Ravnoteža između posla i života te važnost ustanova za djecu

Agnes Simonyi razmatra pitanja koja proizlaze iz rasprave o ‘ravnoteži između posla i života’

An assessment on resveratrol and its cellular protective properties

Abstract only availableResveratrol is a polyphenolic compound found abundantly in plants such as knotweed or grapes. Under normal circumstances, it is in plants as a phytoalexin - an antibiotic produced by plants for defense under insults. Based on previous studies, it it also has properties that protect cells from damage caused by free radicals or reactive oxygen species In order to test the effectiveness of resveratrol as a protective agent, an experiment was designed using an immortalized astrocyte cell line, DiTNC. These cells were divided into control, resveratrol and non-resveratrol groups. They were cultured in a standard six well plate, with two wells per group. Menadione (25 and 50 micromolar), a compound that generates reactive oxygen species in cells, was added to four wells, two of which contained pre-incubated resveratrol (50 micromolar). The cells were left to incubate in an incubating oven at 37 Celsius. Photos are taken at intervals of 30 minutes, 60 minutes, and 120 minutes. Menadione caused cells to project processes and then causes them to become shrunken and rounded within 30 min. When the pictures from the resveratrol group was compared to the non-resveratrol group, a significantly less amount of cells from the resveratrol group were found to be either free floating or shrunken, suggesting that these cells survived for a longer period. An MTT test will also be performed in order to quantify the data. The results show that resveratrol has a fair effect on protecting cells from reactive oxygen species and that this chemical warrants further investigations.Alzheimer's disease program project grant 2P01AGO18357 to G. Su

Antioxidants to combat Alzheimer's disease

Abstract only availableAlzheimer's disease (AD) took the life of my grandfather. It is believed that oxidative stress contributes to the progression of this disease. Studies in Dr. Sun's laboratory recognized NADPH oxidase as possible source for production of reactive oxygen species (ROS) which can damage cells in the brain. If this were true then something could be found to stop the activation of NADPH oxidase. This would stop the deterioration of cells, decreasing the amount of Alzheimer's cases. To do my part of this research, we culture immortalized astrocytes (DITNC) and look at how cells react to menadione, a compound that produces ROS, possible through activating NADPH oxidase. We will then see how apocynin stands up against menadione. To test this we had six wells containing the astrocytes. In the first two wells we didn't add anything to the cells, these were our control cells. In the other four wells we added menadione, and then in two of those wells we added apocynin. We then took pictures right after we added the inhibitor, 30 minutes after they were in the incubator, 60 minutes, and then 120 minutes later. Doing this allowed us to see the difference in adding apocynin and not adding it. Looking at our results, we found the wells with only Menadione had more cells that were dead and shriveled up, and there were fewer dying cells in the wells containing Apocynin. This means it is possible for Apocynin to be used to stop the activation of NADPH oxidase; this would keep cells from dying.Missouri Academy at Northwest Missouri State University, Alzheimer disease program project grant 2PO1 AGO18357 to G. Su

MU Neurobehavioral Core Facility: Progressing from Molecules to Behavior

Neuroscience - Vision & Functional Brain Imaging Poster SessionAn important component of modern neuroscience research is the ability to measure systematically and objectively different aspects of behavior. Behavioral analysis is crucial to a strong neuroscience research program because it evaluates the impact of molecular or neurochemical changes on the functioning of the entire organism. Behavioral research can be used to validate the role of a neuroscientist's specific molecular target (e.g., receptor, gene, or enzyme) in a particular behavior (e.g., emotions, learning and memory, or locomotor activity) and subsequently create whole systems that a neuroscientist can use to study a particular pathological state (e.g., depression, drug addiction or obesity). A unique strength of the MU Translational Neuroscience Center is the presence of some “bench” scientists working at the molecular level in pathology, biochemistry and genetics in collaboration with neurobehavioral experts. The Center's modern facilities and trained personnel are available to the MU neuroscience community to help design, conduct and evaluate behavioral research. This will help translate research from the molecular laboratory to the human clinic. This poster will show a summary of the different aspects and tasks we plan to perform at the MU Neurobehavioral Core Facility

ROS from menadione induces astrocytic damage: protective effects of apocynin

Abstract only availableOxidative stress is a core cause of neurodegenerative diseases such as Alzheimer's disease. When cells are under oxidative stress, they will produce a high amount of reactive oxygen species (ROS). ROS are small and highly reactive and include compounds such as oxygen ions, free radicals, and peroxides. Understanding what triggers oxidative stress and how to ameliorate its damaging effects is a crucial step in discovering a cure for Alzheimer's disease. Menadione, a vitamin precursor of K2, is an oxidative compound that is capable of delivering ROS to the cells. Apocynin, a natural organic compound that has been isolated from Picrorhiza curroa grown in the Himalayan Mountains, is an inhibitor of NADPH oxidase, an enzyme for ROS production in cells. In this experiment, we studied whether apocynin may neutralize the effects of menadione using an immortalized astrocyte cell line DITNC. Astrocytes are glial cells that play a crucial role in the brain by providing necessary nutrient to surrounding neurons. We had three sample groups and treated each group with different drugs. The first group was the control, the second group was treated with menadione, and the third group was treated with both menadione and apocynin. After treating the cells, we recorded morphological changes of the cells by taking pictures of each sample group at three different time intervals (30 min, 1 and 2 hours). In addition to the morphological evidence, we also did a MTT assay to assess cell viability and later a data analysis based on the result from the MTT test. MTT assay measures mitochondrial activity and thus indirectly measures cell viability. Both morphological data and MTT analysis showed menadione caused DITNC cell damage with decreased mitochondrial activity. When cells are treated with menadione, they formed processes, shrink, and then round up. We also found apocynin protects against the oxidative damage caused by menadione to a certain extent. Since apocynin is an inhibitor of NADPH oxidase, this also indicates oxidative stress is generated by NADPH oxidase, suggesting apocynin may be a potential means to treat Alzheimer's disease.Alzheimer disease program project grant 2P01 AG018357 to G. Su

Cytosolic phospholipase A 2 plays a crucial role in ROS/NO signaling during microglial activation through the lipoxygenase pathway

BACKGROUND: Oxidative stress and inflammation are important factors contributing to the pathophysiology of numerous neurological disorders, including Alzheimer’s disease, Parkinson’s disease, acute stroke, and infections of the brain. There is well-established evidence that proinflammatory cytokines and glutamate, as well as reactive oxygen species (ROS) and nitric oxide (NO), are produced upon microglia activation, and these are important factors contributing to inflammatory responses and cytotoxic damage to surrounding neurons and neighboring cells. Microglial cells express relatively high levels of cytosolic phospholipase A(2) (cPLA(2)), an enzyme known to regulate membrane phospholipid homeostasis and release of arachidonic acid (AA) for synthesis of eicosanoids. The goal for this study is to elucidate the role of cPLA(2)IV in mediating the oxidative and inflammatory responses in microglial cells. METHODS: Experiments involved primary microglia cells isolated from transgenic mice deficient in cPLA(2)α or iPLA(2)β, as well as murine immortalized BV-2 microglial cells. Inhibitors of cPLA(2)/iPLA(2)/cyclooxygenase (COX)/lipoxygenase (LOX) were used in BV-2 microglial cell line. siRNA transfection was employed to knockdown cPLA(2) expression in BV-2 cells. Griess reaction protocol was used to determine NO concentration, and CM-H2DCF-DA was used to detect ROS production in primary microglia and BV-2 cells. WST-1 assay was used to assess cell viability. Western blotting was used to assess protein expression levels. Immunocytochemical staining for phalloidin against F-actin was used to demonstrate cell morphology. RESULTS: In both primary and BV-2 microglial cells, stimulation with lipopolysaccharide (LPS) or interferon gamma (IFNγ) resulted in a time-dependent increase in phosphorylation of cPLA(2) together with ERK1/2. In BV-2 cells, LPS- and IFNγ-induced ROS and NO production was inhibited by arachidonyl trifluoromethyl ketone (AACOCF3) and pyrrophenone as well as RNA interference, but not BEL, suggesting a link between cPLA(2), and not iPLA(2), on LPS/IFNγ-induced nitrosative and oxidative stress in microglial cells. Primary microglial cells isolated from cPLA(2)α-deficient mice generated significantly less NO and ROS as compared with the wild-type mice. Microglia isolated from iPLA(2)β-deficient mice did not show a decrease in LPS-induced NO and ROS production. LPS/IFNγ induced morphological changes in primary microglia, and these changes were mitigated by AACOCF3. Interestingly, despite that LPS and IFNγ induced an increase in phospho-cPLA(2) and prostaglandin E2 (PGE2) release, LPS- and IFNγ-induced NO and ROS production were not altered by the COX-1/2 inhibitor but were suppressed by the LOX-12 and LOX-15 inhibitors instead. CONCLUSIONS: In summary, the results in this study demonstrated the role of cPLA(2) in microglial activation with metabolic links to oxidative and inflammatory responses, and this was in part regulated by the AA metabolic pathways, namely the LOXs. Further studies with targeted inhibition of cPLA(2)/LOX in microglia during neuroinflammatory conditions can be valuable to investigate the therapeutic potential in ameliorating neurological disease pathology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12974-015-0419-0) contains supplementary material, which is available to authorized users

A[beta] toxicity to SHSY-5Y human neuroblastoma cells

Abstract only availableAlzheimer's disease (AD) is a progressive neurodegenerative disorder that results in the loss of memory, language deterioration, confusion, restlessness, and eventually destroyed cognition. It is characterized by an accumulation of beta amyloid (Aβ) plaques and tangels in the brain. Increased oxidative stress has been regarded as an early event underlying the progression of AD and there is evidence that Aβ can cause oxidative stress. In our lab, we are using SHSY-5Y human neuroblastoma cells (SH cells) as a model to look at NADPH-oxidase, an enzyme that may be involved Aβ induced toxicity to the cells. NADPH-oxidase is a multi-subunit enzyme that catalyzes molecular oxygen to form reactive oxygen species (ROS). In order for this enzyme to be activated, its cytosolic components must translocate to the membrane. One of its cytosolic components is p47-phox. We stimulate cells with oxidative agents such as menadione and H2O2 to monitor the translocation of p47-phox to the membrane. Because the production of ROS may be one of the factors leading to neuronal death, we chose to look at the activation of NADPH-oxidase. SH cells were treated with menadione (stimulator), H2O2 (stimulator) and DPI (inhibitor) for different time intervals. After treatments, the cells were harvested and treated with a lysis buffer to lyse the cells. After the cells were lysed, we put them through ultra-centrifugation and collected the membrane and cytosolic fractions. Western Blots were performed on both the membrane and the cytosolic fractions to look for the presence of p47-phox. From the Western Blot we saw that there was an increase in the p47-phox levels in the membrane fraction after exposing the cells to menadione and H2O2. Our results show that treating the cells with oxidative agents caused an increase in the p47-phox level in the membrane fraction thus leading to the activation of NADPH-oxidase.NSF-REU Program in Biological Sciences & Biochemistr

Prolonged exposure of cortical neurons to oligomeric amyloid-β impairs NMDA receptor function via NADPH oxidase-mediated ROS production: protective effect of green tea (–)-epigallocatechin-3-gallate

Excessive production of Aβ (amyloid β-peptide) has been shown to play an important role in the pathogenesis of AD (Alzheimer's disease). Although not yet well understood, aggregation of Aβ is known to cause toxicity to neurons. Our recent study demonstrated the ability for oligomeric Aβ to stimulate the production of ROS (reactive oxygen species) in neurons through an NMDA (N-methyl-d-aspartate)-dependent pathway. However, whether prolonged exposure of neurons to aggregated Aβ is associated with impairment of NMDA receptor function has not been extensively investigated. In the present study, we show that prolonged exposure of primary cortical neurons to Aβ oligomers caused mitochondrial dysfunction, an attenuation of NMDA receptor-mediated Ca2+ influx and inhibition of NMDA-induced AA (arachidonic acid) release. Mitochondrial dysfunction and the decrease in NMDA receptor activity due to oligomeric Aβ are associated with an increase in ROS production. Gp91ds-tat, a specific peptide inhibitor of NADPH oxidase, and Mn(III)-tetrakis(4-benzoic acid)-porphyrin chloride, an ROS scavenger, effectively abrogated Aβ-induced ROS production. Furthermore, Aβ-induced mitochondrial dysfunction, impairment of NMDA Ca2+ influx and ROS production were prevented by pre-treatment of neurons with EGCG [(−)-epigallocatechin-3-gallate], a major polyphenolic component of green tea. Taken together, these results support a role for NADPH oxidase-mediated ROS production in the cytotoxic effects of Aβ, and demonstrate the therapeutic potential of EGCG and other dietary polyphenols in delaying onset or retarding the progression of AD