Cerebral vascular and blood brain-barrier abnormalities in a mouse model of epilepsy and tuberous sclerosis complex.

OBJECTIVE: Tuberous sclerosis complex (TSC) is a genetic disorder, characterized by tumor formation in the brain and other organs, and severe neurological symptoms, such as epilepsy. Abnormal vascular endothelial growth factor (VEGF) expression may promote angiogenesis in kidney and lung tumors in TSC and has been identified in brain specimens from TSC patients, but the role of VEGF and vascular abnormalities in neurological manifestations of TSC is poorly defined. In this study, we investigated abnormalities in brain VEGF expression, cerebral blood vessel anatomy, and blood-brain barrier (BBB) structure and function in a mouse model of TSC. METHODS: Tsc1GFAP CKO mice were used to investigate VEGF expression and vascular abnormalities in the brain by Western blotting and immunohistochemical analysis of vascular and BBB markers. In vivo two-photon imaging was used to assess BBB permeability to normally impenetrable fluorescently labeled compounds. The effect of mechanistic target of rapamycin (mTOR) pathway inhibitors, VEGF receptor antagonists (apatinib), or BBB stabilizers (RepSox) was assessed in some of these assays, as well as on seizures by video-electroencephalography. RESULTS: VEGF expression was elevated in cortex of Tsc1GFAP CKO mice, which was reversed by the mTOR inhibitor rapamycin. Tsc1GFAP CKO mice exhibited increased cerebral angiogenesis and vascular complexity in cortex and hippocampus, which were reversed by the VEGF receptor antagonist apatinib. BBB permeability was abnormally increased and BBB-related tight junction proteins occludin and claudin-5 were decreased in Tsc1GFAP CKO mice, also in an apatinib- and RepSox-dependent manner. The BBB stabilizer (RepSox), but not the VEGF receptor antagonist (apatinib), decreased seizures and improved survival in Tsc1GFAP CKO mice. SIGNIFICANCE: Increased brain VEGF expression is dependent on mTOR pathway activation and promotes cerebral vascular abnormalities and increased BBB permeability in a mouse model of TSC. BBB modulation may affect epileptogenesis and represent a rational treatment for epilepsy in TSC.

A role of dentate gyrus mechanistic target of rapamycin activation in epileptogenesis in a mouse model of posttraumatic epilepsy.

OBJECTIVE: The mechanistic target of rapamycin (mTOR) pathway has been implicated in promoting epileptogenesis in animal models of acquired epilepsy, such as posttraumatic epilepsy (PTE) following traumatic brain injury (TBI). However, the specific anatomical regions and neuronal populations mediating mTOR's role in epileptogenesis are not well defined. In this study, we tested the hypothesis that mTOR activation in dentate gyrus granule cells promotes neuronal death, mossy fiber sprouting, and PTE in the controlled cortical impact (CCI) model of TBI. METHODS: An adeno-associated virus (AAV)-Cre viral vector was injected into the hippocampus of Rptorflox/flox (regulatory-associated protein of mTOR) mutant mice to inhibit mTOR activation in dentate gyrus granule cells. Four weeks after AAV-Cre or AAV-vehicle injection, mice underwent CCI injury and were subsequently assessed for mTOR pathway activation by Western blotting, neuronal death, and mossy fiber sprouting by immunopathological analysis, and posttraumatic seizures by video-electroencephalographic monitoring. RESULTS: AAV-Cre injection primarily affected the dentate gyrus and inhibited hippocampal mTOR activation following CCI injury. AAV-Cre-injected mice had reduced neuronal death in dentate gyrus detected by Fluoro-Jade B staining and decreased mossy fiber sprouting by ZnT3 immunostaining. Finally, AAV-Cre-injected mice exhibited a decrease in incidence of PTE. SIGNIFICANCE: mTOR pathway activation in dentate gyrus granule cells may at least partly mediate pathological abnormalities and epileptogenesis in models of TBI and PTE. Targeted modulation of mTOR activity in this hippocampal network may represent a focused therapeutic approach for antiepileptogenesis and prevention of PTE.

HpaII-assisted and linear amplification-enhanced isothermal exponential amplification fluorescent strategy for rapid and sensitive detection of DNA methyltransferase activity.

The detection of methyltransferase (MTase) activity is of great significance in methylation-related disease diagnosis and drug screening. Herein, a HpaII-assisted and linear amplification-enhanced exponential amplification strategy is proposed for sensitive and label-free detection of M.SssI MTase activity. The P1 probe contains self-complementary sequence 5'-CTAGCCGGCTAG-3' at 3'-terminal. After denaturation and annealing, P1 probes hybridize with itself to generate P1 duplexes. M.SssI MTase induces methylation of cytosine at 5'-CG-3' in P1 duplexes, and thus, HpaII fails to cleave at 5'-CCGG-3' due to methylation sensitivity, leaving P1 duplex intact. Then, these intact P1 duplexes are extended along 3'-terminal through Vent (exo-) DNA polymerase to generate dsDNA, which is recognized and nicked at the recognition sites by Nt.BstNBI, releasing two copies of primer X. Primer X hybridizes with X' at the amplification template T1 (X'-Y'-X') and then serves as primers to trigger the exponential amplification reaction (EXPAR). The point of inflection (POI) values of real-time fluorescence curves is linearly correlated with the logarithm of M.SssI MTase concentration in the range of 0.125 [Formula: see text] 8 U mL-1 with a low detection limit of 0.034 U mL-1. In the absence of M.SssI, P1 duplexes are cut by HpaII and separated into ssDNA under the executed temperature of EXPAR and thus unable to trigger the amplification. The strategy provides good selectivity against other types of MTases and protein and is able to detect M.SssI activity in human serum. Furthermore, the analytical method has the generality and can be extended to the analysis of other types of DNA MTases.

Streptomyces changanensis sp. nov. Isolated from Soil in China.

An aerobic, Gram-positive, and non-motile actinomycete, designated HL-66 T, was isolated from a soil sample collected in the Meridian Valley, Shaanxi Province, China. Morphological, chemotaxonomic, and phylogenetic characteristics showed a high similarity to the genus Streptomyces. Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbour of HL-66 T were Streptomyces lavendofoliae NBRC 12882 T (99.17%), Streptomyces gobitricini NBRC 15419 T (99.03%) and Streptomyces roseolilacinus NBRC 12815 T (98.96%). Genome relatedness indexes revealed that the average nucleotide identity and digital DNA-DNA hybridization values between HL-66 T and its closest phylogenomic relative (S. roseolilacinus JCM 4335 T) were 88.61% and 32.10%, respectively. The cell-wall peptidoglycan contains LL-diaminopimelic acid. Predominant menaquinones are MK-9 (H6), MK-9(H4) and MK-9(H8). The major cellular fatty acids were iso-C16:0, anteiso-C15:0, iso-C16:1 H, and C16:1 ω7c. The polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides, and an unknown phospholipid. Based on phylogenetic analyses, genome-genome distance calculation, and average nucleotide identity, strain HL-66 T represents a novel species of the genus Streptomyces. Therefore, a new species Streptomyces changanensis sp. nov. is proposed with strain HL-66 T (= CGMCC 22674 = JCM 35800) as the type strain.

Protective effect of vitamin C against tetrachlorobenzoquinone-induced 5-hydroxymethylation-dependent apoptosis in HepG2 cells mainly via the mitochondrial apoptosis pathway.

Tetrachlorobenzoquinone (TCBQ) is an active metabolite of pentachlorophenol, and stimulates the accumulation of ROS to trigger apoptosis. The preventive effect of vitamin C (Vc) against TCBQ-induced apoptosis in HepG2 cells is unknown. And there is little known about TCBQ-triggered 5-hydromethylcytosine (5hmC)-dependent apoptosis. Here, we confirmed that Vc alleviated TCBQ-induced apoptosis. Through investigating the underlying mechanism, we found TCBQ downregulated 5hmC levels of genomic DNA in a Tet-dependent manner, with a particularly pronounced decrease in the promoter region, using UHPLC-MS-MS analysis and hydroxymethylated DNA immunoprecipitation sequencing. Notably, TCBQ exposure resulted in alterations of 5hmC abundance to ∼91% of key genes at promoters in the mitochondrial apoptosis pathway, along with changes of mRNA expression in 87% of genes. By contrast, 5hmC abundance of genes only exhibited slight changes in the death receptor/ligand pathway. Interestingly, the pretreatment with Vc, a positive stimulator of 5hmC generation, restored 5hmC in the genomic DNA to near-normal levels. More notably, Vc pretreatment further counter-regulated TCBQ-induced alteration of 5hmC abundance in the promoter with 100% of genes, accompanying the reverse modulation of mRNA expressions in 89% of genes. These data from Vc pretreatment supported the relationship between TCBQ-induced apoptosis and the altered 5hmC abundance. Additionally, Vc also suppressed TCBQ-stimulated generation of ROS, and further increased the stability of mitochondria. Our study illuminates a new mechanism of TCBQ-induced 5hmC-dependent apoptosis, and the dual mechanisms of Vc against TCBQ-stimulated apoptosis via reversely regulating 5hmC levels and scavenging ROS. The work also provided a possible strategy for the detoxification of TCBQ.

Effects of indole derivatives from Purpureocillium lilacinum in controlling tobacco mosaic virus.

There are various types of compounds studied and applied for plant disease management, and some of them are environment friendly and suitable in organic production. An example is indole-3-carboxaldehyde (A1) and indole-3-carboxylic acid (A2) derived from Purpureocillium lilacinum H1463, which have shown a strong activity in the control of tobacco mosaic virus (TMV). In this study, the effects of these compounds were studied on suppressing TMV and corresponding mechanism. Both A1 and A2 exhibited strong anti-TMV activities in vitro and in vivo. They fractured TMV virions and forced the fractured particles agglomerated. A1 and A2 also induced immune responses or resistance of tobacco to TMV infection, including expressing hypersensitive reaction (HR), increasing defense-related enzymes and overexpressing pathogenesis-related (PR) proteins. The upregulation of salicylic acid (SA) biosynthesis genes PAL, ICS, and PBS3 confirmed that SA served as a defense-related signal molecule. Therefore, indole derivatives have a potential for activating defense of tobacco against TMV and other pathogens and can be used for disease control.

H2O2 signaling modulates Glycoprotein-1 induced programmed cell death in tobacco suspension cells.

Glycoprotein (GP)-1 is a glycoprotein elicitor with antiviral activity found in Streptomyces kanasensis zx01. GP-1 can induce programmed cell death (PCD) in vitro; however, the underlying mechanism is unclear. In the present study, we demonstrated that GP-1 induced PCD in tobacco suspension cells, which was modulated by hydrogen peroxide (H2O2). GP-1 participated in and modulated biologically relevant signaling in plant cells. GP-1 induced tobacco cell death in a dose- and time-dependent manner; affected the expression of BRI1-associated receptor kinase 1 (BAK1) and the accumulation of salicylic acid (SA), which are related to PCD; and enzymatic activities and mitochondrial functions. In conclusion, GP-1-induced PCD in tobacco may be mediated by H2O2 which alters BAK1 and SA levels, as well as mitochondrial and gene function. This cell signal cascade played an important role in the process of GP-1 induced plant disease resistance.

A novel glycoprotein from Streptomyces sp. triggers early responses of plant defense.

GP-1, a novel glycoprotein from Streptomyces sp. ZX01 has a plant immunity-inducing effect. GP-1-treated plants exhibited enhanced systemic resistance with a significant reduction in TMV lesions on tobacco leaves, but its antiviral mechanism remains unclear. In this study, early plant defense-related responses, such as Ca2+ influx, callose apposition, oxidative burst, hypersensitive response, programmed cell death, increase in nitric oxide (NO), and stomatal closure, were investigated under GP-1 treatment, and the mechanism of how GP-1 induces viral resistance in Nicotiana benthamiana was studied. Results showed that GP-1 induced [Ca2+]cyt rapidly in tobacco leaves and suspended cells, followed by reactive oxygen species (ROS) and NO elevation. Transcriptome analysis showed significant differences in expression levels between the GP-1-treated N. benthamiana and the control and showed significantly upregulated and enriched pathways including defense and immune reaction. Similar to typical pathogen-associated molecular patterns, GP-1 induced callose deposition and stomatal closure to form defense barriers against pathogen invasion. The expression of defense-related genes further confirmed the above conclusions. By analyzing transcriptome in N. benthamiana and the contents of salicylic acid (SA) and jasmonic acid (JA), GP-1 enhanced viral resistance of tobacco by improving the SA and JA contents, strengthening plant secondary metabolites activities, promoting systemic accumulation of pathogenesis-related proteins in TMV- inoculated tobacco there by producing antiviral activity.

Hypothalamic orexin and mechanistic target of rapamycin activation mediate sleep dysfunction in a mouse model of tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a genetic disease related to hyperactivation of the mechanistic target of rapamycin (mTOR) pathway and manifested by neurological symptoms, such as epilepsy and sleep disorders. The pathophysiology of sleep dysfunction is poorly understood and is likely multifactorial, but may involve intrinsic biological regulators in the brain. Here, we characterized a mouse model of sleep disorders in TSC and investigated mechanisms of sleep dysfunction in this conditional knockout model involving inactivation of the Tsc1 gene in neurons and astrocytes (Tsc1GFAPCKO mice). Sleep studies utilizing EEG, EMG, and behavioral analysis found that Tsc1GFAPCKO mice have decreased REM sleep and impaired sleep-wake differentiation between light and dark phases. mTOR activity and orexin expression were increased in hypothalamic sections and cultured hypothalamic neurons from Tsc1GFAPCKO mice. Both the sleep abnormalities and increased orexin expression in Tsc1GFAPCKO mice were reversed by rapamycin treatment, indicating their dependence on mTOR activation. An orexin antagonist, suvorexant, also restored normal REM levels in Tsc1GFAPCKO mice. These results identify a novel mechanistic link between mTOR and orexin in the hypothalamus related to sleep dysfunction and suggest a targeted therapeutic approach to sleep disorders in TSC.

The specificity and role of microglia in epileptogenesis in mouse models of tuberous sclerosis complex.

OBJECTIVE: Microglial abnormalities have been reported in pathologic specimens from patients with tuberous sclerosis complex (TSC), a genetic disorder characterized by epilepsy, intellectual disability, and autism. However, the pathogenic role of microglia in epilepsy in TSC is poorly understood, particularly whether microglia defects may be a primary contributor to epileptogenesis or are secondary to seizures or simply epiphenomena. In this study, we tested the hypothesis that Tsc1 gene inactivation in microglia is sufficient to cause epilepsy in mouse models of TSC. METHODS: Using a chemokine receptor, Cx3cr1, to target microglia, conventional Tsc1Cx3cr1-Cre CKO (conditional knockout) mice and postnatal-inducible Tsc1Cx3cr1-CreER CKO mice were generated and assessed for molecular and histopathologic evidence of microglial abnormalities, mechanistic target of rapamycin 1 (mTORC1) pathway activation, and epilepsy. RESULTS: Tsc1Cx3cr1-Cre CKO mice exhibited a high efficiency of microglia Tsc1 inactivation, mTORC1 activation, increased microglial size and number, and robust epilepsy, which were rapamycin-dependent. However, Cre reporter studies demonstrated that constitutive Cx3cr1 expression affected not only microglia, but also a large percentage of cortical neurons, confounding the role of microglia in epileptogenesis in Tsc1 Cx3cr1-Cre CKO mice. In contrast, postnatal inactivation of Tsc1 utilizing a tamoxifen-inducible Cx3cr1-CreER resulted in a more-selective microglia Tsc1 inactivation with high efficiency, mTORC1 activation, and increased microglial size and number, but no documented epilepsy. SIGNIFICANCE: Microglia abnormalities may contribute to epileptogenesis in the context of neuronal involvement in TSC mouse models, but selective Tsc1 gene inactivation in microglia alone may not be sufficient to cause epilepsy, suggesting that microglia have more supportive roles in the pathogenesis of seizures in TSC.

Optimization of Production Conditions for Protoplasts and Polyethylene Glycol-Mediated Transformation of Gaeumannomyces tritici.

Take-all, caused by Gaeumannomyces tritici, is one of the most important wheat root diseases worldwide, as it results in serious yield losses. In this study, G. tritici was transformed to express the hygromycin B phosphotransferase using a combined protoplast and polyethylene glycol (PEG)-mediated transformation technique. Based on a series of single-factor experimental results, three major factors-temperature, enzyme lysis time, and concentration of the lysing enzyme-were selected as the independent variables, which were optimized using the response surface methodology. A higher protoplast yield of 9.83 × 107 protoplasts/mL was observed, and the protoplast vitality was also high, reaching 96.27% after optimization. Protoplasts were isolated under the optimal conditions, with the highest transformation frequency (46⁻54 transformants/µg DNA). Polymerase chain reaction and Southern blotting detection indicated that the genes of hygromycin phosphotransferase were successfully inserted into the genome of G. tritici. An optimised PEG-mediated protoplast transformation system for G. tritici was established. The techniques and procedures described will lay the foundation for establishing a good mutation library of G. tritici and could be used to transform other fungi.

Optimization of Fermentation Conditions and Bench-Scale for Improvement of a Novel Glycoprotein GP-1 Production by Streptomyces kanasenisi ZX01.

GP-1 is a novel glycoprotein produced by Streptomyces kanasenisi ZX01 that was isolated from soil near Kanas Lake with significant bioactivity against tobacco mosaic virus. However, extremely low fermentation production has largely hindered further research and market applications on glycoprotein GP-1. In this study, response surface methodology was used to optimize fermentation conditions in a shake flask for higher glycoprotein GP-1 production. When the optimized fermentation conditions were inoculum volume of 6%, initial pH of 6.5, and rotating speed of 150 rpm, glycoprotein GP-1 production could reach 0.9253 mg/L, which was increased by 52.14% compared to the original conditions. In addition, scale-up fermentation was conducted in a 5-L bioreactor to preliminarily explore the feasibility for mass production of glycoprotein GP-1 in a large fermentor, obtaining GP-1 production of 2.54 mg/L under the same conditions, which was 2.75 times higher than the production obtained from a shake flask of 0.9253 mg/L. This work will be helpful to improve GP-1 production on a large scale and lay the foundations for developing it as a novel agent against plant virus.

Effects of Agitation, Aeration and Temperature on Production of a Novel Glycoprotein GP-1 by Streptomyces kanasenisi ZX01 and Scale-Up Based on Volumetric Oxygen Transfer Coefficient.

The effects of temperature, agitation and aeration on glycoprotein GP-1 production by Streptomyces kanasenisi ZX01 in bench-scale fermentors were systematically investigated. The maximum final GP-1 production was achieved at an agitation speed of 200 rpm, aeration rate of 2.0 vvm and temperature of 30 °C. By using a dynamic gassing out method, the effects of agitation and aeration on volumetric oxygen transfer coefficient (kLa) were also studied. The values of volumetric oxygen transfer coefficient in the logarithmic phase increased with increase of agitation speed (from 14.53 to 32.82 h-1) and aeration rate (from 13.21 to 22.43 h-1). In addition, a successful scale-up from bench-scale to pilot-scale was performed based on volumetric oxygen transfer coefficient, resulting in final GP-1 production of 3.92, 4.03, 3.82 and 4.20 mg/L in 5 L, 15 L, 70 L and 500 L fermentors, respectively. These results indicated that constant volumetric oxygen transfer coefficient was appropriate for the scale-up of batch fermentation of glycoprotein GP-1 by Streptomyces kanasenisi ZX01, and this scale-up strategy successfully achieved 100-fold scale-up from bench-scale to pilot-scale fermentor.

Potential use of cuminic acid as a botanical fungicide against Valsa mali.

Valsa canker caused by Valsa mali is commonly present in eastern Asia and cause large economic losses. Because of limited agricultural measures and chemical residues of commonly used fungicides there is an urgent need of alternative plant protecting agents. On this background the activity of cuminic acid, a plant extract from the seed of Cuminum cyminum L, was assessed. The median effective concentration (EC50) values for inhibition of mycelial growth of seven V. mali strains ranged from 3.046 to 8.342 µg/mL, with an average EC50 value of 4.956 ± 0.281 µg/mL. The antifungal activity was the direct activity of cuminic acid instead of the influence on the pH of media by cuminic acid. After treated with cuminic acid, mycelia dissolved with decreased branches and swelling; cell membrane permeability increased while pectinases activity decreased significantly. Moreover, peroxidase (POD) activity of the apple leaves increased after treated with cuminic acid. Importantly, on detached branches of apple tree, cuminic acid exhibited both protective and curative activity. These results indicated that cuminic acid not only showed the antifungal activity, but also could improve the defense capacity of the plants. Taken together, cuminic acid showed the potential as a natural alternative to commercial fungicides or a lead compound to develop new fungicides for the control of Valsa canker.

Sensitivity and biochemical characteristics of Sclerotinia sclerotiorum to propamidine.

Propamidine is an aromatic diamidine compound. In the current study, baseline sensitivity of Sclerotinia sclerotiorum to propamidine was determined using 78 strains collected from the oilseed rape fields without a previous history of propamidine usage. The median effective concentration (EC50) values for propamidine inhibiting mycelial growth ranged from 0.406 to 3.647µg/mL, with a mean of 1.616±0.217µg/mL. There was no correlation between sensitivity to propamidine and sensitivity to dimethachlon or carbendazim. After treated with propamidine, mycelia were thinner with irregular distortion and more branches; cell wall became thicker with uneven distribution of cytoplasm than untreated control. In addition, sclerotia production, cell membrane permeability and oxalic acid content significantly decreased. On detached oilseed rape leaves, propamidine exhibited better control efficacy than carbendazim at the same concentration whether the leaves were inoculated with carbendazim-sensitive or resistant strains. All the results showed that propamidine exhibited strong antifungal activity and potential application in controlling S. sclerotiorum. Importantly, these data will provide more information on understanding the mode of action of propamidine against S. sclerotiorum and should be valuable for development of new antifungal drugs.

Microglial activation during epileptogenesis in a mouse model of tuberous sclerosis complex.

OBJECTIVE: Tuberous sclerosis complex (TSC) is a genetic disorder, characterized by tumor formation in multiple organs and severe neurologic manifestations, including epilepsy, intellectual disability, and autism. Abnormalities of both neurons and astrocytes have been implicated in contributing to the neurologic phenotype of TSC, but the role of microglia in TSC has not been investigated. The objectives of this study were to characterize microglial activation in a mouse model of TSC, involving conditional inactivation of the Tsc1 gene predominantly in glial cells (Tsc1(GFAP) CKO mice), and to test the hypothesis that microglial activation contributes to epileptogenesis in this mouse model. METHODS: Microglial and astrocyte activation was examined in Tsc1(GFAP) CKO mice by ionized calcium binding adaptor molecule 1 and glial fibrillary acidic protein immunohistochemistry. Cytokine and chemokine expression was evaluated with quantitative polymerase chain reaction. Seizures were monitored by video-electroencephalography (EEG). The effect of minocycline in inhibiting microglial and astrocyte activation, cytokine expression, and seizures was tested. RESULTS: Microglial cell number and size were increased in cortex and hippocampus of 3- to 4-week-old Tsc1(GFAP) CKO mice, which correlated with the onset of seizures. Minocycline treatment prevented the increase in number and cell size of microglia in 4-week-old Tsc1(GFAP) CKO mice. However, minocycline treatment had no effect on astrocyte proliferation and cytokine/chemokine expression and the progression of seizures in Tsc1(GFAP) CKO mice. SIGNIFICANCE: Microglia cell number and size are abnormal in Tsc1(GFAP) CKO mice, and minocycline treatment inhibits this microglia activation, but does not suppress seizures. Microglia may play a role in the neurologic manifestations of TSC, but additional studies are needed in other models and human studies to determine whether microglia are critical for epileptogenesis in TSC.

Eicosapentaenoic acid induced SKOV-3 cell apoptosis through ERK1/2-mTOR-NF-κB pathways.

Eicosapentaenoic acid (EPA), a typical kind of n-3 polyunsaturated fatty acids, has been considered to be a potent antitumor adjuvant. However, the mechanism related to EPA-induced SKOV-3 cell apoptosis has not been investigated. In this study, we elucidated the anticancer effect of EPA on SKOV-3 cells and its molecular mechanisms. The results of fluorescence microscopy showed that EPA induced typical apoptotic morphological features in SKOV-3 cells. Flow cytometric analysis indicated that EPA induced apoptosis of SKOV-3 cells through cells arrested at the S phase. Western blotting results showed that EPA could inhibit the phosphorylation of ERK1/2 and Akt, which restrained mammalian target of rapamycin (mTOR) phosphorylated. Simultaneously, EPA downregulated the phosphorylation status of mTOR, which may act as an upstream regulator of EPA-blocked nuclear factor κB (NF-κB) p65 translocation from the cytoplasm into the nucleus; the apoptotic mechanism of SKOV-3 cells induced by EPA was associated with the release of cytochrome c, Bax-to-Bcl-2 expression ratio, and activation of caspase-3 and caspase-9. The results suggested that EPA induced SKOV-3 cell apoptosis through ERK1/2, Akt-mTOR-NF-κB pathways.

Identification of potential cargo proteins of transportin protein AtTRN1 in Arabidopsis thaliana.

KEY MESSAGE: We identified 23 novel proteins that can interact with At TRN1. These proteins are potential candidates of At TRN1 cargo proteins, which will facilitate our comprehending of At TRN1 functions in Arabidopsis. Tranportin 1 (TRN1) carries out the nucleo-cytoplasmic transport of many proteins, thereby ensuring that each of them is delivered to the right compartment for its proper function. These cargo proteins involved in lots of important processes, such as alternative pre-mRNA splicing, transcriptional regulation, and protein translation. Current understanding of cargo proteins transported by Arabidopsis thaliana transportin 1 (AtTRN1) is limited. Here, first we employed the yeast two-hybrid (Y2H) screening to identify proteins that can interact with AtTRN1 in Arabidopsis, and 12 novel proteins were found. Searching for PY-NLS motif in these 12 proteins suggested that no typical PY-NLS motif was present. We next investigated the specific motifs that will mediate the interactions in these sequences, and found that thirteen truncated fragments interacted with AtTRN1, containing 8 acidic and 5 basic fragments, respectively. We also searched the Arabidopsis proteome for homologs of cargo proteins of yeast Kapl04p and mammalian Kapß2, and PY-NLS motif-containing proteins. Among these proteins, 11 were identified to interact with AtTRN1. The interactions between all the 23 proteins and AtTRN1 were confirmed by both Y2H and bimolecular fluorescence complementation (BiFC) assays. Our results show that AtTRN1 recognizes a broad spectrum of proteins having diverse functions, which will potentially be the cargoes of AtTRN1. Taken together, these results demonstrate the feasibility and potential power of these methods to identify cargo proteins of AtTRN1, and represent a primary and significant step in interpretation of AtTRN1 functionalities.

Eicosapentaenoic acid (EPA) induced apoptosis in HepG2 cells through ROS-Ca(2+)-JNK mitochondrial pathways.

Eicosapentaenoic acid (EPA), a well-known dietary n-3 PUFAS, has been considered to inhibit proliferation of tumor cells. However, the molecular mechanism related to EPA-induced liver cancer cells apoptosis has not been reported. In this study, we investigated the effect of EPA on HepG2 cells proliferation and apoptosis mechanism through mitochondrial pathways. EPA inhibited proliferation of HepG2 cells in a dose-dependent manner and had no significant effect on the cell viability of humor normal liver L-02 cells. It was found that EPA initially evoked ROS formation, leading to [Ca(2+)]c accumulation and the mitochondrial permeability transition pore (MPTP) opening; EPA-induced HepG2 cells apoptosis was inhibited by N-acetylcysteine (NAC, an inhibitor of ROS), 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM, a chelator of calcium) and CsA (inhibitor of MPTP). The relationship between ROS production, the increase of cytoplasmic Ca and MPTP opening was detected. It seems that ROS may act as an upstream regulator of EPA-induced [Ca(2+)]c generation, moreover, generation of ROS, overload of mitochondrial [Ca(2+)]c, and JNK activated cause the opening of MPTP. Western blotting results showed that EPA elevated the phosphorylation status of JNK, processes associated with the ROS generation. Simultaneously, the apoptosis induced by EPA was related to release of cytochrome C from mitochondria to cytoplasm through the MPTP and activation of caspase-9 and caspase-3. These results suggest that EPA induces apoptosis through ROS-Ca(2+)-JNK mitochondrial pathways.

Intermittent dosing of rapamycin maintains antiepileptogenic effects in a mouse model of tuberous sclerosis complex.

OBJECTIVE: Inhibitors of the mechanistic target of rapamycin (mTOR) pathway have antiepileptogenic effects in preventing epilepsy and pathologic and molecular mechanisms of epileptogenesis in mouse models of tuberous sclerosis complex (TSC). However, long-term treatment with mTOR inhibitors may be required to maintain efficacy and potentially has chronic side effects, such as immunosuppression. Attempts to minimize drug exposure will facilitate translational efforts to develop mTOR inhibitors as antiepileptogenic agents for patients with TSC. In this study, we tested intermittent dosing paradigms of mTOR inhibitors for antiepileptogenic properties in a TSC mouse model. METHODS: Western blot analysis of phosphorylation of S6 protein was used to assess the dose- and time-dependence of mTOR inhibition by rapamycin in control mice and conditional knockout mice with inactivation of the Tsc1 gene in glial fibrillary acidic protein (GFAP)-expressing cells (Tsc1(GFAP)CKO mice). Based on the Western blot studies, different dosing paradigms of rapamycin starting at postnatal day 21 were tested for their ability to prevent epilepsy or pathologic abnormalities in Tsc1(GFAP)CKO mice: 4 days of rapamycin only (4-∞), 4 days on-24 days off (4-24), and 4 days on-10 days off (4-10). RESULTS: mTOR activity was inhibited by rapamycin in a dose-dependent fashion and recovered to baseline by about 10 days after the last rapamycin dose. The 4-10 and 4-24 dosing paradigms almost completely prevented epilepsy and the 4-10 paradigm inhibited glial proliferation and megalencephaly in Tsc1(GFAP)CKO mice. SIGNIFICANCE: Intermittent dosing of rapamycin, with drug holidays of more than 3 weeks, maintains significant antiepileptogenic properties in mouse models of TSC. These findings have important translational applications in developing mTOR inhibitors as antiepileptogenic agents in TSC patients by minimizing drug exposure and potential side effects.