Author: neuroscience research

Superoxide dismutase is an enzyme responsible for dismutating superoxide radicals, which are generated in the mitochondria by ETC complex I and complex III. Over-expression of SOD has been shown in lung tumors as compared to normal tissues suggesting its role in lung carcinogenesis. Moreover, SOD was recently identified as a target for the selective killing of cancer cells. Our results clearly show that capsaicin treatment significantly decreased SOD activity in BxPC-3 cells and AsPC-1 tumor xenografts. Glutathione peroxidase is an important enzyme that utilizes GSH as a substrate to detoxify intracellular 4-(Benzyloxy)phenol peroxides Atropine sulfate including hydrogen peroxide. Capsaicin treatment resulted in the significant inhibition of GPx activity and expression in BxPC-3 cells. These results indicate that capsaicin deplete GSH level and inhibit GSH dependent anti-oxidant enzymes resulting in the accumulation of ROS in pancreatic cancer cells leading to mitochondrial damage. In addition catalase is another important enzyme which is responsible for detoxifying hydrogen peroxide to water. Consistently, we observed that PEG-SOD, PEG-catalase, catalase or EUK-134 prevented capsaicin mediated ROS generation by complex-I and complex-III, ATP depletion, mitochondrial damage and apoptosis, indicating the involvement of catalase. As a proof-of-concept, over-expression of catalase by transient transfection completely blocked capsaicin mediated ROS generation and apoptosis in BxPC-3 cells demonstrating its critical role in the survival of pancreatic cancer cells. Most of the cancer cells have higher levels of ROS that helps in proliferation and cell growth. Due to elevated ROS, cancer cells are highly dependent on their antioxidant system to maintain redox balance and hence are more susceptible to further oxidative stress. In contrast, normal cells are more resistant to oxidative stress due to the fact that these cells have lower levels of ROS and increased levels of antioxidants. Hence any agent that increases intracellular ROS in cancer cells may increase ROS to a toxic level resulting in mitochondrial damage and cell death as shown in our model. It is noteworthy that several agents such as Elesclomol or Trisenx are currently being used for the treatment of metastatic melanoma and acute promyelocytic leukemia respectively. Both of these agents selectively kill cancer cells by increasing ROS generation. We and others have shown previously that administration of 2.5 or 5 mg/kg capsaicin orally or subcutaneously suppress pancreatic and prostate tumor xenografts in vivo respectively. In the present study, 2.5 mg/kg capsaicin was given to mice by oral gavage, which is 0.202 mg/kg when converted to human equivalent dose and equates to 13.2 mg dose of capsaicin for a 60 kg person. However, further pharmacokinetic, bioavailability and clinical studies are needed to validate these correlations. Taken together our studies provide detailed mechanism how capsaicin treatment causes ROS generation through mitochondria and depleted intracellular antioxidants resulting in mitochondrial damage and apoptosis in pancreatic cancer cells. On the other hand, normal pancreatic epithelial cells were resistant to the effects of capsaicin. CNTF promotes the survival of a variety of neurons and oligodendrocytes, and induces neurite outgrowth and axon regeneration in both developing and mature nervous system. In addition, it appears to be an effective neuroprotective agent in animal models of CNS neurodegenerative diseases. CNTF has also been reported to activate leptin-like pathways in the brain and reduce body fat and stress in a leptin-independent manner. In the vertebrate retina, CNTF exhibits numerous effects on the development, differentiation and survival of retinal neurons. CNTF appears to play a critical role in progenitor commitment to the rod photoreceptor cell fate and in photoreceptor differentiation. It is reported to increase the long-term survival of retinal ganglion cells after axotomy. Furthermore, CNTF is capable of retarding retinal degeneration in several animal models of retinitis pigmentosa.

NC markers Sox10, p75 and HNK-1 was nearly ablated compared to controls. Treatment with the Wnt/b-catenin antagonist Dkk1 resulted in the ablation of Sox10 and HNK1, but not p75. These results are consistent with the distinct mechanisms of action of Noggin and Dkk1. While Noggin re-specifies the dorsal neuroepithelium into ventral fates, Dkk1 Pimozide inhibits the early steps of NC induction, without altering the axial cell fates or general patterning. In our hands, the dorsal Butenafine hydrochloride neuroepithelium-like clusters are positive for p75. The loss of Sox10 and p75 after the addition of Noggin is consistent with re-specification of dorsal neuroepithelium into ventral neuroepithelium, which typically does not express NC markers. Following the same logic, the addition of Dkk1 may inhibit the NC specification, i.e. appearance of Sox10- and HNK-1-positive emNCSCs, but does not re-specify the dorsal neuroepithelium-like cells, which remain positive for p75. Indeed, the human neural tube cells were found positive for the p75 antigen at the time when the NC is generated. It remains to be determined if this expression is localized to the premigratory neural crest in the dorsal part of human neural tube. In addition to the patterning growth factors discussed above, matrix elasticity may affect hESC differentiation towards NC lineages, similar to that seen for muscle differentiation. It will be important to investigate hESC differentiation into emNCSC using various elasticity matrixes. Finally, we assessed the ability of emNCSCs to migrate in vivo, incorporate into NC derivatives and differentiate appropriately. Grafted emNCSCs efficiently contributed to a variety of NCderived tissues and differentiated appropriately. This finding demonstrates that emNCSCs are competent to contributing to NC derivatives such as the trigeminal ganglia, as cells that do not normally incorporate into the trigeminal ganglia are excluded from the developing ganglia even when they are immediately adjacent to it. Furthermore, emNCSCs contributed to connective tissues including smooth muscle. Therefore, emNCSCs are capable of extensive migration to appropriate NC cell destinations and appear to have the ability to interact with adjacent host NC cells and differentiate efficiently compared to late emNCSCs, in vivo. Critically, transplanted human emNCSCs do not contribute to the non-NC cell types, such as CNS tissue. Given the fact that these cells are derived in an antigen free environment the protocol for the derivation of emNCSCs is ideal for generating emNCSC-derived tissues in the culture dish that can subsequently be used for patient treatment. To determine the therapeutic potential of these cells for treating a disease model, we investigated the ability of emNCSCs to colonize aganglionic embryonic guts in organotypic cultures. EmNCSCs were found to be capable of colonizing aganglionic guts and differentiating into neurofilamentpositive cells, presumably enteric neurons, in ex vivo gut cultures. This suggests that emNCSCs might be useful in cell replacement therapies to treat neurocristopathies. Genome-wide significant SNPs in complex traits generally explain only a proportion of the heritability of that disorder. Much of the residual heritability underlying common traits appears to lie in SNPs that do not achieve genome-wide significance, meaning that a substantial proportion of the associated genetic signal in current GWAS is hidden below the genome-wide significance threshold. We know that SNPs that are robustly associated with particular common disorders are not randomly distributed across all genes. Instead, the implicated genes show biologically relevant relationships between each other. This is also true for SNPs in genes for which there is weaker individual evidence for association that falls short of stringent levels of genome-wide significance and statistical approaches have recently been developed to identify sets of functionally related genes containing genetic variants that collectively show evidence for association.

Cardiolipin is exclusively present in mitochondria and after being labeled with NAO and exhibits yellow fluorescence. When we analyzed our cells under the fluorescent Lomitapide Mesylate microscope, we observed that almost all the cells from control group were exhibiting yellow color. However, the yellow staining decreased and turned into green in capsaicin treated cells indicating drastic oxidation of cardiolipin. Nonetheless, catalase and EUK-134 completely prevented the oxidation of cardiolipin. These results were confirmed by flow cytometry where we observed that capsaicin causes cardiolipin oxidation in BxPC-3 cells as shown by a shift of NAO fluorescence towards left. We further used catalase and EUK-134 to see whether the oxidation of cardiolipin can be prevented. We found that addition of catalase or EUK-134 almost completely blocked the shift of NAO staining suggesting that the decrease of NAO fluorescence was due to oxidation of mitochondrial lipid cardiolipin by mitochondrial ROS. Mitochondria are a major physiological source of ROS, which are generated due to incomplete reduction of oxygen during normal mitochondrial respiration. Excessive ROS that are generated under certain pathological conditions acts as mediator of apoptotic signaling pathway. Under normal physiological conditions, mitochondria contain sufficient levels of antioxidants that prevent ROS generation and Gomisin-D oxidative damage. However, under circumstances in which excessive mitochondrial ROS are produced or when antioxidant levels are depleted, oxidative damage to mitochondria occurs. Our current results shows that capsaicin induced apoptosis in BxPC-3 and AsPC-1 cells but not in HPDE-6 cells was associated with ROS generation. The ROS generation by capsaicin was due to marked inhibition of mitochondrial electron transport chain complexes-I and III and downregulation of antioxidants such as GSH, catalase, SOD and GPx indicating the involvement of mitochondria. On the other hand, r0 cells derived from BxPC-3 cells, which lack normal oxidative phosphorylation were unable to cause ROS generation and were totally resistant to the apoptosis inducing effects of capsaicin. ROS once generated cause oxidation of critical redox sensitive proteins and lipids leading to mitochondrial damage. Our results clearly show that capsaicin treatment, cause massive oxidation of cardiolipin, which is specifically present in the mitochondria. Mitochondrial damage due to oxidation of cardiolipin has been documented in a recent study. Cytochrome c preferentially binds to cardiolipin and is liberated upon oxidation of cardiolipin. In agreement, our results show the release of cytochrome c into cytosol by capsaicin treatment, which could be due to cardiolipin oxidation. Our results also demonstrate massive depletion of ATP as evaluated by complex-V ATP synthase activity. ETC complex forms a transmembrane potential. ATP synthase uses potential energy stored in Dy to phosphorylate ADP. However, under certain pathological conditions, the Dy can collapse resulting in the release of molecules from the mitochondria into the cytosol. Our result do show decrease in Dy and release of cytochrome c into the cytosol in response to capsaicin treatment. Further, ATP production was shown to be highly sensitive to complex-III inhibition in a previous report. In agreement, our results also show a relationship between complex III inhibition and ATP depletion. Cellular redox homeostasis is maintained by a fine balance between antioxidants and pro-oxidants. Glutathione is a critical intracellular antioxidant responsible for maintaining redox balance. GSH can be oxidized to formGSSG and the ratio of GSH/GSSG is an indicator of oxidative stress in the cells. High concentrations of GSSG can oxidatively damage many critical enzymes. Our results reveal that capsaicin treatment caused time dependent increase in the levels of GSSG and decrease in GSH levels in BxPC3 cells. Similar observations were made in the tumors of capsaicin treated mice as compared to the tumors from control mice.

To these surfactants than polarized epithelial cells. We interpret this result to mean that TX-100, DDPS and SDS act mainly at the level of the plasma membrane of the cells probably by causing structural changes at the level of the membrane or even its dissolution, as expected at concentrations close to the surfactant CMC. On the other hand, the cationic surfactants are probably toxic at a more subtle level, toxicity being at concentrations that are not sufficient to cause significant damage to the physical integrity of the membranes. These effects could even be at the intracellular level, conditioned by membrane partitioning and/or translocation across the membranes. The highly ordered apical membranes of fully polarized cells are the only membrane exposed to the surfactants in confluent polarized cell cultures and intact epithelia whereas non-polarized cells have significant amounts of less-ordered membrane domains exposed. For the homologous series of cationic surfactants examined, the results show that the toxicity to mammalian cells was not linearly dependent upon the surfactant hydrophobic chain length. This observation may have complex reasons related to different affinities of the surfactant for the different, possibly multiple, sites of their action. Without precise information concerning these affinities, something that we are working to obtain, any further discussion of this aspect would be speculative. The effect of the polar head group of the cationic surfactants was also evaluated; C12BZK and C12PB, which have the larger polar head groups and more delocalized charge, were between 2 to 5 times more toxic than C12TAB in all cell lines tested. Delocalized charge on the surfactant head group makes its ionic radius considerably larger and reduces the work required for translocation of the polar group from one side of the membrane to the other. Though there have been a very large number of reports concerning the disinfectant properties of surfactants, their mechanism of action is still not fully understood. Attempts to use surfactants in STI prophylaxis Catharanthine sulfate relied upon their capacity to destroy viral and bacterial membranes but did not seem to take into Benzethonium Chloride account, what in hindsight appears all too obvious, that if they destroyed those membranes they would also destroy the membranes of cells of the vaginal epithelium. However, destruction of cell membranes is not the only mechanism of surfactant toxicity as is evidenced in the case of cationic surfactants. As argued above, their toxic effects probably do not involve gross disassembly of the cell membrane but rather some more subtle effects. Candidate mechanisms that have been proposed in the literature include modulation of membrane curvature elastic stress and consequent reduction of membranebound protein activity, alteration of the electrostatic surface potential of membranes, or interaction with anionic polymers in the cytoplasm or cell nucleus following translocation across the cell plasma membrane. Cationic surfactants are known to bind strongly to DNA and RNA and induce drastic conformational changes in the structure of these polymers. The cell viability results obtained with the MTT assay correlated well with the observed release of LDH from the cells. However, in the case of C10TAB and C12TAB, the LD50 values obtained with the LDH leakage assay were slightly but significantly higher than with the MTT assay. This can be explained by the nature of each assay: the LDH leakage assay, which evaluates the loss of intracellular LDH and its release into the culture medium, is an indicator of irreversible cell death either due to cell membrane damage directly caused by the surfactants or due to loss of plasma membrane integrity posterior to cell death due to reasons that have nothing to do with direct membrane damage by the surfactants. On the other hand, the MTT assay evaluates the metabolic capacity of the cell in reducing MTT to formazan. Our results suggest that the molecular targets of the CnTAB surfactants may be different depending on the length of their hydrophobic chain.

To test for coordinate regulation of gene homologs or modules across different stem cell types, we developed a pattern recognition algorithm capable of combining the results of any number of experiments to identify significantly and recurrently up- or down-regulated genes and gene modules in stem cells. To obtain a global overview of stem cell expression patterns, we assembled a compendium of data from 30 different studies assaying gene expression of 49 stem cell populations representing twelve different types of stem cells including hematopoietic, retinal, neural, embryonic, and intestinal. From each of the 49 datasets we collected genes up-regulated in stem cells into a stem cell gene list and, where available, a corresponding set of genes up-regulated in differentiated cells into a differentiated gene list. The use of gene lists facilitated the straightforward integration of results from the variety of experimental test platforms, which has proven effective for meta-analysis compared to alternative approaches. A permissive, or poised, chromatin structure may underlie stem cell multipotency. S-MAP detected several chromatin-related modules Tulathromycin B associated with stemness including those involved in imprinting, chromatin-dependent silencing, heterochromatin and the nuclear lamina, which may indicate widespread suppression of lineage-associated genes. Stemness contributors include the Chd/Smarca family, nucleosome assembly protein like proteins; and histone variants H2afz and H2afv. Indeed, Chd1 was recently shown to be important in ESC multipotency by regulating chromatin structure; other Amikacin hydrate family members may fill this function in adult stem cells. S-MAP also revealed a number of Wnt signaling modules with alternating patterns of specificity in different stem cell types. Wnt-related stemness modules include the secreted Frizzled-like proteins of the Sfrp family; the Frizzled receptors; a subfamily of the TCF/LEF transcriptional regulators; the Enhancer of Split/Groucho-related Tle factors; and both alpha- and delta-catenins. Whether Wnt signaling regulates stem cell maintenance or differentiation has been extensively debated, and some genes have both inhibitory and activating abilities dependent on the state of Wnt signals. While functional consequences cannot be resolved by expression data alone, our analysis demonstrates that all stem cells tightly regulate the Wnt pathway at the transcriptional level. The identified S-MAP patterns provide specific candidates for functional interrogation in different stem cell types. DNA repair is uniquely critical in stem cells as mutations accumulated in stem cells amplify in differentiated daughters. SMAP identified many stemness modules associated with DNA repair, including the Terf, p53, and Rad families. Terf1 and 2 are highly stem cell-specific and are expressed in alternating patterns in different stem cells. S-MAP classified the p53 family as a stemness-on module. While p53 was found to be expressed in several tissues, p63 was upregulated in gastric and intestinal cells consistent with its known role in the development and maintenance of epithelial stem cells. Likewise, Brca1 and its homolog Mcph1, the Msh family of proteins, and a Rad/Dmc module were detected as stemness modules by S-MAP. Several transcriptional regulators, including the Myb family of oncogenes, were among the highest scoring stemness-on modules. c-myb was enriched in hematopoietic stem cells, consistent with its known role in differentiation control, as well as neural, embryonic, intestinal and retinal stem cells. a-myb complements the expression of its partner genes by significant upregulation in gastric stem cells, while b-myb was up-regulated in liver and trophoblast stem cells. The Pbx and Id families have also been implicated in maintaining stem cell function. The myc family displayed a strongly complementary stemness expression pattern. c-myc has been implicated in reprogramming of differentiated cells into pluripotent cells.