through a 22-amino-acid peptide (22aa). By immunostaining, Draxin was positively expressed in small cell carcinoma, adenocarcinoma (ADC), and squamous cell carcinoma of the lung. In addition, western blot analysis revealed that Draxin was expressed in all histological types of lung cancer cell lines examined. Knockdown of Draxin in an ADC cell line H358 resulted in altered expression of molecules associated with proliferation and apoptosis. The Ki-67 labeling index of Draxin-knockdown ADC cells was increased compared to that of control ADC cells. In H358 cells, treatment of 22aa induced phosphorylation of histone H3, but did not change apoptosis-associated enzymes. These data suggest that Draxin might be involved in cell proliferation and apoptosis in lung adenocarcinoma cells.