The green tea compound, (-)-epigallocatechin-3-gallate downregulates N-cadherin and suppresses migration of bladder carcinoma cells

Green tea has been reported as potential dietary protection against numerous cancers and has been shown to have activity in bladder tumor inhibition in different animal models. The goal of this study was to examine the effects of (-)-epigallocatechin gallate (EGCG-the major phytochemical in green tea) on growth inhibition and behavior of human bladder carcinoma cells and to identify the altered signaling pathway(s) underlying the response to EGCG exposure. EGCG inhibited the in vitro growth of invasive bladder carcinoma cells with an IC(50) range of 70-87 microM. At a concentration of 20 microM, EGCG decreased the migratory potential of bladder carcinoma cells with concomitant activation of p42/44 MAPK and STAT3 and inactivation of Akt. Using biochemical inhibitors of MAPK/ERK, and siRNA to knockdown STAT3 and Akt, inhibition of migration was recorded associated with Akt but not MAPK/ERK or STAT3 signaling in bladder cells. In addition, EGCG downregulated N-cadherin in a dose-dependent manner where reduction in N-cadherin expression paralleled declining migratory potential. Continuous feeding of EGCG to mice prior to and during the establishment of bladder carcinoma xenografts in vivo revealed >50% reduction in mean final tumor volume (P 相似文献   

Enhancer and promoter elements directing activation and glucocorticoid repression of the alpha 1-fetoprotein gene in hepatocytes.

Mutations were introduced in 7 kilobases of 5'-flanking rat alpha 1-fetoprotein (AFP) genomic DNA, linked to the chloramphenicol acetyltransferase gene. AFP promoter activity and its repression by a glucocorticoid hormone were assessed by stable and transient expression assays. Stable transfection assays were more sensitive and accurate than transient expression assays in a Morris 7777 rat hepatoma recipient (Hepa7.6), selected for its strong AFP repression by dexamethasone. The segment of DNA encompassing a hepatocyte-constitutive chromatin DNase I-hypersensitive site at -3.7 kilobases and a liver developmental stage-specific site at -2.5 kilobases contains interacting enhancer elements sufficient for high AFP promoter activity in Hepa7.6 or HepG2 cells. Deletions and point mutations define an upstream promoter domain of AFP gene activation, operating with at least three distinct promoter-activating elements, PEI at -65 base pairs, PEII at -120 base pairs, and DE at -160 base pairs. PEI and PEII share homologies with albumin promoter sequences, PEII is a near-consensus nuclear factor I recognition sequence, and DE overlaps a glucocorticoid receptor recognition sequence. An element conferring glucocorticoid repression of AFP gene activity is located in the upstream AFP promoter domain. Receptor-binding assays indicate that this element is the glucocorticoid receptor recognition sequence which overlaps with promoter-activating element DE.     

DING proteins; novel members of a prokaryotic phosphate-binding protein superfamily which extends into the eukaryotic kingdom

PstS proteins are the cell-bound phosphate-binding elements of the ubiquitous bacterial ABC phosphate uptake mechanisms. Primary and tertiary structures, characteristic of pstS proteins, are conserved in proteins, which are expressed in secretory operons and induced by phosphate deprivation, in Pseudomonas species. There are two subsets of these proteins; AP proteins, which are alkaline phosphatases, and DING proteins, named for their N-terminal sequence, which are phosphate-binding proteins. Both form elements of a proposed phosphate-scavenging system in pseudomonads. DING proteins have also been isolated from many eukaryotic sources, and are associated with both normal and pathological functions in mammals. Their phosphate-binding function suggests a role in biomineralization, but the ability to bind other ligands may be related to signal transduction in eukaryotes. Though it has been claimed that all such proteins may originate from pseudomonads, many eukaryotic DING proteins have unique features which are incompatible with a bacterial origin.     

Using Fecal Hormonal and Behavioral Analyses to Evaluate the Introduction of Two Sable Antelope at Lincoln Park Zoo

Introductions of sable antelope (Hippotragus niger) can be difficult due to the potential ensuing aggression compounded by their large horns. The goal was to use hormonal assays and behavioral analyses to evaluate the success of an introduction of 2 adult females at Lincoln Park Zoo. The objectives were to (a) document behavioral and hormonal changes in 2 female sable antelope during the introduction, (b) compare fecal glucocorticoid metabolites (FGM) in each individual during the introduction stages, (c) measure fecal androgen metabolites (FAM) during introduction and compare with dominance rank and observed aggression, and (d) monitor estrous cycle synchronization. Results demonstrate that FGM were higher before than during and after the introduction. Behavioral observations indicated limited aggression between females, although the keeper survey results revealed that the new female was more dominant and had higher mean FGM and FAM than the resident. Both sable antelope were reproductively active throughout the year. Results indicate that fecal hormone analysis can provide zoo management with valuable information to minimize the risk of aggression, injury, and stress during introductions of nonhuman animals.     

Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an alpha-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4x10(4) U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.     

Phosphatidylinositol 3-kinase requirement in activation of the ras/C-raf-1/MEK/ERK and p70(s6k) signaling cascade by the insulinomimetic agent vanadyl sulfate.

The mechanisms by which inorganic salts of the trace element vanadium mediate their insulinomimetic effects are not clearly understood and were investigated. We have shown previously that vanadium salts activate mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase activities (PI3-K) via a pathway that does not involve the insulin receptor (IR) tyrosine kinase function [Pandey, S. K., Anand-Srivastava, M. B., and Srivastava, A. K. (1998) Biochemistry 37, 7006-7014]. Herein, we have examined a possible role of PI3-K in the vanadyl sulfate (VS)-mediated increase in the level of ras-MAPK activation as well as the contribution of signaling components upstream to MAPK in this VS response. Treatment of IR-overexpressing cells with VS resulted in an increased level of tyrosine phosphorylation of p44(mapk) (ERK-1) and p42(mapk) (ERK-2) along with stimulation of MAPK, MAPK kinase (MEK), and C-raf-1 activities, and ras activation. Preincubation with wortmannin and LY294002, two structurally and mechanistically different inhibitors of PI3-K, blocked the VS-mediated increase in MAPK activity and phosphorylation of ERK-1 and ERK-2. Furthermore, wortmannin inhibited activation of ras, C-raf-1, and MEK in response to VS. The addition of a farnesyltransferase inhibitor, B581, to cells reduced the level of MAPK activation as well as ERK-1 and ERK-2 phosphorylation stimulated by VS. Finally, VS increased PI3-K activity in ras immunoprecipitates. A VS-mediated increase in p70(s6k) activity was also found to be inhibited by wortmannin. Taken together, these results demonstrate that the insulinomimetic effects of VS may be mediated, in part, by PI3-K-dependent stimulation of the ras-MAPK and p70(s6k) pathways.     

Partial characterization of anEscherichia coll strain harboring a xylanase encoding plasmid

Summary The plasmid pRH271, harboring a xylanase gene cioned fromBacilius subtilis, has been transferred into a mutant ofE. coli SK2284 which allowed the release of part of the xylanase in the culture supernatant. Kinetic parameters of this recombinantE. coll strain were determined in microscale batch culture with and without the selective pressure of antibiotics. No significant difference in µ_max was observed for the nontransformedE. coli strain when compared to the recombinant strain. However, K₅ values for glucose were two times higher in the case of the recombinant strain. Preliminary study of xylanase production in a large batch farmenter was also described.     

Electrophoretic karyotypes of the elm tree pathogen Ophiostoma ulmi (sensu lato)

Pulsed field gel electrophoresis using OFAGE, TAFE, and CHEF systems has been used to more fully characterize karyotypic variation within the two closely related fungal species of Ophiostoma ulmi sensu lato. Twelve wild-type and laboratory strains, representing the less agressive species O. ulmi and both of the biotypes of the more aggressive species O. novo-ulmi were studied and their karyotypes determined. Depending on the strain, a minimum of four to a minimum of eight chromosomal DNA bands were present that fall into three distinct size classes, with one exception. Strain CESSI6K (O. novo-ulmi, North American aggressive subgroup) contains a unique chromosomal DNA band which comigrated near a Saccharomyces cerevisiae chromosome of 0.95 Mb. This unique band was the smallest O. ulmi s. l. chromosomal DNA observed. Seven of the twelve strains shared a common chromosomal DNA banding pattern, whereas each of the other five had a unique karyotype. There was no correlation between chromosome profile and species, as some O. novo-ulmi and O. ulmi strains shared common electrophoretic karyotypes.