Glucuronidation of the green tea catechins, (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate, by rat hepatic and intestinal microsomes

The flavonoids (-)-epigallocatechin-3-gallate (EGCg) and (-)-epicatechin-3-gallate (ECg) are major components of green tea and show numerous biological effects. We investigated the glucuronidation of these compounds and of quercetin by microsomes. Quercetin was almost fully glucuronidated by liver microsomes after 3 h, whereas ECg and ECGg were conjugated to a lesser extent ([Formula: See Text] and [Formula: See Text] respectively). The intestinal microsomes also glucuronidated quercetin much more efficiently than ECg and EGCg. Although the rates were lower than quercetin, intestinal microsomes exhibited higher activity on the galloyl group of ECg and EGCg compared to the flavonoid ring, whereas hepatic glucuronidation was higher on the flavonoid ring of EGCg and ECg compared to the galloyl groups. The low glucuronidation rates could partially explain why these flavanols are present in plasma as unconjugated forms.     

Hyperphosphorylation of extracellular regulated kinase 2 (ERK2) and inhibition of JNK2 phosphorylation are associated with increased S-phase during transformation of Syrian hamster embryo cells by Malachite Green

Malachite Green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumour promoter. In view of its industrial importance and possible exposure to human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells in primary culture by MG. In this study, we have studied the mitogen activated protein (MAP) kinase signal transduction pathway in preneoplastic cells induced by MG. Western blots of MG induced preneoplastic cells showed no phosphorylation of ERK1, an increased phosphoactive ERK2 associated with a decreased expression of phosphoactive JNK2. However, total forms of ERKs, JNKs and p38 Kinases showed similar levels of expression in control and preneoplastic SHE cells. Indirect immunofluorescence studies have shown a distinct nuclear localisation of phosphoactive ERKs in MG induced preneoplastic cells. Flow cytometric analysis showed an increase of S-phase cells in preneoplastic cells compared to control SHE cells. The present study indicates that hyperphosphorylation of ERK2, decreased JNK2 phosphorylation and an increase in S-phase cells seems to be the early changes associated with the MG induced malignant transformation of SHE cells in primary culture.     

Continual assembly of desmosomes within stable intercellular contacts of epithelial A-431 cells

Subclones of human carcinoma-derived A-431 cell line stably producing fusion proteins consisting of the enhanced green fluorescent protein and either human desmoglein 2 (Dsg-GFP) or human plakoglobin (GFP-Pg) were used to examine the behavior of desmosomes in living cells. Immunofluorescence microscopy of the fixed cells showed that both fusion proteins, which were expressed in significantly lower levels relative to their endogenous counterparts, were efficiently recruited into desmosomes. Time-lapse confocal imaging of these cells reveals that such GFP-labeled desmosomes (GFP desmosomes) are stable structures which exhibit various dynamic and motile activities. The most notable are independent lateral mobility and fusion. Furthermore, the continual assembly of new nascent desmosomes is observed within stable contacts located at the middle of the epithelial sheet. A new GFP desmosome appears as a closely apposed group of fine patches which after a few minutes aggregate into a single structure. These three dynamic processes resulted in constant changes of desmosome distribution, numbers, and sizes. In addition, fluorescence recovery after photobleaching experiments showed that fine patches of desmosomal proteins may participate in desmosome maintenance. Such a diverse range of dynamic activities of desmosomes apparently produces flexible but tight cell-cell adhesion required for different morphogenetic events in epithelial structures.This work was supported by grant AR44016-04 from the National Institutes of Health     

Fluorometric quantification of RNA and DNA in solutions containing both nucleic acids

A fluorescence-based method for quantitative determination of RNA and DNA in probes containing both nucleic acids has been developed. The total concentration of nucleic acids is determined using SYBR Green II dye under conditions providing independent binding of the fluorophore with DNA and RNA. The concentration of DNA is specifically measured using the Hoechst 33258 dye and the RNA concentration is calculated from these data. The procedure allows for accurate determination of DNA concentration in the range 10-1000 ng/ml in the presence of 200-fold excess of RNA and determination of RNA concentrations in the range 10-1000 ng/ml in the presence of large excess of DNA. An absence of the treatment of mixed samples with RNase-free DNase I provides rapid, reproducible, and accurate RNA quantification.     

Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.     

Molecular pathway for (-)-epigallocatechin-3-gallate-induced cell cycle arrest and apoptosis of human prostate carcinoma cells

Epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent present in green tea, is a promising chemopreventive agent. We recently showed that green tea polyphenols exert remarkable preventive effects against prostate cancer in a mouse model and many of these effects are mediated by the ability of polyphenols to induce apoptosis in cancer cells [Proc. Natl. Acad. Sci. USA 98 (2001) 10350]. Earlier, we showed that EGCG causes a G0/G1 phase cell cycle arrest and apoptosis of both androgen-sensitive LNCaP and androgen-insensitive DU145 human prostate carcinoma cells, irrespective of p53 status [Toxicol. Appl. Pharmacol. 164 (2000) 82]. Here, we provide molecular understanding of this effect. We tested a hypothesis that EGCG-mediated cell cycle dysregulation and apoptosis is mediated via modulation of cyclin kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery. As shown by immunoblot analysis, EGCG treatment of LNCaP and DU145 cells resulted in significant dose- and time-dependent (i) upregulation of the protein expression of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18, (ii) down-modulation of the protein expression of cyclin D1, cyclin E, cdk2, cdk4, and cdk6, but not of cyclin D2, (iii) increase in the binding of cyclin D1 toward WAF1/p21 and KIP1/p27, and (iv) decrease in the binding of cyclin E toward cdk2. Taken together, our results suggest that EGCG causes an induction of G1 phase ckis, which inhibits the cyclin-cdk complexes operative in the G0/G1 phase of the cell cycle, thereby causing an arrest, which may be an irreversible process ultimately leading to apoptotic cell death. This is the first systematic study showing the involvement of each component of cdk inhibitor-cyclin-cdk machinery during cell cycle arrest and apoptosis of human prostate carcinoma cells by EGCG.     

Na+/Mg2+ transporter acts as a Mg2+ buffering mechanism in PC12 cells

Mg(2+) buffering mechanisms in PC12 cells were demonstrated with particular focus on the role of the Na(+)/Mg(2+) transporter by using a newly developed Mg(2+) indicator, KMG-20, and also a Na(+) indicator, Sodium Green. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a protonophore, induced a transient increase in the intracellular Mg(2+) concentration ([Mg(2+)](i)). The rate of decrease of [Mg(2+)](i) was slower in a Na(+)-free extracellular medium, suggesting the coupling of Na(+) influx and Mg(2+) efflux. Na(+) influxes were different for normal and imipramine- (a putative inhibitor of the Na(+)/Mg(2+) transporter) containing solutions. FCCP induced a rapid increase in [Na(+)](i) in the normal solution, while the increase was gradual in the imipramine-containing solution. The rate of decrease of [Mg(2+)](i) in the imipramine-containing solution was also slower than that in the normal solution. From these results, we show that the main buffering mechanism for excess Mg(2+) depends on the Na(+)/Mg(2+) transporter in PC12 cells.     

Intracellular redistribution of protein kinase D2 in response to G-protein-coupled receptor agonists

The protein kinase D (PKD) family consists of three serine/threonine protein kinases: PKC mu/PKD, PKD2, and PKC nu/PKD3. While PKD has been the focus of most studies to date, no information is available on the intracellular distribution of PKD2. Consequently, we examined the mechanism that regulates its intracellular distribution in human pancreatic carcinoma Panc-1 cells. Analysis of the intracellular steady-state distribution of fluorescent-tagged PKD2 in unstimulated cells indicated that this kinase is predominantly cytoplasmic. Cell stimulation with the G protein-coupled receptor agonist neurotensin induced a rapid and reversible plasma membrane translocation of PKD2 by a mechanism that requires PKC activity. In contrast to the other PKD isoenzymes, PKD2 activation did not induce its redistribution from the cytoplasm to the nucleus. Thus, this study demonstrates that the regulation of the distribution of PKD2 is distinct from other PKD isoenzymes, and suggests that the differential spatio-temporal localization of these signaling molecules regulates their specific signaling properties.     

Prolonged gene expression in primary porcine pancreatic cells using an Epstein-Barr virus-based episomal vector

Epstein-Barr virus (EBV)-based plasmids containing the origin of replication (oriP) and EBV nuclear antigen 1 (EBNA-1) are well known for the stable episomal maintenance in human cells. In order to clarify whether an EBV-based plasmid can be maintained stably in the porcine pancreatic cells which are the primary candidate sources of islet xenotransplantation, we constructed pEBVGFP encoding the green fluorescent protein (GFP). Monolayer culture of the porcine neonatal pancreatic cells was lipofected with pEBVGFP or pGFP which was derived from pEBVGFP by deleting out oriP and EBNA-1. pEBVGFP significantly prolonged GFP expression not only in human cell lines but also in the primary porcine pancreatic cells compared with pGFP. Interestingly, the duct cells that are believed as the pancreatic precursor cells were preferentially transfected and conveniently enriched among the mixed primary cell populations using a hygromycin B selection. To our knowledge, this is the first report suggesting the potential application of an EBV-based plasmid for the extended gene expression in the primary porcine pancreatic duct cells.     

A system using convertible vectors for screening soluble recombinant proteins produced in Escherichia coli from randomly fragmented cDNAs

Protein insolubility is a major problem when producing recombinant proteins (e.g., to be used as antigens) from large cDNAs in Escherichia coli. Here, we describe a system using three convertible plasmid vectors to screen for soluble proteins produced in E. coli. This system experimentally identified any random cDNA fragments producing soluble protein domains. Shotgun fragments introduced into any of our three plasmids, which contain Gateway recombination sites, fused in-frame to the ORF of the protein tag. These plasmids produced N-terminal GST- and C-terminal three-frame-adaptive FLAG-tagged proteins, kanamycin-resistant gene-tagged proteins (which were pre-selected for in-frame fused cDNAs), or GFP-tagged fusion proteins. The latter is useful as a fluorescence indicator of protein folding. The Gateway recombination sites promote smooth conversion for enrichment of in-frame clones and facilitate both protein solubility assays and final production of proteins without the C-terminal tag. This high-throughput screening method is particularly useful for procedures that require the handling of many cDNAs in parallel.