Enrichment of electrochemically active bacteria using a three-electrode electrochemical cell

Electrochemically active bacteria were successfully enriched in an electrochemical cell using a positively poised working electrode. The positively poised working electrode (+0.7 V vs. Ag/AgCl) was used as an electron acceptor for enrichment and growth of electrochemically active bacteria. When activated sludge and synthetic wastewater were fed to the electrochemical cell, a gradual increase in amperometric current was observed. After a period of time in which the amperometric current was stabilized (generally 8 days), linear correlations between the amperometric signals from the electrochemical cell and added BOD (biochemical oxygen demand) concentrations were established. Cyclic voltammetry of the enriched electrode also showed prominent electrochemical activity. When the enriched electrodes were examined with electron microscopy and confocal scanning laser microscopy, a biofilm on the enriched electrode surface and bacterium-like particles were observed. These experimental results indicate that the electrochemical system in this study is a useful tool for the enrichment of an electrochemically active bacterial consortium and could be used as a novel microbial biosensor.     

Egr-1, a new downstream molecule of Epstein-Barr virus latent membrane protein 1

To investigate the effect of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) on human cancer cells, we sought to identify and analyze potential target genes that were differentially expressed in the presence and absence of LMP1. Our cDNA microarray analysis revealed that expression of early growth response gene-1 (Egr-1) was increased by LMP1 expression in MCF7 and Jurkat cells. An NFkappaB inhibitor (SN50) antagonized LMP1-induced enhancement of Egr-1 expression, indicating that LMP1 induced Egr-1 via NFkappaB. Furthermore, three lines of evidence indicated that Egr-1 was required for LMP1-induced cancer cell survival. First, Egr-1 expression enhanced the survival of doxorubicin-treated MCF7 cells. Second, inhibition of Egr-1 expression by siRNA (siEgr-1) effectively suppressed LMP-1-induced survival of MCF7 cells. Third, Egr-1 knockdown decreased LMP1-induced expression of Bfl-1. Similar relationships among EBV infection, Egr-1 and drug resistance were also observed in tissues of peripheral T-cell lymphoma-unspecified (PTCL-u) patients.     

Heterologous Aggregates Promote De Novo Prion Appearance via More than One Mechanism

Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q)/asparagine (N)-rich region in their prion domains. [PSI⁺], the prion form of Sup35, appears de novo at dramatically enhanced rates following transient overproduction of Sup35 in the presence of [PIN⁺], the prion form of Rnq1. Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [PIN⁺] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI ⁺][PIN ⁺] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI ⁺], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [PIN⁺], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI ⁺]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [PSI⁺] induction in the absence of [PIN ⁺]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI ⁺], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [PSI⁺] aggregates in a [PIN ⁺]-independent pathway.     

Production of agarase from a novel <Emphasis Type=Italic>Micrococcus</Emphasis> sp. GNUM-08124 strain isolated from the East Sea of Korea

The agar degrading bacterial strain GNUM-08124 was isolated from Enteromorpha compressa collected in the East Sea of Korea by using a selective artificial sea water (ASW) agar plate containing agar as the sole carbon source. GNUM-08124 grows to produce a circular, smooth, yellow-colored, and raised colony. Its ability to hydrolyze agar was confirmed by staining the ASW agar plate with Lugol’s solution. In liquid culture, the cell density (A₆₀₀) increased exponentially and reached a maximum level on the third day of cultivation. The specific agarase activity also increased in proportion to the cell density and reached maximum agarolytic activity on the third day. The 16S rRNA sequence of GNUM-08124 showed a close relationship to Micrococcus luteus (99.65%) and Micrococcus endophyticus (99.15%), which led us to assign it to the genus Micrococcus. Physiological studies indicated that optimal growth conditions were between 30 and 40°C, pH 4 and 7, using media containing between 5 and 10% NaCl (w/v), respectively. The GNUM-08124 strain was a grampositive, urease-positive, and catalase-positive bacterium. It could not hydrolyze gelatin, cellulose, xylan, or starch, but fermented a broader range of substrates, including Dglucose, D-galactose, D-fructose, D-lactose, D-trehalose, D-mannitol, D-melibiose, D-raffinose, D-xylose, methyl-α-D-glucopyranoside, N-acetyl-glucosamine, and xylitol, than those fermented by M. luteus or M. endophyticus, suggesting GNUM-08124 is a novel agar hydrolyzing microorganism belonging to Genus Micrococcus. Micrococcus sp. GNUM-08124 showed the highest agarase activity when it was cultured in ASW-YP medium supplemented with 0.4% glucose, but demonstrated lower activity in rich media (LB or TSB), in spite of superior cell growth, implying that agarase production is tightly regulated in an agar-dependent manner and repressed in rich conditions.     

3D-QSAR study of microsomal prostaglandin E<Subscript>2</Subscript> synthase(mPGES-1) inhibitors

Microsomal prostaglandin E₂ synthase (mPGES-1) has been identified recently as a novel target for treating pain and inflammation. The aim of this study is to understand the binding affinities of reported inhibitors for mPGES-1 and further to design potential new mPGES-1 inhibitors. 3D-QSAR-CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) - techniques were employed on a series of indole derivatives that act as selective mPGES-1 inhibitors. The lowest energy conformer of the most active compound obtained from systematic conformational search was used as a template for the alignment of 32 compounds. The models obtained were used to predict the activities of the test set of eight compounds, and the predicted values were in good agreement with the experimental results. The 3D-QSAR models derived from the training set of 24 compounds were all statistically significant (CoMFA; q ² = 0.89, r ² = 0.95, , and CoMSIA; q ² = 0.84, r ² = 0.93, , ). Contour plots generated for the CoMFA and CoMSIA models reveal useful clues for improving the activity of mPGES-1 inhibitors. In particular, substitutions of an electronegative fluorine atom or a bulky hydrophilic phenoxy group at the meta or para positions of the biphenyl rings might improve inhibitory activity. A plausible binding mode between the ligands and mPGES-1 is also proposed.     

Inhibition of fipronil and nonane metabolism in human liver microsomes and human cytochrome P450 isoforms by chlorpyrifos

Previous studies have established that chlorpyrifos (CPS), fipronil, and nonane can all be metabolized by human liver microsomes (HLM) and a number of cytochrome P450 (CYP) isoforms. However, metabolic interactions between these three substrates have not been described. In this study the effect of either coincubation or preincubation of CPS with HLM or CYP isoforms with either fipronil or nonane as substrate was investigated. In both co- and preincubation experiments, CPS significantly inhibited the metabolism of fipronil or nonane by HLM although CPS inhibited the metabolism of fipronil more effectively than that of nonane. CPS significantly inhibited the metabolism of fipronil by CYP3A4 as well as the metabolism of nonane by CYP2B6. In both cases, preincubation with CPS caused greater inhibition than coincubation, suggesting that the inhibition is mechanism based.     

Effect of sodium butyrate on the production, heterogeneity and biological activity of human thrombopoietin by recombinant Chinese hamster ovary cells

Human thrombopoietin (hTPO) is a heavily glycosylated protein with 6 and 24 potential N- and O-glycosylation sites, respectively. To determine the effect of sodium butyrate (NaBu) on the production and quality of hTPO in recombinant Chinese hamster ovary (rCHO) cells, NaBu (0-10 mM) was added to the cultures of exponentially growing cells. NaBu addition significantly increased both the specific and volumetric hTPO production, although it decreased the cell viability by apoptosis in a dose-dependent manner. The highest hTPO concentration of 82.2 +/- 5.6 microgml-1 was obtained in the culture with 3 mM NaBu addition. Compared with the culture without NaBu addition, the culture with 3 mM NaBu resulted in a 6.4-fold increase in qTPO and a 3.3-fold increase in the final hTPO concentration on day 7. However, NaBu deteriorated the quality of hTPO, resulting from increased heterogeneity, reduced acidic hTPO isoforms, reduced alpha(2 --> 3) sialylation, and decreased in vivo biological activity. We also found that the biological activity of hTPO in the culture with 3 mM NaBu addition collected on day 7 was 72% of that in the culture without NaBu addition. Taken together, the use of NaBu or its optimal concentration for high-level expression of a heavily glycosylated protein like hTPO should be determined by considering its detrimental effect on the quality of glycoprotein.     

Effects of green tea polyphenol on cognitive and acetylcholinesterase activities

The effect of tea polyphenol (TP) on cognitive and anti-cholinesterase activity was examined in scopolamine-treated mice. Chronic administration of TP significantly reversed scopolamine-induced retention deficits in both step-through passive avoidance and spontaneous alternation behavior tasks. Furthermore, TP exhibited a dramatic inhibitory effect on acetylcholinesterase activity. This finding suggests that TP might be useful in the treatment of Alzheimer's disease.