Survivin mutant protects differentiated dopaminergic SK-N-SH cells against oxidative stress

Oxidative stress is due to an imbalance of antioxidant/pro-oxidant homeostasis and is associated with the progression of several neurological diseases, including Parkinson’s and Alzheimer’s disease and amyotrophic lateral sclerosis. Furthermore, oxidative stress is responsible for the neuronal loss and dysfunction associated with disease pathogenesis. Survivin is a member of the inhibitors of the apoptosis (IAP) family of proteins, but its neuroprotective effects have not been studied. Here, we demonstrate that SurR9-C84A, a survivin mutant, has neuroprotective effects against H2O2-induced neurotoxicity. Our results show that H2O2 toxicity is associated with an increase in cell death, mitochondrial membrane depolarisation, and the expression of cyclin D1 and caspases 9 and 3. In addition, pre-treatment with SurR9-C84A reduces cell death by decreasing both the level of mitochondrial depolarisation and the expression of cyclin D1 and caspases 9 and 3. We further show that SurR9-C84A increases the antioxidant activity of GSH-peroxidase and catalase, and effectively counteracts oxidant activity following exposure to H2O2. These results suggest for the first time that SurR9-C84A is a promising treatment to protect neuronal cells against H2O2-induced neurotoxicity.

Apoptotic and necrotic effects of hexanedione derivatives on the human neuroblastoma line SK-N-SH

The potential cytotoxicity of two hexanedione food additives (2,3 and 3,4 isomers) was evaluated in comparison with the neurotoxic hexane metabolite 2,5-hexanedione in the human SK-N-SH neuroblastoma line using the MTT assay to indicate mitochondrial dehydrogenase activity and flow cytometry to monitor the cell cycle over 48 h. The IC50s of the 2,3-hexanedione (3.3 ± 0.1 mM) and 3,4-hexanedione (3.5 ± 0.1 mM), indicated that the sensitivity of the cells was approximately seven-fold greater to these toxins compared with the 2,5 derivative (IC50 of 22.4 ± 0.2 mM). Comparison between the respective IC50s of the 2,3-hexanedione and 3,4-hexanedione revealed no difference between the two isomers in terms of their effects on MTT turnover. With flow cytometry analysis, all three hexanediones showed increases in apoptosis within their respective concentration ranges of toxicity shown previously by MTT. In the presence of 2,5-hexanedione, between 8.5 and 17 mM concentrations, there was a significant increase in apoptotic nucleoids which was accompanied by a significant fall in the percentage of nucleoids in the G0/G1 phase (72.4 ± 0.3-45.3 ± 0.6%,), and a rise in the numbers of cells in the G2/M phase. This is likely to indicate growth arrest at cell cycle G2/M checkpoint in response to toxin damage. G2/M accumulation was also shown with 3,4 and 2,3 HD, which was maximal at much lower concentrations (approximately 4 and 3 mM, respectively). Arrest at G1 and G2/M phase is indicative of inhibition of the cell cycle at the stages of DNA replication and chromosome segregation, respectively. It was also apparent that flow cytometry, rather than the MTT assay, did distinguish between the effects of the α-diketones 2,3-hexanedione and 3,4-hexanedione on the cell cycle. At a concentration of 5.8 mM 3,4-hexanedione, the percentage of apoptotic nucleoids was 10.9 ± 0.8% whilst apoptosis induced by 3,4-hexanedione had already reached a maximal level of 60.4 ± 0.5%. In summary, flow cytometry indicated that the 3,4-hexanedione derivative was more toxic than its 2,3 isomer and that both food additives caused interruption in the neuroblastoma cell cycle and further investigation may be required to assess if these α-diketones present in diets pose any possible risks to human health. © 2006 Elsevier Ireland Ltd. All rights reserved.

DE-71-induced apoptosis involving intracellular calcium and the Bax-mitochondria-caspase protease pathway in human neuroblastoma cells In vitro

Polybrominated diphenyl ethers (PBDEs) are used extensively as flame-retardants and are ubiquitous in the environment and in wildlife and human tissue. Recent studies have shown that PBDEs induce neurotoxic effects in vivo and apoptosis in vitro. However, the signaling mechanisms responsible for these events are still unclear. In this study, we investigated the action of a commercial mixture of PBDEs (pentabrominated diphenyl ether, DE-71) on a human neuroblastoma cell line, SK-N-SH. A cell viability test showed a dose-dependent increase in lactate dehydrogenase leakage and 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl-tetrazolium bromide reduction. Cell apoptosis was observed through morphological examination, and DNA degradation in the cell cycle and cell apoptosis were demonstrated using flow cytometry and DNA laddering. The formation of reactive oxygen species was not observed, but DE-71 was found to significantly induce caspase-3, -8, and -9 activity, which suggests that apoptosis is not induced by oxidative stress but via a caspase-dependent pathway. We further investigated the intracellular calcium ([Ca2+](i)) levels using flow cytometry and observed an increase in the intracellular Ca2+ concentration with a time-dependent trend. We also found that the N-methyl d-aspartate (NMDA) receptor antagonist MK801 (3 mu M) significantly reduced DE-71-induced cell apoptosis. The results of a Western blotting test demonstrated that DE-71 treatment increases the level of Bax translocation to the mitochondria in a dose-dependent fashion and stimulates the release of cytochrome c (Cyt c) from the mitochondria into the cytoplasm. Overall, our results indicate that DE-71 induces the apoptosis of ([Ca2+](i)) in SK-N-SH cells via Bax insertion, Cyt c release in the mitochondria, and the caspase activation pathway.

Nanoformulated cell-penetrating survivin mutant and its dual actions

In this study, we investigated the differential actions of a dominant-negative survivin mutant (SurR9-C84A) against cancerous SK-N-SH neuroblastoma cell lines and differentiated SK-N-SH neurons. In both the cases, the mutant protein displayed dual actions, where its effects were cytotoxic toward cancerous cells and proliferative toward the differentiated neurons. This can be explained by the fact that tumorous (undifferentiated SK-N-SH) cells have a high endogenous survivin pool and upon treatment with mutant SuR9-C84A causes forceful survivin expression. These events significantly lowered the microtubule dynamics and stability, eventually leading to apoptosis. In the case of differentiated SK-N-SH neurons that express negligible levels of wild-type survivin, the mutant indistinguishably behaved in a wild-type fashion. It also favored cell-cycle progression, forming the chromosome-passenger complex, and stabilized the microtubule-organizing center. Therefore, mutant SurR9-C84A represents a novel therapeutic with its dual actions (cytotoxic toward tumor cells and protective and proliferative toward neuronal cells), and hence finds potential applications against a variety of neurological disorders. In this study, we also developed a novel poly(lactic-co-glycolic acid) nanoparticulate formulation to surmount the hurdles associated with the delivery of SurR9-C84A, thus enhancing its effective therapeutic outcome.

Nanoformulated mutant SurR9-C84A: a possible key for alzheimer's and its associated inflammation

PURPOSE: Alzheimer's disease (AD) is one of the untreatable neurodegenerative diseases characterised by the pathologic amyloid plaque deposition and inflammation. The aim of this study is to evaluate the neuroprotective effects of nanoformulated SurR9-C84A, a survivin mutant belonging to the inhibitors of the apoptosis (IAP) protein family. The effect of SurR9-C84A was studied against the β-amyloid toxicity and various inflammatory insults in the differentiated SK-N-SH neurons. METHOD: SurR9-C84A loaded poly(lactic-co-glycolic acid) nanoparticles were prepared following the modified double emulsion technique. The neuroprotective effect of SurR9-C84A was evaluated against the amyloid-β (Aβ) peptide fragment, N-methyl-D-aspartate (NMDA) toxicity and the inflammatory assaults. To mimic the in vivo situation, a co-culture of neurons and microglia was also studied to validate these results. RESULTS: SurR9-C84A treatments showed improved neuronal health following Aβ, and NMDA toxicity in addition to inflammatory insults induced in mono and co-cultures. The neuroprotective effect was evident with the reduced neuronal death, accelerated expression of neuronal integrity markers (neurofilaments, beta-tubulin III etc.,) and the neuroprotective ERK/MAPK signalling. CONCLUSION: The current results demonstrated that the SurR9-C84A nanoformulation was very effective in rescuing the neurons and holds a potential future application against AD.

Evaluating the potential neurotoxicity of hexanedione isomers:an in vitro approach

2,5-hexanedione (2,5HD) is the neurotoxic metabolite of the aliphatic hydrocarbon n-Hexane. The isomers, 2,3-hexanedione (2,3HD) and 3,4-hexanedione (3,4HD) are used as food additives. Although the neurotoxicity of 2,5HD is well established, there are no human data of the possible toxicity of the 2,3- and 3,4- isomers. MTT and flow cytometry were utilised to determine the cytotoxicity of hexanedione isomers in neuroblastoma cells. The neuroblastoma cell lines SK-N-SH and SH-SY5Y are sufficiently neuron-like to provide preliminary assessment of the neurotoxic potential of these isomers, in comparison with toxicity towards human non-neuronal cells. Initial studies showed that 2,5HD was the least toxic in all cell lines at all times (4, 24 and 48h). Although considerably lower than for 2,5HD, in general the IC50s for the α isomers were not significantly different from each other and, besides 4h exposure, the SH-SY5Y cells were significantly more sensitive to 2,3HD and 3,4HD than the SK-N-SH cells. All three isomers caused varying degrees of apoptosis in the neuroblastoma lines, with 3,4HD more potent than 2,3HD. Flow cytometry highlighted cell cycle arrest indicative of DNA damage with 2,3- and 3,4HD. The toxicity of the isomers towards 3 non-neuronal cell lines (MCF7, HepG2 and CaCo-2) was assessed by MTT assay. All 3 hexanedione isomers proved to be cytotoxic in all non-neuronal cell lines at all time points. These data suggest cytotoxicity of 2,3- and 3,4HD (mM range), but it is difficult to define this as specific neurotoxicity in the absence of specific neurotoxic endpoints. However, the neuroblastomas were significantly more susceptible to the cytotoxic effects of the α hexanedione isomers at exposures of 4 and 24 hours, compared to non-neuronal lines. Finally, a mechanism of toxicity is suggested for the α HD isomers whereby inhibition of the oxoglutarate carrier (OGC) releases apoptosis inducing factor (AIF), causing apoptosis-like cell death.

The toxicity of hexanedione isomers in neural and astrocytic cell lines

The metabolite 2,5-hexanedione (HD) is the cause of neurotoxicity linked with chronic n-hexane exposure. Acute exposure to high levels of 2,5-HD, have also shown toxic effects in neuronal cells and non-neuronal cells. Isomers of 2,5-HD, 2,3- and 3,4-HD, added to foodstuffs, are reported to be non-toxic. The acute cytotoxic effects of 2,5-, 2,3- and 3,4-HD were evaluated in neural (NT2.N, SK-N-SH), astrocytic (CCF-STTG1) and non-neural (NT2.D1) cell lines. All the cell lines were highly resistant to 2,5-HD (34-426 mM) at 4-h exposure, although sensitivity was greatest with NT2.D1, then SK-N-SH, NT2.N and finally the CCF-STTG1 line. At 24-h exposure, cell vulnerability increased 5-10-fold. The NT2.D1 cells were again the most sensitive, followed by NT2.N, SK-N-SH and then the CCF-STTG1 cells. 2,3- and 3,4-HD (8-84 mM), were significantly more toxic towards all four cell lines compared with 2,5-HD, after 4-h exposure. After 24-h exposure there was a 12-fold increase in inhibition of MTT turnover in the SK-N-SH cells and a 4-fold increase in the CCF-STTG1 cells, compared with 2,5-HD exposure. 2,3- and 3,4-HD, were significantly less toxic to the NT2.N cells than the SK-N-SH cells after 24-h exposure to the compounds, demonstrating a differing toxin vulnerability between these neural and neuroblastoma cell lines. This study indicates that these non-neuronal and neuronal cells are acutely resistant to 2,5-HD cytotoxicity, whilst the previously unreported sensitivity of all four cell lines to the 2,3- and 3,4- isomers of HD to has been shown to be significantly greater than that of 2,5-HD. © 2006 Elsevier B.V. All rights reserved.

Chlorzoxazone, an SK-type potassium channel activator used in humans, reduces excessive alcohol intake in rats

Background Alcoholism imposes a tremendous social and economic burden. There are relatively few pharmacological treatments for alcoholism, with only moderate efficacy, and there is considerable interest in identifying additional therapeutic options. Alcohol exposure alters SK-type potassium channel (SK) function in limbic brain regions. Thus, positive SK modulators such as chlorzoxazone (CZX), a US Food and Drug Administration–approved centrally acting myorelaxant, might enhance SK function and decrease neuronal activity, resulting in reduced alcohol intake. Methods We examined whether CZX reduced alcohol consumption under two-bottle choice (20% alcohol and water) in rats with intermittent access to alcohol (IAA) or continuous access to alcohol (CAA). In addition, we used ex vivo electrophysiology to determine whether SK inhibition and activation can alter firing of nucleus accumbens (NAcb) core medium spiny neurons. Results Chlorzoxazone significantly and dose-dependently decreased alcohol but not water intake in IAA rats, with no effects in CAA rats. Chlorzoxazone also reduced alcohol preference in IAA but not CAA rats and reduced the tendency for rapid initial alcohol consumption in IAA rats. Chlorzoxazone reduction of IAA drinking was not explained by locomotor effects. Finally, NAcb core neurons ex vivo showed enhanced firing, reduced SK regulation of firing, and greater CZX inhibition of firing in IAA versus CAA rats. Conclusions The potent CZX-induced reduction of excessive IAA alcohol intake, with no effect on the more moderate intake in CAA rats, might reflect the greater CZX reduction in IAA NAcb core firing observed ex vivo. Thus, CZX could represent a novel and immediately accessible pharmacotherapeutic intervention for human alcoholism. Key Words: Alcohol intake; intermittent; neuro-adaptation; nucleus accumbens; SK potassium channel

Sh-h-h-h : representations of perpetrators of sexual child abuse in picturebooks

Children’s picturebooks dealing with the topic of child sexual abuse first appeared in the early 1980s with the aim of addressing the need for age-appropriate texts to teach sexual abuse prevention concepts and to provide support for young children who may be at risk of or have already experienced sexual abuse. Despite the apparent potential of children’s picturebooks to convey child sexual abuse prevention concepts, very few studies have addressed the topic of child sexual abuse in children’s literature. Based on a larger study of 60 picturebooks about sexual child abuse published over the past 25 years, this paper critically examines eight picturebook representations of the perpetrators of sexual child abuse as a way to understand how potentially dangerous adults are explained to the young readers of these texts.

Ethanol exposure alters monoamine oxidase gene-expression in neuronal SH-SY5Y cells

Abstract: Monoamine Oxidase (MAO) enzymes catabolise, and thus modulate abundance of, neurotransmitters in the brain. Variation in MAO enzyme activity has been linked to alcohol abuse behaviour, although the molecular mechanisms underlying this association are not understood. The present study evaluated relative gene-transcript abundance of MAO-A and MAO-B in the SH-SY5Y human neuroblastoma cell-line in response to ethanol exposure and following ethanol withdrawal. We found that each isoform of MAO was significantly transcriptionally up-regulated 55-80% in response to 100mM ethanol exposure. This trend was maintained following prolonged exposures (24 h-72 h) and with short exposures (24 h) followed by a period of ethanol withdrawal, suggesting that the transcriptional regulation is the result of a cellular change occurring within the first 24 hours of ethanol exposure. These results suggest a role for MAO transcriptional regulation in the complex neurobiochemical changes underlying alcohol addiction.

Expression patterns of vascular-specific promoters RolC and Sh in transgenic potatoes and their use in engineering PLRV-resistant plants

The expression patterns of GUS fusion constructs driven by the Agrobacterium rhizogenes RolC and the maize Sh (Shrunken: sucrose synthase-1) promoters were examined in transgenic potatoes (cv. Atlantic). RolC drove high-level gene expression in phloem tissue, bundle sheath cells and vascular parenchyma, but not in xylem or non-vascular tissues. Sh expression was exclusively confined to phloem tissue. Potato leafroll luteovirus (PLRV) replicates only in phloem tissues, and we show that when RolC is used to drive expression of the PLRV coat protein gene, virus-resistant lines can be obtained. In contrast, no significant resistance was observed when the Sh promoter was used.

Plan zemelʻnogo uchastka poselka Ėrstmaĭsk N 8 Leninskogo fonda

Digital image

Karnaugh map analysis and synthesis of threshold functions

A unate function can easily be identified on a Karnaugh map from the well-known property that it cons ist s only ofess en ti al prime implicante which intersect at a common implicant. The additional property that the plot of a unate function F(x, ... XII) on a Karnaugh map should possess in order that F may also be Ivrealizable (n';:; 6) has been found. It has been sh own that the I- realizability of a unate function F corresponds to the ' compac tness' of the plot of F. No resort to tho inequalities is made, and no pre-processing such as positivizing and ordering of the given function is required.

Relaxation behavior of twin nonlinear optical chromophores: Effect of the spacer length

A new series of twin nonlinear optical (NLO) molecules, having two 4-nitrophenol chromophores that are linked via a flexible polymethylene spacer of varying length [(CH2)(n), n = 1-12], were synthesized. Powder second harmonic generation measurements of these twin samples indicated a pronounced odd-even oscillation, with the odd twins exhibiting a high SHG value while the even ones gave no measurable SH signal. This behavior reflects the crystal packing preferences in such twin NLO systems that have odd and even numbers of atoms linking them - the even ones appear to prefer a centrosymmetric packing arrangement. The orientational/disordering dynamics of these twin NLO molecules, doped in a polymer (poly(methyl methacrylate)) matrix, has also been studied using SHG in electric field poled samples. Interestingly, the maximum attainable SH signal, chi((2)), in, the poled samples also showed an odd-even oscillation; the odd ones again having a higher value of chi((2)) This unprecedented odd-even oscillation in such molecularly doped systems is rationalized as being due to the intrinsically greater ease of a parallel alignment of the two chromophores in the twins with an odd spacer than in those with an even one. Further, the temporal stability of the SHG intensity at 70 degrees C, after the removal of the applied corona, was also studied. The relaxation of all the twin chromophores followed a biexponential decay; the characteristic relaxation time (tau(2)) for the slow decay component suggests that while the twin with a single methylene unit relaxes relatively slowly, the relaxation is significantly faster in cases where n = 2 and 3. In the twins with even longer spacer segments, the relaxation again becomes slower and reaches a saturation value. The observed minimum appears to reflect the interplay of two competing factors that affect the chromophore alignment in such twin systems, namely, the electrostatic repulsion between neighboring oriented dipoles and the intrinsic flexibility of the spacer.