Mutually antagonistic effects of androgen and activin in the regulation of prostate cancer cell growth

Activin, a member of the TGFbeta superfamily, is expressed in the prostate and inhibits growth. We demonstrate that the effects of activin and androgen on regulation of prostate cancer cell growth are mutually antagonistic. In the absence of androgen, activin induced apoptosis in the androgen-dependent human prostate cancer cell line LNCaP, an effect suppressed by androgen administration. Although activin by itself did not alter the cell cycle distribution, it potently suppressed androgen- induced progression of cells into S-phase of the cell cycle and thus inhibited androgen-stimulated growth of LNCaP cells. Expression changes in cell cycle regulatory proteins such as Rb, E2F-1, and p27 demonstrated a strong correlation with the mutually antagonistic growth regulatory effects of activin and androgen. The inhibitory effect of activin on growth was independent of serine, serine, valine, serine motif phosphorylation of Smad3. Despite their antagonistic effect on growth, activin and androgen costimulated the expression of prostate-specific antigen through a Smad3-mediated mechanism. These observations indicate the existence of a complex cross talk between activin and androgen signaling in regulation of gene expression and growth of the prostate.     

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.     

Nitrous oxide-forming codenitrification catalyzed by cytochrome P450nor

Intact cells of the denitrifying fungus Fusarium oxysporum were previously shown to catalyze codenitrification to form a hybrid nitrous oxide (N2O) species from nitrite and other nitrogen compounds such as azide and ammonia. Here we show that cytochrome P450nor can catalyze the codenitrification reaction to form N2O from nitric oxide (NO) but not nitrite, and azide or ammonia. The results show that the direct substrate of the codenitrification by intact cells should not be nitrite but NO, which is formed from nitrite by the reaction of a dissimilatory nitrite reductase.     

Electron acquisition system constructed from an NAD-independent D-lactate dehydrogenase and cytochrome c2 in Rhodopseudomonas palustris No. 7

The activities of NAD-independent D- and L-lactate dehydrogenases (D-LDH, L-LDH) were detected in Rhodopseudomonas palustris No. 7 grown photoanaerobically on lactate. One of these enzymes, D-LDH, was purified as an electrophoretically homogeneous protein (M(r), about 235,000; subunit M(r) about 57,000). The pI was 5.0. The optimum pH and temperature of the enzyme were pH 8.5 and 50 degrees C, respectively. The Km of the enzyme for D-lactate was 0.8 mM. The enzyme had narrow substrate specificity (D-lactate and DL-2-hydroxybutyrate). The enzymatic activity was competitively inhibited by oxalate (Ki, 0.12 mM). The enzyme contained a FAD cofactor. Cytochrome c(2) was purified from strain No. 7 as an electrophoretically homogeneous protein. Its pI was 9.4. Cytochrome c(2) was reduced by incubating with D-LDH and D-lactate.     

Loss of PGC-specific expression of the orphan nuclear receptor ERR-beta results in reduction of germ cell number in mouse embryos

Estrogen related receptor beta (ERR-beta) is an orphan nuclear receptor specifically expressed in a subset of extra-embryonic ectoderm of post-implantation embryos. ERR-beta is essential for placental development since the ERR-beta null mutants die at 10.5dpc due to the placenta abnormality. Here, we show that the ERR-beta is specifically expressed in primordial germ cells (PGC), obviously another important cell type for reproduction. Expression of the ERR-beta mRNA in embryonic germ cells started at E11.5 as soon as PGC reached genital ridges, and persisted until E15-E16 in both sexes. Immunostaining with anti-ERR-beta antibody revealed that the ERR-beta protein is exclusively expressed in germ cells in both male and female gonads from E11.5 to E16. 5. To study function of the ERR-beta in PGC, we complemented placental defects of the ERR-beta null mutants with wild-type tetraploid embryos, and analyzed germ cell development in the rescued embryos. It was found that development of gonad and PGC was not apparently affected, but number of germ cells was significantly reduced in male and female gonads, suggesting that the ERR-beta appears to be involved in proliferation of gonadal germ cells. The rescued embryos could develop to term and grow up to adulthood. The rescued ERR-beta null male were found to be fertile, but both male and female null mutants exhibited behavioural abnormalities, implying that the ERR-beta plays important roles in wider biological processes than previously thought.     

The Caenorhabditis elegans eukaryotic initiation factor 5A homologue, IFF-1, is required for germ cell proliferation, gametogenesis and localization of the P-granule component PGL-1

Eukaryotic initiation factor 5A (eIF-5A) was originally isolated as a translation initiation factor. However, this function has since been reconsidered, with recent studies pointing to roles for eIF-5A in mRNA metabolism and trafficking [Microbiol. Mol. Biol. Rev. 66 (2002) 460; Eur. Mol. Biol. Org. J. 17 (1998) 2914]. The Caenorhabditis elegans genome contains two eIF-5A homologues, iff-1 and iff-2, whose functions in vivo were examined in this study. The iff-2 mutation causes somatic defects that include slow larval growth and disorganized somatic gonadal structures in hermaphrodites. iff-2 males show disorganized tail sensory rays and spicules. On the other hand, iff-1 mRNA is expressed in the gonad, and the lack of iff-1 activity causes sterility with an underproliferated germline resulting from impaired mitotic proliferation in both hermaphrodites and males. In spite of underproliferation, meiotic nuclei are observed, as revealed by presence of immunoreactivity to the anti-HIM-3 antibody; however, no gametogenesis occurs in the iff-1 gonads. These phenotypes are in part similar to the mutants affected in the components of P granules, which are the C. elegans counterparts of germ granules [Curr. Top Dev. Biol. 50 (2000) 155]. We found that localization of the P-granule component PGL-1 to P granules is disrupted in the iff-1 mutant. In summary, the two C. elegans homologues of eIF-5A act in different tissues: IFF-2 is required in the soma, and IFF-1 is required in the germline for germ cell proliferation, for gametogenesis after entry into meiosis, and for proper PGL-1 localization on P granules.     

Antibacterial activity of hydrolyzable tannins derived from medicinal plants against Helicobacter pylori

Helicobacter pylori is a major etiological agent in gastroduodenal disorders. In this study, we isolated 36 polyphenols and 4 terpenoids from medicinal plants, and investigated their antibacterial activity against H. pylori in vitro. All hydrolyzable tannins tested demonstrated promising antibacterial activity against H. pylori. Monomeric hydrolyzable tannins revealed especially strong activity. Other compounds demonstrated minimal antibacterial activity with a few exceptions. A monomeric hydrolyzable tannin, Tellimagrandin I demonstrated time- and dose-dependent bactericidal activity against H. pylori in vitro. On the other hand, hydrolyzable tannins did not affect the viability of MKN-28 cells derived from human gastric epithelium. Hydrolyzable tannins, therefore, have potential as new and safe therapeutic regimens against H. pylori infection. Furthermore, we investigated effects of hydrolyzable tannins on lipid bilayer membranes. All the hydrolyzable tannins tested demonstrated dose-dependent membrane-damaging activity. However, it remains to be elucidated whether their membrane-damaging activity directly contributes to their antibacterial action.     

Heavy metal stress. Activation of distinct mitogen-activated protein kinase pathways by copper and cadmium

Excessive amounts of heavy metals adversely affect plant growth and development. Whereas some regions naturally contain high levels of heavy metals, anthropogenic release of heavy metals into the environment continuously increases soil contamination. The presence of elevated levels of heavy metal ions triggers a wide range of cellular responses including changes in gene expression and synthesis of metal-detoxifying peptides. To elucidate signal transduction events leading to the cellular response to heavy metal stress we analyzed protein phosphorylation induced by elevated levels of copper and cadmium ions as examples for heavy metals with different physiochemical properties and functions. Exposure of alfalfa (Medicago sativa) seedlings to excess copper or cadmium ions activated four distinct mitogen-activated protein kinases (MAPKs): SIMK, MMK2, MMK3, and SAMK. Comparison of the kinetics of MAPK activation revealed that SIMK, MMK2, MMK3, and SAMK are very rapidly activated by copper ions, while cadmium ions induced delayed MAPK activation. In protoplasts, the MAPK kinase SIMKK specifically mediated activation of SIMK and SAMK but not of MMK2 and MMK3. Moreover, SIMKK only conveyed MAPK activation by CuCl(2) but not by CdCl(2). These results suggest that plants respond to heavy metal stress by induction of several distinct MAPK pathways and that excess amounts of copper and cadmium ions induce different cellular signaling mechanisms in roots.     

New highly active taxoids from 9 beta-dihydrobaccatin-9,10-acetals. Part 5

To improve the metabolic stability of 3, which exhibited both in vitro antitumor activity and in vivo efficacy by both iv and po administration, we designed and synthesized new taxane analogues. Most of the synthetic compounds maintained excellent antitumor activity and were scarcely metabolized by human liver microsomes. And some compounds exhibited potent antitumor effects against B16 melanoma BL6 in vivo by both iv and po administration similarly to 3.