Origin of changes in electrical impedance during the growth and fermentation process of yeast in batch culture

The electrical impedance of the culture medium shows complex changes during the growth and fermentation process of yeast, and this prevents its possible application for the monitoring of certain yeast activities. Clarification of the mechanism of such changes is thus essential for practical use. As a first step toward this aim, the impedance, yeast concentration, and pH of a batch culture medium were measured using special cells with two compartments and also the usual type of cell with one compartment. In the special cells, the yeast was cultured in one compartment only. Conducting ions and nonconducting substances diffused through an intermediate porous membrane sandwiched between the two compartments. The impedances of the two compartments were measured simultaneously by the four-electrode method. The main mechanism responsible for increasing the impedance was the conducting ions produced by the yeast extract added as a nutrient to the culture broth by certain nonconducting substances during the process of growth. The increase in the yeast concentration was also a minor factor increasing the impedance. These increases surpassed the impedance decrease caused by the increase of H(+) ions produced by some accumulated acidic substances, and the impedance thus increased.     

Plasmid-encoded regulation of colicin E1 gene expression.

A plasmid-encoded factor that regulates the expression of the colicin E1 gene was found in molecular cloning experiments. The 2,294-base-pair AvaII fragment of the colicin E1 plasmid (ColE1) carrying the colicin E1 structural gene and the promoter-operator region had the same information with respect to the repressibility and inducibility of colicin E1 synthesis as the original ColE1 plasmid. An operon fusion was constructed between the 204-bp fragment containing the colicin E1 promoter-operator and xylE, the structural gene for catechol 2,3-dioxygenase encoded on the TOL plasmid of Pseudomonas putida. The synthesis of the dioxygenase from the resulting plasmid occurred in recA+, but not in recA- cells and was derepressed in the recA lexA(Def) double mutant. These results indicate that the ColE1 plasmid has no repressor gene for colicin E1 synthesis and that the lexA protein functions as a repressor. Colicin E1 gene expression was adenosine 3',5'-phosphate (cAMP) dependent. Upon the removal of two PvuII fragments (2,000 bp in length) from the ColE1 plasmid, the induced synthesis of colicin E1 occurred in the adenylate-cyclase mutant even without cAMP. The 3,100-bp Tth111I fragment of the ColE1 plasmid cloned on pACYC177 restored the cAMP dependency of the deleted ColE1 plasmid. Since the deleted fragments correspond to the mobility region of ColE1, the cAMP dependency of the gene expression should be somehow related to the plasmid mobilization function.     

Weak Light-Induced Oxygen Consumption Observed during Photoreactivation Is Coupled to the Recovery of Oxygen Evolving Activity

During photoreactivation of the O₂-evolving center in Tris-inactivated/Mn-depletedthylakoids, a slow O₂-consumption occurred. This O₂-consumptionbecame detectable when the O₂-evolving activity of thylakoidswas inactivated by Tris-treatment and decreased as photoreactivationproceeded. The O₂-consumption and photoreactivation similarlyrequired Mn²⁺ at µM levels in addition to PSII electrondonors and shared severa common characteristics. Stimulationof O₂-consumption and photoreactivation by these cofactors werealways accompanied by enhancement in chlorophyll fluorescenceinduction, suggesting the involvement of a Mehler-type reactionin photoreactivation. Although the electron transport due tothis O₂-consumption was rapid enough to oxidize 4 Mn²⁺ ionsto reconstitute the tetranuclear Mn-cluster in each O₂-evolvingcenter in a few seconds, actual recovery of O₂-evolving activityoccurred more slowly in a few minutes. It was inferred thatphotoreactivation in Tris-inactivated thylakoids is not a simplephotooxidation of Mn2²⁺ but involves more complicated processeswhich are coupled to the Mehlertype electron transport fromPSII to oxygen via PSI. (Received July 11, 1994; Accepted August 23, 1996)     

Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.     

Copper(II) complexes of 1,10-phenanthroline-derived ligands: studies on DNA binding properties and nuclease activity

A series of copper(II) complexes of the type [Cu(L)]2+, where L = N,N'-dialkyl-1,10-phenanthroline-2,9-dimethanamine and R = methyl (L1), n-propyl (L2), isopropyl (L3), sec-butyl (L4), or tert-butyl (L5) group, have been synthesized. The interaction of the complexes with DNA has been studied by DNA fiber electron paramagnetic resonance (EPR) spectroscopy, emission, viscosity and electrochemical measurements and agarose gel electrophoresis. In the X-ray crystal structure of [Cu(HL2)Cl2]NO3, copper(II) is coordinated to two ring nitrogens and one of the two secondary amine nitrogens of the side chains and two chloride ions as well and the coordination geometry is best described as trigonal bipyramidal distorted square based pyramidal (TBDSBP). Electronic and EPR spectral studies reveal that all the complexes in aqueous solution around pH 7 possess CuN3O2 rather than CuN4O chromophore with one of the alkylamino side chain not involved in coordination. The structures of the complexes in aqueous solution around pH 7 change from distorted tetragonal to trigonal bipyramidal as the size of the alkyl group is increased. The observed changes in the physicochemical features of the complexes on binding to DNA suggest that the complexes, except [Cu(L5)]2+, bind to DNA with partial intercalation of the derivatised phen ring in between the DNA base pairs. Electrochemical studies reveal that the complexes prefer to bind to DNA in Cu(II) rather than Cu(I) oxidation state. Interestingly, [Cu(L5)]2+ shows the highest DNA cleavage activity among all the present copper(II) complexes suggesting that the bulky N-tert-butyl group plays an important role in modifying the coordination environment around the copper(II) center, the Cu(II)/Cu(I) redox potential and hence the formation of activated oxidant responsible for the cleavage. These results were compared with those for bis(1,10-phenanthroline)copper(II), [Cu(phen)2]2+.     

Expression and methylation status of 14-3-3 sigma gene can characterize the different histological features of ovarian cancer

We hypothesize that 14-3-3 sigma gene expression and its regulation by methylation can characterize histological types of primary human epithelial ovarian cancer. To test this hypothesis, ovarian cancer cell lines and 54 ovarian cancer tissue samples were analyzed for expression and methylation of 14-3-3 sigma gene using methylation specific PCR. The results of our experiments demonstrate that 14-3-3 sigma gene was methylated and inactivated in ES-2 ovarian cell line, which was derived from clear cell adenocarcinoma. Treatment of this cell line with demethylating agent 5-aza-2'-deoxycytidine restored the expression of 14-3-3 sigma gene. In human ovarian cancer tissues, the expression of 14-3-3 sigma protein was inactivated in most of the ovarian clear cell carcinoma tissues. Interestingly, 14-3-3 sigma protein expression was positive in significantly higher percentages of serous (89.5%), endometrioid (90%), and mucinous (81.8%) ovarian adenocarcinoma tissues. The ovarian clear cell carcinoma samples with inactivated 14-3-3 sigma protein were highly methylated, suggesting that inactivation of 14-3-3 sigma gene is through DNA methylation. Using direct DNA sequencing, 14-3-3 sigma gene methylation on all the 17 CpG sites was significantly higher in ovarian clear cell carcinoma as compared to other histological types of ovarian cancer (serous, endometrioid, and mucinous). This is the first report suggesting that 14-3-3 sigma gene expression and methylation status can characterize histological features of different types of ovarian cancer.     

Intratracheal gene transfer of decorin reduces subpleural fibroproliferation induced by bleomycin

Decorin, a small leucin-rich proteoglycan, is a negative regulator of transforming growth factor-beta, but the antifibrotic effect of decorin gene transfer has not been examined in a mouse model of usual interstitial pneumonia (UIP). We constructed a replication-defective recombinant adenovirus harboring human decorin gene (AdCMV.DC) and administered 1 x l0(9) plaque-forming units of AdCMV.DC intratracheally or intravenously to C57BL/6 mice with intraperitoneal injection of bleomycin, which induces a subpleural fibroproliferation, mimicking UIP, by day 28. Only intratracheal administration of AdCMV.DC increased decorin mRNA expression in the lung and decreased the hydroxyproline content augmented in bleomycin-induced pulmonary fibrosis (1.13 +/- 0.02 to 0.96 +/- 0.02, P = 0.006). In contrast, intravenous administration of AdCMV.DC increased the decorin expression only in the liver, but not in the lung, and without reducing lung fibrosis. These results indicate that adenoviral decorin gene transfer is effective only by direct administration to fibrosing lungs.