Pre-conditions for eliminating mitochondrial dysfunction and maintaining liver function after hepatic ischaemia reperfusion.

The liver, the largest organ with multiple synthesis and secretion functions in mammals, consists of hepatocytes and Kupffer, stem, endothelial, stellate and other parenchymal cells. Because of early and extensive contact with the external environment, hepatic ischaemia reperfusion (IR) may result in mitochondrial dysfunction, autophagy and apoptosis of cells and tissues under various pathological conditions. Because the liver requires a high oxygen supply to maintain normal detoxification and synthesis functions, it is extremely susceptible to ischaemia and subsequent reperfusion with blood. Consequently, hepatic IR leads to acute or chronic liver failure and significantly increases the total rate of morbidity and mortality through multiple regulatory mechanisms. An increasing number of studies indicate that mitochondrial structure and function are impaired after hepatic IR, but that the health of liver tissues or liver grafts can be effectively rescued by attenuation of mitochondrial dysfunction. In this review, we mainly focus on the subsequent therapeutic interventions related to the conservation of mitochondrial function involved in mitigating hepatic IR injury and the potential mechanisms of protection. Because mitochondria are abundant in liver tissue, clarification of the regulatory mechanisms between mitochondrial dysfunction and hepatic IR should shed light on clinical therapies for alleviating hepatic IR-induced injury.

Tetrazolium test to evaluate the viability of oil radish seeds / Teste de tetrazólio para avaliação da viabilidade de sementes de nabo forrageiro

The use of tetrazolium test is important in the evaluation of seeds lot quality and it has been adopted for vigor and viability identification for several species. The interest on the production of oil radish is increasing since the seeds were considered a good source of oil for biofuel production. The development of the tetrazolium test methodology for seeds of this species can improve the seed quality control process, and additionally will provide information for the characterization of remaining seeds (dead or dormant) in the germination tests. To verify the ideal conditions to tetrazolium test was conducted two experiments. At the first, oil radish seeds cultivar CATI AL-1000, lots from 2001 and 2006 were submitted to imbibition between paper in water for 6 hours. After the longitudinal cut in the longest direction, the seeds were immersed in the tetrazolium solution at the concentrations of 0,075%, 0,5% and 1% at 25°C for 3h, 12h and 18h. In the second experiment, oil radish seeds cultivar CATI AL-1000 lot from 2005 and IPR 116 cultivar, lots from 2004 and 2005 were immersed in the concentrations of ,1%; 0,2%; 0,3% and 0,4% of tetrazolium solution for 12 hours at a 25°C, 30°C, 35°C and 40°C. At the first experiment was observed the necessity of test intermediate concentrations between 0,075% and 0,5%, since with 0,075% the seeds stained weakly and with 0,5% the test results, were overestimated. In the second experiment was observed that the 0,3% concentration at 30°C can be recommended for the utilization of tetrazolium test to evaluation of oil radish seeds viability.

A utilização do teste de tetrazólio é importante na avaliação da qualidade de lotes de sementes e vem sendo adotado para várias espécies na identificação do vigor e viabilidade. A adequação da metodologia do teste de tetrazólio para sementes de nabo forrageiro, espécie que vem se destacando como fonte de óleo para produção de biocombustíveis, poderia melhorar o processo de controle de qualidade. Além disso, a utilização do teste poderá fornecer subsídios para identificação de sementes remanescentes (mortas e dormentes) nos testes de germinação. Para verificar as condições ideais para a realização do teste de tetrazólio, em um 1º experimento as sementes de nabo forrageiro da cultivar CATI AL-1000, lotes de 2001 e 2006, foram submetidas à embebição entre papel em água por 6 horas. Após corte longitudinal no maior sentido as sementes foram imersas nas concentrações de 0,075%; 0,5% e 1,0% de solução de tetrazólio a 25ºC por 3 h, 12 h e 18 horas. Em um 2º experimento sementes de nabo forrageiro da cultivar CATI AL-1000, lote de 2005 e cultivar IPR 116 lotes de 2004 e 2005, foram imersas nas concentrações de 0,1%; 0,2%; 0,3% e 0,4% de solução de tetrazólio a 25°C, 30°C, 35°C e 40°C por 12 horas. Pelo 1º experimento foram observados a necessidade de testar concentrações intermediárias entre 0,075% e 0,5%, visto que, com 0,075% as sementes coloriram fracamente e com 0,5% os resultados do teste foram superestimados. No 2º experimento observou-se que a concentração 0,3% a 30°C pode ser recomendada para utilização no teste de tetrazólio para avaliação da viabilidade de sementes de nabo forrageiro.

Impact of hyperthermia before and during ischemia-reperfusion on neuronal damage and gliosis in the gerbil hippocampus induced by transient cerebral ischemia.

Hyperthermia can exacerbate the brain damage produced by ischemia. In the present study, we investigated the effects of hyperthermia before and during ischemia-reperfusion on neuronal damage and glial changes in the gerbil hippocampus following transient cerebral ischemia using cresyl violet staining, NeuN immunohistochemistry and Fluoro-Jade B histofluorescence staining. The animals were randomly assigned to 4 groups: (1) sham-operated animals with normothermia (normothermia + sham group); (2) ischemia-operated animals with normothermia (normothermia + ischemia group); (3) sham-operated animals with hyperthermia (hyperthermia + sham group); and (4) ischemia-operated animals with hyperthermia (hyperthermia + ischemia group). Hyperthermia (39.5 ± 0.2°C) was induced by exposing the gerbils to a heating pad connected to a rectal thermistor for 30 min before and during ischemia-reperfusion. In the normothermia+ischemia groups, a significant delayed neuronal death was observed in the stratum pyramidale (SP) of the hippocampal CA1 region (CA1) 5 days after ischemia-reperfusion. In the hyperthermia+ischemia groups, neuronal death in the SP of the CA1 occurred at 1 day post-ischemia, and neuronal death was observed in the SP of the CA2/3 region at 2 days post-ischemia. In addition, we examined activations of astrocytes and microglia using immunohistochemistry for anti-glial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1). GFAP-positive astrocytes and Iba-1-positive microglia in the ischemic hippocampus were activated much earlier and much more accelerated in the hyperthermia+ischemia groups than those in the normothermia+ischemia groups. Based on our findings, we suggest that an experimentally hyperthermic pre-condition before cerebral ischemic insult produces more extensive neuronal damage and glial activation in the ischemic hippocampus.

Untangling the Roles of Anti-Apoptosis in Regulating Programmed Cell Death using Humanized Yeast Cells.

Genetically programmed cell death (PCD) mechanisms, including apoptosis, are important for the survival of metazoans since it allows, among things, the removal of damaged cells that interfere with normal function. Cell death due to PCD is observed in normal processes such as aging and in a number of pathophysiologies including hypoxia (common causes of heart attacks and strokes) and subsequent tissue reperfusion. Conversely, the loss of normal apoptotic responses is associated with the development of tumors. So far, limited success in preventing unwanted PCD has been reported with current therapeutic approaches despite the fact that inhibitors of key apoptotic inducers such as caspases have been developed. Alternative approaches have focused on mimicking anti-apoptotic processes observed in cells displaying increased resistance to apoptotic stimuli. Hormesis and pre-conditioning are commonly observed cellular strategies where sub-lethal levels of pro-apoptotic stimuli lead to increased resistance to higher or lethal levels of stress. Increased expression of anti-apoptotic sequences is a common mechanism mediating these protective effects. The relevance of the latter observation is exemplified by the observation that transgenic mice overexpressing anti-apoptotic genes show significant reductions in tissue damage following ischemia. Thus strategies aimed at increasing the levels of anti-apoptotic proteins, using gene therapy or cell penetrating recombinant proteins are being evaluated as novel therapeutics to decrease cell death following acute periods of cell death inducing stress. In spite of its functional and therapeutic importance, more is known regarding the processes involved in apoptosis than anti-apoptosis. The genetically tractable yeast Saccharomyces cerevisiae has emerged as an exceptional model to study multiple aspects of PCD including the mitochondrial mediated apoptosis observed in metazoans. To increase our knowledge of the process of anti-apoptosis, we screened a human heart cDNA expression library in yeast cells undergoing PCD due to the conditional expression of a mammalian pro-apoptotic Bax cDNA. Analysis of the multiple Bax suppressors identified revealed several previously known as well as a large number of clones representing potential novel anti-apoptotic sequences. The focus of this review is to report on recent achievements in the use of humanized yeast in genetic screens to identify novel stress-induced PCD suppressors, supporting the use of yeast as a unicellular model organism to elucidate anti-apoptotic and cell survival mechanisms.