Mineralization of Trichloroethylene by Heterotrophic Enrichment Cultures

Microbial consortia capable of aerobically degrading more than 99% of exogenous trichloroethylene (TCE) (50 mg/liter) were collected from TCE-contaminated subsurface sediments and grown in enrichment cultures. TCE at concentrations greater than 300 mg/liter was not degraded, nor was TCE used by the consortia as a sole energy source. Energy sources which permitted growth included tryptone-yeast extract, methanol, methane, and propane. The optimum temperature range for growth and subsequent TCE consumption was 22 to 37°C, and the pH optimum was 7.0 to 8.1. Utilization of TCE occurred only after apparent microbial growth had ceased. The major end products recovered were hydrochloric acid and carbon dioxide. Minor products included dichloroethylene, vinylidine chloride, and, possibly, chloroform.     

Factors Affecting High-Oxygen Survival of Heterotrophic Microorganisms from an Antarctic Lake

We sought to determine factors relating to the survival of heterotrophic microorganisms from the high-dissolved-oxygen (HDO) waters of Lake Hoare, Antarctica. This lake contains perpetual HDO about three times that of normal saturation (40 to 50 mg liter⁻¹). Five isolates, one yeast and four bacteria, were selected from Lake Hoare waters by growth with the membrane filter technique with oxygen added to yield dissolved concentrations 14 times that in situ, 175 mg liter⁻¹. One bacterial isolate was obtained from the microbial mat beneath the HDO waters. This organism was isolated at normal atmospheric oxygen saturation. The bacteria were gram-negative rods, motile, oxidase positive, catalase positive, and superoxide dismutase positive; they contained carotenoids. The planktonic isolates grew in media containing 10 mg of Trypticase soy (BBL Microbiology Systems)-peptone (2:1) liter⁻¹ but not at 10 g liter⁻¹. Under low-nutrient levels simulating Lake Hoare waters (10 mg liter⁻¹), two of the planktonic isolates tested were not inhibited by HDO. Growth inhibition by HDO increased as nutrient concentration was increased. A carotenoid-negative mutant of one isolate demonstrated a decreased growth rate, maximal cell density, and increased cell lysis in the death phase under HDO compared with the parent strain. The specific activity of superoxide dismutase was increased by HDO in four of the five bacterial isolates. The superoxide dismutase was of the manganese type on the basis of inhibition and electrophoretic studies. The bacterial isolates from Lake Hoare possess several adaptations which may aid their survival in the HDO waters, as well as protection due to the oligotrophic nature of the lake.     

Testing biological activity of model Maillard reaction products: studies on gastric smooth muscle tissues

Water-soluble Maillard reaction products obtained from five different model systems were investigated for their effects upon the mechanical activity of rat gastric smooth muscle. Most of the total Maillard reaction products applied at concentration of 1.5 mg/ml evoked contractions; among them the product obtained from arginine and glucose (Arg-Glc) produced the most powerful contractions. The product obtained from glycine and ascorbic acid (Gly-AsA) was the only one that brought about relaxation response. The high molecular weight fractions (>3,500 Da) isolated from the reaction systems Arg-Glc and Gly-AsA demonstrated effects similar in type and amplitude to those evoked by non-fractioned reaction products. The results obtained suggest that moieties of molecules acting upon the muscle tonus originate mainly from lysine and arginine residues; that these structures are available in both low and high molecular pools in similar concentrations, and most likely these fragments act upon membrane-located cellular structures involved in calcium transport.     

Fluorescent cyclin-dependent kinase inhibitors block the proliferation of human breast cancer cells

Inhibitors of cyclin-dependent kinases (CDKs) are an emerging class of drugs for the treatment of cancers. CDK inhibitors are currently under evaluation in clinical trials as single agents and as sensitizers in combination with radiation therapy and chemotherapies. Drugs that target CDKs could have important inhibitory effects on cancer cell cycle progression, an extremely important mechanism in the control of cancer cell growth. Using rational drug design, we designed and synthesized fluorescent CDK inhibitors (VMY-1-101 and VMY-1-103) based on a purvalanol B scaffold. The new agents demonstrated more potent CDK inhibitory activity, enhanced induction of G2/M arrest and modest apoptosis as compared to purvalanol B. Intracellular imaging of the CDK inhibitor distribution was performed to reveal drug retention in the cytoplasm of treated breast cancer cells. In human breast cancer tissue, the compounds demonstrated increased binding as compared to the fluorophore. The new fluorescent CDK inhibitors showed undiminished activity in multidrug resistance (MDR) positive breast cancer cells, indicating that they are not a substrate for p-glycoprotein. Fluorescent CDK inhibitors offer potential as novel theranostic agents, combining therapeutic and diagnostic properties in the same molecule.     

ATM-dependent CHK2 activation induced by anticancer agent, irofulven

Irofulven (6-hydroxymethylacylfulvene, HMAF, MGI 114) is one of a new class of anticancer agents that are semisynthetic derivatives of the mushroom toxin illudin S. Preclinical studies and clinical trials have demonstrated that irofulven is effective against several tumor types. Mechanisms of action studies indicate that irofulven induces DNA damage, MAPK activation, and apoptosis. In this study we found that in ovarian cancer cells, CHK2 kinase is activated by irofulven while CHK1 kinase is not activated even when treated at higher concentrations of the drug. By using GM00847 human fibroblast expressing tetracycline-controlled, FLAG-tagged kinase-dead ATR (ATR.kd), it was demonstrated that ATR kinase does not play a major role in irofulven-induced CHK2 activation. Results from human fibroblasts proficient or deficient in ATM function (GM00637 and GM05849) indicated that CHK2 activation by irofulven is mediated by the upstream ATM kinase. Phosphorylation of ATM on Ser(1981), which is critical for kinase activation, was observed in ovarian cancer cell lines treated with irofulven. RNA interference results confirmed that CHK2 activation was inhibited after introducing siRNA for ATM. Finally, experiments done with human colon cancer cell line HCT116 and its isogenic CHK2 knockout derivative; and experiments done by expressing kinase-dead CHK2 in an ovarian cancer cell line demonstrated that CHK2 activation contributes to irofulven-induced S phase arrest. In addition, it was shown that NBS1, SMC1, and p53 were phosphorylated in an ATM-dependent manner, and p53 phosphorylation on serine 20 is dependent on CHK2 after irofulven treatment. In summary, we found that the anticancer agent, irofulven, activates the ATM-CHK2 DNA damage-signaling pathway, and CHK2 activation contributes to S phase cell cycle arrest induced by irofulven.     

Efficient reversal of phiC31 integrase recombination in mammalian cells

Over the past decade, the integrase enzyme from phage phiC31 has proven to be a useful genome engineering tool in a wide variety of species, including mammalian cells. The enzyme efficiently mediates recombination between two distinct sequences, attP and attB, producing recombinant product sites, attL and attR. The reaction proceeds exclusively in a unidirectional manner, because integrase is unable to synapse attL and attR. To date, use of phiC31 integrase has been limited to attP × attB recombination. The factor needed for the reverse reaction – the excisionase or recombination directionality factor (RDF) – was identified recently and shown to function in vitro and in bacterial cells. To determine whether the phiC31 RDF could also function in mammalian cells, we cloned and tested several vectors that permit assessment of phiC31 RDF activity in mammalian environments. In the human and mouse cell lines tested (HeLa, HEK293, and NIH3T3), we observed robust RDF activity, using plasmid and/or genomic assays. This work is the first to demonstrate attL–attR serine integrase activity in mammalian cells and validates phiC31 RDF as a new tool that will enable future studies to take advantage of phiC31 integrase recombination in the forward or reverse direction.     

Bioconversion of silybin to phase I and II microbial metabolites with retained antioxidant activity

Microbial transformation of silybin (1), the major flavonolignan of milk thistle (Silybum marianum, Asteraceae), resulted in the isolation of four metabolites. The structures of the isolated metabolites were determined by spectroscopic methods. One phase I metabolite was produced by Beauveria bassiana and was characterized as 8-hydroxysilybin (2). Three phase II metabolites were produced by two Cunninghamella species and were identified as 2,3-dehydrosilybin-3-O-β-d-glucoside (3), obtained from Cunninghamella species; and silybin-7-sulfate (4) and 2,3-dehydrosilybin-7-sulfate (5), obtained from Cunninghamella blakesleana. Compared to 1 (IC(50) 284 μg/mL), the generated metabolites displayed varying levels of antioxidant activities in the DPPH free-radical scavenging assay.