Characterization of a novel murine preadipocyte line,AP‐18, isolated from subcutaneous tissue: Analysis of adipocyte‐related gene expressions

Adipocyte lines are a useful tool for adipocyte research. Recently, a new preadipocyte line designated AP‐18 was established from subcutaneous tissue of the C3H/He mouse. In this study, we further characterized AP‐18 cells. Adipocyte differentiation was assessed by accumulation of fat droplets stained by Oil Red O. The expression of the preadipocyte‐ or adipocyte‐specific genes and adipocytokine genes was analysed qualitatively by RT‐PCR and quantitatively by real‐time PCR in comparison with the LM cell, a murine fibroblast line, and the 3T3‐L1 cell, respectively. AP‐18 cells were fibroblastoid in maintenance culture. After the confluence, fat droplets were accumulated in 50–60% of the cells cultured in the medium alone and in 70–90% of the cells cultured with insulin within 2 to 3 weeks. The fat accumulation was not promoted by the addition of dexamethazone, IBMX (3‐isobutyl‐1‐methylxanthine) or troglitazone in combination with insulin, which were obligatory for differentiation of the 3T3‐L1 cell, a murine preadipocyte line. Throughout the differentiation, AP‐18 cells expressed Pref‐1, LPL, C/EBPβ, C/EBPδ, RXRα, C/EBPα, PPARγ, RXRγ, aP2, GLUT4, SCD1, UCP2, UCP3, TNFα, resistin, leptin, adiponectin and PAI‐1 genes, but not the UCP1 gene, indicating that the cell is derived from WAT (white adipose tissue). The time course of these gene expressions was similar to that of 3T3‐L1 cells, although the expressions were slower and lower in AP‐18 cells. These data indicate that AP‐18 cells are preadipocytes originated from WAT and differentiate into adipocytes under more physiological conditions than 3T3‐L1 cells. AP‐18 may be useful in adipocyte research.     

Synthesis and evaluation of the anti-hepatitis B virus activity of 4′-Azido-thymidine analogs and 4′-Azido-2′-deoxy-5-methylcytidine analogs: structural insights for the development of a novel anti-HBV agent

Abstract

Hepatitis B virus (HBV) infection is a major worldwide health problem that requires the development of improved antiviral therapies. Here, a series of 4′-Azido-thymidine/4′-Azido-2′-deoxy-5-methylcytidine derivatives (6, 10–15) were synthesized, and their anti-HBV activities evaluated. Compounds 10–15 were synthesized via an S_NAr reaction of 18, in which the 4-position of the thymine moiety was activated as the 2,4,6-triisopropylbenzenesulfonate. Compounds 11–15 showed no antiviral activity. However, 4′-Azido thymidine (6) and 4′-Azido-2′-deoxy-5-methylcytidine (10) displayed significant anti-HBV activity (EC₅₀ = 0.63 and 5.99?µM, respectively) with no detectable cytotoxicity against MT-2 cells up to 100?µM.     

Neonatal estrogenization leads to increased expression of cellular retinol binding protein 2 in the mouse reproductive tract

Exposure to estrogenic substances during a time window, the so-called critical period, in perinatal life causes an irregular development of the genital tract that leads to ovary-independent proliferation and cornification in the vaginal epithelium in mice. We have previously demonstrated that retinol inhibits the irreversible effects of estrogen on the vagina. Here, mice kept in a vitamin-A-deficient condition during perinatal life were shown to be more sensitive to the harmful effects of estrogen. In addition, expression of mRNA for retinol binding protein type 2 (CRBP2), a small intestine-specific cytosolic protein that captures intracellular retinal and retinol, was detected in the vaginal epithelium. Induction of increased expression of CRBP2 mRNA by estrogen was also evident in the uterus and epididymis. Both estradiol-17 and diethylstilbestrol markedly increased the tissue content of CRBP2 mRNA in the vagina and uterus during the neonatal critical period but not after 15 days of age. These results taken together imply that estrogen disrupts the local vitamin A balance by an induction of CRBP2 gene expression in the epithelium in the developing mouse genital tract, and that retinoid imbalance may contribute to the genesis of irreversible effects of estrogen on the vagina.This work was supported by Grants-in-Aid for Priority Area B (to T.M. and M.M.) and for Young Scientists (to M.M.) from the Ministry of Culture, Sports, Science and Technology, Japan     

Tacrolimus potently inhibits human osteoclastogenesis induced by IL-17 from human monocytes alone and suppresses human Th17 differentiation

Tacrolimus (FK506, Prograf?) is an orally available, T cell specific and anti-inflammatory agent that has been proposed as a therapeutic drug in rheumatoid arthritis (RA) patients. It has been known that T cells have a critical role in the pathogenesis of RA. Recent studies suggest that Th17 cells, which mainly produce IL-17, are involved in many autoimmune inflammatory disease including RA. The present study was undertaken to assess the effect of tacrolimus on IL-17-induced human osteoclastogenesis and human Th17 differentiation. Human CD14(+) monocytes were cultured in the presence of macrophage-colony stimulating factor (M-CSF) and IL-17. From day 4, tacrolimus was added to these cultures. Osteoclasts were immunohistologically stained for vitronectin receptor 10days later. IL-17 production from activated T cells stimulated with IL-23 was measured by enzyme-linked immunosorbent assay (ELISA). Th17 differentiation from na?ve T cells was assayed by flow cytometry. Tacrolimus potently inhibited IL-17-induced osteoclastogenesis from human monocytes and osteoclast activation. Addition of tacrolimus also reduced production of IL-17 in human activated T cells stimulated with IL-23. Interestingly, the population of human IL-17(+)IFN-γ(-) CD4 T cells or IL-17(+)TNF-α(+) CD4 T cells were decreased by adding of tacrolimus. The present study demonstrates that the inhibitory effect of tacrolimus on IL-17-induced osteoclastogenesis from human monocytes. Tacrolimus also inhibited expression of IL-17 or TNF-α by reducing the proportion of Th17, suggesting that therapeutic effect on Th17-associated disease such as RA, inflammatory bowel disease, multiple sclerosis, psoriasis, or allograft rejection.     

Semaphorin 4D/Plexin-B1-mediated R-Ras GAP activity inhibits cell migration by regulating beta(1) integrin activity

Plexins are cell surface receptors for semaphorins and regulate cell migration in many cell types. We recently reported that the semaphorin 4D (Sema4D) receptor Plexin-B1 functions as a GTPase-activating protein (GAP) for R-Ras, a member of Ras family GTPases implicated in regulation of integrin activity and cell migration. We characterized the role of R-Ras downstream of Sema4D/Plexin-B1 in cell migration. Activation of Plexin-B1 by Sema4D suppressed the ECM-dependent R-Ras activation, R-Ras-mediated phosphatydylinositol 3-kinase activation, and beta(1) integrin activation through its R-Ras GAP domain, leading to inhibition of cell migration. In addition, inactivation of R-Ras by overexpression of the R-Ras-specific GAP or knockdown of R-Ras by RNA interference was sufficient for suppressing beta(1) integrin activation and cell migration in response to the ECM stimulation. Thus, we conclude that R-Ras activity is critical for ECM-mediated beta(1) integrin activation and cell migration and that inactivation of R-Ras by Sema4D/Plexin-B1-mediated R-Ras GAP activity controls cell migration by modulating the activity of beta(1) integrins.     

Phospholipase Cdelta4 is required for Ca2+ mobilization essential for acrosome reaction in sperm

Zona pellucida (ZP)-induced acrosome reaction in sperm is a required step for mammalian fertilization. However, the precise mechanism of the acrosome reaction remains unclear. We previously reported that PLCdelta4 is involved in the ZP-induced acrosome reaction in mouse sperm. Here we have monitored Ca2+ responses in single sperm, and we report that the [Ca2+]i increase in response to ZP, which is essential for driving the acrosome reaction in vivo, is absent in PLCdelta4-/- sperm. Progesterone, another physiological inducer of the acrosome reaction, failed to induce sustained [Ca2+]i increases in PLCdelta4-/- sperm, and consequently the acrosome reaction was partially inhibited. In addition, we observed oscillatory [Ca2+]i increases in wild-type sperm in response to these acrosome inducers. Calcium imaging studies revealed that the [Ca2+]i increases induced by exposure to ZP and progesterone started at different sites within the sperm head, indicating that these agonists induce the acrosome reaction via different Ca2+ mechanisms. Furthermore, store-operated channel (SOC) activity was severely impaired in PLCdelta4-/- sperm. These results indicate that PLCdelta4 is an important enzyme for intracellular [Ca2+]i mobilization in the ZP-induced acrosome reaction and for sustained [Ca2+]i increases through SOC induced by ZP and progesterone in sperm.     

Co-operative effect of the isoforms of type III antifreeze protein expressed in Notched-fin eelpout, Zoarces elongatus Kner

We found that Notched-fin eelpout, which lives off the north east coast of Japan, expresses an antifreeze protein (AFP). The liver of this fish contains DNAs that encode at least 13 type III AFP isoforms (denoted nfeAFPs). The primary sequences of the nfeAFP isoforms were categorized into SP- and QAE-sephadex binding groups, and the latter were further divided into two subgroups, QAE1 and QAE2 groups. Ice crystals observed in HPLC-pure nfeAFP fractions are bipyramidal in shape with different ratios of c and a axes, suggesting that all the isoforms are able to bind ice. We expressed five recombinant isoforms of nfeAFP and analyzed the thermal hysteresis (TH) activity of each as a function of protein concentration. We also examined the change in activity on mixing the isoforms. TH was estimated to be 0.60 degrees C for the QAE1 isoform, 0.11 degrees C for QAE2, and almost zero for the SP isoforms when the concentrations of these isoforms was standardized to 1.0 mm. Significantly, the TH activity of the SP isoforms showed concentration dependence in the presence of 0.2 mm QAE1, indicating that the less active SP isoform becomes 'active' when a small amount of QAE1 is added. In contrast, it does not become active on the addition of another SP isoform. These results suggest that the SP and QAE isoforms of type III AFP have different levels of TH activity, and they accomplish the antifreeze function in a co-operative manner.     

Interaction between noradrenaline or adrenaline and the beta 1-adrenergic receptor in the nervous system triggers early metamorphosis of larvae in the ascidian,Ciona savignyi

Molecular mechanisms underlying the metamorphosis of larvae, e.g., ligand and receptor interaction, have to be determined and roles for the nervous system in marine invertebrates are not well understood. We report here that treatment of swimming larvae of the ascidian Ciona savignyi with noradrenaline or adrenaline promoted morphological changes in early metamorphosis, e.g., tail resorption. Antagonists of the beta-adrenergic receptor, propranolol, and the beta(1)-adrenergic receptor, metoprolol, inhibited the noradrenaline-induced tail resorption, while an antagonist of the alpha-adrenergic receptor, phentolamine, and of the beta(2)- adrenergic receptor, butoxamine, had no inhibitory effects. In addition, a selective agonist of the beta-adrenergic receptor, isoproterenol, the concentration of which was lower than the effective concentration of the neurotransmitters, facilitated tail resorption. Immunohistochemical studies, using an anti-dopamine-hydroxylase antibody, showed that neurotransmitters such as noradrenaline and adrenaline localized around the brain vesicle of the larvae during metamorphosis. The beta(1)-adrenergic receptor stained with antibodies was localized on the nervous system. Temporal expression of the beta(1)-adrenergic receptor was intense in the nervous system in the larvae competent for metamorphosis. We propose that interactions between noradrenaline or adrenaline and the beta(1)-adrenergic receptor in the nervous system mediate the process of metamorphosis of Ciona larvae.