Exposure of yeast cells to anoxia induces transient oxidative stress. Implications for the induction of hypoxic genes

The mitochondrial respiratory chain is required for the induction of some yeast hypoxic nuclear genes. Because the respiratory chain produces reactive oxygen species (ROS), which can mediate intracellular signal cascades, we addressed the possibility that ROS are involved in hypoxic gene induction. Recent studies with mammalian cells have produced conflicting results concerning this question. These studies have relied almost exclusively on fluorescent dyes to measure ROS levels. Insofar as ROS are very reactive and inherently unstable, a more reliable method for measuring changes in their intracellular levels is to measure their damage (e.g. the accumulation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in DNA, and oxidative protein carbonylation) or to measure the expression of an oxidative stress-induced gene, e.g. SOD1. Here we used these approaches as well as a fluorescent dye, carboxy-H(2)-dichloro-dihydrofluorescein diacetate (carboxy-H(2)-DCFDA), to determine whether ROS levels change in yeast cells exposed to anoxia. These studies reveal that the level of mitochondrial and cytosolic protein carbonylation, the level of 8-OH-dG in mitochondrial and nuclear DNA, and the expression of SOD1 all increase transiently during a shift to anoxia. These studies also reveal that carboxy-H(2)-DCFDA is an unreliable reporter of ROS levels in yeast cells shifted to anoxia. By using two-dimensional electrophoresis and mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), we have found that specific proteins become carbonylated during a shift to anoxia and that some of these proteins are the same proteins that become carbonylated during peroxidative stress. The mitochondrial respiratory chain is responsible for much of this carbonylation. Together, these findings indicate that yeast cells exposed to anoxia experience transient oxidative stress and raise the possibility that this initiates the induction of hypoxic genes.     

New solid supports linking nucleoside scaffolds

An easy and efficient strategy to obtain new nucleoside based solid supports in which the nucleoside moieties have been anchored to the solid support through the nucleobase is here proposed. A simple and efficient solid-phase synthesis of 5' and 3'-derivatized uridine analogues has so been developed, following methodologies well established in organic chemistry.     

Two alternative conformations of S-adenosyl-L-homocysteine bound to Escherichia coli DNA adenine methyltransferase and the implication of conformational changes in regulating the catalytic cycle

The crystal structure of the Escherichia coli DNA adenine methyltransferase (EcoDam) in a binary complex with the cofactor product S-adenosyl-L-homocysteine (AdoHcy) unexpectedly showed the bound AdoHcy in two alternative conformations, extended or folded. The extended conformation represents the catalytically competent conformation, identical to that of EcoDam-DNA-AdoHcy ternary complex. The folded conformation prevents catalysis, because the homocysteine moiety occupies the target Ade binding pocket. The largest difference between the binary and ternary structures is in the conformation of the N-terminal hexapeptide ((9)KWAGGK(14)). Cofactor binding leads to a strong change in the fluorescence of Trp(10), whose indole ring approaches the cofactor by 3.3A(.) Stopped-flow kinetics and AdoMet cross-linking studies indicate that the cofactor prefers binding to the enzyme after preincubation with DNA. In the presence of DNA, AdoMet binding is approximately 2-fold stronger than AdoHcy binding. In the binary complex the side chain of Lys(14) is disordered, whereas Lys(14) stabilizes the active site in the ternary complex. Fluorescence stopped-flow experiments indicate that Lys(14) is important for EcoDam binding of the extrahelical target base into the active site pocket. This suggests that the hexapeptide couples specific DNA binding (Lys(9)), AdoMet binding (Trp(10)), and insertion of the flipped target base into the active site pocket (Lys(14)).     

Omega-alkoxy analogues of SAHA (vorinostat) as inhibitors of HDAC: a study of chain-length and stereochemical dependence

A series of omega-alkoxy ethers were prepared with variation of the length of the aliphatic chain of suberoylanilide hydroxamic acid (SAHA, vorinostat). Eight carbon long chain analogues showed the best activity, among which several substituted benzyl ether derivatives exhibited inhibitory activity on HDAC comparable to SAHA, and antiproliferative activity on three human cell lines (NB4, H460, and HCT-116) better than SAHA. However, no significant difference in antiproliferative activity was observed between two enantiomers bearing the benzyl ether moiety.     

Total extract of Beta vulgaris var. cicla seeds versus its purified phenolic components: antioxidant activities and antiproliferative effects against colon cancer cells

Introduction – Beta vulgaris var. cicla (BV) leaves contain chemopreventive compounds that have been investigated for new drug discovery. These compounds belong to the family of the apigenin‐glycosides. Since the leaves are seasonal products containing high percentages of water, they are easily degradable during storage in fresh conditions. To be stored they require a drying process, consuming time and a large amount of energy. The extraction of apigenin‐glycosides may also be conveniently performed from BV seeds, which represent a stable and year‐long available biomass. Objectives – The present report was undertaken to find a strategy of purification of bioactive flavonoids from BV seeds and test their ability to inhibit proliferation both on human colon cancer (RKO) cells and normal human fibroblasts (HF). Materials and methods – The ethyl‐acetate extract of BV seeds was fractionated on a Sephadex LH 20 column. A fraction of this extract, labeled as P4, exploited a marked antiproliferative activity on RKO cells. The components of P4 were purified on an RP₁₈ column chromatography and identified by HPLC‐ESI‐MS as 2,4,5‐trihydroxybenzaldehyde, 2,5‐dihydroxybenzaldehyde, vanillic acid, xylosylvitexin, glucopyranosyl‐glucopyrasyl‐rhamnetin and glucopyranosyl‐xylosyl‐rhamnetin. All of them were tested for cytostatic and cytotoxic activity on RKO and HF cells. Results – Xylosylvitexin exhibited the strongest antiproliferative activity on RKO cells, together with an enhancement of the apoptosis, an increase of cells in the G₁ phase and a reduction of cells in the S phase; on the contrary, the proliferation of HF was significantly stimulated. Conclusion – Xylosylvitexin is the main and more efficient chemopreventive compound in BV seeds, but the natural cocktail of molecules, represented by P4 fraction, showed a better compromise between the antiproliferative activity on RKO cells and the enhancement of HF proliferation. Copyright © 2011 John Wiley & Sons, Ltd.