A new Hyphessobrycon (Characiformes: Characidae) of the Hyphessobrycon heterorhabdus species-group from the lower Amazon basin, Brazil

A new species of Hyphessobrycon belonging to the Hyphessobrycon heterorhabdus species-group from the lower rio Tapajós, state of Pará, Brazil, is described. The new species is allocated into the Hyphessobrycon heterorhabdus species-group due to its color pattern, composed by an anteriorly well-defined, horizontally elongated humeral blotch that becomes diffuse and blurred posteriorly, where it overlaps with a conspicuous midlateral dark stripe that becomes blurred towards the caudal peduncle and the presence, in living specimens, of a tricolored longitudinal pattern composed by a dorsal red or reddish longitudinal stripe, a middle iridescent, golden or silvery longitudinal stripe, and a more ventrally-lying longitudinal dark pattern composed by the humeral blotch and dark midlateral stripe. It can be distinguished from all other species of the group by possessing humeral blotch with a straight or slightly rounded ventral profile, lacking a ventral expansion present in all other species of the group. The new species is also distinguished from Hyphessobrycon heterorhabdus by a 9.6% genetic distance in the cytochrome c oxidase I gene. The little morphological distinction of the new species when compared with its most similar congener, H. heterorhabdus, indicates that the new species is one of the first truly cryptic fish species described from the Amazon basin.(AU)

Uma nova espécie de Hyphessobrycon pertencente ao grupo Hyphessobrycon heterorhabdus é descrita da região do baixo rio Tapajós, estado do Pará, Brasil. A nova espécie é incluída no grupo Hyphessobrycon heterorhabdus devido ao seu padrão de coloração, composto por uma mancha umeral alongada, anteriormente bem definida, que se torna difusa e borrada posteriormente, onde se sobrepõe a uma conspícua faixa escura médio-lateral que se torna borrada próxima ao pedúnculo caudal, e pela presença, em exemplares vivos, de um padrão longitudinal tricolor, composto por uma faixa longitudinal vermelha ou avermelhada dorsal, uma faixa média iridescente dourada ou prateada e, mais ventralmente, o padrão longitudinal escuro composto pela faixa escura médio-lateral e mancha umeral. A espécie pode ser distinguida das outras espécies pertencentes ao grupo por possuir uma mancha umeral com região ventral retilínea ou levemente arredondada, sem uma expansão ventral presente nas demais espécies do grupo. A espécie também se diferencia de Hyphessobrycon heterorhabdus por uma distância genética de 9,6% no gene citocromo c oxidase I. A sutil diferença morfológica da nova espécie quando comparada ao seu congênere mais similar, H. heterorhabdus, indica que a nova espécie é uma das primeiras espécies de peixes verdadeiramente crípticas descritas da Bacia Amazônica.(AU)

Allergens of the urushiol family promote mitochondrial dysfunction by inhibiting the electron transport at the level of cytochromes b and chemically modify cytochrome c1

BACKGROUND: Urushiols are pro-electrophilic haptens that cause severe contact dermatitis mediated by CD8+ effector T-cells and downregulated by CD4+ T-cells. However, the molecular mechanism by which urushiols stimulate innate immunity in the initial stages of this allergic reaction is poorly understood. Here we explore the sub-cellular mechanisms by which urushiols initiate the allergic response. RESULTS: Electron microscopy observations of mouse ears exposed to litreol (3-n-pentadecyl-10-enyl-catechol]) showed keratinocytes containing swollen mitochondria with round electron-dense inclusion bodies in the matrix. Biochemical analyses of sub-mitochondrial fractions revealed an inhibitory effect of urushiols on electron flow through the mitochondrial respiratory chain, which requires both the aliphatic and catecholic moieties of these allergens. Moreover, urushiols extracted from poison ivy/oak (mixtures of 3-n-pentadecyl-8,11,13 enyl/3-n-heptadecyl-8,11 enyl catechol) exerted a higher inhibitory effect on mitochondrial respiration than did pentadecyl catechol or litreol, indicating that the higher number of unsaturations in the aliphatic chain, stronger the allergenicity of urushiols. Furthermore, the analysis of radioactive proteins isolated from mitochondria incubated with 3H-litreol, indicated that this urushiol was bound to cytochrome c1. According to the proximity of cytochromes c1 and b, functional evidence indicated the site of electron flow inhibition was within complex III, in between cytochromes bL (cyt b566) and bH (cyt b562). CONCLUSION: Our data provide functional and molecular evidence indicating that the interruption of the mitochondrial electron transport chain constitutes an important mechanism by which urushiols initiates the allergic response. Thus, mitochondria may constitute a source of cellular targets for generating neoantigens involved in the T-cell mediated allergy induced by urushiols.

PEGylation strategy to the development of analytical and therapeutic proteins / Potencial da PEGuilação para o desenvolvimento de proteínas para fins terapêuticos e analíticos

Protein PEGylation is the covalent bonding of polyethylene glycol (PEG) polymers to amino acid residues of the protein and it is one of the most promising techniques for improving the therapeutic effect of biopharmaceuticals and long-term stability of protein-based biosensors. This chemical modification brings advantages to biopharmaceuticals, such as an increased half-life, enhanced stability, and reduced immunogenicity. Moreover, in the analytical field, PEGylation improves the multiple properties of protein-based biosensors including biocompatibility, thermal and long-term stability, and solubility in organic solvents. However, the use of PEGylated conjugates in the analytical and therapeutic fields has not been widely explored. The limited industrial application of PEGylated bioconjugates can be attributed to the fact that the reaction and separation steps are currently a challenge. The correct selection of the PEGylation reaction design and the purification process are important challenges in the field of bioconjugation. In this sense, the design and optimization of site-specific PEGylation reactions and application of aqueous biphasic systems (ABS) as purification platforms for PEGylated conjugates are the two main objectives of this thesis. Regarding the purification step, the efficient fractionation (i) of the PEGylated conjugates from the native protein and (ii) of the PEGylated conjugates based on their degree of PEGylation was studied. Centrifugal partition chromatography (CPC) was applied as a continuous regime platform based on ABS technology to efficiently purify the PEGylated proteins. The two proteins under study are L-asparaginase, an important biopharmaceutical applied in the treatment of acute lymphoblastic leukemia and cytochrome c, a promising biosensor. The current work developed in this thesis demonstrates the great potential of ABS in the fractionation of PEGylated proteins, under batch and continuous regime. In addition, in situ recovery of the PEGylated products through one-pot bioconjugation and ABS purification was successfully demonstrated for both enzymes studied. Although further research on scale-up is still required, the results presented show the relevance of ABS platforms for the development of separation processes of PEGylated proteins

A PEGuilação de proteínas é a ligação covalente de polímeros de polietilenoglicol (PEG) a resíduos de aminoácidos da proteína e é uma das técnicas mais promissoras para melhorar o efeito terapêutico dos biofármacos e a estabilidade a longo prazo de biossensores proteícos. Esta modificação química traz vantagens aos produtos biofarmacêuticos, como um aumento da meia-vida, maior estabilidade e imunogenicidade reduzida. Além disso, no campo analítico, a PEGuilação melhora as múltiplas propriedades dos biossensores baseados em proteínas, incluindo biocompatibilidade, estabilidade térmica e a longo prazo, e solubilidade em solventes orgânicos. No entanto, o uso de conjugados PEGuilados em campos analíticos e terapêuticos não tem sido amplamente explorado. A aplicação industrial limitada dos bioconjugados PEGuilados pode ser atribuída ao facto de as etapas de reacção e separação serem atualmente um desafio. A seleção correcta do design da reacção de PEGuilação e do processo de purificação são importantes desafios no campo da bioconjugação. Neste sentido, a concepção e otimização de reações de PEGuilação sítio-específicas e aplicação de sistemas aquosos bifásicos (ABS) como plataformas de purificação de conjugados PEGuilados são os dois principais objetivos desta tese. No que concerne à etapa de purificação foi estudado o eficiente fracionamento (i) dos conjugados PEGuilados, da proteína nativa e (ii) dos conjugados PEGuilados baseados no seu grau de PEGuilação. A cromatografia por partição centrífuga (CPC) foi aplicada como uma plataforma de regime contínuo baseada na tecnologia de ABS para purificar eficientemente as proteínas PEGuiladas. As duas proteínas em estudo são a L-asparaginase, importante biofármaco aplicado no tratamento da leucemia linfoblástica aguda e o citocromo c, um potencial biossensor. A partir dos trabalhos desenvolvidos, é possível confirmar o grande potencial dos ABS no fracionamento de proteínas PEGuiladas, em regime contínuo e descontínuo. Além disso, a recuperação in situ dos produtos PEGuilados através da integração em uma única etapa de bioconjugação e purificação por ABS foi comprovada com sucesso para ambas as enzimas estudadas. Embora ainda sejam necessários estudos adicionais sobre a viabilidade destes sistemas em larga escala, os resultados aqui apresentados demonstram a relevância dos ABS para o desenvolvimento de processos de separação de proteínas PEGuiladas

Taxonomia integrativa de espécies, com fêmeas morfologicamente similares, do gênero Psychodopygus (Diptera, Psychodidae), Série Chagasi, registradas no Brasil / Integrative taxonomy of morphologically indistinguishable species of the genus Psychodopygus (Diptera, Psychodidae), Chagasi series, registered in Brazil

Introdução. A identificação dos flebotomíneos baseia-se principalmente na morfologia do adulto, o que pode ser problemático quando as espécies são morfologicamente muito semelhantes. Psychodopygus é um gênero de flebotomíneos de grande interesse em saúde pública devido ao papel de algumas espécies na veiculação de Leishmania spp. no Brasil. No entanto, este gênero inclui espécies com fêmeas morfologicamente indistinguíveis que pertencem à Série Chagasi, sendo elas: P. chagasi, P. complexus, P. squamiventris maripaensis, P. squamiventris squamiventris e P. wellcomei. Objetivos. Investigar a possibilidade de distinguir essas espécies por meio de análises morfométrica e molecular, além de produzir uma distribuição geográfica atualizada para o grupo analisando a probabilidade de ocorrência das espécies através da análise de modelagem de nicho ecológico. Material e Métodos. Foi realizada a análise discriminante na morfometria geométrica (cabeça e asa) e linear, morfologia (usando microscopia óptica e eletrônica de varredura) e a análise do citocromo c oxidase subunidade 1 (COI), avaliando-se um total de 752 espécimes (460 fêmeas e 292 machos) dos seguintes estados Amapá, Amazonas, Ceará, Mato Grosso, Pará, Rondônia, Roraima e Tocantins. Mapas de distribuição foram produzidos através de dados obtidos do material analisado e de revisão bibliográfica. Resultados. A análise discriminante usando caracteres morfométricos lineares mostrou-se capaz de diferenciar todas as espécies, exceto P. complexus, que apresentou 2,2% de erro de identificação. A morfometria geométrica das asas foi incapaz de separar completamente as espécies através da conformação, mas o tamanho do centróide dos espécimes fêmeas falhou apenas em distinguir P. complexus de P. s. maripaensis. Por outro lado, a morfometria geométrica das cabeças foi capaz de distinguir todas as espécies com grande eficiência ao usar tanto a forma como o tamanho do centróide. A análise morfológica revelou que a coloração torácica, principalmente do pronoto e do pós-noto, pode ser usada para separar as cinco espécies em três grupos: P. chagasi, P. wellcomei / P. complexus e P. s. mariapaensis / P. s. squamiventris. Os resultados da análise de DNA Barcoding, mostraram um agrupamento semelhante ao observado na morfologia; embora os espécimes de P. wellcomei do estado do Ceará mostrem uma grande distância genética da população do estado do Pará, evidenciando que essa espécie possa representar um complexo. Quanto à microscopia eletrônica de varredura, foram avaliadas detalhadamente as estruturas das antenas, tórax e genitália masculina. Salientamos que no anepímero (tórax) foi observada uma escama tipo \"raquete\" modificada apenas em Psychodopygus s. squamiventris. A revisão da distribuição geográfica mostrou que as espécies possuem uma distribuição cis-andina, ocorrendo principalmente no bioma Amazônico. A nítida separação de algumas espécies pelo rio Amazonas, sugere que o surgimento do grupo ocorreu no período que se estende da orogênese dos Andes até a formação deste rio. Conclusões. O estudo possibilitou diferenciar completamente as fêmeas das cinco espécies da Série Chagasi utilizando o conjunto de dados obtidos por morfometria linear e geométrica e análises morfológicas e também apresentar novos caracteres morfológicos e padrões distribucionais que facilitarão a identificação de machos e fêmeas dessas espécies

Introduction. The identification of sand flies is mainly based on adult morphology, which can be problematic when species are morphologically very similar. Psychodopygus is one of the sand fly genera of great interest in public health, due to the role of some species in the transmission of Leishmania spp. in Brazil. However, this genus includes species with morphologically indistinguishable females that belong to the Chagasi series, which includes: P. chagasi, P. complexus, P. squamiventris maripaensis, P. squamiventris squamiventris and P. wellcomei. Objectives. To investigate the possibility of distinguishing among these species by means of morphometric and molecular analyses in addition to producing an updated geographical distribution for the group, analyzing the probability of the occurrence of the species by the analysis of ecological niche modeling. Material and methods. The analyses of the cytochrome c oxidase subunit 1 (COI), geometrical (head and wing) and of linear morphometry and morphology (using optical microscopy and scanning electron microscopy) were carried out using a total of 752 specimens (460 females and 292 males) from the following states: Amapá, Amazonas, Ceará, Mato Grosso, Pará, Rondônia, Roraima e Tocantins. Distribution maps were produced on the basis of data obtained from the material analyzed and a bibliographical review. Results. The discriminant analysis using linear morphometric characters was able to differentiate among all the species, except for P. complexus, which presented a 2.2% error of identification. The geometric morphometry of the wings was unable to completely separate the species by means of the shape analyses, but the centroid size of the female specimens only failed to distinguish P. complexus from P. s. maripaensis. Otherwise, the geometric morphometry of the heads was sufficient to distinguish all the species with great efficiency, when using both the head-shape and the centroid size. The morphological analysis revealed that the thoracic coloration, mainly of the pronotum and the post-notum, can be used to separate the five species into three groups: P. chagasi, P. wellcomei / P. complexus, P. s. mariapaensis / P. s. squamiventris. The results of the Barcoding DNA analyses showed a cluster similar to that observed in the morphology; however, P. wellcomei specimens from the Ceará population showed a great genetic distance from the population of Pará, evidencing that this species may represent a complex. As for the scanning electron microscopy, the structures of the antennae, thorax and male genitalia were evaluated in detail. In the anepimerum (thorax) a modified \"racket\"-type scale was observed only in Psychodopygus s. squamiventris. The review of the geographical distribution showed that the species have a cis-Andean distribution, occurring mainly in the Amazonian biome. The separation of some species from the others by the Amazon river suggests that the appearance of the Chagasi series occurred in the period from the orogenesis of the Andes to the formation of this river. Conclusions. The results clearly differentiate the females of the five species of the Chagasi series using the data set of linear and geometric morphometry and morphological analyses, providing new morphological and distributional data that will facilitate the identification of the males and females of this group

Modificações em proteínas induzidas por compostos eletrofílicos. possível papel em esclerose lateral amiotrófica / Modifications in proteins induced by electrophilic compounds. Possible role in amyotrophic lateral

Danos em biomoléculas podem ocorrer a partir de uma interação direta entre as biomoléculas e espécies reativas de oxigênio e nitrogênio como também, pela reação de produtos secundários dessas espécies como eletrófilos gerados na peroxidação lipídica. Alguns desses produtos secundários possuem estabilidade química maior que as espécies reativas das quais foram derivadas e podem se ligar covalentemente as biomoléculas comprometendo o funcionamento normal das mesmas. Portanto, modificações em proteínas por aldeídos gerados na lipoperoxidação têm sido investigadas por suas implicações com desordens patológicas relacionadas à agregação proteica, e modificações em diversas vias de sinalização amplificando os efeitos deletérios em sistemas biológicos. Os objetivos desse trabalho foi contribuir na elucidação dos mecanismos moleculares associados ao desenvolvimento da esclerose lateral amiotrófica (ELA) através da identificação, caracterização e quantificação de modificações póstraducionais em proteínas pelos aldeídos 4-hidroxi-2-hexenal (HHE) e trans-4-hidroxi-2-nonenal (HNE) in vitro (citocromo c) e in vivo (modelo ELA) a partir de técnicas de Western blot, imunoprecipitação e espectrometria de massa com abordagem proteômica de "shotgun" em ratosSOD1G93A modelo de esclerose lateral amiotrófica (ELA). Estudos com citocromo c mostraram a ligação dos aldeídos ao citocromo c e mecanismos de reação foram propostos. Foram encontrados seis peptídeos modificados por HHE e um para o HNE, e o peptídeo TGPNLHGLFGR se mostrou modificado pelos dois aldeídos paralelamente. Foi demonstrado que a histidina 33 é um "hot spot" frente as adições pelos aldeídos. Nas análises por western blot das proteínas ligadas a aldeídos foi possível observar uma tendência de aumento na concentração de proteínas ligadas ao HNE nos animais ELA, mais acentuada nas amostras de 70 dias comparadas ao controle. Com relação aos resultados obtidos com HHE tanto os animais pré-sintomáticos quanto os sintomáticos não apresentaram diferenças de HHE-proteína, tantonos controles quanto nos animais ELA. Nas amostras dos animais sintomáticos não detectamos diferença significativa na concentração de aldeído-proteína entre os grupos. Já as análises proteômicas revelaram 24 proteínas diferencialmente expressas, com destaque para proteínas com os maiores valores de significância (p-value), como a ubiquitina no grupo dos pré- sintomáticos e a neurogranina, no grupo dos animais sintomáticos e várias proteínas de metabolismo energético, de neurofilamentos, proteínas de processos redox e proteínas ligadas o metabolismo de cálcio (fundamentais na fisiopatologia em ELA). Algumas proteínas importantes foram encontradas com exclusividades nos grupos pré-sintomáticos e sintomáticos pelo diagrama de Venn. Com relação a proteínas modificadas pelos aldeídos, foram encontradas algumas relevantes como a proteína 2 de interação com a polimerase delta que foi modificada por HNE via adição de Michael encontrada nos animais ELA pré-sintomáticos e sintomáticos, a catalase que foi encontrada modificada por HNE via base de Schiff apenas nos ELA pré- sintomáticos, e a tiol redutase induzível por interferon gama no grupo dos animais ELA sintomáticos. Com relação a proteínas modificadas por HHE, foram encontradas a Janus quinase e proteína 3 de interação com microtúbulo, modificadas tanto por adição de Michael quanto via base de Schiff nos animais ELA sintomáticos. É interessante ressaltar que algumas modificações encontradas em proteínas não caracterizadas podem indicar proteínas novas ainda não descritas como modificadas por esses aldeídos. Os resultados mostram que algumas das proteínas modificadas por HNE e HHE encontradas neste trabalho, estão relacionadas ao estresse redox, vias metabólicas energéticas, proteínas envolvidas na resposta a danos oxidativos, e processos inflamatórios. Tais modificações ocorrem não só no modelo de neurodegeneração, mas foram previamente descritas em outros processos patológicos, como doença cardiovascular, lesão hepática por uso crônico de álcool

Damage to biomolecules can occur from a direct interaction between biomolecules and reactive of oxygen and nitrogen species as well as from the reaction of secondary products of these species as electrophiles generated in lipid peroxidation. Some of these by-products have greater chemical stability than the derived reactive species and can bind to biomolecules compromising their normal function. Therefore, protein modifications by aldehydes generated during lipoperoxidation have been investigated for their implications with pathological disorders related to protein aggregation and modifications in signaling pathways amplifying the deleterious effects in biological systems. The aim of this work was to contribute to the elucidation of the molecular mechanisms associated with the development of amyotrophic lateral sclerosis (ALS) through the identification, characterization and quantification of posttranslational modifications in proteins by 4-hydroxy-2-hexenal (HHE) and trans-4-hydroxy-2- nonenal (HNE) in vitro, cytochrome c, and in vivo, rat model (SOD1G93A) of amyotrophic lateral sclerosis (ALS), throught Western blot techniques, and mass spectrometry with shotgun proteomics approach. The results showed the binding of aldehydes to cytochrome c. Six peptides were modified by HHE and one by HNE. The peptide TGPNLHGLFGR was modified by the two aldehydes. Histidine 33 has been shown to be a hot spot against aldehydes additions. By western blot analysis of the aldehyde-bound proteins, it was possible to observe a tendency of increase in the concentration of HNE-bound proteins in the ALS animals, more pronounced in the samples of 70 days compared to control samples. Regarding the results obtained with HHE, both pre-symptomatic and symptomatic animals did not show HHE-protein differences, both in controls and in ALS animals. We did not detect a significant difference in the aldehyde-protein concentration between the groups in the samples of the symptomatic animals. Proteomic analysis revealed 24 differentially expressed proteins, with emphasis on proteins with thehighest values of significance (p-value), such as the ubiquitin in the pre-symptomatic group and neurogranin in the group of the symptomatic animals and several proteins of the energetic metabolism pathways, neurofilaments, proteins of redox processes and proteins linked to calcium metabolism (fundamental in the pathophysiology of ALS). Some important proteins were found exclusivity in the pre-symptomatic and symptomatic groups by the Venn diagram. With regard to aldehyde-modified proteins, some relevant ones such as Delta-2 polymerase interaction protein, that was modified by HNE via the addition of Michael found in presymptomatic and symptomatic ELA animals, catalase that was found to be modified by HNE via Schiff's base only in pre-symptomatic ALS, and gamma interferon-inducible thiol reductase in the group of symptomatic ALS animals. Janus kinase and microtubule interaction protein 3, were found to be modified by Michael addition and Schiff base pathway respectively in symptomatic ALS animals. It is interesting to note that some modifications found in uncharacterized proteins may indicate new proteins not yet described as modified by these aldehydes. The results show that some of the proteins modified by HNE and HHE found in this work are related to redox stress, energetic metabolic pathways, proteins involved in the response to oxidative damage, and inflammatory processes. Such modifications occur not only in the neurodegeneration model, but were previously described in other pathological processes, such as cardiovascular disease, liver injury due to chronic alcohol use

Effects of endurance training on reduction of plasma glucose during high intensity constant and incremental speed tests in Wistar rats

The aim of this research was to investigate the effects of endurance training on reduction of plasma glucose during high intensity constant and incremental speed tests in Wistar rats. We hypothesized that plasma glucose might be decreased in the exercised group during heavy (more intense) exercise. Twenty-four 10-week-old male Wistar rats were randomly assigned to sedentary and exercised groups. The prescription of endurance exercise training intensity was determined as 60% of the maximum intensity reached at the incremental speed test. The animals were trained by running on a motorized treadmill, five days/week for a total period of 67 weeks. Plasma glucose during the constant speed test in the exercised group at 20 m/min was reduced at the 14th, 21st and 28th min compared to the sedentary group, as well at 25 m/min at the 21st and 28th min. Plasma glucose during the incremental speed test was decreased in the exercised group at the moment of exhaustion (48th min) compared to the sedentary group (27th min). Endurance training positively modulates the mitochondrial activity and capacity of substrate oxidation in muscle and liver. Thus, in contrast to other studies on high load of exercise, the effects of endurance training on the decrease of plasma glucose during constant and incremental speed tests was significantly higher in exercised than in sedentary rats and associated with improved muscle and hepatic oxidative capacity, constituting an important non-pharmacological intervention tool for the prevention of insulin resistance, including type 2 diabetes mellitus.

Glutamine treatment attenuates hyperglycemia-induced mitochondrial stress and apoptosis in umbilical vein endothelial cells

OBJECTIVE: The aim of this study was to determine the in vitro effect of glutamine and insulin on apoptosis, mitochondrial membrane potential, cell permeability, and inflammatory cytokines in hyperglycemic umbilical vein endothelial cells. MATERIALS AND METHODS: Human umbilical vein endothelial cells were grown and subjected to glutamine and insulin to examine the effects of these agents on the hyperglycemic state. Mitochondrial function and the production of inflammatory cytokines were assessed using fluorescence analysis and multiple cytotoxicity assays. Apoptosis was analyzed by the terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. RESULTS: Glutamine maintains the integrity of the mitochondria by reducing the cell permeability and cytochrome c levels and increasing the mitochondrial membrane potential. The cytochrome c level was significantly (p<0.005) reduced when the cells were treated with glutamine. An apoptosis assay revealed significantly reduced apoptosis (p<0.005) in the glutamine-treated cells. Moreover, glutamine alone or in combination with insulin modulated inflammatory cytokine levels. Interleukin-10, interleukin-6, and vascular endothelial growth factor were up-regulated while tumor necrosis factor-α was down-regulated after treatment with glutamine. CONCLUSION: Glutamine, either alone or in combination with insulin, can positively modulate the mitochondrial stress and cell permeability in umbilical vein endothelial cells. Glutamine regulates the expression of inflammatory cytokines and maintains the balance of the mitochondria in a cytoprotective manner. .

Suicide attempts among children and adolescents: partial or total injury? / Tentativa de suicídio infanto-juvenil: lesão da parte ou do todo?

This study sought to verify the records on file and the number of cases of attempted suicide among children and adolescents who were attended by Emergency Care health professionals in the municipality of Matozinhos, Minas Gerais, Brazil. Documentary and descriptive research was conducted, the data for which was collected by means of an investigation of Outpatient Records from 2008 to 2010. Of the 73,000 files evaluated, those dealing with cases of attempted suicide among children and adolescents between the age of 3 and 18 years were selected. It was revealed that the health professionals, particularly physicians and nurses, fail to register the cases appropriately, invalidating information about the problem and potential prevention measures. The conclusion reached was that underreporting and the discrepancy of the diagnoses which were not duly referred to the competent agencies require rethinking and reviewing medical practices, and taking a systematic and careful look to address the individual as a complex whole.

Neste estudo procurou-se verificar o registro e o número de casos de tentativa de suicídio entre crianças e adolescentes do município de Matozinhos, Minas Gerais, Brasil, que foram atendidos pelos profissionais de saúde do Pronto-Atendimento. Trata-se de uma pesquisa documental e descritiva, cuja coleta dos dados ocorreu por meio de investigação nas Fichas Ambulatoriais, no período de 2008 a 2010. Das 73.000 fichas levantadas, selecionaram-se aquelas que tratavam de casos de tentativa de suicídio entre crianças e adolescentes do município, com idades entre três e 18 anos. Percebeu-se que os profissionais de saúde, mais especificamente os médicos e enfermeiros, não registram os casos de forma adequada, inviabilizando a informação sobre o problema e as medidas de prevenção. Concluiu-se que a subnotificação, a discrepância dos diagnósticos e o não encaminhamento aos órgãos competentes exigem repensar e rever a prática médica e dirigir um olhar sistematizado e cuidadoso para perceber o sujeito como um todo complexo.

ERKs and mitochondria-related pathways are essential for glycyrrhizic acid-mediated neuroprotection against glutamate-induced toxicity in differentiated PC12 cells

The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamate-enhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondria-related pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases.

Modificação de proteínas por produtos de oxidação do colesterol: mecanismos e implicações biológicas / Modification of proteins by oxidation products of cholesterol: mechanisms and biological implications

O colesterol é um importante componente das membranas celulares em eucariotos superiores, desempenhando papéis estruturais e funcionais. O colesterol possui uma insaturação em sua estrutura sendo, portanto, alvo de oxidação mediada por espécies reativas de oxigênio e/ou nitrogênio. A oxidação não enzimática do colesterol gera, como produtos primários, os hidroperóxidos de colesterol. Tais moléculas, por sua vez, são altamente reativas e podem reagir com metais livres e/ou metaloproteínas, trazendo consequências à celula. Neste sentido, o primeiro capítulo deste trabalho tem como objetivo estudar a reação dos hidroperóxidos de colesterol (ChOOH) com o citocromo c (citc), uma heme proteína envolvida no transporte de elétrons na mitocôndria. Análises de espectroscopia no UV-Vis mostraram que o ChOOH promove o bleaching da banda Soret do citc de uma maneira dose-dependente. Mais ainda, esta reação leva à formação de radicais centrados em carbono tanto na proteína como no lipídeo, sugerindo uma redução homolítica do ChOOH. Como consequências, pode-se observar a oligomerização do citc, um processo que pode influenciar no transporte de elétrons bem como na sinalização para a apoptose. A partir da reação do citc com ChOOH podem surgir, direta ou indiretamente, outras espécies reativas, como aldeídos, cetonas e epóxidos. Dentre estas, destacam-se os aldeídos de colesterol, em particular o colesterol secoaldeído (CSec) e o carboxialdeído (ChAld), uma vez que foram encontrados elevados em placas ateroscleróticas e em tecidos cerebrais de pacientes com doenças neurodegenerativas. Tais espécies podem reagir com resíduos de aminoácidos provocando alterações estruturais e funcionais em proteínas. Neste sentido, o segundo capítulo deste trabalho tem como objetivo estudar a reação do ChAld com citc. Usando modelos mimétivos de membrana e espectrometria de massas, foi mostrado que o ChAld modifica covalentemente o citc por um mecanismo consistente com a formação de bases de Schiff. Tal modificação ocorre preferencialmente em resíduos de lisina que interagem com a membrana. Estas modificações influenciam na afinidade do citc pela membrana, aumentando sua aderência, o que pode ter influência no transporte de elétrons e sinalização para a apoptose. No terceiro e último capítulo deste trabalho nós buscamos uma ferramente analítica que permitisse analisar modificação de proteínas promovidas por produtos de oxidação de colesterol e outros esteróis. Em um estudo realizado em colaboração com o grupo do professor Porter na Universidade de Vanderbilt, utilizamos ensaios baseados em click chemistry para buscar proteínas modificadas. Para isso, foram sintetizados derivados de colesterol e 7-deidrocolesterol (7-DHC, precursor imediato do colesterol) contendo um grupo alquinil na sua cadeia lateral. Este grupo pode ser ligado a um grupo azida por meio de uma reação de cicloadição, em um processo conhecido como click chemistry. Após a síntese e caracterização dos derivados lipídicos contendo o grupo alquinil na cadeia lateral, células Neuro2a foram tratadas com o alquinil-7-DHC e o alquinil-colesterol para averiguar seu metabolismo. Análises por HPLC-MS/MS mostraram que ambos derivados contendo o grupo alquinil foram metabolisados e convertdos nos respectivos ésteres. Usando um modelo celular para a doença conhecida como Sindrome de Smith-Lemli-Opitz (SLOS), doença caracterizada pela deficiência na enzima 7-deidrocolesterol redutase, foi mostrado que o acúmulo característico de 7-DHC nos pacientes pode levar a uma maior modificação de proteínas promovidas por seus derivados, o que pode contribuir para o desenvolvimento da doença

Cholesterol is an important component of eukaryotic cellular membranes, where it has an influence in the fluidity and stability. Due to the presence of a double bond in its structure, cholesterol can be oxidized by reactive oxygen and nitrogen species. This non-enzymatic oxidation generates, as primary products, cholesterol hydroperoxides. Such molecules, in turn, are highly reactive and can react with free metal ions and/or metalloproteins, affecting cell metabolism. Therefore, the first chapter of the present study aims to investigate the reaction of cholesterol hydroperoxides (ChOOH) with cytochrome c (cytc), a heme protein involved in the mitochondrial electron transport. Spectroscopic analyses in the UV-Vis region showed that ChOOH induces a dose-dependent bleaching of cytc's Soret band. In addition, this reaction leads to the formation of carbon-centered radicals on both protein and lipid, suggesting a homolytic reduction of ChOOH. As consequences, cytc undergoes oligomerization, a process that can influence electron transport and apoptosis signaling. The reaction of cytc and ChOOH can produce, directly or indirectly, reactive species such as epoxides, aldehydes and ketones. Among them, cholesterol aldehydes, such as cholesterol secoaldehyde (CSec) and cholesterol carboxyaldehyde (ChAld), are of particular interest, since they were previously found elevated in atherosclerotic plaques and brain tissue of patients bearing neurodegenerative diseases. These species can also react with amino acid residues leading to protein denaturation and malfunction. With that in mind, the second chapter of this study aims to investigate the reaction of ChAld and cytc. Using mimetic membrane models and mass spectrometry analyses, we showed that ChAld covalently modifies cytc through a mechanism consistent with the formation of Schiff base adducts. Such modification occurs mostly at lysine residues that are known to interact with the membrane. The modifications have an influence in the affinity of cytc to the membrane, where they increase its binding to the membrane, a process that could affect the electron transport and apoptosis signaling. In the last and third chapter of this study we wanted an analytical tool that allowed the investigation of protein adduction promoted by cholesterol and other sterols-derived oxidation products. In a study performed in collaboration with the Porter group from Vanderbilt University, we used analyses based on click chemistry to search for protein adduction. To address that, we first synthesized derivatives of cholesterol and 7-dehydrocholesterol (7-DHC, the immediate precursor of cholesterol) containing an alkynyl group in the side chain. The alkynyl group can be ligated to an azide group through a cycloaddition reaction, in a process known as click chemistry. After the synthesis and characterization of alkynyl derivatives, Neuro2a cells were treated with alkynyl-7-DHC and alkynyl-cholesterol to check their metabolism. HPLC-MS/MS analyses showed that both alkynyl derivatives are metabolized and converted into their respective esters. In addition, using a cell model for Smith-Lemli-Optiz Syndrome (SLOS), a disease characterized by the deficiency in the dehydrocholesterol reductase 7, we showed that the characteristic accumulation of 7-DHC in SLOS patients might be associated with protein adduction promoted by its oxidation products, which might contribute to the development of the disease

Hydrogen/rydride ion relay - a mechanism for early electron transfer in cytochrome c oxidases / El reléuónico hidrógeno-hidruro: un mecanismo de transferencia temprana de electrones en las citocromo c oxidasas

Cytochrome c oxidase (COX) employs electrons obtained from cytochrome c to bring about the reduction of oxygen to water. It is known that the electrons originate from the haem edge of cytochrome c and enters bovine COX at Trp-104. It is also known that Tyr-105, Glu-198 and Asp-158 of COX subunit II play roles in the enzyme's catalysis but how these roles are linked to electron transfer remain unclear. Recently, we proposed that electrons travel from the haem edge of cytochrome c to CuA, the first metal redox centre of COX, by a hydrogen/hydride ion relay using six residues. Now using a similar computer assisted approach, we investigate the extent to which this hydride/hydrogen ion mechanism is common amongst oxidases. The crystal structures of COX from P denitrificans, R sphaeroides and T thermophilus and quinol oxidase from E coli were downloaded and their binding domains analysed. As with bovine, all four oxidases had only nine amino acid residues in that region and both the sequences and three-dimensional structures were highly conserved. We propose that these residues function as a hydrogen/hydride ion relay, participating directly in electron transfer to CuA. We further suggest that this electron transfer mechanism might be a common feature in oxidases.

La citocromo c oxidasa (COX) emplea electrones obtenidos del citocromo c para producir la reducción del oxígeno a agua. Se sabe que los electrones originan a partir del hemo del citocromo c, y entran en la COX bovina en Trp-104. También se conoce que Tyr-105, Glu-198 y Asp-158 de la subunidad II de COX, desempeñan papeles en la catálisis de la enzima, pero no hay todavía claridad en cuanto a cómo estos papeles se hallan vinculados con la transferencia de electrones. Recientemente, sugerimos que los electrones viajan del borde del hemo del citocromo c al CuA, el primer centro metálico de reacción redox de la COX, por un relé iónico hidrógeno-hidruro, usando seis residuos. Ahora, usando un enfoque similar computarizado, investigamos hasta que punto este mecanismo de iones hidrógeno/hidruro es común entre las oxidasas. Se bajaron y analizaron los dominios de unión de las estructuras cristalinas de la COX de P denitrificans, R sphaeroides, y T thermophilus, y de la quinol oxidasa de la E coli. Como en el caso de la bovina, las cuatro oxidasas tenían sólo nueve residuos de aminoácido en esa región, y tanto las secuencias como las estructuras tridimensionales presentaban un alto grado de conservación. Proponemos que estos residuos funcionan como un relé iónico hidrógeno-hidruro, participando directamente en una transferencia de electrones al CuA. Asimismo, sugerimos que este mecanismo de transferencia de electrones podría ser un rasgo común de las oxidasas.

Modulation of ROS production in human leukocytes by ganglioside micelles

Recent studies have reported that exogenous gangliosides, the sialic acid-containing glycosphingolipids, are able to modulate many cellular functions. We examined the effect of micelles of mono- and trisialoganglioside GM1 and GT1b on the production of reactive oxygen species by stimulated human polymorphonuclear neutrophils using different spectroscopic methods. The results indicated that exogenous gangliosides did not influence extracellular superoxide anion (O2.-) generation by polymorphonuclear neutrophils activated by receptor-dependent formyl-methionyl-leucyl-phenylalanine. However, when neutrophils were stimulated by receptor-bypassing phorbol 12-myristate 13-acetate (PMA), gangliosides above their critical micellar concentrations prolonged the lag time preceding the production in a concentration-dependent way, without affecting total extracellular O2.- generation detected by superoxide dismutase-inhibitable cytochrome c reduction. The effect of ganglioside GT1b (100 µM) on the increase in lag time was shown to be significant by means of both superoxide dismutase-inhibitable cytochrome c reduction assay and electron paramagnetic resonance spectroscopy (P < 0.0001 and P < 0.005, respectively). The observed phenomena can be attributed to the ability of ganglioside micelles attached to the cell surface to slow down PMA uptake, thus increasing the diffusion barrier and consequently delaying membrane events responsible for PMA-stimulated O2.- production.

Infección por el virus de la Lengua azul: activación de señales celulares que inducen apoptosis / Bluetongue virus infection: signaling pathway activated during apoptosis

El virus de la Lengua azul (VLA) es un ARN virus de doble cadena que induce apoptosis tanto en cultivos celulares como en tejidos blanco. Con el fin de dilucidar el mecanismo de apoptosis en la infección por el VLA, en el presente trabajo examinamos en detalle, por la técnica de Western blot, las señales celulares de caspasas, Bax, citocromo c, Smac/DIABLO y factor nuclear kappa B (NF-kB) que se activan en la infección viral. Hemos comprobado que luego de la infección in vitro con el VLA, se detectó la activación de la caspasa 8 y con ello el mecanismo extrínseco de la apoptosis. También detectamos por primera vez no sólo la activación de miembros de la familia Bcl-2 (Bax), sino también la liberación del citocromo c y la proteína Smac/DIABLO, confirmando que en la infección por el VLA está involucrado el mecanismo secuencial intrínseco de la apoptosis. Asimismo, demostramos que la infección por el VLA activa el NF-kB y que la apoptosis es sustancialmente reducida mediante la inhibición del mismo. La activación de las señales celulares tales como Bax, citocromo c, Smac/DIABLO y NF-kB presentados en este trabajo, esclarecen los mecanismos apoptóticos durante la infección por el VLA para una mayor comprensión del papel primario que juega la apoptosis en la patogénesis del virus.

Bluetongue (BTV) is a double-stranded RNA virus that induces apoptosis both in mammalian cell cultures and in target tissues. To elucidate the apoptosis pathways in BTV infection, we have examined in detail the apoptosis mechanism by examination of caspases, Bax, cytochrome c, Smac/DIABLO and NF-kB signalling pathways. In this report, after cell infection with BTV, the activation of caspase 8 was detected, proving the extrinsic receptor binding apoptotic pathway. Apoptosis followed a sequential pathway involving the detection of activated Bcl-2 family members. Furthermore, its translocation to the mitochondria, as well as the release of cytochrome c and Smac/Diablo confirmed that BTV apoptosis involves the sequential intrinsic pathway. In addition, we demonstrated that NF-kB was activated following BTV infection and cell treatment with an inhibitor peptide before BTV infection, prevented NF-kB activation and substantially reduced cellular apoptosis. Our accumulating data concerning the activation of Bax, cytochrome c, Smac/DIABLO and NF-kB clarify the mechanism of apoptosis during BTV infection, and confer a better understanding of the primary role of apoptosis in BTV pathogenesis.

Probing the specifics of substrate binding for cytochrome c oxidase: a computer assisted approach / Investigación de las especifidades de la unión de substrato para la citocromo-c-oxidasa: un enfoque asistido por computadora

A deficiency of cytochrome c oxidase (COX) is associated with a number of diseases but details of the enzyme's mechanism of action especially the interaction with its substrate, ferrocytochrome c, remain unclear. It is known that the transfer of electrons from ferrocytochrome c to COX is facilitated by the formation of enzyme-substrate (ES) complexes which are stabilized by intermolecular salt bridges, however the identity of residues participating in the salt bridges remains obscure. Using the published structures of the two proteins, computer simulations were employed to model their interactions and to attempt to identify residues that participate in intermolecular salt bridges. The simulation process was guided in the main by cross-linking studies which proposed that Lys-13 of cytochrome c is paired with Asp-158 of COX. The initial enzyme-substrate complex, created by computer assisted manipulation of the two structures exhibited three salt bridges; following the application of energy minimization procedures, the number of salt bridges increased to seven and there were twenty-four intermolecular hydrogen bonds. The salt bridges emanated from: Glu-119 and Asp-221 of subunit I; Glu-114, Asp-115 and Asp-158 of subunit II and Asp-73 and Glu-78 of subunit VIb. These were paired with Lys-87, 8, 25, 27, 13, 22 and 100 respectively of cytochrome c. These results suggest that subunits I, II and VIb play direct roles in substrate binding. The results also suggest that hydrogen bonds contribute significantly to the stability of the ES-complex.

La deficiencia de la citocromo-c-oxidasa (COX) se halla asociada con un número de enfermedades, pero los detalles del mecanismo de acción - especialmente la interacción con su substrato, el ferrocitocromo c - no está aún claro. Se sabe que la transferencia de electrones del ferrocitocromo c a la COX, es facilitada por la formación de los complejos enzima-substrato (ES), los cuales son estabilizados por puentes intermoleculares de sal. No obstante, la identidad de los residuos que participan en los puentes sigue sin estar clara. Recurriendo a las estructuras publicadas de dos proteínas, se emplearon simulaciones por computadora a fin de obtener un modelo de sus interacciones, en un intento por identificar los residuos que toman parte en los puentes de sal. El proceso de simulación fue guiado principalmente por estudios de reticulación, que proponen que el Lys-13 del citocromo c está pareado con el Asp-18 de la COX. El complejo enzima-sustrato inicial creado mediante la manipulación asistida por computadora de las dos estructuras, exhibía tres puentes de sal. Tras aplicar los procedimientos de minimización de la energía, el número de puentes de sal aumentó a siete y hubo veinticuatro enlaces intermoleculares de hidrógeno. Los puentes de sal emanaron de: Glu-119 y Asp-221 de la subunidad I; Glu-114, Asp-115 y Asp-158 de la subunidad II y Asp-73 y Glu-78 de la subunidad VIb. Estos fueron pareados con Lys-87, 8, 25, 27, 13, 22 y 100 respectivamente del citocromo c. Estos resultados sugieren que las subunidades I, II y VIb juegan un papel directo en la unión del substrato. Los resultados también sugieren que los enlaces de hidrógeno contribuyen significativamente a la estabilidad del complejo-ES.

Mitocondria y muerte celular: [revisión] / Mitochondria and cell death: [review]

Recientemente se ha definido el papel decisivo que tienen las mitocondrias en los mecanismos de muerte celular, los cuales se han explicado en diversos modelos, donde la permeabilización de la membrana externa mitocondrial y la liberación de proteínas importantes del espacio intermembranal de la mitocondria son características importantes que definen este proceso. Específicamente proteínas pro-apoptóticas tales como Citocromo c, Smac/diablo entre otras, son liberadas durante estadios tempranos del proceso apoptótico. Los mecanismos por los cuales estas proteínas son liberadas dependen presumiblemente del tipo celular y la naturaleza del estimulo. La activación de las caspasas (proteasas de cisteína) durante la apoptosis temprana parece estar regulada principalmente por la familia de las proteínas Bcl-2, cuya función principal es el control de la permeabilidad de la membrana mitocondrial a través de la formación o regulación de poros, en particular el poro de permeabilidad transicional mitocondrial. La presente revisión busca mostrar una visión global del papel de la mitocondria en los procesos de muerte celular, en la apoptosis, y en algunos de los mecanismos moleculares involucrados en su regulación.

A mechanistic view of mitochondrial death decision pores

Mitochondria increase their outer and inner membrane permeability to solutes, protons and metabolites in response to a variety of extrinsic and intrinsic signaling events. The maintenance of cellular and intraorganelle ionic homeostasis, particularly for Ca2+, can determine cell survival or death. Mitochondrial death decision is centered on two processes: inner membrane permeabilization, such as that promoted by the mitochondrial permeability transition pore, formed across inner membranes when Ca2+ reaches a critical threshold, and mitochondrial outer membrane permeabilization, in which the pro-apoptotic proteins BID, BAX, and BAK play active roles. Membrane permeabilization leads to the release of apoptogenic proteins: cytochrome c, apoptosis-inducing factor, Smac/Diablo, HtrA2/Omi, and endonuclease G. Cytochrome c initiates the proteolytic activation of caspases, which in turn cleave hundreds of proteins to produce the morphological and biochemical changes of apoptosis. Voltage-dependent anion channel, cyclophilin D, adenine nucleotide translocase, and the pro-apoptotic proteins BID, BAX, and BAK may be part of the molecular composition of membrane pores leading to mitochondrial permeabilization, but this remains a central question to be resolved. Other transporting pores and channels, including the ceramide channel, the mitochondrial apoptosis-induced channel, as well as a non-specific outer membrane rupture may also be potential release pathways for these apoptogenic factors. In this review, we discuss the mechanistic models by which reactive oxygen species and caspases, via structural and conformational changes of membrane lipids and proteins, promote conditions for inner/outer membrane permeabilization, which may be followed by either opening of pores or a rupture of the outer mitochondrial membrane.

Pitfalls in the use of electron microscopy to study the mitochondrial membrane permeability transition in apoptotic cells and pellets: where do we stand in relation to the incidence of mitochondrial swelling in apoptosis?

The importance of apoptosis as a form of programmed cell death was recognized in the 1980s, whereas the central role of mitochondria in controlling this process was identifi ed in the mid-1990s. An important event in apoptosis is the collapse of the mitochondrial transmembrane potential (ÃØm), with the ensuing loss of the selective permeability of the inner membrane resulting in swelling of the hyperosmolar mitochondrial matrix. This event is known as the mitochondrial permeability transition (MPT). After swelling of the intermembrane space, the outer membrane ruptures, exposing the permeable inner membrane. An increasingly swollen matrix covered by the inner membrane eventually herniates into the cytoplasm through the breach formed in the outer membrane (OM). The increase in surface area of the inner mitochondrial membrane (IMM) involves the unfolding of membrane stored in the cristae. This membrane movement is osmotically driven since the cytoplasm has a lower osmolality. The proteins partly embedded in the inner membrane are thus exposed to the cytoplasm. In nine out of ten electron microscopy studies of isolated mitochondria expressing the permeability transition, the existing ruptures of the OMM were overlooked. The MPT can also be recognized in individual mitochondria by using fl uorescent probes that are not retained in these organelles once the ÃØm is lost. In cases in which there is no rupture of the OMM, cytochrome c must be released from mitochondria with impermeable inner membranes. Examination of several hundred of the more than 61,000 published papers on programmed cell death revealed that the key signaling events of apoptosis, such as the onset of the MPT, mitochondrial swelling and cytochrome c release to the cytoplasm, are infl uenced by factors such as the cell type and presence of apoptogenic agents...

Inactivación de la dihidrolipoamida deshidrogenasa de Trypanosoma cruzi por fenotiazinas en presencia de citocromo c y peróxido de hidrógeno: efectos de los antioxidantes / Trypanosoma cruzi dihydrolipoamide dehydrogenase is inactivated by phenothiazines in the presence of cytochrome c and hydrogen peroxide: effects of antioxidants

El citocromo c catalizó la oxidación de las fenotiazinas (FTZ) en presencia de peróxido de hidrógeno. La formación del radical catiónico de promazina (PZ+.) se demostró por espectrofo-tometría y por su conversión a promazina sulfóxido La dihidrolipoamida deshidrogenasa (LADH) del Trypanosoma cruzi es inhibida irreversiblemente por el sistema citocromo c/H2O2 complementado con fenotiazinas. La inactivación de la LADH del parásito varía según la estructura de las FTZ, el tiempo de incubación del sistema pro-oxidante con la LADH, y la presencia de un antioxidante supresor de radicales FTZ+. Entre las 12 FTZ ensayadas, la promazina (PZ), tioridazina (TRDZ) y trimeprazina (TMPZ) fueron las más efectivas produciendo inactivaciones de 82 por ciento,76 por ciento y 72 por ciento, respectivamente, a los 90 min de incubación. El efecto de PZ (con grupo alquilamino en la posición N 10) disminuyó por modificación de su estructura en la posición 2 (efecto inactivante de PZ > cloropromazina (CPZ) > propionilpromazina (PPZ) > trifluopromazina (TFPZ) o en la posición 10 ( efecto inactivante de PZ > TMPZ > prometazina (PMTZ).El efecto de las FTZ con sustituyente piperidinil en N 10 dependió del grupo de la posición 2 ( SCH3, en TRDZ de mayor efecto; CN, en propericiazina (PCYZ), la de menor efecto entre las FTZ estudiadas). Parece que la presencia del sustituyente piperazinil en posición N 10 no tiene función importante en el efecto inactivante de las FTZ, el cual dependió de la estructura del grupo en la posición 2. El efecto de los compuestos con Cl en posición 2 (CPZ, procloroperazina (PCP), perfenazina (PFZ)) fue mayor que el obtenido con los compuestos CF3 (TFPZ, trifluoroperazina (TFP), flufenazina (FFZ), e independiente de la estructura del sustituyente N 10.El efecto de las FTZ sobre la LADH de T. cruzi depende, por lo menos en parte, de la estabilidad de los radicales FTZ+. generados por la actividad peroxidasa. La LADH T c, en comparación con la LADH de mamífero...

Cytochrome c catalyzed the oxidation of phenothiazines (PTZ) in the presence of hydrogen peroxide. The transient formation of the promazine radical cation (PZ+.) has been demonstrated by light absorption measurements as well as by its conversión to promazine sulfoxide. Trypanosoma cruzi dihydrolipoamide dehydrogenase (LADH T c) was irreversibly inhibited by treatment with cytochrome c (cyt c)/H2O2 system supplemented with PTZ. LADH T c inactivation depended on a) The PTZ structure b) Time of incubación with the complete oxidant system c) The presence of an antioxidant that intercept free radicals. PZ, thioridazine (TRDZ) and trimeprazine (TMPZ), were the most effective systems out of twelve PTZ studied, with inactivation values of 82, 76 and 72%, respectively, after 90 min of incubation. LADH T c inactivation by PZ (with alkylamine substituent at N 10 position) decreased by its structural modification at 2 position (inactivation PZ > chlorpromazine (CPZ) > propionylpromazine (PPZ)>trifluopromazine (TFPZ)) or at N 10 position (inactivation PZ > TMPZ > promethazine (PMTZ)) PTZ activity with piperidinyl substituent at N10 position depended on the group at 2 position (TRDZ, with thiomethyl group, has high inactivating effect on LADH T c; propericyazine (PCYZ), with cyano group, is much less active). Apparently, piperazinyl substituent at the N10 position on the phenothiazine have not an important function in the compound's inactivating effect on LADH T c. The effect of PTZ with Cl at 2 position (CPZ, prochlorperazine (PCP), perphenazine (PFZ)) was higher than the effect of compounds with CF3 in the same position (TFPZ,trifluoperazine (TFP),fluphenazine (FFZ) ) independent on the structure of substituents at N10 position. Production of PTZ+. radicals was essential for LADH T c inactivation and this effect depended on the stability of these free radicals. Comparision of inactivation values for LADH T c and mammalian LADH demonstrated...

Mitochondrial apoptotic pathways

Apoptosis or programmed cell death (PCD) is a physiological process characteristic of pluricellular organisms leading to self-destruction of the cell. It is therefore involved in development, homeostasis and host defense. However, a significant difference has been shown between mammalian cell apoptosis and non-mammalian cell apoptosis: mitochondria are implicated only in the former. Execution of PCD includes the release of several proapoptotic proteins from the intermembrane space of mitochondria. They could exert their actions through a caspase dependent as well as a caspase independent way. On the other hand, regulation of PCD is mainly given by the Bcl-2 family members, which are in turn essentially regulated by activation of death receptors and/or DNA damage. Nowadays, execution of apoptosis is better known than its regulation. Nevertheless, we are still far of a complete understanding of the apoptotic process

La apoptosis y su importancia biomédica / Apoptosis and its biomedical importance

En este trabajo, revisaremos las características morfológicas y las fases en que se ha dividido a la apoptosis, así como la importancia de su presencia en algunas enfermedades.La apoptosis y la necrosis son dos mecanismos mediante los cuales puede ocurrir muerte celular. La apoptosis constituye una medida fisiológica de remoción celular, bajo control genético, que se caracteriza por colapso celular, condensación de la cromatina y fragmentación del ácido desoxirribonucleico (ADN). Las células apoptóticas son rápidamente fagocitadas por células vecinas o macrófagos, previniendo así una reacción inflamatoria. La apoptosis se ha propuesto como un evento crítico para mantener la homeostasis tisular que asegura el estado de salud de los organismos.La necrosis implica la ruptura de la membrana e hipoxia, lo que conduce a la disminución en las concentraciones de adenosin trifosfato (ATP), colapso metabólico, edematización y disolución de la célula originando un proceso inflamatorio.