Estrogen-producing steroidogenic pathways in parietal cells of the rat gastric mucosa

Recently we demonstrated the presence of aromatase (P450(arom)), estrogen synthetase, and the active production of estrogen in parietal cells of the rat stomach. We therefore investigated the steroidogenic pathways of estrogen and also other steroid metabolites in the gastric mucosa of male rats, by showing the mRNA expression of steroidogenic enzymes using RT-PCR and in situ hybridization histochemistry, and by measuring the blood concentration of steroids in the artery and the portal vein. RT-PCR analysis showed the strong mRNA expression of 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), 17beta-hydroxysteroid dehydrogenase (HSD) type III and P450(arom), and the weak mRNA expression of 17beta-HSD type II, 5alpha-reductase type I and 3alpha-HSD. The other mRNAs of steroidogenic enzymes examined were not detected. In situ hybridization histochemistry demonstrated the localization of mRNAs for P450(17alpha), 17beta-HSD type III and P450(arom) in the parietal cells. Higher levels of progesterone and testosterone were found in the artery compared with the portal vein. Higher amounts of estrone and 17beta-estradiol, by contrast, were present in the portal vein compared with the artery. These results indicate that parietal cells of rat stomach convert circulating progesterone and/through androstenedione and testosterone to synthesize both estrone and 17beta-estradiol, which then enter the liver via the portal vein.     

Ethanol-induced pseudohyphal transition in the cells of Candida tropicalis: participation of phosphoinositide signal transduction

Ethanol-induced pseudohyphal development in the cells of Candida tropicalis Pk233 was accompanied by the transient accumulation of inositol 1,4,5-trisphosphate (IP3) that occurred at an early growth stage. The concomitant addition of myo-inositol prevented the activation of IP3 accumulation and cancelled pseudohyphal development in the presence of ethanol. The addition of a specific phospholipase C inhibitor, U73 122, inhibited ethanol-induced pseudohyphal transition at the concentrations of subinhibitory levels of cell growth. Pseudohyphal development was also induced by the Ca2+ ionophore, A23 187 in the absence of ethanol. The effect of A23 187 on the development of pseudohyphae was little influenced by myo-inositol, but stimulated by concomitant addition of 12-O-tetradecanoylphorbol 13-acetate. These results suggest that ethanol activated phospholipase C in competition with myo-inositol, and the resulting IP3-Ca2+ and protein kinase C pathways of PI signal transduction may work in pseudohyphal transition.     

Quantitative characterization of airspace enlargement in emphysema.

The mean linear intercept (L(m)) can be used to estimate the surface area for gas exchange in the lung. However, in recent years, it is most commonly used as an index for characterizing the enlargement of airspaces in emphysema and the associated severity of structural destruction in the lung. Specifically, an increase in L(m) is thought to result from an increase in airspace sizes. In this paper, we examined how accurately L(m) measures the linear dimensions of airspaces from histological sections and a variety of computer-generated test images. To this end, we developed an automated method for measuring linear intercepts from digitized images of tissue sections and calculate L(m) as their mean. We examined how the shape of airspaces and the variability of their sizes influence L(m) as well as the distribution of linear intercepts. We found that, for a relatively homogeneous enlargement of airspaces, L(m) was a reliable index for detecting emphysema. However, in the presence of spatial heterogeneities with a large variability of airspace sizes, L(m) did not significantly increase and sometimes even decreased compared with its value in normal tissue. We also developed an automated method for measuring the area and computed an equivalent diameter of each individual airspace that is independent of shape. Finally, we introduced new indexes based on the moments of diameter that we found to be more reliable than L(m) to characterize airspace enlargement in the presence of heterogeneities.     

Preparative fractionation of protein, RNA, and plasmid DNA using centrifugal precipitation chromatography with tubular dialysis membrane inside a convoluted tubing as separation channel

Fractionation of clarified E. coli lysate components in bench-scale and preparative-scale centrifugal precipitation chromatography (CPC), using a solution of cationic surfactant cetyltrimethylammonium bromide (CTAB) containing 0.5 M NaCl as precipitant, are compared here. Step gradient of CTAB from 0.50% to 0.16% (w/v) gave a successful fractionation in bench-scale CPC; however, a linear gradient of lower CTAB concentration, 0.20-0% (w/v), was used in the preparative scale and resulted in similar fractionation. The preparative-scale CPC has a superior sample loading capacity by the use of tubular dialysis membrane inside convoluted tubing as the separation channel. In this study, the quantity of the sample loaded into the preparative CPC was about 15 times more than that in the bench scale, and in a single run the preparative CPC could prepare approximately 3 mg of plasmid DNA with about 96% of RNA removed. The higher surface area per length of the separation channel in the preparative CPC was believed to benefit mass transfer of CTAB across the membrane, leading to less CTAB being required in the process.     

Identification of organoselenium compounds that possess chemopreventive properties in human prostate cancer LNCaP cells

The process of cancer development consists of three sequential stages termed initiation, promotion, and progression. Oxidative stress damages DNA and introduces mutations into oncogenes or tumor suppressor genes, thus contributing to cancer development. Cancer chemoprevention is defined to prevent or delay the development of cancer by the use of natural or synthetic substances. In the present study, we synthesized a series of organoselenium compounds and evaluated their possible chemopreventive properties in human prostate cancer LNCaP cells. Among 42 organoselenium compounds tested, two compounds, 3-selena-1-dethiacephem 13 and 3-selena-1-dethiacephem 14 strongly activated the Nrf2/ARE (antioxidant response element) signaling and thus markedly increased expression of heme oxygenase-1 (HO-1), a phase II antioxidant enzyme. Translocation of Nrf2 to the nucleus preceded HO-1 protein induction by two compounds. The intracellular ROS level was strongly reduced immediately after treatment with these compounds, showing that they are potent antioxidants. Finally, both compounds inhibited cell growth via cell cycle arrest. Our findings suggest that compounds 13 and 14 could not only attenuate oxidative stress through Nrf2/ARE activation and direct ROS scavenging but also inhibit cell growth. Thus, these compounds possess the potential as pharmacological agents for chemoprevention of human prostate cancer.     

Synthesis and structural and pharmacological properties of cyclopropane-based conformationally restricted analogs of 4-methylhistamine as histamine H3/H4 receptor ligands

On the basis of the previous results on a histamine H₄ receptor agonist 4-methylhistamine and a cyclopropane-based conformationally restricted analog CEIC (3) with potent H₃/H₄ receptor antagonistic effect, 4-methylhistamine analogs 4 and 5 of CEIC were designed and synthesized. Compound 4 showed strong affinity (K_i = 38.7 nM) for the H₃ receptor, which was more potent than a well-known H₃ antagonist thioperamide. Stable tautomer and conformation of 3 and 4, which can affect the pharmacological activity, were analyzed by ab initio calculations.     

Chemokine CXCL14/BRAK transgenic mice suppress growth of carcinoma cell xenografts

We reported previously that the forced expression of the chemokine BRAK, also called CXCL14 in head and neck squamous cell carcinoma (HNSCC) cells decreased the rate of tumor formation and size of tumor xenografts compared with mock-vector treated cells in athymic nude mice or in severe combined immunodeficiency mice. This suppression occurred even though the growth rates of these cells were the same under in vitro culture conditions, suggesting that a high expression level of the gene in tumor cells is important for the suppression of tumor establishment in vivo. The aim of this study was to determine whether CXCL14/BRAK transgenic mice show resistance to tumor cell xenografts or not. CXCL14/BRAK cDNA was introduced into male C57BL/6 J pronuclei, and 10 founder transgenic mice (Tg) were obtained. Two lines of mice expressed over 10 times higher CXCL14/BRAK protein levels (14 and 11 ng/ml plasma, respectively) than normal blood level (0.9 ng/ml plasma), without apparent abnormality. The sizes of Lewis lung carcinoma and B16 melanoma cell xenografts in Tg mice were significantly smaller than those in control wild-type mice, indicating that CXCL14/BRAK, first found as a suppressor of tumor progression of HNSCC, also suppresses the progression of a carcinoma of other tissue origin. Immunohistochemical studies showed that invasion of blood vessels into tumors was suppressed in tumor xenografts of CXCL14/BRAK Tg mice. These results indicate that CXCL14/BRAK suppressed tumor cell xenografts by functioning paracrine or endocrine fashion and that CXCL14/BRAK is a very promising molecular target for tumor suppression without side effects.     

Production of recombinant human erythropoietin/Fc fusion protein by genetically manipulated chickens

We previously reported the production of human erythropoietin (hEpo) using genetically manipulated (GM) chickens. The recombinant hEpo was produced in the serum and egg white of the GM chickens, and the oligosaccharide chain structures of the serum-derived hEpo were more favorable than those of the egg white-derived hEpo. In the present study, a retroviral vector encoding an expression cassette for a fusion protein of hEpo and the Fc region of human immunoglobulin G (hEpo/Fc) was injected into developing chicken embryos, with the aim of recovering the serum-derived hEpo from egg yolk through the yolk accumulation mechanism of maternal antibodies. The GM chickens that hatched stably produced the hEpo/Fc fusion protein not only in their serum and egg white, but also in the egg yolk as expected. Lectin blot analyses revealed that significant amounts of the oligosaccharide chains of hEpo/Fc produced in the serum and eggs of GM chickens terminated with galactose, and that the oligosaccharide chains of the serum- and yolk-derived hEpo/Fc incorporated sialic acid residues. Moreover, biological activity assessment using Epo-dependent cells revealed that the yolk-derived hEpo/Fc exhibited a comparable performance to the serum- and CHO-derived hEpo/Fc. These results indicate that transport of Fc fusion proteins from the blood circulation to the yolk in chickens represents an effective strategy for the production of pharmaceutical glycoproteins using transgenic chicken bioreactors.     

Mechanism and Role of High Density Lipoprotein-induced Activation of AMP-activated Protein Kinase in Endothelial Cells

The upstream signaling pathway leading to the activation of AMP-activated protein kinase (AMPK) by high density lipoprotein (HDL) and the role of AMPK in HDL-induced antiatherogenic actions were investigated. Experiments using genetic and pharmacological tools showed that HDL-induced activation of AMPK is dependent on both sphingosine 1-phosphate receptors and scavenger receptor class B type I through calcium/calmodulin-dependent protein kinase kinase and, for scavenger receptor class B type I system, additionally serine-threonine kinase LKB1 in human umbilical vein endothelial cells. HDL-induced activation of Akt and endothelial NO synthase, stimulation of migration, and inhibition of monocyte adhesion and adhesion molecule expression were dependent on AMPK activation. The inhibitory role of AMPK in the adhesion molecule expression and monocyte adhesion on endothelium of mouse aorta was confirmed in vivo and ex vivo. On the other hand, stimulation of ERK and proliferation were hardly affected by AMPK knockdown but completely inhibited by an N17Ras, whereas the dominant-negative Ras was ineffective for AMPK activation. In conclusion, dual HDL receptor systems differentially regulate AMPK activity through calcium/calmodulin-dependent protein kinase kinase and/or LKB1. Several HDL-induced antiatherogenic actions are regulated by AMPK, but proliferation-related actions are regulated by Ras rather than AMPK.