The unfolded protein response triggers selective mRNA release from the endoplasmic reticulum.

The unfolded protein response (UPR) is a stress response program that reprograms cellular translation and gene expression in response to proteotoxic stress in the endoplasmic reticulum (ER). One of the primary means by which the UPR alleviates this stress is by reducing protein flux into the ER via a general suppression of protein synthesis and ER-specific mRNA degradation. We report here an additional UPR-induced mechanism for the reduction of protein flux into the ER, where mRNAs that encode signal sequences are released from the ER to the cytosol. By removing mRNAs from the site of translocation, this mechanism may serve as a potent means to transiently reduce ER protein folding load and restore proteostasis. These findings identify the dynamic subcellular localization of mRNAs and translation as a selective and rapid regulatory feature of the cellular response to protein folding stress.

Dithiothreitol reduces oxidative stress and necrosis caused by ultraviolet A radiation in L929 fibroblasts.

Ultraviolet A (UVA) radiation, present in sunlight, can induce cell redox imbalance leading to cellular damage and even cell death, compromising skin health. Here, we evaluated the in vitro antioxidant and photochemoprotective effect of dithiothreitol (DTT). DTT neutralized the free radicals 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS·+), 2,2-diphenyl-1-picrylhydrazyl (DPPH·), and superoxide anion (O2·-) in in vitro assays, as well as the ferric ion (Fe3+) in the ferric reducing antioxidant power (FRAP) assay. We also evaluated the effect of DTT pre-treatment in L929 dermal fibroblasts and DTT (50 and 100 µM) led to greater cell viability following UVA-irradiation compared to cells that were untreated. Furthermore, the pre-treatment of cells with DTT prevented the increase of intracellular reactive oxygen species (ROS) production, including hydrogen peroxide (H2O2), lipid peroxidation, and DNA condensation, as well as the decrease in mitochondrial membrane potential (Δψm), that occurred following irradiation in untreated cells. The endogenous antioxidant system of cells was also improved in irradiated cells that were DTT pre-treated compared to the untreated cells, as the activity of the superoxide dismutase (SOD) and catalase (CAT) enzymes remained as high as non-irradiated cells, while the activity levels were depleted in the untreated irradiated cells. Furthermore, DTT reduced necrosis in UVA-irradiated fibroblasts. Together, these results showed that DTT may have promising use in the prevention of skin photoaging and photodamage induced by UVA, as it provided photochemoprotection against the harmful effects of this radiation, reducing oxidative stress and cell death, due mainly to its antioxidant capacity.

Dietary-derived vitamin B12 protects Caenorhabditis elegans from thiol-reducing agents.

BACKGROUND: One-carbon metabolism, which includes the folate and methionine cycles, involves the transfer of methyl groups which are then utilised as a part of multiple physiological processes including redox defence. During the methionine cycle, the vitamin B12-dependent enzyme methionine synthetase converts homocysteine to methionine. The enzyme S-adenosylmethionine (SAM) synthetase then uses methionine in the production of the reactive methyl carrier SAM. SAM-binding methyltransferases then utilise SAM as a cofactor to methylate proteins, small molecules, lipids, and nucleic acids. RESULTS: We describe a novel SAM methyltransferase, RIPS-1, which was the single gene identified from forward genetic screens in Caenorhabditis elegans looking for resistance to lethal concentrations of the thiol-reducing agent dithiothreitol (DTT). As well as RIPS-1 mutation, we show that in wild-type worms, DTT toxicity can be overcome by modulating vitamin B12 levels, either by using growth media and/or bacterial food that provide higher levels of vitamin B12 or by vitamin B12 supplementation. We show that active methionine synthetase is required for vitamin B12-mediated DTT resistance in wild types but is not required for resistance resulting from RIPS-1 mutation and that susceptibility to DTT is partially suppressed by methionine supplementation. A targeted RNAi modifier screen identified the mitochondrial enzyme methylmalonyl-CoA epimerase as a strong genetic enhancer of DTT resistance in a RIPS-1 mutant. We show that RIPS-1 is expressed in the intestinal and hypodermal tissues of the nematode and that treating with DTT, ß-mercaptoethanol, or hydrogen sulfide induces RIPS-1 expression. We demonstrate that RIPS-1 expression is controlled by the hypoxia-inducible factor pathway and that homologues of RIPS-1 are found in a small subset of eukaryotes and bacteria, many of which can adapt to fluctuations in environmental oxygen levels. CONCLUSIONS: This work highlights the central importance of dietary vitamin B12 in normal metabolic processes in C. elegans, defines a new role for this vitamin in countering reductive stress, and identifies RIPS-1 as a novel methyltransferase in the methionine cycle.

Comparative metabolomics and transcriptomics analyses provide insights into the high-yield mechanism of phenazines biosynthesis in Pseudomonas chlororaphis GP72.

AIMS: Phenazines, such as phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), 2-hydroxyphenazine-1-carboxylic acid (2-OH-PCA), 2-hydroxyphenazine (2-OH-PHZ), are a class of secondary metabolites secreted by plant-beneficial Pseudomonas. Ps. chlororaphis GP72 utilizes glycerol to synthesize PCA, 2-OH-PCA and 2-OH-PHZ, exhibiting broad-spectrum antifungal activity. Previous studies showed that the addition of dithiothreitol (DTT) could increase the phenazines production in Ps. chlororaphis GP72AN. However, the mechanism of high yield of phenazine by adding DTT is still unclear. METHODS AND RESULTS: In this study, untargeted and targeted metabolomic analysis were adopted to determine the content of metabolites. The results showed that the addition of DTT to GP72AN affected the content of metabolites of central carbon metabolism, shikimate pathway and phenazine competitive pathway. Transcriptome analysis was conducted to investigate the changed cellular process, and the result indicated that the addition of DTT affected the expression of genes involved in phenazine biosynthetic cluster and genes involved in phenazine competitive pathway, driving more carbon flux into phenazine biosynthetic pathway. Furthermore, genes involved in antioxidative stress, phosphate transport system and mexGHI-opmD efflux pump were also affected by adding DTT. CONCLUSION: This study demonstrated that the addition of DTT altered the expression of genes related to phenazine biosynthesis, resulting in the change of metabolites involved in central carbon metabolism, shikimate pathway and phenazine competitive pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: This work expands the understanding of high yield of phenazine by the addition of DTT and provides several targets for increasing phenazine production.

Biotransformation of Dec-604 and potential effect on thyroid deiodinase activity in highly flame retardant-exposed gulls.

Several halogenated flame retardants (HFRs) have been identified as thyroid disruptors in birds including the polybrominated diphenyl ether (PBDE) mixtures, which have been replaced with other HFRs such as Dechlorane-604 (Dec-604). Dec-604 Component B (Dec-604 CB), a putative debrominated product of Dec-604, has been frequently reported in urban-adapted ring-billed gulls (Larus delawarensis) breeding in the Montreal area (QC, Canada). The metabolic pathways of Dec-604 are yet to be characterized, although the occurrence of Dec-604 CB in gulls may suggest that enzyme-mediated dehalogenation may occur, potentially involving the thyroid deiodinases. The objective of this study was to investigate the effect of Dec-604 on type 1 deiodinase (DIO1) in the presence of thyroxine (T4) in an in vitro DIO1 assay using liver microsomes of ring-billed gulls that are highly exposed to HFRs in the Montreal area, and to determine whether DIO1 is involved in the in vitro debromination of Dec-604. We tested the in vitro activity of DIO1 in gull liver microsomes in the presence of five concentrations of Dec-604 ranging from 0.86 to 86.21 nM. HFR concentrations (Σ40HFR) were also determined in liver samples of gulls. Results showed that total DIO1 activity in gull liver microsomes was increased by three of the five concentrations of Dec-604. No relationship between liver Σ40HFR concentrations and DIO1 activity was observed, except for T2 formation rates that significantly decreased with increasing liver HFR concentrations. Moreover, greater Dec-604 CB to Dec-604 concentration ratios in activated gull microsomes (with the DIO1 cofactor dithiothreitol) were found at the intermediate Dec-604 concentration compared to controls. These results suggested that liver microsome DIO1 activity may be perturbed in ring-billed gulls exposed to Dec-604, and be involved at least in part, in the debromination of Dec-604 leading to the formation of Dec-604 CB.

Preparations of Terminal Oxidase Cytochrome bd-II Isolated from Escherichia coli Reveal Significant Hydrogen Peroxide Scavenging Activity.

Cytochrome bd-II is one of the three terminal quinol oxidases of the aerobic respiratory chain of Escherichia coli. Preparations of the detergent-solubilized untagged bd-II oxidase isolated from the bacterium were shown to scavenge hydrogen peroxide (H2O2) with high rate producing molecular oxygen (O2). Addition of H2O2 to the same buffer that does not contain enzyme or contains thermally denatured cytochrome bd-II does not lead to any O2 production. The latter observation rules out involvement of adventitious transition metals bound to the protein. The H2O2-induced O2 production is not susceptible to inhibition by N-ethylmaleimide (the sulfhydryl binding compound), antimycin A (the compound that binds specifically to a quinol binding site), and CO (diatomic gas that binds specifically to the reduced heme d). However, O2 formation is inhibited by cyanide (IC50 = 4.5 ± 0.5 µM) and azide. Addition of H2O2 in the presence of dithiothreitol and ubiquinone-1 does not inactivate cytochrome bd-II and apparently does not affect the O2 reductase activity of the enzyme. The ability of cytochrome bd-II to detoxify H2O2 could play a role in bacterial physiology by conferring resistance to the peroxide-mediated stress.

Structural basis of catalysis and substrate recognition by the NAD(H)-dependent α-d-glucuronidase from the glycoside hydrolase family 4.

Members of the glycoside hydrolase family 4 (GH4) employ an unusual glycosidic bond cleavage mechanism utilizing NAD(H) and a divalent metal ion, under reducing conditions. These enzymes act upon a diverse range of glycosides, and unlike most other GH families, homologs here are known to accommodate both α- and ß-anomeric specificities within the same active site. Here, we report the catalytic properties and the crystal structures of TmAgu4B, an α-d-glucuronidase from the hyperthermophile Thermotoga maritima. The structures in three different states include the apo form, the NADH bound holo form, and the ternary complex with NADH and the reaction product d-glucuronic acid, at 2.15, 1.97 and 1.85 Šresolutions, respectively. These structures reveal the step-wise route of conformational changes required in the active site to achieve the catalytically competent state, and illustrate the direct role of residues that determine the reaction mechanism. Furthermore, a structural transition of a helical region in the active site to a turn geometry resulting in the rearrangement of a unique arginine residue governs the exclusive glucopyranosiduronic acid recognition in TmAgu4B. Mutational studies show that modifications of the glycone binding site geometry lead to catalytic failure and indicate overlapping roles of specific residues in catalysis and substrate recognition. The data highlight hitherto unreported molecular features and associated active site dynamics that determine the structure-function relationships within the unique GH4 family.

Synthesis of functionalised organochalcogenides and in vitro evaluation of their antioxidant activity.

Differently substituted ß-hydroxy- and ß-amino dialkyl and alkyl-aryl tellurides and selenides have been prepared through ring-opening reactions of epoxides and aziridines with selenium- or tellurium-centered nucleophiles. The antioxidant properties and the cytotoxicity of such compounds have been investigated on normal human dermal fibroblasts. Most of the studied compounds exhibited a low cytotoxicity and a number of them proved to be non-toxic, not showing any effect on cell viability even at the highest concentration used (100 µM). The obtained results showed a significant antioxidant potential of the selected organotellurium compounds, particularly evident under conditions of exogenously induced oxidative stress. The antioxidant activity of selenium-containing analogues of active tellurides has also been evaluated on cells, highlighting that the replacement of Se with Te brought about a significant increase in the peroxidase activity.

Understanding the role of electron donors in the reaction catalyzed by Tsrm, a cobalamin-dependent radical S-adenosylmethionine methylase.

The cobalamin-dependent radical S-adenosylmethionine (SAM) enzyme TsrM catalyzes the methylation of C2 of L-tryptophan to form 2-methyltryptophan during the biosynthesis of thiostrepton A. Although TsrM is a member of the radical SAM superfamily, unlike all other annotated members, it does not catalyze a reductive cleavage of SAM to a 5'-deoxyadenosyl 5'-radical intermediate. In fact, it has been proposed that TsrM catalyzes its reaction through two polar nucleophilic displacements, with its cobalamin cofactor cycling directly between methylcobalamin (MeCbl) and cob(I)alamin. Nevertheless, the enzyme has been stated to require the action of a reductant, which can be satisfied by dithiothreitol. By contrast, all other annotated RS enzymes require a reductant that exhibits a much lower reduction potential, which is necessary for the reductive cleavage of SAM. Herein, we show that TsrM can catalyze multiple turnovers in the absence of any reducing agent, but only when it is pre-loaded with MeCbl. When hydroxocobalamin (OHCbl) or cob(II)alamin is bound to TsrM, a reductant is required to convert it to cob(I)alamin, which can acquire a methyl group directly from SAM. Our studies suggest that TsrM uses an external reductant to prime its reaction by converting bound OHCbl or cob(II)alamin to MeCbl, and to regenerate the MeCbl form of the cofactor upon adventitious oxidation of the cob(I)alamin intermediate to cob(II)alamin.

Dimeric Structure of the Blue Light Sensor Protein Photozipper in the Active State.

The light oxygen voltage-sensing (LOV) domain plays a crucial role in blue light (BL) sensing in plants and microorganisms. LOV domains are usually associated with the effector domains and regulate the activities of effector domains in a BL-dependent manner. Photozipper (PZ) is monomeric in the dark state. BL induces reversible dimerization of PZ and subsequently increases its affinity for the target DNA sequence. In this study, we report the analyses of PZ by pulsed electron-electron double resonance (PELDOR). The neutral flavin radical was formed by BL illumination in the presence of dithiothreitol in the LOV-C254S (without the bZIP domain) and PZ-C254S mutants, where the cysteine residue responsible for adduct formation was replaced with serine. The magnetic dipole interactions of 3 MHz between the neutral radicals were detected in both LOV-C254S and PZ-C254S, indicating that these mutants are dimeric in the radical state. The PELDOR simulation showed that the distance between the radical pair is close to that estimated from the dimeric crystal structure in the "light state" [Heintz, U., and Schlichting, I. (2016) eLife 5, e11860], suggesting that in the radical state, LOV domains in PZ-C254S form a dimer similar to that of LOV-C254S, which lacks the bZIP domain.

Heme-thiolate sulfenylation of human cytochrome P450 4A11 functions as a redox switch for catalytic inhibition.

Cytochrome P450 (P450, CYP) 4A11 is a human fatty acid ω-hydroxylase that catalyzes the oxidation of arachidonic acid to the eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), which plays important roles in regulating blood pressure regulation. Variants of P450 4A11 have been associated with high blood pressure and resistance to anti-hypertensive drugs, and 20-HETE has both pro- and antihypertensive properties relating to increased vasoconstriction and natriuresis, respectively. These physiological activities are likely influenced by the redox environment, but the mechanisms are unclear. Here, we found that reducing agents (e.g. dithiothreitol and tris(2-carboxyethyl)phosphine) strongly enhanced the catalytic activity of P450 4A11, but not of 10 other human P450s tested. Conversely, added H2O2 attenuated P450 4A11 catalytic activity. Catalytic roles of five of the potentially eight implicated Cys residues of P450 4A11 were eliminated by site-directed mutagenesis. Using an isotope-coded dimedone/iododimedone-labeling strategy and mass spectrometry of peptides, we demonstrated that the heme-thiolate cysteine (Cys-457) is selectively sulfenylated in an H2O2 concentration-dependent manner. This sulfenylation could be reversed by reducing agents, including dithiothreitol and dithionite. Of note, we observed heme ligand cysteine sulfenylation of P450 4A11 ex vivo in kidneys and livers derived from CYP4A11 transgenic mice. We also detected sulfenylation of murine P450 4a12 and 4b1 heme peptides in kidneys. To our knowledge, reversible oxidation of the heme thiolate has not previously been observed in P450s and may have relevance for 20-HETE-mediated functions.

Response of Three Different Viruses to Interferon Priming and Dithiothreitol Treatment of Avian Cells.

UNLABELLED: We have previously shown that the replication of avian reovirus (ARV) in chicken cells is much more resistant to interferon (IFN) than the replication of vesicular stomatitis virus (VSV) or vaccinia virus (VV). In this study, we have investigated the role that the double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays in the sensitivity of these three viruses toward the antiviral action of chicken interferon. Our data suggest that while interferon priming of avian cells blocks vaccinia virus replication by promoting PKR activation, the replication of vesicular stomatitis virus appears to be blocked at a pretranslational step. Our data further suggest that the replication of avian reovirus in chicken cells is quite resistant to interferon priming because this virus uses strategies to downregulate PKR activation and also because translation of avian reovirus mRNAs is more resistant to phosphorylation of the alpha subunit of initiation factor eIF2 than translation of their cellular counterparts. Our results further reveal that the avian reovirus protein sigmaA is able to prevent PKR activation and that this function is dependent on its double-stranded RNA-binding activity. Finally, this study demonstrates that vaccinia virus and avian reovirus, but not vesicular stomatitis virus, express/induce factors that counteract the ability of dithiothreitol to promote eIF2 phosphorylation. Our data demonstrate that each of the three different viruses used in this study elicits distinct responses to interferon and to dithiothreitol-induced eIF2 phosphorylation when infecting avian cells. IMPORTANCE: Type I interferons constitute the first barrier of defense against viral infections, and one of the best characterized antiviral strategies is mediated by the double-stranded RNA-activated protein kinase R (PKR). The results of this study revealed that IFN priming of avian cells has little effect on avian reovirus (ARV) replication but drastically diminishes the replication of vaccinia virus (VV) and vesicular stomatitis virus (VSV) by PKR-dependent and -independent mechanisms, respectively. Our data also demonstrate that the dsRNA-binding ability of ARV protein sigmaA plays a key role in the resistance of ARV toward IFN by preventing PKR activation. Our findings will contribute to improve the current understanding of the interaction of viruses with the host's innate immune system. Finally, it would be of interest to uncover the mechanisms that allow avian reovirus transcripts to be efficiently translated under conditions (moderate eIF2 phosphorylation) that block the synthesis of cellular proteins.

Pharmacologic Studies of a Prodrug of Mitomycin C in Pegylated Liposomes (Promitil(®)): High Stability in Plasma and Rapid Thiolytic Prodrug Activation in Tissues.

PURPOSE: Pegylated liposomal (PL) mitomycin C lipid-based prodrug (MLP) has recently entered clinical testing. We studied here the preclinical pharmacology of PL-MLP. METHODS: The stability, pharmacokinetics, biodistribution, and other pharmacologic parameters of PL-MLP were examined. Thiolytic cleavage of MLP and release of active mitomycin C (MMC) were studied using dithiothreitol (DTT), and by incubation with tissue homogenates. RESULTS: MLP was incorporated in the bilayer at 10% molar ratio with nearly 100% entrapment efficiency, resulting in a formulation with high plasma stability. In vitro, DTT induced cleavage of MLP with predictable kinetics, generating MMC and enhancing pharmacological activity. A long circulation half-life of MLP (10-15 h) was observed in rodents and minipigs. Free MMC is either extremely low or undetectable in plasma. However, urine from PL-MLP injected rats revealed delayed but significant excretion of MMC indicating in vivo activation of MLP. Studies in mice injected with H3-cholesterol radiolabeled PL-MLP demonstrated relatively greater tissue levels of H3-cholesterol than MLP. MLP levels were highest in tumor and spleen, and very low or undetectable in liver and lung. Rapid cleavage of MLP in various tissues, particularly in liver, was shown in ex-vivo experiments of PL-MLP with tissue homogenates. PL-MLP was less toxic in vivo than equivalent doses of MMC. Therapeutic studies in C26 mouse tumor models demonstrated dose-dependent improved efficacy of PL-MLP over MMC. CONCLUSIONS: Thiolytic activation of PL-MLP occurs in tissues but not in plasma. Liposomal delivery of MLP confers a favorable pharmacological profile and greater therapeutic index than MMC.

Affinity gradients drive copper to cellular destinations.

Copper is an essential trace element for eukaryotes and most prokaryotes. However, intracellular free copper must be strictly limited because of its toxic side effects. Complex systems for copper trafficking evolved to satisfy cellular requirements while minimizing toxicity. The factors driving the copper transfer between protein partners along cellular copper routes are, however, not fully rationalized. Until now, inconsistent, scattered and incomparable data on the copper-binding affinities of copper proteins have been reported. Here we determine, through a unified electrospray ionization mass spectrometry (ESI-MS)-based strategy, in an environment that mimics the cellular redox milieu, the apparent Cu(I)-binding affinities for a representative set of intracellular copper proteins involved in enzymatic redox catalysis, in copper trafficking to and within various cellular compartments, and in copper storage. The resulting thermodynamic data show that copper is drawn to the enzymes that require it by passing from one copper protein site to another, exploiting gradients of increasing copper-binding affinity. This result complements the finding that fast copper-transfer pathways require metal-mediated protein-protein interactions and therefore protein-protein specific recognition. Together with Cu,Zn-SOD1, metallothioneins have the highest affinity for copper(I), and may play special roles in the regulation of cellular copper distribution; however, for kinetic reasons they cannot demetallate copper enzymes. Our study provides the thermodynamic basis for the kinetic processes that lead to the distribution of cellular copper.

Going through the barrier: coupled disulfide exchange reactions promote efficient catalysis in quiescin sulfhydryl oxidase.

The quiescin sulfhydryl oxidase (QSOX) family of enzymes generates disulfide bonds in peptides and proteins with the reduction of oxygen to hydrogen peroxide. Determination of the potentials of the redox centers in Trypanosoma brucei QSOX provides a context for understanding catalysis by this facile oxidant of protein thiols. The CXXC motif of the thioredoxin domain is comparatively oxidizing (E'0 of -144 mV), consistent with an ability to transfer disulfide bonds to a broad range of thiol substrates. In contrast, the proximal CXXC disulfide in the ERV (essential for respiration and vegetative growth) domain of TbQSOX is strongly reducing (E'0 of -273 mV), representing a major apparent thermodynamic barrier to overall catalysis. Reduction of the oxidizing FAD cofactor (E'0 of -153 mV) is followed by the strongly favorable reduction of molecular oxygen. The role of a mixed disulfide intermediate between thioredoxin and ERV domains was highlighted by rapid reaction studies in which the wild-type CGAC motif in the thioredoxin domain of TbQSOX was replaced by the more oxidizing CPHC or more reducing CGPC sequence. Mixed disulfide bond formation is accompanied by the generation of a charge transfer complex with the flavin cofactor. This provides thermodynamic coupling among the three redox centers of QSOX and avoids the strongly uphill mismatch between the formal potentials of the thioredoxin and ERV disulfides. This work identifies intriguing mechanistic parallels between the eukaryotic QSOX enzymes and the DsbA/B system catalyzing disulfide bond generation in the bacterial periplasm and suggests that the strategy of linked disulfide exchanges may be exploited in other catalysts of oxidative protein folding.

Fluorescent probes for tracking the transfer of iron-sulfur cluster and other metal cofactors in biosynthetic reaction pathways.

Iron-sulfur (Fe-S) clusters are protein cofactors that are constructed and delivered to target proteins by elaborate biosynthetic machinery. Mechanistic insights into these processes have been limited by the lack of sensitive probes for tracking Fe-S cluster synthesis and transfer reactions. Here we present fusion protein- and intein-based fluorescent labeling strategies that can probe Fe-S cluster binding. The fluorescence is sensitive to different cluster types ([2Fe-2S] and [4Fe-4S] clusters), ligand environments ([2Fe-2S] clusters on Rieske, ferredoxin (Fdx), and glutaredoxin), and cluster oxidation states. The power of this approach is highlighted with an extreme example in which the kinetics of Fe-S cluster transfer reactions are monitored between two Fdx molecules that have identical Fe-S spectroscopic properties. This exchange reaction between labeled and unlabeled Fdx is catalyzed by dithiothreitol (DTT), a result that was confirmed by mass spectrometry. DTT likely functions in a ligand substitution reaction that generates a [2Fe-2S]-DTT species, which can transfer the cluster to either labeled or unlabeled Fdx. The ability to monitor this challenging cluster exchange reaction indicates that real-time Fe-S cluster incorporation can be tracked for a specific labeled protein in multicomponent assays that include several unlabeled Fe-S binding proteins or other chromophores. Such advanced kinetic experiments are required to untangle the intricate networks of transfer pathways and the factors affecting flux through branch points. High sensitivity and suitability with high-throughput methodology are additional benefits of this approach. We anticipate that this cluster detection methodology will transform the study of Fe-S cluster pathways and potentially other metal cofactor biosynthetic pathways.

Gallotannin is a DNA damaging compound that induces senescence independently of p53 and p21 in human colon cancer cells.

The plant secondary metabolite gallotannin (GT) is the simplest hydrolyzable tannin shown to have anti-carcinogenic properties in several cell lines and to inhibit tumor development in different animal models. Here, we determined if GT induces senescence and DNA damage and investigated the involvement of p53 and p21 in this response. Using HCT116 human colon cancer cells wildtype for p53(+/+) /p21(+/+) and null for p53(+/+) /p21(-/-) or p53(-/-) /p21(+/+) , we found that GT induces senescence independently of p21 and p53. GT was found to increase the production of reactive oxygen species (ROS) by altering the redox balance in the cell, mainly by reducing the levels of glutathione and superoxide dismutase (SOD). Using the key antioxidants N-acetyl cysteine, dithiothreitol, SOD, and catalase, we showed that ROS were partially involved in the senescence response. Furthermore, GT-induced cell cycle arrest in S-phase in all HCT116 cell lines. At later time points, we noticed that p53 and p21 null cells escaped complete arrest and re-entered cell cycle provoking higher rates of multinucleation. The senescence induction by GT was irreversible and was accompanied by significant DNA damage as evidenced by p-H2AX staining. Our findings indicate that GT is an interesting anti colon cancer agent which warrants further study.

Tris(3-hydroxypropyl)phosphine is superior to dithiothreitol for in vitro assessment of vitamin K 2,3-epoxide reductase activity.

Use of the reductant dithiothreitol (DTT) as a substrate for measuring vitamin K 2,3-epoxide reductase (VKOR) activity in vitro has been reported to be problematic because it enables side reactions involving the vitamin K1 2,3-epoxide (K1>O) substrate. Here we characterize specific problems when using DTT and show that tris(3-hydroxypropyl)phosphine (THPP) is a reliable alternative to DTT for in vitro assessment of VKOR enzymatic activity. In addition, the pH buffering compound imidazole was found to be problematic in enhancing DTT-dependent non-enzymatic side reactions. Using THPP and phosphate-based pH buffering, we measured apparent Michaelis-Menten constants of 1.20 µM for K1>O and 260 µM for the active neutral form of THPP. The Km value for K1>O is in agreement with the value that we previously obtained using DTT (1.24 µM). Using THPP, we successfully eliminated non-enzymatic production of 3-hydroxyvitamin K1 and its previously reported base-catalyzed conversion to K1, both of which were shown to occur when DTT and imidazole are used as the reductant and pH buffer, respectively, in the in vitro VKOR assay. Accordingly, substitution of THPP for DTT in the in vitro VKOR assay will ensure more accurate enzymatic measurements and assessment of warfarin and other 4-hydroxycoumarin inhibition constants.

A non-pediocin low molecular weight antimicrobial peptide produced by Pediococcus pentosaceus strain IE-3 shows increased activity under reducing environment.

BACKGROUND: Species of the genus Pediococcus are known to produce antimicrobial peptides such as pediocin-like bacteriocins that contain YGNGVXC as a conserved motif at their N-terminus. Until now, the molecular weight of various bacteriocins produced by different strains of the genus Pediococcus have been found to vary between 2.7 to 4.6 kD. In the present study, we characterized an antimicrobial peptide produced by P. pentosaceus strain IE-3. RESULTS: Antimicrobial peptide was isolated and purified from the supernatant of P. pentosaceus strain IE-3 grown for 48 h using cation exchange chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) techniques. While MALDI-TOF MS experiments determined the precise molecular mass of the peptide to be 1701.00 Da, the de novo sequence (APVPFSCTRGCLTHLV) of the peptide revealed no similarity with reported pediocins and did not contain the YGNGVXC conserved motif. Unlike pediocin-like bacteriocins, the low molecular weight peptide (LMW) showed resistance to different proteases. Moreover, peptide treated with reducing agent like dithiothreitol (DTT) exhibited increased activity against both Gram-positive and Gram-negative test strains in comparison to native peptide. However, peptide treated with oxidizing agent such as hydrogen peroxide (H2O2) did not show any antimicrobial activity. CONCLUSION: To our knowledge this is the lowest molecular weight peptide produced by members of the genus Pediococcus. The low molecular weight peptide shared amino acid arrangement with N-terminal sequence of Class IIa, pediocin-like bacteriocins and showed increased activity under reducing conditions. Antimicrobial peptides active under reduced conditions are valuable for the preservation of processed foods like meat, dairy and canned foods where low redox potential prevails.

How to deal with PCR contamination in molecular microbial ecology.

Microbial ecology studies often use broad-range PCR primers to obtain community profiles. Contaminant microbial DNA present in PCR reagents may therefore be amplified together with template DNA, resulting in unrepeatable data which may be difficult to interpret, especially when template DNA is present at low levels. One possible decontamination method consists in pre-treating PCR mixes with restriction enzymes before heat-inactivating those enzymes prior to the start of the PCR. However, this method has given contrasting results, including a reduction in PCR sensitivity. In this study, we tested the efficiency of two different enzymes (DNase 1 and Sau3AI) as well as the effect of dithiothreitol (DTT), a strong reducing agent, in the decontamination procedure. Our results indicate that enzymatic treatment does reduce contamination levels. However, DNase 1 caused substantial reductions in the bacterial richness found in communities, which we interpret as a result of its incomplete inactivation by heat treatment. DTT did help maintain bacterial richness in mixes treated with DNase 1. No such issues arose when using Sau3AI, which therefore seems a more appropriate enzyme. In our study, four operational taxonomic units (OTU) decreased in frequency and relative abundance after treatment with Sau3AI and hence are likely to represent contaminant bacterial DNA. We found higher within-sample similarity in community structure after treatment with Sau3AI, probably better reflecting the initial bacterial communities. We argue that the presence of contaminant bacterial DNA may have consequences in the interpretation of ecological data, especially when using low levels of template DNA from highly diverse communities. We advocate the use of such decontaminating approaches as a standard procedure in microbial ecology.