Establishment and characterization of a clear cell carcinoma cell line,designated NOCC,derived from human ovary

A cell line, designated NOCC, was established from the ascites of a patient with clear cell adenocarcinoma of the ovary. The cell line has been grown without interruption and continuously propagated by serial passaging (more than 76 times) over 7 years. The cells are spherical to polygonal-shaped, display neoplastic, and pleomorphic features, and grow in a jigsaw puzzle-like pattern while forming monolayers without contact inhibition. The cells proliferate rapidly, but are easily floated as a cell sheet. The population doubling time is about 29 h. The number of chromosomes ranges from 60 to 83. The modal number of chromosomes is 70–74 at the 30th passage. NOCC cells secreted 750.5 ng/ml of VEGF over 3 days of culture. Hypoxia inducible factor-1α (HIF-1α) is a primary regulator of VEGF under hypoxic conditions. NOCC cells were not sensitive to the anticancer drugs BEV, DOX, GEM, ETP, CDDP, or TXT. The graft of NOCC cells to a scid mouse displayed similar histological aspects to the original tumor. Both the NOCC cells and the graft of the NOCC cells gave a positive PAS reaction.     

Growing and differentiating characterization of aortic smooth muscle cell line, p53LMAC01 obtained from p53 knock out mice

Recently we have established an aortic smooth muscle cell line, p53LMAC01 obtained from p53 knockout mice. This cell line showed some differentiated properties which were accelerated by 5-azacytidine treatment [1]. In this study, further characterization of p53LMAC01 cell line was investigated according to cell growth and differentiation, and especially focused into the changes of cell feature, actin filaments' formation, and changes of intracellular calcium concentrations to sympathetic nerve transmitter, norepinephrine. While the cell feature was changed from flattened shape to extended form during 4 days, actin filaments were developing, arranging in parallel to longitudinal direction, and gathering under the surface membrane. In 11 days many cells died and detached from substrate, while actin filaments became poor except for the surface membrane in the remained cells. Appearance of calcium response to noradrenalin needed several days after passage as well as a morphological change of the cells for the extended form and development of actin filaments. The calcium response was maintained on 11 days, which coincided with the result that the cells hold actin filaments under the surface membrane. These results suggest that p53LMAC01 cell line maintains several differentiated characters of adult smooth muscle cell and that their expression needs several days after passage.     

Akt and Ca2+ signaling in endothelial cells

The protein kinase Akt participates in such important functions of endothelial cells as nitric oxide production and angiogenesis, activities that involve changes in cytosolic Ca2+ concentration. However, it is not known if activation of Akt is itself involved in the regulation of Ca2+ signals produced in these cells. The objective of this study was to examine if Akt is involved in the regulation of Ca2+ signaling in endothelial cells. Agonist-stimulated Ca2+ signals, assessed using fura-2, were compared in porcine aortic endothelial cells under control conditions or conditions in which Akt was blocked either by different inhibitors of phosphatidylinositol 3-kinase (PI3 kinase)/Akt or by transient expression of a dominant-negative form of Akt (dnAkt). We found that the release of intracellular Ca2+ stores stimulated by bradykinin or thapsigargin is not affected by the PI3 kinase inhibitors LY294002 and wortmannin, or by expression of dnAkt. LY294002 dose-dependently inhibits store-operated Ca2+ entry, an effect not seen with wortmannin. Expression of dnAkt has no effect on store-operated Ca2+ entry. We conclude that Akt is not involved in the regulation of agonist-stimulated Ca2+ signals in endothelial cells. The compound LY294002 inhibits store-operated Ca2+ entry in these cells by a mechanism independent of PI3 kinase/Akt inhibition.     

Biological characteristics of cultured cells derived from various types of human brain tumors

We placed in culture brain tumors from 45 cases (7 cases of astrocytoma, 2 from oligodendrogliomas, 2 glioblastomas, 2 ependymomas, 13 meningiomas, 6 pituitary adenomas, 5 neurinomas, a malignant lymphoma, a choroid plexus papilloma, and 6 metastatic tumors) and succeeded in making a primary culture from 33, and maintained 17 in vitro over a considerable period of time (greater than three months). In the early period of the primary cultures, the astrocytoma cells had cytoplasmic processes which contacted each other, the oligodendroglioma cells were small and spindle-shaped, the glioblastoma cells were neoplastic with pleopmorphic features and possessed cytoplasmic processes, the ependymoma cells formed a rosette-like cell arrangement, the meningioma cells were spindle- or round-shaped cells and characterized as forming psammoma bodies, the pituitary adenoma cells were round- or oval-shaped cells and produced growth hormone (GH), adenocorticoid tropic hormone (ACTH), prolactin, or other hypophyseal hormones, the choroid plexus papilloma cells were round-or polygonal and showed a papillary cell arrangement, the neurinoma cells were spindle- or fibrous-shaped cells, and the malignant lymphoma cells were round and formed cell aggregates floating in the culture medium.     

Simultaneous determination of isoflavones and bisphenol A in rat serum by high-performance liquid chromatography coupled with coulometric array detection

A method for simultaneous detection and quantification is presented to determine the presence of isoflavones and bisphenol A in a biological sample. A coulometric array detector was used with reversed-phase high-performance liquid chromatography (HPLC). Daidzein (1), glycitein (2), genistein (3) and their glucoside conjugates, daidzin (4), glycitin (5) and genistin (6), were measured as phytochemicals. Also assayed here was equol (7), a metabolite from compound 1, and bisphenol A (8), an industrial chemical that acts as an endocrine disrupter. All chemicals were simultaneously detected by using a 600-mV single detection voltage with high efficacy. A mixture of 1, 3 and 8 was orally administered to rats, and the levels of these three chemicals in the serum were clearly increased after a 4 kU beta-glucuronidase treatment. The levels of compounds 1 and 3 in the serum were detected at 1665 and 2040 ng/ml, while 8 was at a low level of 417 ng/ml. Compound 7 in the serum was not detected until after enzymatic hydrolysis (72 ng/ml). These results suggest that this analytical method would be useful for metabolic and pharmacokinetic studies on isoflavones and bisphenol A.     

Effects of soybean beta-conglycinin on hepatic lipid metabolism and fecal lipid excretion in normal adult rats

beta-Conglycinin decreased blood triacylglycerol (TAG) levels in male Wistar adult rats. Liver mitochondrial carnitine palmitoyltransferase activity in the beta-conglycinin-fed group significantly increased as against the casein-fed group. Hepatic fatty acid synthase activity in the beta-conglycinin group significantly decreased as against that of the casein-fed group. Fecal fatty acid excretion in the beta-conglycinin group was significantly higher than in the casein group.     

Effect of n-3 fatty acids on serum lipid levels and hepatic fatty acid metabolism in BALB/c.KOR-Apoeshl mice deficient in apolipoprotein E expression

N-3 fatty acids exert a potent serum lipid-lowering effect in rodents mainly by affecting hepatic fatty acid oxidation and synthesis. However, it has been observed that fish oil and docosahexaenoic acid ethyl ester do not lower serum lipid levels in apolipoprotein E (apoE)-knockout (Apoetm1Unc) mice generated by gene targeting. To test the hypothesis that apoE expression is required for n-3 fatty acid-dependent regulation of serum lipid levels and hepatic fatty acid metabolism, we examined the effect of fish oil and n-3 fatty acid ethyl esters on the activity and gene expression of hepatic enzymes involved in fatty acid oxidation and synthesis using an alternative apoE-deficient mouse model with the BALB/c genetic background (BALB/c.KOR-Apoeshl). ApoE-deficient mice were fed diets containing 9.4% palm oil, fish oil, or 5.4% palm oil and 1% EPA plus 3% DHA ethyl esters for 15 days. In contrast to the reported data on apoE-knockout mice, fish oil and n-3 fatty acid ethyl esters greatly decreased serum triacylglycerol, cholesterol, and phospholipid levels in the Apoeshl mice. The decreases were greater with fish oil than with ethyl esters. The alterations by dietary n-3 fatty acids of serum lipid levels were accompanied by parallel changes in the activity and mRNA levels of enzymes involved in hepatic fatty acid oxidation and synthesis. The reason for the discrepancy between the results of the current study and previous studies is unknown. However, our study at least indicates that a lack of apoE expression does not necessarily accompany deficits in the n-3 fatty acid-dependent regulation of serum lipid levels and hepatic fatty acid metabolism.     

The hypothalamus is involved in the anorexic effect of glucagon-like peptide-1 in chicks

The present study was carried out to investigate whether the hypothalamus is involved in the anorexic effect of glucagon-like peptide-1 (GLP-1) in chicks. To examine this, Fos expression in the chick hypothalamus were immunohistochemically detected after intracerebroventricular (ICV) injection of 30-pmol GLP-1. ICV injection of GLP-1 stimulated the expression of Fos-like immunoreactive (FLI) cells in the ventromedial hypothalamic nucleus (VMN). When 15-pmol GLP-1 was directly injected into the chick VMN, the chick's food intake was significantly decreased compared with the control treatment. Microinjection of GLP-1 into the (LHA) also inhibited feeding in chicks, although ICV injection of GLP-1 did not stimulate FLI expression in the brain area. These results suggest that VMN and some brain regions are involved in the anorexic effect of GLP-1 in chicks.