Ceramide Inhibits Cell Proliferation through Akt/PKB Inactivation and Decreases Melanin Synthesis in Mel‐Ab Cells

Ceramide is a bioactive sphingolipid that mediates a variety of cell functions. However, the effects of ceramide on cell growth and the melanogenesis of melanocytes are not known. In the present study, we investigated the actions of cell‐permeable ceramide and its possible role in the signaling pathway of a spontaneously immortalized mouse melanocyte cell line, Mel‐Ab. Our results show that C₂‐ceramide inhibits DNA synthesis in Mel‐Ab cells and G361 human melanoma cells in a dose‐dependent manner. Cell cycle analysis confirmed the inhibition of DNA synthesis by a reduction in the S phase. To investigate the ceramide signaling pathway, we studied whether C₂‐ceramide is able to influence extracellular signal‐regulated kinase (ERK) and/or Akt/protein kinase B (PKB) activation. We demonstrated that phosphorylated Akt/PKB is decreased by C₂‐ceramide, whereas phosphorylated ERK was only slightly affected. Therefore, the C₂‐ceramide‐induced inactivation of Akt/PKB may be closely related to the reduced cell proliferation of Mel‐Ab cells. Furthermore, we assessed the effects of C₂‐ceramide on the pigmentation of Mel‐Ab cells. The results obtained showed that the melanin content of cells was significantly reduced by C₂‐ceramide at concentrations in the range of 1–10 μM, and that the pigmentation‐inhibiting effect of C₂‐ceramide is much greater than that of kojic acid at 1–100 μM. In addition, we found that the activity of tyrosinase is reduced by C₂‐ceramide treatment. Our results demonstrate that C₂‐ceramide reduces the pigmentation of Mel‐Ab cells by inhibiting tyrosinase activity.     

Karyological studies on the Sino-Himalayan genus, Cremanthodiurn (Asteraceae: Senecioneae)

Twenty-two populations of seven species of Cremanthodium from high altitude regions of western China were observed karyologically. C. ellisii, C. microglossum, C. brunneo-pilosum, C. stenoglossum, C. discoideum and C. lineare all had the same chromosome number of 2 n = 58 whereas C. humile had 2 n = 60. All chromosome numbers of these species are documented here for the first time. The basic number of x = 30 is new for this genus. The karyotypes of all species belong to 2A type according to Stebbins' asymmetry classification of karyotypes. Two basic chromosome numbers, x = 30 and x = 29 in Cremanthodium , correspond exactly to two branching patterns in this genus, sympodial versus monopodial. The systematic and taxonomic statuses of the sympodial species need further study. The karyomorphological data provide no support to the sectional subdivision in Cremanthodium .     

Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels

Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture.     

Effect of adenosine and inosine on ureagenesis in hepatocytes.

Adenosine and inosine produced a dose-dependent stimulation of ureagenesis in isolated rat hepatocytes. Hypoxanthine, xanthine and uric acid were without effect. Half-maximally effective concentrations were 0.08 microM for adenosine and 5 microM for inosine. Activation of ureagenesis by both nucleosides had the following characteristics: (a) it was observed with either glutamine or (NH4)2CO3, provided that glucose was present; (b) it was not detected when glucose was replaced by lactate plus oleate; (c) it was mutually antagonized by glucagon, but not by adrenaline; and (d) it was dependent on Ca2+. We suggest that the action of adenosine and inosine on ureagenesis might be of physiological significance.     

Distribution of alkanes and fatty acids in theCondalia montana (Rhamnaceae) species complex

The alkane and fatty acid composition of the lipid extracts from leaves of 14 populations ofCondalia: C. microphylla f.xanthocarpa, C. microphylla f.erythrocarpa, C. microphylla f.melanocarpa, C. montana, andC. buxifolia, were determined by gas chromatography. Seventeen alkanes and 14 fatty acids were investigated and a predominance of nonacosane, hentriacontane, palmitic and linoleic acids in all species was found.     

Sulfatides Inhibit Binding of Helicobacter pylori to the Gastric Cancer Kato III Cell Line

Helicobacter pylori adhere to Kato III and Hela S3 cells in monolayer cultures. To explore whether cell surface glycoconjugates on these two cell lines mediate binding of H. pylori, various carbohydrates, glycoproteins, and glycolipids were tested to inhibit H. pylori cell adhesion. The adhesion was measured (i) with a urease-based assay and (ii) by cells stained with fluorescein. Sodium periodate and sialidase treatment (but not α- or β-galactosidase, heparitinase, lysozyme, or trypsin) inhibited H. pylori binding to both cell lines. Sulfatides and sulfated glycoconjugates (50 μg/ml) but not heparin or a number of simple carbohydrates inhibited binding (1 mg/ml). The two H. pylori strains studied (CCUG 17874 and strain 25) showed high binding of soluble ¹²⁵I-labeled heparin and other sulfated carbohydrate compounds. Received: 5 July 1996 / Accepted: 17 October 1996     

Effect of TGF-Alpha on growth of normal human breast epithelial cells in serum-free primary culture using 3-dimensional collagen gels.

The mitogenic effect of TGF-alpha, acidic-FGF, basic-FGF and lithium on normal human breast epithelial cells was studied in a collagen gel culture system using a serum-free 1:1 mixture of Ham's F12 and DME medium containing insulin, cholera toxin and bovine serum albumin. TGF-alpha elicited a strong mitogenic response in a dose dependent manner. Addition of cortisol to TGF-alpha stimulated growth over and above that achieved with TGF-alpha alone. A consistent observation has been the effect of a combination of TGF-alpha and cortisol on growth stimulation of normal human breast epithelial cells resulting in 3-12 fold growth after 11-13 days in culture. Acidic-FGF, basic-FGF and lithium were not growth promoting.