Inhibitory effects of U73122 and U73343 on Ca2+ influx and pore formation induced by the activation of P2X7 nucleotide receptors in mouse microglial cell line

P2X7 receptors are ATP-gated ion channels and play important roles in microglial functions in the brain. Activation of P2X7 receptors by ATP or its agonist BzATP induces Ca2+ influx from extracellular space, followed by the formation of non-selective membrane pores that is permeable to larger molecules, such as fluorescent dye. To determine whether phospholipase C (PLC) is involved in the activation of P2X7 receptors in microglial cells, U73122, a specific inhibitor of PLC, and its inactive analogue U73343 were examined on ATP and BzATP-induced channel and pore formation in an immortalized C57BL/6 mouse microglial cell line (MG6-1). ATP induced both a transient and a sustained increase in the intracellular Ca2+ concentration ([Ca2+]i) in MG6-1 cells, whereas BzATP evoked only a sustained increase. U73122, but not U73343, inhibited the transient [Ca2+]i increase involving Ca2+ release from intracellular stores through PLC activation. In contrast, both U73122 and U73343 inhibited the sustained [Ca2+]i increase either prior or after the activation of P2X7 receptor channels by ATP and BzATP. In addition, these U-compounds inhibited the influx of ethidium bromide induced by ATP and BzATP, suggesting possible PLC-independent blockage of the process of P2X7-associated channel and pore formations by U-compounds in C57BL/6 mouse microglial cells.     

Characterization of multipotent adult stem cells from the skin: transforming growth factor-beta (TGF-beta) facilitates cell growth

Recently, adult stem cells have been isolated from the skin and designated as skin-derived precursors (SKPs). These SKPs, cultured in vitro, can give rise to neurons, glia, smooth muscle cells, and adipocytes. In the current study, we confirmed the clonal expansion of SKPs using a sphere-forming culture system in a medium containing methylcellulose. Among the growth factors, only transforming growth factor-beta (TGF-beta) was revealed to uniquely facilitate the sphere formation and proliferation of the SKPs in combination with EGF and bFGF. In addition, TGF-beta did not alter phenotypical characteristics of the SKPs under sphere-forming conditions. The effect of TGF-beta on sphere formation was not observed in neural stem cells, which expressed a different set of cell surface markers from SKPs, suggesting that SKPs have distinct features. Although the number of SKPs decreased with age, TGF-beta increased the sphere colony formation and proliferation in all ages. These results suggest that SKPs maintained in the presence of TGF-beta during culture are of potential use in cell-replacement therapies employing adult tissue sources.     

Effects of the supplementation of trehalose extender containing egg yolk with sodium dodecyl sulfate on the freezability of goat spermatozoa

Two experiments were conducted to determine the effect of sodium dodecyl sulfate (SDS) added to a trehalose-egg yolk extender on the cryopreservation of goat spermatozoa. In Experiment 1, semen from four goats was frozen in trehalose extender (osmolality = 370, pH = 7) containing 4 and 20% (v/v) glycerol and egg yolk, respectively, and 0.035-0.2% SDS. After thawing, sperm motility and acrosome integrity were assessed using a computer-assisted sperm analysis (CASA) system and fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Both motility and progressive motility were improved (P < 0.05) by increasing the concentration of SDS in the trehalose-egg yolk extender, with the best results obtained with SDS at 0.1% (80.0 +/- 1.5% and 65.0 +/- 1.7%, respectively). There were no significant differences in path velocity when spermatozoa were frozen in a diluent containing 0.035, 0.05, 0.075, or 0.1% SDS, but path velocity decreased significantly with 0.2% SDS. The percentage of acrosome-intact sperm were highest (P < 0.05) when 0.05% (74.0 +/- 1.1) and 0.075% (70.0 +/- 1.2) SDS were used. In Experiment 2, the effect of diluent storage time (6, 24, or 48 h) before freezing on the cryoprotective effect of SDS was investigated. Prolonged storage of the diluent had slight cryoprotective effects when 0.2% SDS is used, while motility and the acrosome integrity of the cryopreserved spermatozoa improved slightly when the extender was stored for 48 h at 5 degrees C before use. In conclusion, goat sperm freezability was significantly improved when sperm were frozen in a trehalose-egg yolk extender containing an adequate concentration of SDS.     

Central stresscopin modulates cardiovascular function through the adrenal medulla in conscious rats

Stresscopin (SCP or urocortin III), a member of the corticotropin-releasing factor (CRF) neuropeptide family, is a high-affinity ligand for the type 2 CRF receptor (CRF(2)). When administered peripherally, SCP suppresses food intake, delays gastric emptying and decreases heat-induced edema. Central administration of CRF produces marked hypertension and increased plasma catecholamine. However, the effects of SCP on the cardiovascular system are unknown. Thus, the present study compared the effects of intracerebroventricular (i.c.v.) administration of CRF and SCP on cardiovascular function. Central administration of SCP (0.05 or 0.5 nmol) elicited transient increases in mean arterial blood pressure (MABP) and heart rate (HR), and the higher dose of SCP (0.5 nmol) resulted in increased plasma epinephrine. In contrast, central administration of CRF provoked long-lasting increases in MABP, HR and plasma catecholamine levels (norepinephrine and epinephrine). Intravenously administered CRF and SCP (0.5 nmol) did not elicit significant changes in MABP and HR. Therefore, these data suggest that centrally administered SCP modulates cardiovascular function, likely through the sympatho-adrenal-medullary (SAM) system.     

Co-treatment with dexamethasone and octanoate induces adipogenesis in 3T3-L1 cells

We report here that octanoate, a medium chain fatty acid, induces adipocyte differentiation in 3T3-L1 cells by co-treatment with dexamethasone, although octanoate has been known not to stimulate 3T3-L1 adipogenesis. A low concentration of exogenous glucose prevented 3T3-L1 adipogenesis induced by 1-methyl 3-isobutylxanthine, dexamethasone, and insulin (MDI) treatment (a common protocol for adipocyte differentiation). In contrast, co-treatment with dexamethasone and octanoate (D-OCT) induced adipogenesis under the same conditions. These findings imply that octanoate, rather than glucose, is the source of accumulated lipids in D-OCT-induced adipogenesis. D-OCT increased expression of the differentiation markers peroxisome proliferator-activated receptor (PPAR)gamma2 and caveolin-1. A specific inhibitor of p38 mitogen-activated protein (MAP) kinase inhibited D-OCT-induced adipogenesis. These results suggest that the p38 MAP kinase pathway followed by up-regulation of PPARgamma2 may be involved in 3T3-L1 adipocyte differentiation induced by D-OCT, as well as by MDI.     

Prion Replication Elicits Cytopathic Changes in Differentiated Neurosphere Cultures

The molecular mechanisms of prion-induced cytotoxicity remain largely obscure. Currently, only a few cell culture models have exhibited the cytopathic changes associated with prion infection. In this study, we introduced a cell culture model based on differentiated neurosphere cultures isolated from the brains of neonatal prion protein (PrP)-null mice and transgenic mice expressing murine PrP (dNP0 and dNP20 cultures). Upon exposure to mouse Chandler prions, dNP20 cultures supported the de novo formation of abnormal PrP and the resulting infectivity, as assessed by bioassays. Furthermore, this culture was susceptible to various prion strains, including mouse-adapted scrapie, bovine spongiform encephalopathy, and Gerstmann-Sträussler-Scheinker syndrome prions. Importantly, a subset of the cells in the infected culture that was mainly composed of astrocyte lineage cells consistently displayed late-occurring, progressive signs of cytotoxicity as evidenced by morphological alterations, decreased cell viability, and increased lactate dehydrogenase release. These signs of cytotoxicity were not observed in infected dNP0 cultures, suggesting the requirement of endogenous PrP expression for prion-induced cytotoxicity. Degenerated cells positive for glial fibrillary acidic protein accumulated abnormal PrP and exhibited features of apoptotic death as assessed by active caspase-3 and terminal deoxynucleotidyltransferase nick-end staining. Furthermore, caspase inhibition provided partial protection from prion-mediated cell death. These results suggest that differentiated neurosphere cultures can provide an in vitro bioassay for mouse prions and permit the study of the molecular basis for prion-induced cytotoxicity at the cellular level.     

Hedgehog-Gli Activators Direct Osteo-chondrogenic Function of Bone Morphogenetic Protein toward Osteogenesis in the Perichondrium

Specification of progenitors into the osteoblast lineage is an essential event for skeletogenesis. During endochondral ossification, cells in the perichondrium give rise to osteoblast precursors. Hedgehog (Hh) and bone morphogenetic protein (BMP) are suggested to regulate the commitment of these cells. However, properties of perichondrial cells and regulatory mechanisms of the specification process are still poorly understood. Here, we investigated the machineries by combining a novel organ culture system and single-cell expression analysis with mouse genetics and biochemical analyses. In a metatarsal organ culture reproducing bone collar formation, activation of BMP signaling enhanced the bone collar formation cooperatively with Hh input, whereas the signaling induced ectopic chondrocyte formation in the perichondrium without Hh input. Similar phenotypes were also observed in compound mutant mice, where signaling activities of Hh and BMP were genetically manipulated. Single-cell quantitative RT-PCR analyses showed heterogeneity of perichondrial cells in terms of natural characteristics and responsiveness to Hh input. In vitro analyses revealed that Hh signaling suppressed BMP-induced chondrogenic differentiation; Gli1 inhibited the expression of Sox5, Sox6, and Sox9 (SRY box-containing gene 9) as well as transactivation by Sox9. Indeed, ectopic expression of chondrocyte maker genes were observed in the perichondrium of metatarsals in Gli1^−/− fetuses, and the phenotype was more severe in Gli1^−/−;Gli2^−/− newborns. These data suggest that Hh-Gli activators alter the function of BMP to specify perichondrial cells into osteoblasts; the timing of Hh input and its target populations are critical for BMP function.