Butyrate induces apoptosis in murine macrophages via caspase-3, but independent of autocrine synthesis of tumor necrosis factor and nitric oxide

We demonstrated that 4 mM butyrate induces apoptosis in murine peritoneal macrophages in a dose- and time-dependent manner as indicated by studies of cell viability, flow cytometric analysis of annexin-V binding, DNA ladder pattern and the determination of hypodiploid DNA content. The activity of caspase-3 was enhanced during macrophage apoptosis induced by butyrate and the caspase inhibitor z-VAD-FMK (100 µM) inhibited the butyrate effect, indicating the major role of the caspase cascade in the process. The levels of butyrate-induced apoptosis in macrophages were enhanced by co-treatment with 1 µg/ml bacterial lipopolysaccharide (LPS). However, our data indicate that apoptosis induced by butyrate and LPS involves different mechanisms. Thus, LPS-induced apoptosis was only observed when macrophages were primed with IFN-gamma and was partially dependent on iNOS, TNFR1 and IRF-1 functions as determined in experiments employing macrophages from various knockout mice. In contrast, butyrate-induced macrophage apoptosis was highly independent of IFN-gamma priming and of iNOS, TNFR1 and IRF-1 functions.

Antioxidant dietary deficiency induces caspase activation in chick skeletal muscle cells

Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambdaexc = 320 nm and lambdaem = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway.

Caspase-8 and p38MAPK in DATS-induced apoptosis of human CNE2 cells

Nasopharyngeal carcinoma is a common malignancy in Southern China of uncertain etiologic origin. Diallyl trisulfide (DATS), one of the major components of garlic (Allium sativum), is highly bactericidal and fungicidal. In this study, we investigated the function of p38 mitogen-activated protein kinase (MAPK) and caspase-8 in DATS-induced apoptosis of human CNE2 cells using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], flow cytometry assay, and Western blotting. After CNE2 cells were treated with DATS (50, 100, or 150 μM) for 24 h, cell viability rates were 75.9, 63.4 and 39.6%, and apoptosis rates were 24.5, 36.9, and 62.4%, respectively. The data showed that DATS induced CNE2 cell death in a dose-dependent manner. After human CNE2 cells were treated with 100 μM DATS and inhibitors (10 μM SB203580 and Z-LETD-FMK for p38MAPK and caspase-8, respectively), changes in cell viability and apoptosis and in p38MAPK and caspase-8 activity were detected. Cell viability rates were 66.5 and 68.1% and decreased 9.9 and 11.5% compared with inhibitor treatment alone. Apoptosis rates were 31.53 and 29.98% and increased 9.1 and 10% compared with inhibitor treatment alone. The results indicated that DATS activates p38MAPK and caspase-8, but both inhibitors have an effect on P38MAPK and caspase-8 activity. In conclusion, our data indicate that p38MAPK and caspase-8 are involved in the process of DATS-induced apoptosis in human CNE2 cells and interact with each other.

Caspase dependence of the death of neonatal retinal ganglion cells induced by axon damage and induction of autophagy as a survival mechanism

We examined the degeneration of post-mitotic ganglion cells in ex-vivo neonatal retinal explants following axon damage. Ultrastructural features of both apoptosis and autophagy were detected. Degenerating cells reacted with antibodies specific for activated caspase-3 or -9, consistent with the presence of caspase activity. Furthermore, peptidic inhibitors of caspase-9, -6 or -3 prevented cell death (100 µM Ac-LEDH-CHO, 50 µM Ac-VEID-CHO and 10 µM Z-DEVD-fmk, respectively). Interestingly, inhibition of autophagy by 7-10 mM 3-methyl-adenine increased the rate of cell death. Immunohistochemistry data, caspase activation and caspase inhibition data suggest that axotomy of neonatal retinal ganglion cells triggers the intrinsic apoptotic pathway, which, in turn, is counteracted by a pro-survival autophagic response, demonstrated by electron microscopy profiles and pharmacological autophagy inhibitor.

Paleogenetic and taphonomic analysis of human bones from Moa, Beirada, and Zé Espinho Sambaquis, Rio de Janeiro, Brazil

The present paper discusses mtDNA and taphonomy of human remains from Moa, Beirada, and Zé Espinho sambaquis of Saquarema, state of Rio de Janeiro, Brazil. New human bone dating by 14C-AMS for Moa archeological site (3810+50 BP - GX-31826-AMS) is included. Preservation of microscopic lamellae and DNA is not related to the macroscopic integrity of the bones. Results here suggest that the preservation of amplifiable DNA fragments may have relation to the preservation of the lamellar arrangement as indicated by optical microscopic examination (polarized light). In 13 human bone fragments from Moa, Beirada, and Zé Espinho it was possible to sequence mtDNA from the 3 individuals of Moa, and from 1 of 4 individuals of Beirada, whose bones also show extensive areas with preserved lamellar structures. The 6 human bone fragments of Zé Espinho and 3 of the 4 fragments of Beirada showed extensive destruction of cortical microstructure represented by cavities, intrusive minerals, and agglomerated microscopic bodies of fungi and bacteria; it was not possible to extract mtDNA from these samples. The results support the hypothesis that the preservation of the microscopic osteon organization is a good predictor for DNA preservation. It was also confirmed the C haplogroup antiquity in Brazil.

Mapeamento pedológico digital da folha Botucatu (SF-22-Z-B-VI-3): treinamento de dados em mapa tradicional e validação de campo

O mapeamento digital de solos permite prever padrões de ocorrência de solos com base em áreas de referência e no uso de técnicas de mineração de dados para modelar associações solo-paisagem. Os objetivos deste trabalho foram produzir um mapa pedológico digital por meio de técnicas de mineração de dados aplicadas a variáveis geomorfométricas e de geologia, com base em áreas de referência; e testar a confiabilidade desse mapa por meio de validação em campo com diferentes sistemas de amostragem. O mapeamento foi realizado na folha Botucatu (SF-22-Z-B-VI-3), utilizando-se as folhas 1:50.000, Dois Córregos e São Pedro, como áreas de referência. Variáveis descritoras do relevo e de geologia associadas às unidades de mapeamento pedológico das áreas de referência compuseram a matriz de dados de treinamento. A matriz foi analisada pelo algoritmo PART de árvore de decisão, do aplicativo Weka (Waikato Environment for Knowledge Analysis), que cria regras de classificação. Essas regras foram aplicadas aos dados geomorfométricos e geológicos da folha Botucatu, para predição de unidades de mapeamento pedológico. A validação de campo dos mapas digitais deu-se por meio de amostragem por transectos em uma unidade de mapeamento da folha São Pedro e de forma aleatório-estratificada na folha Botucatu. A avaliação da unidade de mapeamento na folha São Pedro verificou confiabilidade, respectivamente, de 83 e 66 %, para os mapas pedológicos digital e tradicional com legenda simplificada. Apesar de terem sido geradas regras para todas as unidades de mapeamento pedológico das áreas de treinamento, nem todas as unidades de mapeamento foram preditas na folha Botucatu, o que resultou das diferenças de relevo e geologia entre as áreas de treinamento e de mapeamento. A validação de campo do mapa digital da folha Botucatu verificou exatidão global de 52 %, compatível com levantamentos em nível de reconhecimento de baixa intensidade, e kappa de 0,41, indicando qualidade Boa. Unidades de mapeamento mais extensas geraram mais regras, resultando melhor reprodução dos padrões solo-relevo na área a ser mapeada. A validação por transectos na folha São Pedro indicou compatibilidade do mapa digital com o nível de reconhecimento de alta intensidade e compatibilidade do mapa tradicional, após simplificação de sua legenda, com o nível de reconhecimento de baixa intensidade. O treinamento do algoritmo em mapas e não em observações pontuais reduziu em 14 % a exatidão do mapa pedológico digital da folha Botucatu. A amostragem aleatório-estratificada pelo hipercubo latino é apropriada a mapeamentos com extensa base de dados, o que permite avaliar o mapa como um todo, tornando os trabalhos de campo mais eficientes. A amostragem em transectos é compatível com a avaliação da pureza de unidades de mapeamento individualmente, não necessitando de base de dados detalhada e permitindo estudos de associações solo-paisagem em pedossequências.

Seleção de progênies de eucalipto pelo índice Z por MQM e Blup

O objetivo deste trabalho foi avaliar a eficiência do índice Z (índice somatório das variáveis padronizadas) na seleção de progênies de irmãos completos entre Eucalyptus grandis e Eucalyptus urophylla via método dos quadrados mínimos (MQM) e da melhor predição linear não viesada (Blup). Os experimentos foram realizados em dois tipos de solo e de ambiente, em blocos ao acaso com cinco repetições, oito plantas por parcela e espaçamento de 3x2 m. Aos três anos de idade, as plantas foram avaliadas quanto ao incremento médio anual de madeira, à densidade básica, ao rendimento de celulose e ao álcali efetivo. Para a comparação da eficiência da seleção das melhores progênies, pelos procedimentos MQM e Blup, foi obtido o índice Z para cada indivíduo. Posteriormente, foram obtidas as médias preditas das progênies para o índice Z, ranqueadas em sentido favorável ao melhoramento. O índice clássico também foi utilizado. A coincidência entre as melhores progênies selecionadas pelos índices clássico e Z foi boa. No entanto, o índice Z permite visualização gráfica, o que possibilita verificar em quais caracteres uma determinada progênie apresenta alguma deficiência. Não há diferença entre as melhores progênies selecionadas pelo índice Z via MQM e Blup.

Determinacion de la configuracion E-Z de los acidos Fumarico y Maleico. Un experimento orientado a incentivar el desarrollo de la investigacion cientifica en alumnos de Pregrado

In this work we intend to eliminate the idea that laboratory exercises seem like cookbooks. That is, exercises shall be presented as a problematic situation. Based on observation and experimentation, the students should determine the E-Z configuration of maleic and fumaric acids. The basis of this laboratory exercise is the acid-catalyzed isomerization of maleic acid to fumaric acid. Students are given the starting material, reagents and the experimental procedure. They are told that the starting material is a dicarboxylic acid containing a C=C double bond of formula C4H4O4. Students determine melting points, solubilities, acidity and chromatographic patterns for both the starting material and the product, so that a configuration of each acid can be proposed. This type of experiment yields excellent results, because the students are left to deduce that maleic acid is less stable than fumaric acid. Additionally, they conclude that maleic acid is the "Z" isomer and fumaric acid is the "E" isomer. Finally, this laboratory exercise allows the students to develop simultaneously their critical-thinking skills with the respective laboratory techniques and not to see chemistry as recipes to be followed.

Preconcentration, determination and speciation of iron by solid-phase extraction using dimethyl (E)-2-[(Z)-1-acetyl)-2-hydroxy-1-propenyl]-2-butenedioate

A new sensitive and selective procedure for speciation of trace dissolved Fe(III) and Fe(II), using modified octadecyl silica membrane disks and determination by flame atomic absorption spectrometry was developed. A ML3 complex is formed between the ligand and Fe(III) responsible for extraction of metal ion on the disk. Various factors influencing the separation of iron were investigated and the optimized operation conditions were established. Under optimum conditions, an enrichment factor of 166 was obtained for Fe3+ ions. The calibration graph using the preconcentration system for Fe3+ was linear between 40.0 and 1000.0 μg L-1.

Ni(II), Cu(II), AND Zn(II) COMPLEXES DERIVED FROM A NEW SCHIFF BASE 2-((Z)-(3-METHYLPYRIDIN-2- YLEIMINO)METHYL)PHENOL AND SYNTHESIS OF NANO SIZED METAL OXIDE PARTICLES FROM THESE COMPOUNDS

Synthesis, spectral identification, and magnetic properties of three complexes of Ni(II), Cu(II), and Zn(II) are described. All three compounds have the general formula [M(L)2(H2O)2], where L = deprotonated phenol in the Schiff base 2-((z)-(3-methylpyridin-2-yleimino)methyl)phenol. The three complexes were synthesized in a one-step synthesis and characterized by elemental analysis, Fourier transform infrared spectroscopy, electronic spectra, X-ray diffraction (XRD), and room temperature magnetic moments. The Cu(II) and Ni(II) complexes exhibited room temperature magnetic moments of 1.85 B.M. per copper atom and 2.96 B.M. per nickel atom. The X-band electron spin resonance spectra of a Cu(II) sample in dimethylformamide frozen at 77 K (liquid nitrogen temperature) showed a typical ΔMS = ± 1 transition. The complexes ([M(L)2(H2O)2]) were investigated by the cyclic voltammetry technique, which provided information regarding the electrochemical mechanism of redox behavior of the compounds. Thermal decomposition of the complexes at 750 ºC resulted in the formation of metal oxide nanoparticles. XRD analyses indicated that the nanoparticles had a high degree of crystallinity. The average sizes of the nanoparticles were found to be approximately 54.3, 30.1, and 44.4 nm for NiO, CuO, and ZnO, respectively.

La marchitez tardía del maíz (Zea mays L.) causada por Cephalosporium maydis en la Península Ibérica, y otros hongos asociados

Las especies de hongos de suelo asociadas a Cephalosporium maydis como agente causal de la marchitez tardía del maíz en la Península Ibérica se identificaron muestreando 19 campos con síntomas de marchitez en las principales zonas de cultivo entre 2011 y 2012. En el 47% de los campos no se identificó C. maydis, pero sí Fusarium graminearum, F. verticillioides, F. equiseti, F. proliferatum, Macrophomina phaseolina, Rhizoctonia solani y Trichoderma harzianum infectando las plantas de maíz. En los campos restantes, junto a C. maydis se identificaron otros hongos de suelo en porcentajes apreciables: F. verticillioides (19%), F. proliferatum (19%), F.equiseti (9%), F. oxysporum (9%) y Pythium oligandrum (9%). El crecimiento vascular de C. maydis y de otras especies fúngicas en plantas de maíz se confirmó analizando plantas con marchitez procedentes de tres campos diferentes. Tanto C. maydis como F. graminearum, F. equiseti, F. proliferatum y T. harzianum se aislaron de la inserción entre la raíz y tallo y a 10 cm de altura en el tallo de las plantas. El efecto de la infección por C. maydis sobre la producción de las plantas de maíz se cuantificó en macetas y condiciones seminaturales en el 2011. En plantas inoculadas se obtuvo una reducción del peso de las mazorcas del 54%, además de pesos de raíz y de parte aérea (tallo y hojas) significativamente menores en comparación con el control no inoculado, lo que sugiere el gran impacto económico que puede tener la marchitez tardía en condiciones naturales. Asimismo este trabajo pone de manifiesto el grado de complejidad de la etiología de la marchitez tardía, que debería ser estudiado mediante la confirmación de la patogenicidad de los hongos de suelo identificados en maíz, con el fin de determinar el papel que puede jugar cada una de estas especies en el desarrollo de la enfermedad y/o severidad de los síntomas.

Expression of anti-Z-DNA single chain antibody variable fragment on the filamentous phage surface

We describe the expression of an anti-Z-DNA single chain variable region antibody fragment (scFv) on a filamentous phage surface. Four vectors for phage display were constructed. Two of them are able to display multiple copies of the antibody fragment, and the others can be used to make monovalent libraries. The vectors use different promoter/leader sequences to direct the expression of the fused proteins. All were able to promote the assembly of fusion virion particles. In this paper we also show the affinity selection (biopanning) of those phage-antibodies based on the capacity of their products to recognize the antigen. We used biotinylated Z-DNA and the selection was performed in a solution phase fashion. The data presented here indicate that these vectors can be further used to construct anti-nucleic acid antibody fragment libraries that can be used to study the basis of nucleic acid-protein interaction and its role in autoimmunity mechanisms.