Selenoprotein oxidoreductase with specificity for thioredoxin and glutathione systems

Thioredoxin (Trx) and glutathione (GSH) systems are considered to be two major redox systems in animal cells. They are reduced by NADPH via Trx reductase (TR) or oxidized GSH (GSSG) reductase and further supply electrons for deoxyribonucleotide synthesis, antioxidant defense, and redox regulation of signal transduction, transcription, cell growth, and apoptosis. We cloned and characterized a pyridine nucleotide disulfide oxidoreductase, Trx and GSSG reductase (TGR), that exhibits specificity for both redox systems. This enzyme contains a selenocysteine residue encoded by the TGA codon. TGR can reduce Trx, GSSG, and a GSH-linked disulfide in in vitro assays. This unusual substrate specificity is achieved by an evolutionary conserved fusion of the TR and glutaredoxin domains. These observations, together with the biochemical probing and molecular modeling of the TGR structure, suggest a mechanism whereby the C-terminal selenotetrapeptide serves a role of a protein-linked GSSG and shuttles electrons from the disulfide center within the TR domain to either the glutaredoxin domain or Trx.

Carcinogen-specific induction of genetic instability

It has been proposed recently that the type of genetic instability in cancer cells reflects the selection pressures exerted by specific carcinogens. We have tested this hypothesis by treating immortal, genetically stable human cells with representative carcinogens. We found that cells resistant to the bulky-adduct-forming agent 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) exhibited a chromosomal instability (CIN), whereas cells resistant to the methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) exhibited a microsatellite instability (MIN) associated with mismatch repair defects. Conversely, we found that cells purposely made into CIN cells are resistant to PhIP, whereas MIN cells are resistant to MNNG. These data demonstrate that exposure to specific carcinogens can indeed select for tumor cells with distinct forms of genetic instability and vice versa.

Oxidative Damage in Pea Plants Exposed to Water Deficit or Paraquat1

The application of a moderate water deficit (water potential of −1.3 MPa) to pea (Pisum sativum L. cv Lincoln) leaves led to a 75% inhibition of photosynthesis and to increases in zeaxanthin, malondialdehyde, oxidized proteins, and mitochondrial, cytosolic, and chloroplastic superoxide dismutase activities. Severe water deficit (−1.9 MPa) almost completely inhibited photosynthesis, decreased chlorophylls, β-carotene, neoxanthin, and lutein, and caused further conversion of violaxanthin to zeaxanthin, suggesting damage to the photosynthetic apparatus. There were consistent decreases in antioxidants and pyridine nucleotides, and accumulation of catalytic Fe, malondialdehyde, and oxidized proteins. Paraquat (PQ) treatment led to similar major decreases in photosynthesis, water content, proteins, and most antioxidants, and induced the accumulation of zeaxanthin and damaged proteins. PQ decreased markedly ascorbate, NADPH, ascorbate peroxidase, and chloroplastic Fe-superoxide dismutase activity, and caused major increases in oxidized glutathione, NAD+, NADH, and catalytic Fe. It is concluded that, in cv Lincoln, the increase in catalytic Fe and the lowering of antioxidant protection may be involved in the oxidative damage caused by severe water deficit and PQ, but not necessarily in the incipient stress induced by moderate water deficit. Results also indicate that the tolerance to water deficit in terms of oxidative damage largely depends on the legume cultivar.

Direct evidence for secondary loss of mitochondria in Entamoeba histolytica.

Archezoan protists are though to represent lineages that diverged from other eukaryotes before acquisition of the mitochondrion and other organelles. The parasite Entamoeba histolytica was originally included in this group. Ribosomal RNA based phylogenies, however, place E. histolytica on a comparatively recent branch of the eukaryotic tree, implying that its ancestors had these structures. In this study, direct evidence for secondary loss of mitochondrial function was obtained by isolating two E. histolytica genes encoding proteins that in other eukaryotes are localized in the mitochondrion: the enzyme pyridine nucleotide transhydrogenase and the chaperonin cpn60. Phylogenetic analysis of the E. histolytica homolog of cpn60 confirmed that it is specifically related to the mitochondrial lineage. The data suggest that a mitochondrial relic may persist in this organism. Similar studies are needed in archezoan protists to ascertain which, if any, eukaryotic lineages primitively lack mitochondria.

Propriedades fotoquímicas de alguns complexos de ferro(II) e rênio(I)

Neste trabalho, foram estudadas as propriedades fotoquímica e/ou fotofísica de alguns compostos de coordenação de rênio(l) e ferro(I I). A irradiação dos complexos fac-[Re(CO)3(NN)(trans-L)]+, NN= 4,7-difenil-1,10- fenantrolina (ph2phen) ou 5-cloro-1,10-fenantrolina (Clphen) e L = 1,2-bis(4-piridil)etileno (bpe) ou 4-estirilpiridina (stpy), em acetonitrila ou em filme de poli(metacrilato de metila) (PMMA) resulta em variações espectrais condizentes com a fotoisomerização trans-cis do ligante coordenado. A determinação dos rendimentos quânticos para a fotorreação pela variação espectral resultou em valores aparentes, uma vez que o reagente e o fotoproduto absorvem na mesma região. Para a determinação do rendimento quântico real, Φreal, utilizou-se a técnica de 1H RMN, na qual os sinais do fotoproduto e do reagente são observados em regiões distintas com diferentes constantes de acoplamento. Os valores de Φreal obtidos para fac-[Re(CO)3h(NN)(trans-bpe)]+ (ph2phen: Φ313= 0,43 ± 0,03; Φ365= 0,44 ± 0,02; Φ404= 0,43 ± 0,02; Clphen: Φ313= 0,56 ± 0,03; Φ365= 0,55 ± 0,04; Φ404= 0,57 ± 0,06) são independentes do comprimento de onda de irradiação, indicando a existência de um único canal para a população do estado excitado 3ILtrans-bpe. Por outro lado, para fac-[Re(CO)3(NN)(trans-stpy)]+, os valores de Φreal sob irradiação a 404 nm são menores que os determinados para os demais comprimentos de onda de irradiação (ph2phen: Φ313= 0,60 ± 0,05; Φ365= 0,64 ± 0,09; Φ404= 0,42 ± 0,03; Clphen: Φ313= 0,52 ± 0,05; Φ365= 0,58 ± 0,02; Φ404= 0,41 ± 0,06), indicando que, a energias maiores, em que o Iigante absorve significativamente, deve existir a contribuição de outro canal para a população do estado excitado 3ILtrans-stpy. A eficiência do fotoprocesso foi avaliada por meio da substituição dos ligantes NN e/ou L, e a diferença nos valores de Φreal entre os complexos deve estar relacionada principalmente com as distintas eficiências de cruzamento intersistemas. o fotoprocesso altera as propriedades fotofísicas desses complexos. Os isômeros trans apresentam fraca ou nenhuma emissão a 298 K, enquanto os fotoprodutos, fac-[Re(CO)3(NN)(cis-L)]+, apresentam intensa luminescência dominada pelo estado excitado 3MLCTRe→NN, que é sensivel à rigidez do meio. A reatividade fotoquímica dos pentacianoferratos(II) [Fe(CN)5 (NN)]3-, NN= 2aminobenzilamina (aba), 2-aminobenzamida (ab), 2-(dimetilaminometil)-3-hidroxipiridina (dmampy), 2-aminometilpiridina (ampy), 2-aminoetilpiridina (aepy) ou 2-(2metilaminoetil) piridina (maepy), também foi investigada. A irradiação desses complexos resulta na fotossubstituição do CN-, a qual só pode ser detectada quando o ligante possui um segundo grupo coordenante nas proximidades da esfera de coordenação. Os rendimentos quânticos da fotossubstituição são dependentes do comprimento de onda de irradiação (Φ313= 0,13 ± 0,01; Φ334= 0,091 ± 0,001; Φ365= 0,056 ± 0,002; Φ404= 0,022 ± 0,002; Φ436= 0,015 ± 0,001, por exemplo, para NN = aba) e indicam a existência de canais distintos pelos quais a fotorreação ocorre ou as diferentes eficiências de cruzamento intersistema para a população do estado excitado reativo. A eficiência do fotoprocesso também depende do Iigante utilizado (λirr= 365 nm: Φaba= 0,056, Φab= 0,14, Φampy= 0,046, Φaepy= 0,066, Φmaepy= 0,069 e Φdmampy= 0,12). Na série das diaminas, o rendimento quântico é maior para [Fe(CN)5(ab)]3-, que possui dois sítios para ocorrer o fechamento do anel. Na série das aminopiridinas, observa-se a influência do comprimento da cadeia na eficiência do fechamento do anel. A presença de metilas ligadas ao nitrogênio alifático deve ter pouca ou nenhuma influência na eficiência do fotoprocesso.

Avaliação da inervação entérica e análise proteômica do intestino delgado de ratos expostos à dose aguda ou crônica de fluoreto

O trato gastrointestinal (TGI) é a principal rota de exposição ao fluoreto (F) e o seu mais importante sítio de absorção. Acredita-se que a toxicidade do F comprometa a fisiologia do intestino, devido à relevante sintomatologia gastrointestinal relatada em consequência da exposição excessiva ao F. A função intestinal é controlada por uma complexa rede neuronal interligada e incorporada à parede deste órgão, denominada Sistema Nervoso Entérico (SNE). Embora os efeitos tóxicos do F sobre o Sistema Nervoso Central sejam descritos na literatura, não há estudos relacionados à sua toxicidade sobre o SNE. Neste estudo realizado em ratos, foi avaliado o efeito da exposição aguda ou crônica ao F, sobre a população geral de neurônios entéricos e sobre as subpopulações que expressam os principais neurotransmissores entéricos: Acetilcolina (ACh), Óxido Nítrico (NO), Peptídeo Vasoativo Intestinal (VIP), Peptídeo Relacionado ao Gene da Calcitonina (CGRP) e Substância P (SP). Os animais foram divididos em 5 grupos: 3 destinados à exposição crônica (0 ppm, 10 ppm ou 50 ppm de F na água de beber) e 2 à exposição aguda (0 ou 25 mgF/Kg por gavagem gástrica). Foram coletados os 3 segmentos do intestino delgado (duodeno, jejuno e íleo) e processados para a detecção da HuC/D, ChAT, nNOS, VIP, CGRP e SP, através de técnicas de imunofluorescência, no plexo mioentérico. Foram obtidas imagens para a realização da análise quantitativa dos neurônios da população geral (HuC/D) e nitrérgicos (imunorreativos à nNOS); e morfométrica dos neurônios imunorreativos à HuC/D ou nNOS; e das varicosidades imunorreativas à ChAT, VIP, CGRP ou SP. Amostras dos 3 segmentos intestinais foram preparadas e coradas em Hematoxilina e Eosina para análise histológica da morfologia básica. O segmento intestinal considerado mais afetado na análise morfométrica da população geral de neurônios, o duodeno, foi selecionado para a realização da análise proteômica, com o objetivo de oferecer o seu perfil proteico e determinar diferenças na expressão proteica em decorrência da exposição crônica ou aguda ao F. A análise da concentração de F no plasma sanguíneo foi realizada para a confirmação da exposição. Na análise quantitativa, o grupo de 50 ppm F, apresentou uma diminuição significativa na densidade da população geral de neurônios do jejuno e do íleo e na densidade dos neurônios imunorreativos à nNOS no duodeno e no jejuno. Quanto à análise morfométrica, a população geral e as subpopulações neuronais entéricas avaliadas apresentaram alterações morfológicas significativas, tanto após a exposição crônica quanto a aguda. Para a análise proteômica do duodeno, verificou-se que da associação de seus genes a um termo, e assim classificadas de acordo com diferentes processos biológicos. No caso do grupo da dose aguda, o processo biológico com a maior porcentagem de genes associados foi a geração de metabólitos precursores e energia (27% das proteínas); enquanto para os grupos de 10 e 50 ppm F foram o processo metabólico da piridina (41%) e a polimerização proteica (33%), respectivamente.

On the origin of the high capacitance of nitrogen-containing carbon nanotubes in acidic and alkaline electrolytes

The synthesis of nitrogenated carbon nanotubes (N-CNTs) with up to 6.1 wt% N, via the use of pyridine as the nitrogen containing carbon precursor, can provide a facile route to significantly enhance the low intrinsic specific capacitance of carbon nanotubes. The nitrogen functionalities determine this, at least, five-fold increase of the specific capacitance.

Terahertz Response of Excitons in Nanorod Heterostructures

Quantum-confined systems are one of the most promising ways to enable us to control a material's interactions with light. Nanorods in particular offer the right dimensions for exploring and manipulating the terahertz region of the spectrum. In this thesis, we model excitons confined inside a nanorod using the envelope function approximation. A region-matching transfer matrix method allows us to simulate excitonic states inside arbitrary heterostructures grown along the length of the rod. We apply the method to colloidal CdSe rods 70 nm in length and under 10 nm in diameter, capped with ligands of DDPA and pyridine. We extend past studies on these types of rods by taking into account their dielectric permittivity mismatch. Compared to previous calculations and experimentally measured terahertz absorption, we predict a higher energy main 1S$z$ to 2P$z$ transition peak. This indicates that the rods are likely larger in diameter than previously thought. We also investigate a nanorod with GaAs/Al$_{0.3}$Ga$_{0.7}$As coupled double dots. The excitonic transitions were found to be manipulable by varying the strength of an applied electric field. We employ quasi-static state population distributions to simulate the effects of exciton relaxation from optically active states to dim ground states. A critical value of the applied field, corresponding to the exciton binding energy of ~18 meV, was found to dramatically alter the terahertz absorption due to state mixing. Above this critical field, more nuanced shifts in transition energies were observed, and gain from radiative relaxation to the ground state is predicted.

Soft solution processing of cerium hydroxysulfate powders with different morphologies

Polycrystalline cerium hydroxysulfate powders have been prepared by soft solution processing using various basic solvents. The crystals prepared have varying morphologies, spherical and flaky, depending on the solvent used. The crystals obtained from distilled water-pyridine and aqueous ammonia solvent mixtures are spherical, whereas those obtained from mixtures of distilled water and ethylenediamine or hydrazine hydrate are flaky. All the crystalline cerium hydroxysulfate samples display luminescence properties. It was found that the flaky crystals generally show a much stronger luminescence than their spherical counterparts.

Reactions of fac-[PtMe2(OMe)(H2O)3]+ with halide ions: effect of halide trans effect on methoxide hydrolysis

A solution of fac-[PtMe2(OMe)(H2O)(3)](+) (1) in aqueous perchloric acid underwent very slow hydrolysis of the Pt-OMe bond, over many, weeks. When chloride was added to a solution of 1, two interconverting isomers of [PtMe2(OMe)Cl(H2O)(2)] (with chloride trans to methyl) were formed, and with excess chloride, [PtMe2(OMe)Cl-2(H2O)](-) (both chloride ligands trans to methyl). This solution was stable at ambient temperature, but on heating, methanol was formed and [PtMe2Cl2(H2O)(2)] (both chloride ligands cis to methyl) was produced in the solution. It is proposed that this reaction proceeds via an intermediate complex with chloride bound trans to methoxide. Concentration gave solid [{PtMe2Cl2}n], whose identity was confirmed by conversion to [PtMe(2)Cl(2)py(2)] (pyridine, py, trans to methyl). With bromide and iodide, methoxide hydrolysis occurred at ambient temperature, more slowly with bromide than with iodide, to form solid [{PtMe2X2}(n)] without significant concentrations of [PtMe2X2(H2O)(2)] formed as an intermediate. The greater tendency for Pt-OMe bond to hydrolyse trans to halide compared with 1 was ascribed to the higher trans effect of the halide ligand compared with that of water. (C) 2003 Elsevier Science B.V. All rights reserved.

The preparation and characterization of seven-coordinate tin(IV) complexes of the 2,6-diacetylpyridine Schiff bases of S-alkyl/aryl-dithiocarbazates and the X-ray crystal structure of the [Sn(dapsme)I-2] complex (dapsme=doubly deprotonated form of the 2,6

New tin(IV) complexes of empirical formula, Sn(SNNNS)I-2 (SNNNS = anionic form of the 2,6-diacetylpyridine Schiff bases of S-methyl- or S-benzyldithiocarbazate) have been prepared and characterized by a variety of physico-chemical techniques. The structure of Sn(dapsme)I-2 has been determined by single crystal X-ray crystallographic structural analysis. The complex has a seven-coordinate distorted pentagonal-bipyramidal geometry with the Schiff base coordinated to the tin(IV) ion as a dinegatively charged pentadentate chelating agent via the pyridine nitrogen atom, the two azomethine nitrogen atoms and the two thiolate sulfur atoms. The ligand occupies the equatorial plane and the iodo ligands are coordinated to the tin(IV) ion at axial positions. The distortion from an ideal pentagonal bipyramidal geometry is attributed to the restricted bite size of the pentadentate ligands. (C) 2004 Elsevier Ltd. All rights reserved.

Synthesis of 1,3-diazepines and ring contraction to cyanopyrroles

Several tetrazolo[1,5-a] pyridines/2-azidopyridines undergo photochemical nitrogen elimination and ring expansion to 1,3-diazacyclohepta-1,2,4,6-tetraenes (7,10,13,16,19,22) as well as ring cleavage to cyanovinylketenimines (8,17,20b) in low temperature Ar matrices. 6,8-Dichlorotetrazolo[1,5-a] pyridine/2-azido-3,5-dichloropridine 6 undergoes ready exchange of the chlorine in position 8 (3) with ROH/RONa. 8-Chloro-6-trifluoromethyltetrazolo[1,5-a] pyridine 15 undergoes solvolysis of the CF3 group to afford 8-chloro-6-methoxycarbonyltetrazolo[1,5-a] pyridine 18. Several tetrazolopyridines/2-azidopyridines afford 1H- or 5H-1,3-diazepines in good yields on photolysis in the presence of alcohols or amines (11,14,23,25). 5-Chlorotetrazolo[1,5-a] pyridines/2-azido-6-chloropyridines 21 and 38 undergo a rearrangement to 1H- and 3H-3-cyanopyrroles 27 and 45, respectively. The mechanism of this rearrangement was investigated by N-15-labelling and takes place via transient 1,3-diazepines. The structures of 6,8-dichloro-tetrazolo[1,5-a] pyridine 6T, 6-chloro-8-ethoxytetrazolo[1,5-a] pyridine 9Tb, dipyrrolylmethane 28, and 2-isopropoxy-4-dimethylamino-5H-1,3-diazepine 25b were determined by X-ray crystallography. In the latter case, this represents the first reported X-ray crystal structure of a 5H-1,3-diazepine.

A cyclopropabenzenylidenethenone (Propadienone) via a new route to alkylidenecycloproparenes

Reaction of 1,1-dichloro-2,5-diphenylcyclopropabenzene 6 with Meldrum's acid 8 in the presence of pyridine leads to coupling of the cycloproparenyl cation 7 with the stabilized diketo anion 9. Subsequent, spontaneous, base-induced dehydrochlorination gives the alkylidenecyclopropabenzene 11 in a one-pot reaction. Flash vacuum thermolysis of 11 at 650 degreesC ejects acetone and carbon dioxide, giving cyclopropabenzenylldenethenone 12 that is isolated in an Ar matrix at 20 K and characterized by a strong ketene band at 2107 cm(-1) in the IR spectrum.

The crystal structure of a bacterial Class II ketol-acid reductolsomerase: Domain conservation and evolution

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class 1) found in fungi and most bacteria, and a long form (Class 11) typical of plants. Crystal structures of each have been reported previously. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. Here, we report the crystal structure of the E. coli enzyme at 2.6 A resolution, the first three-dimensional structure of any bacterial Class 11 KARI. The enzyme consists of two domains, one with mixed alpha/beta structure, which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly alpha-helical and shows strong evidence of internal duplication. Comparison of the active sites between KARI of E. coli, Pseudomonas aeruginosa, and spinach shows that most residues occupy conserved positions in the active site. E. coli KARI was crystallized as a tetramer, the likely biologically active unit. This contrasts with P. aeruginosa KARI, which forms a dodecamer, and spinach KARI, a dimer. In the E. coli KARI tetramer, a novel subunit-to-subunit interacting surface is formed by a symmetrical pair of bulbous protrusions.

Synthesis, characterization and X-ray crystal structures of dinuclear nickel(II) complexes of a novel potentially hexadentate but functionally tetradentate binucleating ligand

A binucleating potentially hexadentate chelating agent containing oxygen, nitrogen and sulfur as potential donor atoms (H2ONNO) has been synthesized by condensing alpha,alpha-xylenebis(N-methyldithiocarbazate) with 2,4-pentanedione. An X-ray crystallographic structure determination shows that the Schiff base remains in its ketoimine tautomeric form with the protons attached to the imine nitrogen atoms. The reaction of the Schiff base with nickel(II) acetate in a 1:1 stoichiometry leads to the formation of a dinuclear nickel(II) complex [Ni(ONNO)](2) (ONNO2- = dianionic form of the Schiff base) containing N,O-chelated tetradentate ligands, the sulfur donors remaining uncoordinated. A single crystal X-ray structure determination of this dimer reveals that each ligand binds two low spin nickel(II) ions, bridged by a xylyl group. The nickel(II) atoms adopt a distorted square-planar geometry in a trans-N2O2 donor environment. Reaction of the Schiff base with nickel(II) acetate in the presence of excess pyridine leads to the formation of a similar dinuclear complex, [Ni(ONNO)(py)](2), but in this case comprises five coordinate high-spin Ni(II) ions with pyridine ligands occupying the axial coordination sites as revealed by X-ray crystallographic analysis. (c) 2005 Published by Elsevier B.V.