Cyclophilin catalyzes protein folding in yeast mitochondria.

Cyclophilins are a family of ubiquitous proteins that are the intracellular target of the immunosuppressant drug cyclosporin A. Although cyclophilins catalyze peptidylprolyl cis-trans isomerization in vitro, it has remained open whether they also perform this function in vivo. Here we show that Cpr3p, a cyclophilin in the matrix of yeast mitochondria, accelerates the refolding of a fusion protein that was synthesized in a reticulocyte lysate and imported into the matrix of isolated yeast mitochondria. The fusion protein consisted of the matrix-targeting sequence of subunit 9 of F1F0-ATPase fused to mouse dihydrofolate reductase. Refolding of the dihydrofolate reductase moiety in the matrix was monitored by acquisition of resistance to proteinase K. The rate of refolding was reduced by a factor of 2-6 by 2.5 microM cyclosporin A. This reduced rate of folding was also observed with mitochondria lacking Cpr3p. In these mitochondria, protein folding was insensitive to cyclosporin A. The rate of protein import was not affected by cyclosporin A or by deletion of Cpr3p.

Coenzyme Q reductase from liver plasma membrane: purification and role in trans-plasma-membrane electron transport.

A specific requirement for coenzyme Q in the maintenance of trans-plasma-membrane redox activity is demonstrated. Extraction of coenzyme Q from membranes resulted in inhibition of NADH-ascorbate free radical reductase (trans electron transport), and addition of coenzyme Q10 restored the activity. NADH-cytochrome c oxidoreductase (cis electron transport) did not respond to the coenzyme Q status. Quinone analogs inhibited trans-plasma-membrane redox activity, and the inhibition was reversed by coenzyme Q. A 34-kDa coenzyme Q reductase (p34) has been purified from pig-liver plasma membranes. The isolated enzyme was sensitive to quinone-site inhibitors. p34 catalyzed the NADH-dependent reduction of coenzyme Q10 after reconstitution in phospholipid liposomes. When plasma membranes were supplemented with extra p34, NADH-ascorbate free radical reductase was activated but NADH-cytochrome c oxidoreductase was not. These results support the involvement of p34 as a source of electrons for the trans-plasma-membrane redox system oxidizing NADH and support coenzyme Q as an intermediate electron carrier between NADH and the external acceptor ascorbate free radical.

A cyclophilin from the polycentric anaerobic rumen fungus Orpinomyces sp. strain PC-2 is highly homologous to vertebrate cyclophilin B.

A cyclophilin (CyP) purified to homogeneity from the polycentric anaerobic rumen fungus Orpinomyces sp. strain PC-2 had a molecular mass of 20.5 kDa and a pI of 8.1. The protein catalyzed the isomerization of the prolyl peptide bond of N-succinyl-Ala-Ala-(cis,trans)-Pro-Phe p-nitroanilide with a kcat/Km value of 9.3 x 10(6) M-1.s-1 at 10 degrees C and pH 7.8. Cyclosporin A strongly inhibited this peptidylprolyl cis-trans isomerase activity with an IC50 of 19.6 nM. The sequence of the first 30 N-terminal amino acids of this CyP had high homology with the N-terminal sequences of other eukaryotic CyPs. By use of a DNA hybridization probe amplified by PCR with degenerate oligonucleotide primers designed based on the amino acid sequences of the N terminus of this CyP and highly conserved internal regions of other CyPs, a full-length cDNA clone was isolated. It possessed an open reading frame encoding a polypeptide of 203 amino acids with a calculated molecular weight of 21,969, containing a putative hydrophobic signal peptide sequence of 22 amino acids preceding the N terminus of the mature enzyme and a C-terminal sequence, Lys-Ala-Glu-Leu, characteristic of an endoplasmic reticulum retention signal. The Orpinomyces PC-2 CyP is a typical type B CyP. The amino acid sequence of the Orpinomyces CyP exhibits striking degrees of identity with the corresponding human (70%), bovine (69%), mouse (68%), chicken (66%), maize (61%), and yeast (54%) proteins. Phylogenetic analysis based on the CyP sequences indicated that the evolutionary origin of the Orpinomyces CyP was closely related with CyPs of animals.

Estudo dos espectros vibracionais de poliacetilenos substituídos

Neste trabalho foram sintetizados a poli-2-etinilpiridina (P2EP), poli-4etinilpiridina (P4EP), o iodeto de poli(2-N-t-butilpiridiniumilacetileno) (P2EPtBu) e a poli-β-etinilnaftaleno (Pβ:EN), os quais são poliacetilenos substituídos. Estes polímeros, juntamente com o cloreto de poli(2-piridínio-2-piridilacetileno) (P2EPH), foram caracterizados por espectroscopia vibracional no infravermelho e Raman. Estes polímeros apresentaram variação na posição das bandas Raman com a energia da radiação excitante - chamada dispersão Raman ou fotosseletividade - da ordem de 10 cm-1, bem inferior ao apresentado pelo poliacetileno (cerca de 60 cm-1). Este deslocamento foi interpretado utilizando-se dois dos modelos existentes para descrever este fenômeno: o Modelo de Modo de Amplitude (AMM) e o Modelo de Coordenada de Conjugação Efetiva (ECCM), os quais fornecem informações sobre a estrutura polimérica e sobre seus níveis eletrônicos. Utilizando-se o AMM foi possível obter informações sobre os níveis eletrônicos excitados de mesma simetria que o estado eletrônico fundamental. Por outro lado, o ECCM, com a ajuda de cálculos DFT, mostrou diferenças na extensão da conjugação e no grau de dimerização entre o P2EP na forma cis e trans e indicou que este polímero apresentava, predominantemente, a estrutura cis, fato este confirmado pelos espectros no infravermelho. A dopagem com I2 provocou efeitos diferentes na estrutura dos polímeros. Os espectros no infravermelho dos polímeros dopados indicaram que o P2EP e o P2EPH apresentaram aumento na quantidade de segmentos cis enquanto o P2EPtBu apresentou diminuição na quantidade desses segmentos. Os espectros Raman dos polímeros dopados confirmaram os dados dos espectros no infravermelho. Esta diferença foi interpretada como sendo devida à diferença no volume do substituinte, pois grupos volumosos favorecem o isômero trans-cisóide onde a distância entre os substituintes é maior. A dopagem também levou a um aumento na condutividade dos polímeros, porém os valores de condutividade obtidos foram bem inferiores que os apresentados pelo poliacetileno dopado (10-5 a 10-7 contra 102 S cm-1, tipicamente).

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.

Translation of the flavivirus Kunjin NS3 gene in cis but not its RNA sequence or secondary structure is essential for efficient RNA packaging

Our previous studies using trans-complementation analysis of Kunjin virus (KUN) full-length cDNA clones harboring in-frame deletions in the NS3 gene demonstrated the inability of these defective complemented RNAs to be packaged into virus particles (W. J. Liu, P. L. Sedlak, N. Kondratieva, and A. A. Khromykh, J. Virol. 76:10766-10775). In this study we aimed to establish whether this requirement for NS3 in RNA packaging is determined by the secondary RNA structure of the NS3 gene or by the essential role of the translated NS3 gene product. Multiple silent mutations of three computer-predicted stable RNA structures in the NS3 coding region of KUN replicon RNA aimed at disrupting RNA secondary structure without affecting amino acid sequence did not affect RNA replication and packaging into virus-like particles in the packaging cell line, thus demonstrating that the predicted conserved RNA structures in the NS3 gene do not play a role in RNA replication and/or packaging. In contrast, double frameshift mutations in the NS3 coding region of full-length KUN RNA, producing scrambled NS3 protein but retaining secondary RNA structure, resulted in the loss of ability of these defective RNAs to be packaged into virus particles in complementation experiments in KUN replicon-expressing cells. Furthermore, the more robust complementation-packaging system based on established stable cell lines producing large amounts of complemented replicating NS3-deficient replicon RNAs and infection with KUN virus to provide structural proteins also failed to detect any secreted virus-like particles containing packaged NS3-deficient replicon RNAs. These results have now firmly established the requirement of KUN NS3 protein translated in cis for genome packaging into virus particles.

Syntheses of aza analogues of kainic acid

The kainate receptors are one of the three major groups of ionotropic glutamate receptors in the mammalian central nervous system. They are so named after their most potent agonist, kainic acid (KA), a natural product isolated from the seaweed Diginea simplex. This compound shows both neuroexcitatory and excitotoxic activities, and is an important pharmacological tool for neurophysiological studies. We predict that the more synthetically accessible aza analogues of kainic acid, could act as functional mimics of KA. These could be produced by the 1,3-dipolar cycloaddition of diazoalkanes with trans glutaconate esters. ^ 1,3-Dipolar cycloadditions have been shown to produce 1-pyrazolines that isomerize into 2-pyrazolines. The 1- and 2-pyrazolines can be precursors to aza analogs of kainoids. The regioselectivity, relative stereochemistry and isomerization of the 1-pyrazolines into 2-pyrazolines have been evaluated. Reductions of the 1- and 2-pyrazolines produced aza analogs of kainoids. TMS diazomethane was used as the dipole in 1,3-dipolar cycloaddition reactions leading to aza KA analogs via 2-pyrazolines. A systematic study of cycloaddition-isomerization processes involving TMS-diazomethane and various α, β-unsaturated dipolarophiles has been undertaken. 1H-NMR monitoring of the reaction mixture compositions during the cycloaddition reaction revealed evidence of retro-dipolar cycloaddition processes. Faster formation of 4,5- trans-1-pyrazoline at the beginning of the reaction and subsequent isomerization of this product into 4,5-cis-1-pyrazoline via a retro-dipolar cycloaddition has been observed. Increased reaction time and/or reaction temperature preferentially caused the irreversible isomerization of 4,5-cis-1-pyrazoline into 4,5-cis-2-pyrazoline, which led to high yields of 4,5-cis-2-pyrazolines in the overall process. ^ Two syntheses of the 5-unsubstituted aza-kainic acid have been performed; first, via the reduction of the TMS-eliminated 2-pyrazoline from TMS diazomethane; second by the direct reduction of 1-pyrazoline with Hg/Al-amalgam. 5-Phenyl aza-kainic acid has been produced by direct reduction of 1-pyrazoline, obtained in the reaction of phenyldiazomethane and dibenzyl glutaconate, with Hg/Al-amalgam. ^ Current responses to aza kainate analogs in Aplysia whole cell buccal ganglia indicate potent neuroexcitatory activity. The repetitive exposure of neuronal cells to the 5-unsubstituted aza-kainic acid led to non-desensitizing current responses, showing both binding affinity and neuronal ion-channel activation by the synthesized agonist compound. ^

Lésbicas, bissexuais, cis, professoras

Trata-se de diálogos entre ativistas-investigador.a.s4 das questões de gênero e sexualidades com professoras lésbicas e bissexuais da educação pública de Vitória, Espírito Santo, Brasil sobre temas relacionados à diversidade sexual na escola. Apresenta relatos de professoras cisgênero sobre dificuldades em lidar com a diversidade sexual na escola. Relata as impressões das professoras sobre o tema, com foco nas implicações do pertencimento a determinadas categorias sexuais e de gênero no desempenho pedagógico. Informa que o ambiente escolar está permeado por preconceitos, em que a lesbo-homo-trans e bifobia fazem parte dos discursos heteronormativos e que obriga as professoras lésbicas e bissexuais a uma ocultação da sua orientação sexual como espécie de blindagem para garantir o respeito profissional.

Efeito da adição dos azocorantes DR73 e DB79 nas propriedades do poli(metacrilato de metila)

Currently new polymeric materials have been developed to replace other of traditionally materials classes. The use of dyes allows to expand and to diversify the applications in the polymeric materials development. In this work the behavior and ability of azo dyes Disperse Blue 79 (DB79) and Disperse Red 73 (DR73) on poly(methyl methacrylate) (PMMA) were studied. Two types of mixtures were used in the production of masterbatches: 1) rheometer 2) solution. Processing by extrusion-blow molding of PMMA was carried out in order to evaluate the applications of polymeric films. Thermal analysis were performed by thermogravimetry to evaluate polymer and azo dyes thermal stability. Colorimetric analysis were obtained through monitoring the spectral variations associated with sys/trans/anti azo dyes isomerization process Colorimetric data were treated and evaluated in accordance to the color system RGB and CIEL*ab, by monitoring the color change as function of time. Mechanical properties, characterized by tensile tests, were evaluated and correlated with the presence and content of azo dyes in the samples. Analyses by scanning electronic microscopy (SEM) were performed on the surfaces of samples to check the azo dye dispersion after the mixing process. It was concluded that the production of PMMA/azo dyes is possible and feasible, and the mixtures produced had synergy of properties for use in various applications

The Putative Leishmania Telomerase Rna (leishter) Undergoes Trans-splicing And Contains A Conserved Template Sequence.

Telomerase RNAs (TERs) are highly divergent between species, varying in size and sequence composition. Here, we identify a candidate for the telomerase RNA component of Leishmania genus, which includes species that cause leishmaniasis, a neglected tropical disease. Merging a thorough computational screening combined with RNA-seq evidence, we mapped a non-coding RNA gene localized in a syntenic locus on chromosome 25 of five Leishmania species that shares partial synteny with both Trypanosoma brucei TER locus and a putative TER candidate-containing locus of Crithidia fasciculata. Using target-driven molecular biology approaches, we detected a ∼2,100 nt transcript (LeishTER) that contains a 5' spliced leader (SL) cap, a putative 3' polyA tail and a predicted C/D box snoRNA domain. LeishTER is expressed at similar levels in the logarithmic and stationary growth phases of promastigote forms. A 5'SL capped LeishTER co-immunoprecipitated and co-localized with the telomerase protein component (TERT) in a cell cycle-dependent manner. Prediction of its secondary structure strongly suggests the existence of a bona fide single-stranded template sequence and a conserved C[U/C]GUCA motif-containing helix II, representing the template boundary element. This study paves the way for further investigations on the biogenesis of parasite TERT ribonucleoproteins (RNPs) and its role in parasite telomere biology.

A New Platinum Complex With Tryptophan: Synthesis, Structural Characterization, Dft Studies And Biological Assays In Vitro Over Human Tumorigenic Cells.

A new platinum(II) complex with the amino acid L-tryptophan (trp), named Pt-trp, was synthesized and characterized. Elemental, thermogravimetric and ESI-QTOF mass spectrometric analyses led to the composition [Pt(C11H11N2O2)2]⋅6H2O. Infrared spectroscopic data indicate the coordination of trp to Pt(II) through the oxygen of the carboxylate group and also through the nitrogen atom of the amino group. The (13)C CP/MAS NMR spectroscopic data confirm coordination through the oxygen atom of the carboxylate group, while the (15)N CP/MAS NMR data confirm coordination of the nitrogen of the NH2 group to the metal. Density functional theory (DFT) studies were applied to evaluate the cis and trans coordination modes of trp to platinum(II). The trans isomer was shown to be energetically more stable than the cis one. The Pt-trp complex was evaluated as a cytotoxic agent against SK-Mel 103 (human melanoma) and Panc-1 (human pancreatic carcinoma) cell lines. The complex was shown to be cytotoxic over the considered cells.

Interesterificação química: alternativa para obtenção de gorduras zero trans

The function of lipids in human nutrition has been intensively debated in the last decade.This context reinforces the concern about controlling the trans fat ingestion, due to its negative implications on health. Interesterification provides an important alternative to modify the consistency of oils and fats without causing formation of trans isomers. This article reports research done towards production of zero trans fats by chemical interesterification, for different industrial purposes. Aspects related to the effect of trans fats on diet, their impact on health and modifications in Brazilian legislation are also covered.

Reações de oxidação de derivados de cis-octalinas promovidas por trinitrato de tálio (TTN)

The reaction of ten cis-octalins and cis-octalones with thallium trinitrate (TTN) leads to different products, depending mainly on the substitution pattern of the substrate. Functionalized cis-hydrindanes were obtained from the reaction of 1,2,3,4,4a,5,8,8a-octahydro-4a-methylnaphthalene and of 1,2,3,4,4a,5,8,8a-octahydro-4a,7-dimethylnaphthalene with TTN in acetonitrile, whereas a cyclic ether was formed treating 1,2,3,4,4a,5,8,8a-octahydro-6,8a-dimethylnaphthalen-1-ol with TTN in trimethylorthoformate (TMOF).

Trans fatty acid intake among the population of the city of São Paulo, Brazil

OBJECTIVE: To analyze the monounsaturated and polyunsaturated trans fatty acid intake among the general population. METHODS: A cross-sectional study was conducted in São Paulo, Southeastern Brazil, in 2003, on a representative sample of 2,298 male and female subjects, including 803 adolescents (12 to 19 years), 713 adults (20 to 59 years) and 782 elderly people (60 years or over). Food intake was measured using 24-hour recall. Mean trans fatty acid intake was described according to gender and age group. RESULTS: The mean trans fatty acid intake was 5.0 g/day (SE = 0.1), accounting for 2.4% (SE = 0.1) of total energy and 6.8% (SE = 0.1) of total lipids. The adolescents had the highest mean intake levels (7.4 g/day; 2.9% of energy) while the adults and the elderly had similar intake (2.2% of energy for both; 6.4% of lipids and 6.5% of lipids, respectively). The mean trans fatty acid intake among adult and elderly women (approximately 2.5% of energy and 7.0% of lipids) was higher than among men in the same age group. The food item with the highest contribution towards trans fatty acids was margarine, accounting for more than 30% of total intake, followed by filled cookies among adolescents and meat among adults and the elderly. CONCLUSIONS: The trans fatty acid intake is above the level recommended by the World Health Organization. Replacement of the trans fatty acids in manufactured food items may be an effective measure for reducing trans fatty acid intake in Brazil.

Storage stability of snacks with reduced saturated and trans fatty acids contents

A gordura vegetal parcialmente hidrogenada tem sido utilizada na aromatização de snacks. Entretanto, o risco à saúde ocasionado pelo elevado consumo de ácidos graxos saturados e trans (AGT) vem estimulando o desenvolvimento de abordagens alternativas a essa gordura. Substituímos a gordura vegetal parcialmente hidrogenada (F) por óleo de canola (O) na aromatização de snacks. Snacks com diferentes níveis de O foram produzidos, embalados e armazenados em temperatura ambiente durante vinte semanas. Monitoramos o perfil de ácidos graxos, o teor de substâncias reativas ao TBA (TBARS), a força de cisalhamento e a aceitabilidade sensorial. A substituição total reduziu o teor de ácidos graxos saturados em 72,5 por cento, em comparação aos snacks comerciais. Os snacks eram inicialmente isentos de AGT, porém, após 8 semanas, esses compostos surgiram, havendo aumento gradual durante o período de armazenamento. Entretanto, estes níveis mantiveram-se inferiores aos observados em snacks comercializados. Também foram observados baixos teores de TBARS e estabilidade da força de cisalhamento. Snacks aromatizados com F ou O foram igualmente bem aceitos durante as vinte semanas de armazenamento. É possível desenvolver snacks com teores reduzidos de ácidos graxos saturados e trans, estáveis durante o armazenamento, mantendo a elevada aceitabilidade sensorial típica deste tipo de produto