Anti-apoptotic roles of prostaglandin E2 and F2alpha in bovine luteal steroidogenic cells

Production of prostaglandins (PGs) and expression of their receptors have been demonstrated in bovine corpus luteum (CL). The aim of the present study was to determine whether PGE2 and PGF2alpha have roles in bovine luteal steroidogenic cell (LSC) apoptosis. Cultured bovine LSCs obtained at the midluteal stage (Days 8-12 of the cycle) were treated for 24 h with PGE2 (0.001-1 microM) and PGF2alpha (0.001-1 microM). Prostaglandin E2 (1 microM) and PGF2alpha (1 microM) significantly stimulated progesterone (P4) production and reduced the levels of cell death in the cells cultured with or without tumor necrosis factor alpha (TNF)/interferon gamma (IFNG), in the presence and absence of FAS ligand (P < 0.05). Furthermore, DNA fragmentation induced by TNF/IFNG was observed to be suppressed by PGE2 and PGF2alpha. Prostaglandin E2 and PGF2alpha also attenuated mRNA expression of caspase 3 and caspase 8, as well as caspase 3 activity (P < 0.05) in TNF/IFNG-treated cells. FAS mRNA and protein expression were decreased only by PGF2alpha (P < 0.05). A specific P4 receptor antagonist (onapristone) attenuated the apoptosis-inhibitory effects of PGE2 and PGF2alpha in the absence of TNF/IFNG (P < 0.05). A PG synthesis inhibitor (indomethacin) reduced cell viability in PGE2- and PGF2alpha-treated cells (P < 0.05). A specific inhibitor of cyclooxygenase (PTGS), PTGS2 (NS-398), also reduced cell viability, whereas an inhibitor of PTGS1 (FR122047) did not affect it. The overall results suggest that PGE2 and PGF2alpha locally play luteoprotective roles in bovine CL by suppressing apoptosis of LSCs.     

In vivo influence of the anti-B220 monoclonal antibody administration of the T cell differentiation in mice

B220, a pan-B marker, is known to be also expressed on immature T cells of MRL/1pr or other congenic 1pr mice and minor population of immature thymocytes but not on peripheral T cells. In this study, we investigated in vivo the possibility whether B220 is one kind of premature T or prethymic T precursor cell marker as well as a pan-B cell marker. A monoclonal antibody against B220 glycoprotein was intravenously injected every 2 days into various strains of mice. After the third administration of this antibody, thymocytes decreased remarkably compared with those from the rat IgG-treated group, and spleen cells were also reduced significantly. Further, the number of cells expressing Thy-1, Ly-1, Lyt-2, and asialo GM1 (asGM1) in the spleen were significantly reduced. On the contrary, the number of cells expressing surface IgM (sIgM) or B220 were increased by this treatment, especially after the 8th treatment. Some T-dependent immunological functions including mitogenic responses to lectins and cytotoxic T cell activity were markedly suppressed but mixed lymphocyte reaction (MLR) and natural killer (NK) activity were rather augmented. Thus, B220 may be expressed on some kinds of T cell progenitor. Taken together, in vivo treatment with anti-B220 antibody may influence differentiating stages of some T cells from bone marrow progenitors before or just after their homing into the thymus.     

Tumor necrosis factor alpha receptors in microvascular endothelial cells from bovine corpus luteum.

There is sufficient evidence to prove that tumor necrosis factor alpha (TNFalpha) modulates bovine corpus luteum (CL) function. Our previous study demonstrated that functional TNFalpha receptors are present on luteal cells in bovine CL throughout the estrous cycle. The purpose of the present study was to identify the presence of functional TNFalpha receptors on the microvascular endothelial cells derived from developing bovine CL. TNFalpha receptors were analyzed by a radioreceptor assay using (125)I-labeled TNFalpha on two types of cultured endothelial cells. One has a cobblestone appearance (CS cells), and the other has a tube-like structure (TS cells). (125)I-Labeled TNFalpha binding was maximal after incubation for 30 h at 37 degrees C, and the specificity of binding was confirmed. A Scatchard analysis showed the presence of two binding sites (high- and low-affinity) for TNFalpha receptors on both CS and TS cells. The dissociation constant (K(d)) values and concentrations of the high-affinity binding sites for TNF receptors were similar for CS and TS cells. However, K(d) values and concentrations of the low-affinity binding sites in CS cells were significantly higher than those in TS cells (P < 0.05 or lower). The expression of TNF receptor type 1 (TNF-RI) mRNA was determined in both cell types. Furthermore, TNFalpha significantly stimulated prostaglandin E(2) and endothelin-1 secretion by both CS and TS cells (P < 0.05 or lower). These results indicate the presence of two types of TNF receptors and the expression of TNF-RI mRNA in the endothelial cells derived from bovine CL, and suggest that TNFalpha plays two or more roles in regulating the secretory function of the endothelial cells.     

Ultrastructure of mucous cells from the oviduct epithelium of the rabbit (Lepus cuniculus). Preliminary study]

Authors studied the ultrastructural features of the mucous cells present in the three segments of the rabbit oviduct in anoestrous. Results showed that only one kind of mucous cell was present in the isthmus while two different kinds of mucous cells were found in the ampulla and infundibulum. The ultrastructural features observed in the isthmic cells correlated well with the histochemical data already described in that segment. However such correlations could not be made between the ampulla and infundibulum. Authors suggest that the ampulla can be considered a transitional segment between isthmus and infundibulum.     

Sodium induces oxytocin release from superfused rat pituitary

The effects of various osmotic agents on the release of oxytocin were examined in a superfusion system. Oxytocin was released significantly from the rat pituitary by superfusion with medium of an osmolality of 350 mOsm/kg H2O adjusted with NaCl, regardless of the presence of the rat hypothalamus. Media adjusted to an osmolality of 350 mOsm/kg H2O with sucrose, glucose, urea or mannitol had no effect on oxytocin release from the hypothalamo-pituitary complex. Medium containing excess Na2SO4 induced significant release of oxytocin from the pituitary without the hypothalamus. The administration of tetraethylammonium chloride had no oxytocin secretion. These data suggest that oxytocin release from the pituitary is influenced by the level of sodium ion rather than the osmotic pressure.     

Establishment and characterization of the NCC–SS1–C1 synovial sarcoma cell line

Synovial sarcoma is an aggressive mesenchymal malignancy characterized by unique gene fusions. Tissue culture cells are essential tools for further understanding tumorigenesis and anti-cancer drug development; however, only a limited number of well-characterized synovial sarcoma cell lines exist. Thus, the objective of this study was to establish a patient-derived synovial sarcoma cell line. We established a synovial sarcoma cell line from tumor tissue isolated from a 72-year-old female patient. Prepared cells were analyzed for the presence of gene fusions by fluorescence in situ hybridization, RT-PCR, and karyotyping. In addition, the resulting cell line was characterized by viability, short tandem repeat, colony and spheroid formation, and invasion analyses. Differences in gene enrichment between the primary tumor and cell line were examined by mass spectrometric protein expression profiling and KEGG pathway analysis. Our analyses revealed that the primary tumor and NCC–SS1–C1 cell line harbored the SS18–SSX1 fusion gene typical of synovial sarcoma and similar proteomics profiles. In vitro analyses also confirmed that the established cell line harbored invasive, colony-forming, and spheroid-forming potentials. Moreover, drug screening with chemotherapeutic agents and tyrosine kinase inhibitors revealed that doxorubicin, a subset of tyrosine kinase inhibitors, and several molecular targeting drugs markedly decreased NCC–SS1–C1 cell viability. Results from the present study support that the NCC–SS1–C1 cell line will be an effective tool for sarcoma research.     

Establishment of a novel patient-derived Ewing’s sarcoma cell line,NCC-ES1-C1

Ewing’s sarcoma is an aggressive mesenchymal tumor characterized by the presence of a unique EWSR1-FLI1 translocation. Ewing’s sarcoma primarily occurs in the bone and soft tissues. Cell lines enable researchers to investigate the molecular backgrounds of disease and the significance of genetic alterations in relevant cellular contexts. Here, we report the establishment and characterization of a novel Ewing’s sarcoma cell line following primary Ewing’s sarcoma tumor tissue culture. The established cell line was authenticated by DNA microsatellite short tandem repeat analysis, characterized by in vitro assays, and named NCC-ES1-C1. The NCC-ES1-C1 cell line grew well for 15 mo and was subcultured more than 50 times during this period. Characterization of the cells revealed that they were not adherent and showed floating features. In conclusion, we successfully established a novel Ewing’s sarcoma cell line, NCC-ES1-C1, from primary tumor tissue. The cell line has the characteristic EWSR1-FLI1 gene fusion and exhibits aggressive growth in vitro. Thus, the NCC-ES1-C1 cell line will be a useful tool for investigating the mechanisms of disease and the biological role of the EWSR1-FLI1 fusion gene.     

Demonstration of a different localization of perikarya immunoreactive to oxytocin and vasopressin in the cat hypothalamus

Using immunohistochemical techniques, we demonstrated oxytocin (OT) and vasopressin (AVP) neurons in the cat hypothalamus. The OT immunoreactive neurons were found mainly in the paraventricular nucleus, supraoptic nucleus and dorsal accessory group located lateral to the fornix. In addition to these hypothalamic structures, the AVP immunoreactive neurons were observed in the suprachiasmatic nucleus, ventral accessory group located in the retrochiasmatic area and lateral accessory group, dorsal to the supraoptic nucleus caudally, and ventral to the medial part of the internal capsule rostrally. We further demonstrated a different localization of the OT and AVP immunoreactive neurons in the paraventricular and supraoptic nuclei.