Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells

Inhibitors of DNA methyltransferase, typified by 5-aza-2′-deoxycytidine (5-Aza-CdR), induce the expression of genes transcriptionally down-regulated by de novo methylation in tumor cells. We utilized gene expression microarrays to examine the effects of 5-Aza-CdR treatment in HT29 colon adenocarcinoma cells. This analysis revealed the induction of a set of genes that implicated IFN signaling in the HT29 cellular response to 5-Aza-CdR. Subsequent investigations revealed that the induction of this gene set correlates with the induction of signal transducer and activator of transcription (STAT) 1, 2, and 3 genes and their activation by endogenous IFN-α. These observations implicate the induction of the IFN-response pathway as a major cellular response to 5-Aza-CdR and suggests that the expression of STATs 1, 2, and 3 can be regulated by DNA methylation. Consistent with STAT’s limiting cell responsiveness to IFN, we found that 5-Aza-CdR treatment sensitized HT29 cells to growth inhibition by exogenous IFN-α2a, indicating that 5-Aza-CdR should be investigated as a potentiator of IFN responsiveness in certain IFN-resistant tumors.

Regulation of cyclooxygenase-2 (COX-2) in rat renal cortex by adrenal glucocorticoids and mineralocorticoids

Production of prostaglandins involved in renal salt and water homeostasis is modulated by regulated expression of the inducible form of cyclooxygenase-2 (COX-2) at restricted sites in the rat renal cortex. Because inflammatory COX-2 is suppressed by glucocorticoids, and prostaglandin levels in the kidney are sensitive to steroids, the sensitivity of COX expression to adrenalectomy (ADX) was investigated. By 2 weeks after ADX in mature rats, cortical COX-2 immunoreactivity increased 10-fold in the cortical thick ascending limb and macula densa. The constitutive isoform, COX-1, was unchanged. The magnitude of the changes and specificity of COX-2 immunoreactivity were validated by in situ hybridization histochemistry of COX-2 mRNA and Western blot analysis. Increased COX-2 activity (>5-fold) was documented by using a specific COX-2 inhibitor. The COX-2 up-regulation in ADX rats was reversed by replacement therapy with either corticosterone or deoxycorticosterone acetate. In normal rats, inhibition of glucocorticoid receptors with RU486 or mineralocorticoid receptors with spironolactone caused up-regulation of renal cortical COX-2. These results indicate that COX-2 expression in situ is tonically inhibited by adrenal steroids, and COX-2 is regulated by mineralocorticoids as well as glucocorticoids.

IL-4 and -5 prime human mast cells for different profiles of IgE-dependent cytokine production

Mast cells (MC) are stem cell factor-dependent tissue-based hematopoietic cells with substantial functional heterogeneity. Cord blood-derived human MC (hMC) express functional receptors for IL-5, and IL-5 mediates stem cell factor-dependent comitogenesis of hMC in vitro. Although IL-5 is not required for normal hMC development, we considered that it might prime hMC for their high-affinity Fc receptor for IgE (FcɛRI)-dependent generation of cytokines, as previously demonstrated for IL-4. Compared with hMC maintained in stem cell factor alone, hMC primed with IL-5 expressed 2- to 4-fold higher steady-state levels of TNF-α, IL-5, IL-13, macrophage inflammatory protein 1α, and granulocyte-macrophage colony-stimulating factor transcripts 2 h after FcɛRI crosslinking and secreted 2- to 5-fold greater quantities of the corresponding cytokines, except IL-13, at 6 h. Unlike IL-4, IL-5 priming did not enhance FcɛRI-dependent histamine release. Thus, IL-5 augments cytokine production by hMC by a mechanism distinct from that of IL-4 and with a different resultant profile of cytokine production. These observations suggest a potentially autocrine effect of IL-5 on hMC for amplification of allergic immune responses, in addition to its recognized paracrine effects on eosinophils, and implicate both IL-4 and IL-5 in the modulation of the hMC phenotype.

Position-specific trapping of topoisomerase I–DNA cleavage complexes by intercalated benzo[a]- pyrene diol epoxide adducts at the 6-amino group of adenine

DNA topoisomerase I (top1) is the target of potent anticancer agents, including camptothecins and DNA intercalators, which reversibly stabilize (trap) top1 catalytic intermediates (cleavage complexes). The aim of the present study was to define the structural relationship between the site(s) of covalently bound intercalating agents, whose solution conformations in DNA are known, and the site(s) of top1 cleavage. Two diastereomeric pairs of oligonucleotide 22-mers, derived from a sequence used to determine the crystal structure of top1–DNA complexes, were synthesized. One pair contained either a trans-opened 10R- or 10S-benzo[a]pyrene 7,8-diol 9,10-epoxide adduct at the N6-amino group of a central 2′-deoxyadenosine residue in the scissile strand, and the other pair contained the same two adducts in the nonscissile strand. These adducts were derived from the (+)-(7R,8S,9S,10R)- and (−)-(7S,8R,9R,10S)-7,8-diol 9,10-epoxides in which the benzylic 7-hydroxyl group and the epoxide oxygen are trans. On the basis of analogy with known solution conformations of duplex oligonucleotides containing these adducts, we conclude that top1 cleavage complexes are trapped when the hydrocarbon adduct is intercalated between the base pairs flanking a preexisting top1 cleavage site, or between the base pairs immediately downstream (3′ relative to the scissile strand) from this site. We propose a model with the +1 base rotated out of the duplex, and in which the intercalated adduct prevents religation of the corresponding nucleotide at the 5′ end of the cleaved DNA. These results suggest mechanisms whereby intercalating agents interfere with the normal function of human top1.

Uncoupling proteins 2 and 3 are highly active H+ transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone)

Based on the discovery of coenzyme Q (CoQ) as an obligatory cofactor for H+ transport by uncoupling protein 1 (UCP1) [Echtay, K. S., Winkler, E. & Klingenberg, M. (2000) Nature (London) 408, 609–613] we show here that UCP2 and UCP3 are also highly active H+ transporters and require CoQ and fatty acid for H+ transport, which is inhibited by low concentrations of nucleotides. CoQ is proposed to facilitate injection of H+ from fatty acid into UCP. Human UCP2 and 3 expressed in Escherichia coli inclusion bodies are solubilized, and by exchange of sarcosyl against digitonin, nucleotide binding as measured with 2′-O-[5-(dimethylamino)naphthalene-1-sulfonyl]-GTP can be restored. After reconstitution into vesicles, Cl− but no H+ are transported. The addition of CoQ initiates H+ transport in conjunction with fatty acids. This increase is fully sensitive to nucleotides. The rates are as high as with reconstituted UCP1 from mitochondria. Maximum activity is at a molar ratio of 1:300 of CoQ:phospholipid. In UCP2 as in UCP1, ATP is a stronger inhibitor than ADP, but in UCP3 ADP inhibits more strongly than ATP. Thus UCP2 and UCP3 are regulated differently by nucleotides, in line with their different physiological contexts. These results confirm the regulation of UCP2 and UCP3 by the same factors CoQ, fatty acids, and nucleotides as UCP1. They supersede reports that UCP2 and UCP3 may not be H+ transporters.

2-Arachidonyl glyceryl ether, an endogenous agonist of the cannabinoid CB1 receptor

Two types of endogenous cannabinoid-receptor agonists have been identified thus far. They are the ethanolamides of polyunsaturated fatty acids—arachidonoyl ethanolamide (anandamide) is the best known compound in the amide series—and 2-arachidonoyl glycerol, the only known endocannabinoid in the ester series. We report now an example of a third, ether-type endocannabinoid, 2-arachidonyl glyceryl ether (noladin ether), isolated from porcine brain. The structure of noladin ether was determined by mass spectrometry and nuclear magnetic resonance spectroscopy and was confirmed by comparison with a synthetic sample. It binds to the CB1 cannabinoid receptor (Ki = 21.2 ± 0.5 nM) and causes sedation, hypothermia, intestinal immobility, and mild antinociception in mice. It binds weakly to the CB2 receptor (Ki > 3 μM).

Metabolism of Uridine 5′-Diphosphate-Glucose in Golgi Vesicles from Pea Stems1

Uridine 5′-diphosphate-glucose (UDP-Glc) is transported into the lumen of the Golgi cisternae, where is used for polysaccharide biosynthesis. When Golgi vesicles were incubated with UDP-[3H]Glc, [3H]Glc was rapidly transferred to endogenous acceptors and UDP-Glc was undetectable in Golgi vesicles. This result indicated that a uridine-containing nucleotide was rapidly formed in the Golgi vesicles. Since little is known about the fate of the nucleotide derived from UDP-Glc, we analyzed the metabolism of the nucleotide moiety of UDP-Glc by incubating Golgi vesicles with [α-32P]UDP-Glc, [β-32P]UDP-Glc, and [3H]UDP-Glc and identifying the resulting products. After incubation of Golgi vesicles with these radiolabeled substrates we could detect only uridine 5′-monophosphate (UMP) and inorganic phosphate (Pi). UDP could not be detected, suggesting a rapid hydrolysis of UDP by the Golgi UDPase. The by-products of UDP hydrolysis, UMP and Pi, did not accumulate in the lumen, indicating that they were able to exit the Golgi lumen. The exit of UMP was stimulated by UDP-Glc, suggesting the presence of a putative UDP-Glc/UMP antiporter in the Golgi membrane. However, the exit of Pi was not stimulated by UDP-Glc, suggesting that the exit of Pi occurs via an independent membrane transporter.

The mode of action and the structure of a herbicide in complex with its target: binding of activated hydantocidin to the feedback regulation site of adenylosuccinate synthetase.

(+)-Hydantocidin, a recently discovered natural spironucleoside with potent herbicidal activity, is shown to be a proherbicide that, after phosphorylation at the 5' position, inhibits adenylosuccinate synthetase, an enzyme involved in de novo purine synthesis. The mode of binding of hydantocidin 5'-monophosphate to the target enzyme was analyzed by determining the crystal structure of the enzyme-inhibitor complex at 2.6-A resolution. It was found that adenylosuccinate synthetase binds the phosphorylated compound in the same fashion as it does adenosine 5'-monophosphate, the natural feedback regulator of this enzyme. This work provides the first crystal structure of a herbicide-target complex reported to date.

Mechanism of anti-HIV action of masked alaninyl d4T-MP derivatives.

So324 is a 2',3'-dideoxy-2',3'-didehydrothymidine-5'-monophosphate (d4T-MP) prodrug containing at the phosphate moiety a phenyl group and the methylester of alanine linked to the phosphate through a phosphoramidate linkage. So324 has anti-HIV activity in human CEM, MT4, and monocyte/macrophage cells that is superior to that of d4T. In contrast to d4T, So324 is also able to inhibit HIV replication in thymidine kinase-deficient CEM cells. After uptake of So324 by intact human lymphocytes, d4T-MP is released and subsequently converted intracellularly to d4T-TP. In addition, accumulation of substantial amounts of a novel d4T derivative has been found. This d4T metabolite has been characterized as alaninyl d4T-MP. The latter metabolite accumulates at approximately 13- to 200-fold higher levels than d4T-TP depending the experimental conditions. Alaninyl d4T-MP should be considered as an intra- and/or extracellular depot form of d4T and/or d4T-MP. These findings may explain the superior anti-retroviral activity of So324 over d4T in cell culture.

Latent N-methyl-D-aspartate receptors in the recurrent excitatory pathway between hippocampal CA1 pyramidal neurons: Ca(2+)-dependent activation by blocking A1 adenosine receptors.

When performed at increased external [Ca2+]/[Mg2+] ratio (2.5 mM/0.5 mM), temporary block of A1 adenosine receptors in hippocampus [by 8-cyclopentyltheophylline (CPT)] leads to a dramatic and irreversible change in the excitatory postsynaptic current (EPSC) evoked by Schaffer collateral/commissural (SCC) stimulation and recorded by in situ patch clamp in CA1 pyramidal neurons. The duration of the EPSC becomes stimulus dependent, increasing with increase in stimulus strength. The later occurring component of the EPSC is carried through N-methyl-D-aspartate (NMDA) receptor-operated channels but disappears under either the NMDA antagonist 2-amino-5-phosphonovaleric acid (APV) or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These findings indicate that the late component of the SCC-evoked EPSC is polysynaptic: predominantly non-NMDA receptor-mediated SCC inputs excite CA1 neurons that recurrently excite each other by predominantly NDMA receptor-mediated synapses. These recurrent connections are normally silent but become active after CPT treatment, leading to enhancement of the late component of the EPSC. The activity of these connections is maintained for at least 2 hr after CPT removal. When all functional NMDA receptors are blocked by dizocilpine maleate (MK-801), subsequent application of CPT leads to a partial reappearance of NMDA receptor-mediated EPSCs evoked by SCC stimulation, indicating that latent NMDA receptors are recruited. Altogether, these findings indicate the existence of a powerful system of NMDA receptor-mediated synaptic contacts in SCC input to hippocampal CA1 pyramidal neurons and probably also in reciprocal connections between these neurons, which in the usual preparation are kept latent by activity of A1 receptors.

The benzene metabolite p-benzoquinone forms adducts with DNA bases that are excised by a repair activity from human cells that differs from an ethenoadenine glycosylase.

Benzene is a ubitiquous human environment mental carcinogen. One of the major metabolites is hydroquinone, which is oxidized in vivo to give p-benzoquinone (p-BQ). Both metabolites are toxic to human cells. p-BQ reacts with DNA to form benzetheno adducts with deoxycytidine, deoxyadenosine, and deoxyguanosine. In this study we have synthesized the exocyclic compounds 3-hydroxy-3-N4-benzetheno-2'-deoxycytidine (p-BQ-dCyd) and 9-hydroxy-1,N6-benzetheno-2'-deoxyadenosine (p-BQ-dAdo), respectively, by reacting deoxycytidine and deoxyadenosine with p-BQ. These were converted to the phosphoamidites, which were then used to prepare site-specific oligonucleotides with either the p-BQ-dCyd or p-BQ-dAdo adduct (pbqC or pbqA in sequences) at two different defined positions. These oligonucleotides were efficiently nicked 5' to the adduct by partially purified HeLa cell extracts--the pbqC-containing oligomer more rapidly than the pbqA-containing oligomer. In contrast to the enzyme binding to derivatives produced by the vinyl chloride metabolite chloroacetaldehyde, the oligonucleotides up to 60-mer containing p-BQ adducts did not bind measurably to the same enzyme preparation in a gel retardation assay. Furthermore, there was no competition for the binding observed between oligonucleotides containing 1,N6-etheno A deoxyadenosine (1,N6-etheno-dAdo; epsilon A in sequences) and these oligomers containing either of the p-BQ adducts, even at 120-fold excess. When highly purified fast protein liquid chromatography (FPLC) enzyme fractions were obtained, there appeared to be two closely eluting nicking activities. One of these enzymes bound and cleaved the epsilon A-containing deoxyoligonucleotide. The other enzyme cleaved the pbqA- and pbqC-containing deoxyoligonucleotides. One additional unexpected fact was that bulk p-BQ-treated salmon sperm DNA did compete effectively with the epsilon A-containing oligonucleotide for protein binding. This raises the possibility that such DNA contains other, as-yet-uncharacterized adducts that are recognized by the same enzyme that recognizes the etheno adducts. In summary, we describe a previously undescribed human DNA repair activity, possibly a glycosylase, that excises from DNA pbqC and pbqA, exocyclic adducts resulting from reaction of deoxycytidine and deoxyadenosine with the benzene metabolite, p-BQ. This glycosylase activity is not identical to the one previously reported from this laboratory as excising the four etheno bases from DNA.

Caderno de Formação (5) - Formação de Professores - Educação, Cultura e Desenvolvimento

Trata-se do caderno de formação de número 5 (v. 3, bloco 1, módulo 2). Reúne as disciplinas 09 (Sociologia da Educação), Eixo Articulador Memória do Professor e 10 (Introdução à pesquisa científica em educação).

Nd isotope ratios of surface sediments from the Pacific Ocean (Table 2)

The neodymium isotopic composition of the silicate fraction of Holocene pelagic sediments from the North Pacific define two provinces: a central North Pacific province characterized by unradiogenic and remarkably homogeneous end (-10.2 +/- 0.5) and a narrow circum-Pacific marginal province characterized by more radiogenic and variable end (-4.2 +/- 3.8). The silicate fraction in the central North Pacific is exclusively eolian; based on prevailing wind patterns, meteorological data, and neodymium isotopic data, the only significant sediment source is Chinese loess. Leaching experiments on Chinese loess confirm that leachable Nd is isotopically indistinguishable from bulk and residual silicate Nd. Silicates in the circum-North Pacific marginal province comprise eolian loess, volcanic ash, and hemipelagic sediments derived from volcanic arcs. A compilation of Pacific seawater and Mn nodule epsilon-Nd data shows no clear spatial variation except for a general decrease from surface to deep waters from -3 to -4 and slightly lower epsilon-Nd in bottom waters along the western North Pacific due to the incursion of Antarctic Bottom Water. The relative homogeneity of bottom water epsilon-Nd, which contrasts sharply with the distinctive variation in sediment epsilon-Nd, plus the large difference between the average end of bottom waters and the central North Pacific eolian silicates (-4 vs. -10), suggests that any contribution of REE to seawater from eolian materials is insignificant. Furthermore, leaching of REE from eolian particles as they sink though the water column must be insignificant because Nd in shallow waters is more radiogenic than Nd in deeper waters. That there is no contrast in the Nd isotopic composition of bottom waters that overlie the central and marginal sediment provinces suggests that the ash and hemipelagic sediments derived from Pacific rim volcanic arcs also contribute minimal REE to seawater. The elimination of eolian, ash, and hemipelagic sediments leaves only near-shore riverine particulates as a possibly significant particulate source of REE to seawater.

Compilation of North Atlantic albacore tuna (Thunnus alalunga) fork length frequencies in 5 centimeter intervals from catches in the North Atlantic for the period 1956-2010

The development of the ecosystem approach and models for the management of ocean marine resources requires easy access to standard validated datasets of historical catch data for the main exploited species. They are used to measure the impact of biomass removal by fisheries and to evaluate the models skills, while the use of standard dataset facilitates models inter-comparison. North Atlantic albacore tuna is exploited all year round by longline and in summer and autumn by surface fisheries and fishery statistics compiled by the International Commission for the Conservation of Atlantic Tunas (ICCAT). Catch and effort with geographical coordinates at monthly spatial resolution of 1° or 5° squares were extracted for this species with a careful definition of fisheries and data screening. In total, thirteen fisheries were defined for the period 1956-2010, with fishing gears longline, troll, mid-water trawl and bait fishing. However, the spatialized catch effort data available in ICCAT database represent a fraction of the entire total catch. Length frequencies of catch were also extracted according to the definition of fisheries above for the period 1956-2010 with a quarterly temporal resolution and spatial resolutions varying from 1°x 1° to 10°x 20°. The resolution used to measure the fish also varies with size-bins of 1, 2 or 5 cm (Fork Length). The screening of data allowed detecting inconsistencies with a relatively large number of samples larger than 150 cm while all studies on the growth of albacore suggest that fish rarely grow up over 130 cm. Therefore, a threshold value of 130 cm has been arbitrarily fixed and all length frequency data above this value removed from the original data set.