Asymmetric localization of natural antisense RNA of neuropeptide sensorin in Aplysia sensory neurons during aging and activity

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Beena eKadakkuzha

2014-04-01

Full Text Available Despite the advances in our understanding of transcriptome, regulation and function of its noncoding components continue to be poorly understood. Here we searched for natural antisense transcript for sensorin (NAT-SRN, a neuropeptide expressed in the presynaptic sensory neurons of gill-withdrawal reflex of the marine snail Aplysia californica. Sensorin (SRN has a key role in learning and long-term memory storage in Aplysia. We have identified NAT-SRN in the central nervous system (CNS and have confirmed its expression by northern blotting and fluorescent RNA in situ hybridization. Quantitative analysis of NAT-SRN in micro dissected cell bodies and processes of sensory neurons suggest that NAT-SRN is present in the distal neuronal processes along with sense transcripts. Importantly, aging is associated with reduced levels of NAT-SRN in sensory neuron processes. Furthermore, we find that forskolin, an activator of CREB signaling, differentially alters the distribution of SRN and NAT-SRN. These studies reveal novel insights into physiological regulation of natural antisense RNAs.

Analysis list: CREB1 [Chip-atlas[Archive

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Full Text Available CREB1 Blood,Digestive tract,Liver,Pluripotent stem cell,Prostate,Uterus + hg19 http...://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/CREB1.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/CRE...B1.5.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/CREB1.10.tsv http://dbarchive.b...iosciencedbc.jp/kyushu-u/hg19/colo/CREB1.Blood.tsv,http://dbarchive.biosciencedbc....jp/kyushu-u/hg19/colo/CREB1.Digestive_tract.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/CREB1.

Reversible antisense inhibition of Shaker-like Kv1.1 potassium channel expression impairs associative memory in mouse and rat

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Meiri, Noam; Ghelardini, Carla; Tesco, Giuseppina; Galeotti, Nicoletta; Dahl, Dennis; Tomsic, Daniel; Cavallaro, Sebastiano; Quattrone, Alessandro; Capaccioli, Sergio; Bartolini, Alessandro; Alkon, Daniel L.

1997-01-01

Long-term memory is thought to be subserved by functional remodeling of neuronal circuits. Changes in the weights of existing synapses in networks might depend on voltage-gated potassium currents. We therefore studied the physiological role of potassium channels in memory, concentrating on the Shaker-like Kv1.1, a late rectifying potassium channel that is highly localized within dendrites of hippocampal CA3 pyramidal and dentate gyrus granular cells. Repeated intracerebroventricular injection of antisense oligodeoxyribonucleotide to Kv1.1 reduces expression of its particular intracellular mRNA target, decreases late rectifying K+ current(s) in dentate granule cells, and impairs memory but not other motor or sensory behaviors, in two different learning paradigms, mouse passive avoidance and rat spatial memory. The latter, hippocampal-dependent memory loss occurred in the absence of long-term potentiation changes recorded both from the dentate gyrus or CA1. The specificity of the reversible antisense targeting of mRNA in adult animal brains may avoid irreversible developmental and genetic background effects that accompany transgenic “knockouts”. PMID:9114006

Reversible antisense inhibition of Shaker-like Kv1.1 potassium channel expression impairs associative memory in mouse and rat.

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Meiri, N; Ghelardini, C; Tesco, G; Galeotti, N; Dahl, D; Tomsic, D; Cavallaro, S; Quattrone, A; Capaccioli, S; Bartolini, A; Alkon, D L

1997-04-29

Long-term memory is thought to be subserved by functional remodeling of neuronal circuits. Changes in the weights of existing synapses in networks might depend on voltage-gated potassium currents. We therefore studied the physiological role of potassium channels in memory, concentrating on the Shaker-like Kv1.1, a late rectifying potassium channel that is highly localized within dendrites of hippocampal CA3 pyramidal and dentate gyrus granular cells. Repeated intracerebroventricular injection of antisense oligodeoxyribonucleotide to Kv1.1 reduces expression of its particular intracellular mRNA target, decreases late rectifying K+ current(s) in dentate granule cells, and impairs memory but not other motor or sensory behaviors, in two different learning paradigms, mouse passive avoidance and rat spatial memory. The latter, hippocampal-dependent memory loss occurred in the absence of long-term potentiation changes recorded both from the dentate gyrus or CA1. The specificity of the reversible antisense targeting of mRNA in adult animal brains may avoid irreversible developmental and genetic background effects that accompany transgenic "knockouts".

Electroacupuncture Ameliorates Learning and Memory and Improves Synaptic Plasticity via Activation of the PKA/CREB Signaling Pathway in Cerebral Hypoperfusion

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Cai-Xia Zheng

2016-01-01

Full Text Available Electroacupuncture (EA has shown protective effects on cognitive decline. However, the underlying molecular mechanisms are ill-understood. The present study was undertaken to determine whether the cognitive function was ameliorated in cerebral hypoperfusion rats following EA and to investigate the role of PKA/CREB pathway. We used a rat 2-vessel occlusion (2VO model and delivered EA at Baihui (GV20 and Dazhui (GV14 acupoints. Morris water maze (MWM task, electrophysiological recording, Golgi silver stain, Nissl stain, Western blot, and real-time PCR were employed. EA significantly (1 ameliorated the spatial learning and memory deficits, (2 alleviated long-term potentiation (LTP impairment and the reduction of dendritic spine density, (3 suppressed the decline of phospho-CREB (pCREB protein, brain-derived neurotrophic factor (BDNF protein, and microRNA132 (miR132, and (4 reduced the increase of p250GAP protein of 2VO rats. These changes were partially blocked by a selective protein kinase A (PKA inhibitor, N-[2-(p-bromocinnamylaminoethyl]-5-isoquinoline-sulfonamide (H89, suggesting that the PKA/CREB pathway is potentially involved in the effects of EA. Moreover, any significant damage to the pyramidal cell layer of CA1 subregion was absent. These results demonstrated that EA could ameliorate learning and memory deficits and alleviate hippocampal synaptic plasticity impairment of cerebral hypoperfusion rats, potentially mediated by PKA/CREB signaling pathway.

Early-onset sleep defects in Drosophila models of Huntington's disease reflect alterations of PKA/CREB signaling

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Gonzales, Erin D.; Tanenhaus, Anne K.; Zhang, Jiabin; Chaffee, Ryan P.; Yin, Jerry C.P.

2016-01-01

Huntington's disease (HD) is a progressive neurological disorder whose non-motor symptoms include sleep disturbances. Whether sleep and activity abnormalities are primary molecular disruptions of mutant Huntingtin (mutHtt) expression or result from neurodegeneration is unclear. Here, we report Drosophila models of HD exhibit sleep and activity disruptions very early in adulthood, as soon as sleep patterns have developed. Pan-neuronal expression of full-length or N-terminally truncated mutHtt recapitulates sleep phenotypes of HD patients: impaired sleep initiation, fragmented and diminished sleep, and nighttime hyperactivity. Sleep deprivation of HD model flies results in exacerbated sleep deficits, indicating that homeostatic regulation of sleep is impaired. Elevated PKA/CREB activity in healthy flies produces patterns of sleep and activity similar to those in our HD models. We were curious whether aberrations in PKA/CREB signaling were responsible for our early-onset sleep/activity phenotypes. Decreasing signaling through the cAMP/PKA pathway suppresses mutHtt-induced developmental lethality. Genetically reducing PKA abolishes sleep/activity deficits in HD model flies, restores the homeostatic response and extends median lifespan. In vivo reporters, however, show dCREB2 activity is unchanged, or decreased when sleep/activity patterns are abnormal, suggesting dissociation of PKA and dCREB2 occurs early in pathogenesis. Collectively, our data suggest that sleep defects may reflect a primary pathological process in HD, and that measurements of sleep and cAMP/PKA could be prodromal indicators of disease, and serve as therapeutic targets for intervention. PMID:26604145

Expression profile of CREB knockdown in myeloid leukemia cells

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Pellegrini, Matteo; Cheng, Jerry C; Voutila, Jon; Judelson, Dejah; Taylor, Julie; Nelson, Stanley F; Sakamoto, Kathleen M

2008-01-01

The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, differentiation, and survival in several model systems, including neuronal and hematopoietic cells. We demonstrated that CREB is overexpressed in acute myeloid and leukemia cells compared to normal hematopoietic stem cells. CREB knockdown inhibits leukemic cell proliferation in vitro and in vivo, but does not affect long-term hematopoietic reconstitution. To understand downstream pathways regulating CREB, we performed expression profiling with RNA from the K562 myeloid leukemia cell line transduced with CREB shRNA. By combining our expression data from CREB knockdown cells with prior ChIP data on CREB binding we were able to identify a list of putative CREB regulated genes. We performed extensive analyses on the top genes in this list as high confidence CREB targets. We found that this list is enriched for genes involved in cancer, and unexpectedly, highly enriched for histone genes. Furthermore, histone genes regulated by CREB were more likely to be specifically expressed in hematopoietic lineages. Decreased expression of specific histone genes was validated in K562, TF-1, and primary AML cells transduced with CREB shRNA. We have identified a high confidence list of CREB targets in K562 cells. These genes allow us to begin to understand the mechanisms by which CREB contributes to acute leukemia. We speculate that regulation of histone genes may play an important role by possibly altering the regulation of DNA replication during the cell cycle

Cyclic AMP-Responsive Element-Binding Protein (CREB is Critical in Autoimmunity by Promoting Th17 but Inhibiting Treg Cell Differentiation

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Xiaohu Wang

2017-11-01

Full Text Available The molecular mechanisms that govern differential T cell development into pro-inflammatory Th17 vs. regulatory T (Treg cells remain unclear. Here, we show that selective deletion of CREB in T cells or Th17 cells impaired Th17 cell differentiation in vitro and in vivo, and led to resistance to autoimmune diseases. Mechanistically, CREB, activated by CD3-PKC-ϴ signaling, plays a key role in regulating Th17 cell differentiation, at least in part through directly binding to the Il17-Il17f gene locus. Unexpectedly, although dispensable for FOXP3 expression and for the homeostasis and suppressive function of thymus-derived Treg cells, CREB negatively regulates the survival of TGF-β-induced Treg cells, and deletion of CREB resulted in increased FOXP3+ Treg cells in the intestine and protection in a colitis model. Thus, CREB is critical in autoimmune diseases by promoting Th17 cell and inhibiting de novo Treg cell generation.

GSK-3α Is a Novel Target of CREB and CREB-GSK-3α Signaling Participates in Cell Viability in Lung Cancer.

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Sin-Aye Park

Full Text Available Overexpression or activation of cyclic AMP-response element-binding protein (CREB has been known to be involved in several human malignancies, including lung cancer. Genes regulated by CREB have been reported to suppress apoptosis, induce cell proliferation, inflammation, and tumor metastasis. However, the critical target genes of CREB in lung cancer have not been well understood. Here, we identified GSK-3α as one of the CREB target genes which is critical for the viability of lung cancer cells. The CREB knockdown significantly reduced the expression of GSK-3α and the direct binding of CREB on the promoter of GSK3A was identified. Kaplan-Meier analysis with a public database showed a prognostic significance of aberrant GSK-3α expression in lung cancer. Inhibition of GSK-3α suppressed cell viability, colony formation, and tumor growth. For the first time, we demonstrated that GSK-3α is regulated by CREB in lung cancer and is required for the cell viability. These findings implicate CREB-GSK-3α axis as a novel therapeutic target for lung cancer treatment.

The Traditional Japanese Herbal Medicine Hachimijiogan Elicits Neurite Outgrowth Effects in PC12 Cells and Improves Cognitive in AD Model Rats via Phosphorylation of CREB

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Kaori Kubota

2017-11-01

Full Text Available Hachimijiogan (HJG is a traditional herbal medicine that improves anxiety disorders in patients with dementia. In this study, we tested the hypothesis that HJG exerts neurotrophic factor-like effects to ameliorate memory impairment in Alzheimer disease (AD model rats. First, we describe that HJG acts to induce neurite outgrowth in PC12 cells (a rat pheochromocytoma cell line like nerve growth factor (NGF in a concentration-dependent manner (3 μg/ml HJG, p < 0.05; 10–500 μg/ml HJG, p < 0.001. While six herbal constituents of HJG, Rehmannia root, Dioscorea rhizome, Rhizoma Alismatis, Poria sclerotium, Moutan bark, and Cinnamon bark, could induce neurite outgrowth effects, the effect was strongest with HJG (500 μg/ml. Second, we demonstrated that HJG-induced neurite outgrowth was blocked by an inhibitor of cAMP response element binding protein (CREB, KG-501 (10 μM, p < 0.001. Moreover, HJG was observed to induce CREB phosphorylation 20–90 min after treatment (20 min, 2.50 ± 0.58-fold and CRE-mediated transcription in cultured PC12 cells (500 μg/ml, p < 0.01; 1000 μg/ml, p < 0.001. These results suggest a CREB-dependent mechanism underlies the neurotrophic effects of HJG. Finally, we examined improvements of memory impairment following HJG treatment using a Morris water maze in AD model animals (CI + Aβ rats. Repeated oral administration of HJG improved memory impairment (300 mg/kg, p < 0.05; 1000 mg/kg, p < 0.001 and induced CREB phosphorylation within the hippocampus (1000 mg/kg, p < 0.01. Together, our results suggest that HJG possesses neurotrophic effects similar to those of NGF, and can ameliorate cognitive dysfunction in a rat dementia model via CREB activation. Thus, HJG could potentially be a substitute for neurotrophic factors as a treatment for dementia.

Cyclic adenosine 3',5'-monophosphate (cAMP) enhances cAMP-responsive element binding (CREB) protein phosphorylation and phospho-CREB interaction with the mouse steroidogenic acute regulatory protein gene promoter.

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Clem, Brian F; Hudson, Elizabeth A; Clark, Barbara J

2005-03-01

Steroidogenic acute regulatory protein (StAR) transcription is regulated through cAMP-protein kinase A-dependent mechanisms that involve multiple transcription factors including the cAMP-responsive element binding protein (CREB) family members. Classically, binding of phosphorylated CREB to cis-acting cAMP-responsive elements (5'-TGACGTCA-3') within target gene promoters leads to recruitment of the coactivator CREB binding protein (CBP). Herein we examined the extent of CREB family member phosphorylation on protein-DNA interactions and CBP recruitment with the StAR promoter. Immunoblot analysis revealed that CREB, cAMP-responsive element modulator (CREM), and activating transcription factor (ATF)-1 are expressed in MA-10 mouse Leydig tumor cells, yet only CREB and ATF-1 are phosphorylated. (Bu)2cAMP treatment of MA-10 cells increased CREB phosphorylation approximately 2.3-fold within 30 min but did not change total nuclear CREB expression levels. Using DNA-affinity chromatography, we now show that CREB and ATF-1, but not CREM, interact with the StAR promoter, and this interaction is dependent on the activator protein-1 (AP-1) cis-acting element within the cAMP-responsive region. In addition, (Bu)2cAMP-treatment increased phosphorylated CREB (P-CREB) association with the StAR promoter but did not influence total CREB interaction. In vivo chromatin immunoprecipitation assays demonstrated CREB binding to the StAR proximal promoter is independent of (Bu)2cAMP-treatment, confirming our in vitro analysis. However, (Bu)2cAMP-treatment increased P-CREB and CBP interaction with the StAR promoter, demonstrating for the first time the physical role of P-CREB:DNA interactions in CBP recruitment to the StAR proximal promoter.

Taurine promotes cognitive function in prenatally stressed juvenile rats via activating the Akt-CREB-PGC1α pathway.

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Jia, Ning; Sun, Qinru; Su, Qian; Dang, Shaokang; Chen, Guomin

2016-12-01

Substantial evidence has shown that the oxidative damage to hippocampal neurons is associated with the cognitive impairment induced by adverse stimuli during gestation named prenatal stress (PS). Taurine, a conditionally essential amino acid, possesses multiple roles in the brain as a neuromodulator or antioxidant. In this study, to explore the roles of taurine in PS-induced learning and memory impairment, prenatal restraint stress was set up and Morris water maze (MWM) was employed for testing the cognitive function in the one-month-old rat offspring. The mitochondrial reactive oxygen species (ROS) level，mitochondrial membrane potential (MMP), ATP and cytochrome c oxidase (CcO) activity and apoptosis-related proteins in the hippocampus were detected. The activity of the Akt-cyclic AMP response element-binding protein (CREB)-peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) pathway in the hippocampus was measured. The results showed that high dosage of taurine administration in the early postnatal period attenuated impairment of spatial learning and memory induced by PS. Meanwhile, taurine administration diminished the increase in mitochondrial ROS, and recovered the reduction of MMP, ATP level and the activities of CcO, superoxide dismutase 2 (SOD2) and catalase induced by PS in the hippocampus. In addition, taurine administration recovered PS-suppressed SOD2 expression level. Taurine administration blocked PS-induced decrease in the ratio of Bcl-2/Bax and increase in the ratio of cleaved caspase-3/full-length caspase-3. Notably, taurine inhibited PS-decreased phosphorylation of Akt (pAkt) and phosphorylation of CREB (pCREB), which consequently enhanced the mRNA and protein levels of PGC1α. Taken together, these results suggest that high dosage of taurine administration during the early postnatal period can significantly improve the cognitive function in prenatally stressed juvenile rats via activating the Akt-CREB-PGC1α pathway. Therefore

Taurine promotes cognitive function in prenatally stressed juvenile rats via activating the Akt-CREB-PGC1α pathway

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Ning Jia

2016-12-01

Full Text Available Substantial evidence has shown that the oxidative damage to hippocampal neurons is associated with the cognitive impairment induced by adverse stimuli during gestation named prenatal stress (PS. Taurine, a conditionally essential amino acid, possesses multiple roles in the brain as a neuromodulator or antioxidant. In this study, to explore the roles of taurine in PS-induced learning and memory impairment, prenatal restraint stress was set up and Morris water maze (MWM was employed for testing the cognitive function in the one-month-old rat offspring. The mitochondrial reactive oxygen species (ROS level，mitochondrial membrane potential (MMP, ATP and cytochrome c oxidase (CcO activity and apoptosis-related proteins in the hippocampus were detected. The activity of the Akt-cyclic AMP response element-binding protein (CREB-peroxisome proliferator-activated receptor–γ coactivator-1α (PGC1α pathway in the hippocampus was measured. The results showed that high dosage of taurine administration in the early postnatal period attenuated impairment of spatial learning and memory induced by PS. Meanwhile, taurine administration diminished the increase in mitochondrial ROS, and recovered the reduction of MMP, ATP level and the activities of CcO, superoxide dismutase 2 (SOD2 and catalase induced by PS in the hippocampus. In addition, taurine administration recovered PS-suppressed SOD2 expression level. Taurine administration blocked PS-induced decrease in the ratio of Bcl-2/Bax and increase in the ratio of cleaved caspase-3/full-length caspase-3. Notably, taurine inhibited PS-decreased phosphorylation of Akt (pAkt and phosphorylation of CREB (pCREB, which consequently enhanced the mRNA and protein levels of PGC1α. Taken together, these results suggest that high dosage of taurine administration during the early postnatal period can significantly improve the cognitive function in prenatally stressed juvenile rats via activating the Akt-CREB-PGC1�

Viral Vector Induction of CREB Expression in the Periaqueductal Gray Induces a Predator Stress-Like Pattern of Changes in pCREB Expression, Neuroplasticity, and Anxiety in Rodents

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Robert Adamec

2009-01-01

Full Text Available Predator stress is lastingly anxiogenic. Phosphorylation of CREB to pCREB (phosphorylated cyclic AMP response element binding protein is increased after predator stress in fear circuitry, including in the right lateral column of the PAG (periaqueductal gray. Predator stress also potentiates right but not left CeA-PAG (central amygdala-PAG transmission up to 12 days after stress. The present study explored the functional significance of pCREB changes by increasing CREB expression in non-predator stressed rats through viral vectoring, and assessing the behavioral, electrophysiological and pCREB expression changes in comparison with handled and predator stressed controls. Increasing CREB expression in right PAG was anxiogenic in the elevated plus maze, had no effect on risk assessment, and increased acoustic startle response while delaying startle habituation. Potentiation of the right but not left CeA-PAG pathway was also observed. pCREB expression was slightly elevated in the right lateral column of the PAG, while the dorsal and ventral columns were not affected. The findings of this study suggest that by increasing CREB and pCREB in the right lateral PAG, it is possible to produce rats that exhibit behavioral, brain, and molecular changes that closely resemble those seen in predator stressed rats.

Regulation of anxiety and initiation of sexual behavior by CREB in the nucleus accumbens

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Barrot, Michel; Wallace, Deanna L.; Bolaños, Carlos A.; Graham, Danielle L.; Perrotti, Linda I.; Neve, Rachael L.; Chambliss, Heather; Yin, Jerry C.; Nestler, Eric J.

2005-01-01

Sexual deficits and other behavioral disturbances such as anxiety-like behaviors can be observed in animals that have undergone social isolation, especially in species having important social interactions. Using a model of protracted social isolation in adult rats, we observed increased anxiety-like behavior and deficits in both the latency to initiate sexual behavior and the latency to ejaculate. We show, using transgenic cAMP response element (CRE)-LacZ reporter mice, that protracted social isolation also reduces CRE-dependent transcription within the nucleus accumbens. This decrease in CRE-dependent transcription can be mimicked in nonisolated animals by local viral gene transfer of a dominant negative mutant of CRE-binding protein (CREB). We previously showed that this manipulation increases anxiety-like behavior. We show here that it also impairs initiation of sexual behavior in nonisolated animals, a deficit that can be corrected by anxiolytic drug treatment. This local reduction in CREB activity, however, has no influence on ejaculation parameters. Reciprocally, we used the viral transgenic approach to overexpress CREB in the nucleus accumbens of isolated animals. We show that this local increase in CREB activity completely rescued the anxiety phenotype of the isolated animals, as well as their deficit in initiating sexual behavior, but failed to rescue the deficit in ejaculation. Our data suggest a role for the nucleus accumbens in anxiety responses and in specific aspects of sexual behavior. The results also provide insight into the molecular mechanisms by which social interactions affect brain plasticity and behavior. PMID:15923261

Impairment of object recognition memory by maternal bisphenol A exposure is associated with inhibition of Akt and ERK/CREB/BDNF pathway in the male offspring hippocampus.

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Wang, Chong; Li, Zhihui; Han, Haijun; Luo, Guangying; Zhou, Bingrui; Wang, Shaolin; Wang, Jundong

2016-02-03

Bisphenol A (BPA) is a commonly used endocrine-disrupting chemical used as a component of polycarbonates plastics that has potential adverse effects on human health. Exposure to BPA during development has been implicated in memory deficits, but the mechanism of action underlying the effect is not fully understood. In this study, we investigated the effect of maternal exposure to BPA on object recognition memory and the expressions of proteins important for memory, especially focusing on the ERK/CREB/BDNF pathway. Pregnant Sprague-Dawley female rats were orally treated with either vehicle or BPA (0.05, 0.5, 5 or 50 mg/kg BW/day) during days 9-20 of gestation. Male offspring were tested on postnatal day 21 with the object recognition task. Recognition memory was assessed using the object recognition index (index=the time spent exploring the novel object/(the time spent exploring the novel object+the time spent exploring the familiar object)). In the test session performed 90 min after the training session, BPA-exposed male offspring not only spent more time in exploring the familiar object at the highest dose than the control, but also displayed a significantly decreased the object recognition index at the doses of 0.5, 5 and 50 mg/kg BW/day. During the test session performed 24h after the training session, BPA-treated males did not change the time spent exploring the familiar object, but had a decreased object recognition index at 5 and 50 mg/kg BW/day, when compared to control group. These findings indicate that object recognition memory was susceptible to maternal BPA exposure. Western blot analysis of hippocampi from BPA-treated male offspring revealed a decrease in Akt, phospho-Akt, p44/42 MAPK and phospho-p44/42 MAPK protein levels, compared to controls. In addition, BPA significantly inhibited the levels of phosphorylation of CREB and BDNF in the hippocampus. Our results show that maternal BPA exposure may full impair object recognition memory, and that

Knock-in Luciferase Reporter Mice for In Vivo Monitoring of CREB Activity.

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Dmitry Akhmedov

Full Text Available The cAMP response element binding protein (CREB is induced during fasting in the liver, where it stimulates transcription of rate-limiting gluconeogenic genes to maintain metabolic homeostasis. Adenoviral and transgenic CREB reporters have been used to monitor hepatic CREB activity non-invasively using bioluminescence reporter imaging. However, adenoviral vectors and randomly inserted transgenes have several limitations. To overcome disadvantages of the currently used strategies, we created a ROSA26 knock-in CREB reporter mouse line (ROSA26-CRE-luc. cAMP-inducing ligands stimulate the reporter in primary hepatocytes and myocytes from ROSA26-CRE-luc animals. In vivo, these animals exhibit little hepatic CREB activity in the ad libitum fed state but robust induction after fasting. Strikingly, CREB was markedly stimulated in liver, but not in skeletal muscle, after overnight voluntary wheel-running exercise, uncovering differential regulation of CREB in these tissues under catabolic states. The ROSA26-CRE-luc mouse line is a useful resource to study dynamics of CREB activity longitudinally in vivo and can be used as a source of primary cells for analysis of CREB regulatory pathways ex vivo.

cAMP/PKA-CREB-BDNF signaling pathway in hippocampus mediates cyclooxygenase 2-induced learning/memory deficits of rats subjected to chronic unpredictable mild stress.

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Luo, Ying; Kuang, Shengnan; Li, Huan; Ran, Dongzhi; Yang, Junqing

2017-05-30

To investigate the mechanism of cyclooxygenase 2 (COX2) in learning and memory impairments in rats subjected to chronic unpredictable mild stress (CUMS), meloxicam was used intragastrically to inhibit the activity of cyclooxygenase 2. Moreover, cyclooxygenase 2 over-expressing or RNA interfere lentivirus was injected intraventricularly to increase or decrease the enzyme's expression, respectively. The body weights and sucrose consumption were used to analyze depressive behaviors, while the Morris water maze and step-down-type passive avoidance tests were carried out to evaluate the learning-memory functions. The levels of inflammatory cytokines were measured to estimate inflammation and the contents of cyclic adenosine monophosphate (cAMP) were used to measure the levels of the second messenger. Changes in cyclooxygenase 2 mRNA levels were analyzed using reverse transcription polymerase chain reaction. Moreover, the expression of cyclooxygenase 2, brain-derived neurotrophic factor (BDNF), prostaglandins receptor 3 (EP3), protein kinase A (PKA), cAMP response element binding protein (CREB), and phosphorylated CREB were estimated using immunohistochemical staining or western blotting. The results showed that CUMS led to significant depressive-like behaviors and learning and memory dysfunctions. Also, the cAMP levels decreased significantly, while levels of inflammatory cytokines and prostaglandins E2 increased significantly. The expressions of PKA, BDNF, phosphorylated CREB/CREB declined and cyclooxygenase 2 was increased. Meloxicam and cyclooxygenase 2 RNA interfere lentivirus reversed the changes caused by CUMS while cyclooxygenase 2-overexpressing lentivirus worsened these abnormalities. The findings also showed that CUMS increased cyclooxygenase 2 expression, which can cause learning and memory impairments, mainly through activating the hippocampal neuronal cAMP/PKA-CREB-BDNF signaling pathways.

CREB regulates spine density of lateral amygdala neurons: implications for memory allocation

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Derya eSargin

2013-12-01

Full Text Available Neurons may compete against one another for integration into a memory trace. Specifically, neurons in the lateral nucleus of the amygdala with relatively higher levels of CREB seem to be preferentially allocated to a fear memory trace, while neurons with relatively decreased CREB function seem to be excluded from a fear memory trace. CREB is a ubiquitous transcription factor that modulates many diverse cellular processes, raising the question as to which of these CREB-mediated processes underlie memory allocation. CREB is implicated in modulating dendritic spine number and morphology. As dendritic spines are intimately involved in memory formation, we investigated whether manipulations of CREB function alter spine number or morphology of neurons at the time of fear conditioning. We used viral vectors to manipulate CREB function in the lateral amygdala principal neurons in mice maintained in their homecages. At the time that fear conditioning normally occurs, we observed that neurons with high levels of CREB had more dendritic spines, while neurons with low CREB function had relatively fewer spines compared to control neurons. These results suggest that the modulation of spine density provides a potential mechanism for preferential allocation of a subset of neurons to the memory trace.

Sleep deprivation prevents stimulation-induced increases of levels of P-CREB and BDNF: protection by caffeine.

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Alhaider, Ibrahim A; Aleisa, Abdulaziz M; Tran, Trinh T; Alkadhi, Karim A

2011-04-01

It is well known that caffeine and sleep deprivation have opposing effects on learning and memory; therefore, this study was undertaken to determine the effects of chronic (4wks) caffeine treatment (0.3g/l in drinking water) on long-term memory deficit associated with 24h sleep deprivation. Animals were sleep deprived using the modified multiple platform method. The results showed that chronic caffeine treatment prevented the impairment of long-term memory as measured by performance in the radial arm water maze task and normalized L-LTP in area CA1 of the hippocampi of sleep-deprived anesthetized rats. Sleep deprivation prevents the high frequency stimulation-induced increases in the levels of phosphorylated-cAMP response element binding protein (P-CREB) and brain-derived neurotrophic factor (BDNF) seen during the expression of late phase long-term potentiation (L-LTP). However, chronic caffeine treatment prevented the effect of sleep-deprivation on the stimulated levels of P-CREB and BDNF. The results suggest that chronic caffeine treatment may protect the sleep-deprived brain probably by preserving the levels of P-CREB and BDNF. Copyright © 2011 Elsevier Inc. All rights reserved.

Dopamine modulates acetylcholine release via octopamine and CREB signaling in Caenorhabditis elegans.

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Satoshi Suo

Full Text Available Animals change their behavior and metabolism in response to external stimuli. cAMP response element binding protein (CREB is a signal-activated transcription factor that enables the coupling of extracellular signals and gene expression to induce adaptive changes. Biogenic amine neurotransmitters regulate CREB and such regulation is important for long-term changes in various nervous system functions, including learning and drug addiction. In Caenorhabditis elegans, the amine neurotransmitter octopamine activates a CREB homolog, CRH-1, in cholinergic SIA neurons, whereas dopamine suppresses CREB activation by inhibiting octopamine signaling in response to food stimuli. However, the physiological role of this activation is unknown. In this study, the effect of dopamine, octopamine, and CREB on acetylcholine signaling was analyzed using the acetylcholinesterase inhibitor aldicarb. Mutants with decreased dopamine signaling exhibited reduced acetylcholine signaling, and octopamine and CREB functioned downstream of dopamine in this regulation. This study demonstrates that the regulation of CREB by amine neurotransmitters modulates acetylcholine release from the neurons of C. elegans.

Upregulation of CREB-mediated transcription enhances both short- and long-term memory.

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Suzuki, Akinobu; Fukushima, Hotaka; Mukawa, Takuya; Toyoda, Hiroki; Wu, Long-Jun; Zhao, Ming-Gao; Xu, Hui; Shang, Yuze; Endoh, Kengo; Iwamoto, Taku; Mamiya, Nori; Okano, Emiko; Hasegawa, Shunsuke; Mercaldo, Valentina; Zhang, Yue; Maeda, Ryouta; Ohta, Miho; Josselyn, Sheena A; Zhuo, Min; Kida, Satoshi

2011-06-15

Unraveling the mechanisms by which the molecular manipulation of genes of interest enhances cognitive function is important to establish genetic therapies for cognitive disorders. Although CREB is thought to positively regulate formation of long-term memory (LTM), gain-of-function effects of CREB remain poorly understood, especially at the behavioral level. To address this, we generated four lines of transgenic mice expressing dominant active CREB mutants (CREB-Y134F or CREB-DIEDML) in the forebrain that exhibited moderate upregulation of CREB activity. These transgenic lines improved not only LTM but also long-lasting long-term potentiation in the CA1 area in the hippocampus. However, we also observed enhanced short-term memory (STM) in contextual fear-conditioning and social recognition tasks. Enhanced LTM and STM could be dissociated behaviorally in these four lines of transgenic mice, suggesting that the underlying mechanism for enhanced STM and LTM are distinct. LTM enhancement seems to be attributable to the improvement of memory consolidation by the upregulation of CREB transcriptional activity, whereas higher basal levels of BDNF, a CREB target gene, predicted enhanced shorter-term memory. The importance of BDNF in STM was verified by microinfusing BDNF or BDNF inhibitors into the hippocampus of wild-type or transgenic mice. Additionally, increasing BDNF further enhanced LTM in one of the lines of transgenic mice that displayed a normal BDNF level but enhanced LTM, suggesting that upregulation of BDNF and CREB activity cooperatively enhances LTM formation. Our findings suggest that CREB positively regulates memory consolidation and affects memory performance by regulating BDNF expression.

Learning-dependent gene expression of CREB1 isoforms in the molluscan brain

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Hisayo Sadamoto

2010-05-01

Full Text Available Cyclic AMP-responsive element binding protein1 (CREB1 has multiple functions in gene regulation. Various studies have reported that CREB1-dependent gene induction is necessary for memory formation and long-lasting behavioral changes in both vertebrates and invertebrates. In the present study, we characterized Lymnaea CREB1 (LymCREB1 mRNA isoforms of spliced variants in the central nervous system (CNS of the pond snail Lymnaea stagnalis. Among these spliced variants, the three isoforms that code a whole LymCREB1 protein are considered to be the activators for gene regulation. The other four isoforms, which code truncated LymCREB1 proteins with no kinase inducible domain, are the repressors. For a better understanding of the possible roles of different LymCREB1 isoforms, the expression level of these isoform mRNAs was investigated by a real-time quantitative RT-PCR method. Further, we examined the changes in gene expression for all the isoforms in the CNS after conditioned taste aversion (CTA learning or backward conditioning as a control. The results showed that CTA learning increased LymCREB1 gene expression, but it did not change the activator/repressor ratio. Our findings showed that the repressor isoforms, as well as the activator ones, are expressed in large amounts in the CNS, and the gene expression of CREB1 isoforms appeared to be specific for the given stimulus. This was the first quantitative analysis of the expression patterns of CREB1 isoforms at the mRNA level and their association with learning behavior.

Ethanol sensitivity: a central role for CREB transcription regulation in the cerebellum

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Biswal Shyam

2006-12-01

Full Text Available Abstract Background Lowered sensitivity to the effects of ethanol increases the risk of developing alcoholism. Inbred mouse strains have been useful for the study of the genetic basis of various drug addiction-related phenotypes. Inbred Long-Sleep (ILS and Inbred Short-Sleep (ISS mice differentially express a number of genes thought to be implicated in sensitivity to the effects of ethanol. Concomitantly, there is evidence for a mediating role of cAMP/PKA/CREB signalling in aspects of alcoholism modelled in animals. In this report, the extent to which CREB signalling impacts the differential expression of genes in ILS and ISS mouse cerebella is examined. Results A training dataset for Machine Learning (ML and Exploratory Data Analyses (EDA was generated from promoter region sequences of a set of genes known to be targets of CREB transcription regulation and a set of genes whose transcription regulations are potentially CREB-independent. For each promoter sequence, a vector of size 132, with elements characterizing nucleotide composition features was generated. Genes whose expressions have been previously determined to be increased in ILS or ISS cerebella were identified, and their CREB regulation status predicted using the ML scheme C4.5. The C4.5 learning scheme was used because, of four ML schemes evaluated, it had the lowest predicted error rate. On an independent evaluation set of 21 genes of known CREB regulation status, C4.5 correctly classified 81% of instances with F-measures of 0.87 and 0.67 respectively for the CREB-regulated and CREB-independent classes. Additionally, six out of eight genes previously determined by two independent microarray platforms to be up-regulated in the ILS or ISS cerebellum were predicted by C4.5 to be transcriptionally regulated by CREB. Furthermore, 64% and 52% of a cross-section of other up-regulated cerebellar genes in ILS and ISS mice, respectively, were deemed to be CREB-regulated. Conclusion These

Rescue of cAMP response element-binding protein signaling reversed spatial memory retention impairments induced by subanesthetic dose of propofol.

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Zhang, Hao; Zhang, Shao-Bo; Zhang, Qing-Qing; Liu, Meng; He, Xing-Ying; Zou, Zui; Sun, Hai-Jing; You, Zhen-Dong; Shi, Xue-Yin

2013-07-01

The intravenous anesthetic propofol caused episodic memory impairments in human. We hypothesized propofol caused episodic-like spatial memory retention but not acquisition impairments in rats and rescuing cAMP response element-binding protein (CREB) signaling using selective type IV phosphodiesterase (PDEIV) inhibitor rolipram reversed these effects. Male Sprague-Dawley rats were randomized into four groups: control; propofol (25 mg/kg, intraperitoneal); rolipram; and rolipram + propofol (pretreatment of rolipram 25 min before propofol, 0.3 mg/kg, intraperitoneal). Sedation and motor coordination were evaluated 5, 15, and 25 min after propofol injection. Invisible Morris water maze (MWM) acquisition and probe test (memory retention) were performed 5 min and 24 h after propofol injection. Visible MWM training was simultaneously performed to resist nonspatial effects. Hippocampal CREB signaling was detected 5 min, 50 min, and 24 h after propofol administration. Rolipram did not change propofol-induced anesthetic/sedative states or impair motor skills. No difference was found on the latency to the platform during the visible MWM. Propofol impaired spatial memory retention but not acquisition. Rolipram reversed propofol-induced spatial memory impairments and suppression on cAMP levels, CaMKIIα and CREB phosphorylation, brain-derived neurotropic factor (BDNF) and Arc protein expression. Propofol caused spatial memory retention impairments but not acquisition inability possibly by inhibiting CREB signaling. © 2013 John Wiley & Sons Ltd.

Identification of sequence motifs significantly associated with antisense activity

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Peek Andrew S

2007-06-01

Full Text Available Abstract Background Predicting the suppression activity of antisense oligonucleotide sequences is the main goal of the rational design of nucleic acids. To create an effective predictive model, it is important to know what properties of an oligonucleotide sequence associate significantly with antisense activity. Also, for the model to be efficient we must know what properties do not associate significantly and can be omitted from the model. This paper will discuss the results of a randomization procedure to find motifs that associate significantly with either high or low antisense suppression activity, analysis of their properties, as well as the results of support vector machine modelling using these significant motifs as features. Results We discovered 155 motifs that associate significantly with high antisense suppression activity and 202 motifs that associate significantly with low suppression activity. The motifs range in length from 2 to 5 bases, contain several motifs that have been previously discovered as associating highly with antisense activity, and have thermodynamic properties consistent with previous work associating thermodynamic properties of sequences with their antisense activity. Statistical analysis revealed no correlation between a motif's position within an antisense sequence and that sequences antisense activity. Also, many significant motifs existed as subwords of other significant motifs. Support vector regression experiments indicated that the feature set of significant motifs increased correlation compared to all possible motifs as well as several subsets of the significant motifs. Conclusion The thermodynamic properties of the significantly associated motifs support existing data correlating the thermodynamic properties of the antisense oligonucleotide with antisense efficiency, reinforcing our hypothesis that antisense suppression is strongly associated with probe/target thermodynamics, as there are no enzymatic

Regulation of Energy Stores and Feeding by Neuronal and Peripheral CREB Activity in Drosophila

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Iijima, Koichi; Zhao, LiJuan; Shenton, Christopher; Iijima-Ando, Kanae

2009-01-01

The cAMP-responsive transcription factor CREB functions in adipose tissue and liver to regulate glycogen and lipid metabolism in mammals. While Drosophila has a homolog of mammalian CREB, dCREB2, its role in energy metabolism is not fully understood. Using tissue-specific expression of a dominant-negative form of CREB (DN-CREB), we have examined the effect of blocking CREB activity in neurons and in the fat body, the primary energy storage depot with functions of adipose tissue and the liver in flies, on energy balance, stress resistance and feeding behavior. We found that disruption of CREB function in neurons reduced glycogen and lipid stores and increased sensitivity to starvation. Expression of DN-CREB in the fat body also reduced glycogen levels, while it did not affect starvation sensitivity, presumably due to increased lipid levels in these flies. Interestingly, blocking CREB activity in the fat body increased food intake. These flies did not show a significant change in overall body size, suggesting that disruption of CREB activity in the fat body caused an obese-like phenotype. Using a transgenic CRE-luciferase reporter, we further demonstrated that disruption of the adipokinetic hormone receptor, which is functionally related to mammalian glucagon and β-adrenergic signaling, in the fat body reduced CRE-mediated transcription in flies. This study demonstrates that CREB activity in either neuronal or peripheral tissues regulates energy balance in Drosophila, and that the key signaling pathway regulating CREB activity in peripheral tissue is evolutionarily conserved. PMID:20041126

CREB Regulates Experience-Dependent Spine Formation and Enlargement in Mouse Barrel Cortex

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Annabella Pignataro

2015-01-01

Full Text Available Experience modifies synaptic connectivity through processes that involve dendritic spine rearrangements in neuronal circuits. Although cAMP response element binding protein (CREB has a key function in spines changes, its role in activity-dependent rearrangements in brain regions of rodents interacting with the surrounding environment has received little attention so far. Here we studied the effects of vibrissae trimming, a widely used model of sensory deprivation-induced cortical plasticity, on processes associated with dendritic spine rearrangements in the barrel cortex of a transgenic mouse model of CREB downregulation (mCREB mice. We found that sensory deprivation through prolonged whisker trimming leads to an increased number of thin spines in the layer V of related barrel cortex (Contra in wild type but not mCREB mice. In the barrel field controlling spared whiskers (Ipsi, the same trimming protocol results in a CREB-dependent enlargement of dendritic spines. Last, we demonstrated that CREB regulates structural rearrangements of synapses that associate with dynamic changes of dendritic spines. Our findings suggest that CREB plays a key role in dendritic spine dynamics and synaptic circuits rearrangements that account for new brain connectivity in response to changes in the environment.

Sweet Dream Liquid Chinese Medicine Ameliorates Learning and Memory Deficit in a Rat Model of Paradoxical Sleep Deprivation through the ERK/CREB Signaling Pathway.

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Su, Xinyun; Wang, Chunhua; Wang, Xiuhua; Han, Fang; Lv, Changjun; Zhang, Xiuli

2016-05-01

Sweet dream oral liquid (SDOL), a traditional Chinese herbal compound contains 17 traditional Chinese medicines. It has various pharmacological effects, such as improving brain dysfunction and increasing sleeping quality. This study investigated the neuroprotective effect and the underlying mechanisms of SDOL-impaired hippocampus learning and memory-induced paradoxical sleep deprivation (PSD) in rats. Sixty Male Wistar rats were randomly divided into six groups. Before PSD, SDOL treatment group rats were intragastrically administered SDOL for 25 days at dose of 2.1, 4.2, and 8.4 mL/kg body weight per day. Normal control group, large platform control group, and PSD groups were treated with normal saline instead of SDOL. After 25 days treatment, PSD and SDOL groups were deprived of paradoxical sleep for 72 h. Then two behavioral studies were conducted to test the spatial learning and memory ability using the open field test and Morris water maze test. Expression of the c-fos, c-jun, cyclic AMP response element binding protein (CREB), extracellular signal-regulated protein kinase (ERK), mitogen-activated protein kinases (MAPK)/ERK kinase (MEK), and p-CREB, p-ERK, and p-MEK in the hippocampus were also assayed by western blot. In this study, PSD decreased the levels of p-CREB, p-ERK, p-MEK, c-fos, and c-jun. However, SDOL treatment increased expressions of these proteins. Our results showed that SDOL improved 72-h PSD-induced cognitive impairment. These affects may be mediated by increasing the contents of c-fos, c-jun, and p-CREB/ERK signaling.

Appetitive Pavlovian conditioned stimuli increase CREB phosphorylation in the nucleus accumbens.

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Shiflett, Michael W; Mauna, Jocelyn C; Chipman, Amanda M; Peet, Eloise; Thiels, Edda

2009-10-01

The transcription factor cAMP response element-binding protein (CREB) in the nucleus accumbens (NAc) has been shown to regulate an animal's behavioral responsiveness to emotionally salient stimuli, and an increase in CREB phosphorylation in the NAc has been observed during exposure to rewarding stimuli, such as drugs of abuse. Here we show that CREB phosphorylation increases in the NAc also during exposure to cues that an animal has associated with delivery of natural rewards. Adult male Sprague-Dawley rats (rattus norvegicus) were trained to associate an auditory stimulus with delivery of food pellets, and CREB phosphorylation was examined in the striatum following training. We found that repeated tone-food pairings resulted in an increase in CREB phosphorylation in the NAc but not in the adjacent dorsal striatum or in the NAc 3h after the final training session. We further found that the cue itself, as opposed to the food pellets, the training context, or tone-food pairings, was sufficient to increase CREB phosphorylation in the NAc. These results suggest that the processing of primary rewarding stimuli and of environmental cues that predict them triggers similar accumbal signaling mechanisms.

Distinctive Roles for Amygdalar CREB in Reconsolidation and Extinction of Fear Memory

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Tronson, Natalie C.; Wiseman, Shari L.; Neve, Rachael L.; Nestler, Eric J.; Olausson, Peter; Taylor, Jane R.

2012-01-01

Cyclic AMP response element binding protein (CREB) plays a critical role in fear memory formation. Here we determined the role of CREB selectively within the amygdala in reconsolidation and extinction of auditory fear. Viral overexpression of the inducible cAMP early repressor (ICER) or the dominant-negative mCREB, specifically within the lateral…

Involvement of phosphorylated Apis mellifera CREB in gating a honeybee's behavioral response to an external stimulus

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Gehring, Katrin B.; Heufelder, Karin; Feige, Janina; Bauer, Paul; Dyck, Yan; Ehrhardt, Lea; Kühnemund, Johannes; Bergmann, Anja; Göbel, Josefine; Isecke, Marlene

2016-01-01

The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees (Apis mellifera) we recently demonstrated a particular high abundance of the phosphorylated honeybee CREB homolog (pAmCREB) in the central brain and in a subpopulation of mushroom body neurons. We hypothesize that these high pAmCREB levels are related to learning and memory formation. Here, we tested this hypothesis by analyzing brain pAmCREB levels in classically conditioned bees and bees experiencing unpaired presentations of conditioned stimulus (CS) and unconditioned stimulus (US). We demonstrate that both behavioral protocols display differences in memory formation but do not alter the level of pAmCREB in bee brains directly after training. Nevertheless, we report that bees responding to the CS during unpaired stimulus presentations exhibit higher levels of pAmCREB than nonresponding bees. In addition, Trichostatin A, a histone deacetylase inhibitor that is thought to enhance histone acetylation by CREB-binding protein, increases the bees’ CS responsiveness. We conclude that pAmCREB is involved in gating a bee's behavioral response driven by an external stimulus. PMID:27084927

IGF-1 protects cardiac myocytes from hyperosmotic stress-induced apoptosis via CREB

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Maldonado, Carola; Cea, Paola; Adasme, Tatiana; Collao, Andres; Diaz-Araya, Guillermo; Chiong, Mario; Lavandero, Sergio

2005-01-01

Hyperosmotic stress stimulates a rapid and pronounced apoptosis in cardiac myocytes which is attenuated by insulin-like growth factor-1 (IGF-1). Because in these cells IGF-1 induces intracellular Ca 2+ increase, we assessed whether the cyclic AMP response element-binding protein (CREB) is activated by IGF-1 through Ca 2+ -dependent signalling pathways. In cultured cardiac myocytes, IGF-1 induced phosphorylation (6.5 ± 1.0-fold at 5 min), nuclear translocation (30 min post-stimulus) and DNA binding activity of CREB. IGF-1-induced CREB phosphorylation was mediated by MEK1/ERK, PI3-K, p38-MAPK, as well as Ca 2+ /calmodulin kinase and calcineurin. Exposure of cardiac myocytes to hyperosmotic stress (sorbitol 600 mOsm) decreased IGF-1-induced CREB activation Moreover, overexpression of a dominant negative CREB abolished the anti-apoptotic effects of IGF-1. Our results suggest that IGF-1 activates CREB through a complex signalling pathway, and this transcription factor plays an important role in the anti-apoptotic action of IGF-1 in cultured cardiac myocytes

Identification of CREB3L1 as a Biomarker Predicting Doxorubicin Treatment Outcome.

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Bray Denard

Full Text Available Doxorubicin has been shown to inhibit proliferation of cancer cells through proteolytic activation of CREB3L1 (cAMP response element binding protein 3-like 1, a transcription factor synthesized as a membrane-bound precursor. Upon doxorubicin treatment, CREB3L1 is cleaved so that the N-terminal domain of the protein can reach the nucleus where it activates transcription of genes that inhibit cell proliferation. These results suggest that the level of CREB3L1 in cancer cells may determine their sensitivity to doxorubicin.Mice transplanted with 6 lines of renal cell carcinoma (RCC were injected with doxorubicin to observe the effect of the chemotherapy on tumor growth. Immunohistochemistry and bioinformatics analyses were performed to compare CREB3L1 levels in types of cancer known to respond to doxorubicin versus those resistant to doxorubicin.Higher levels of CREB3L1 protein are correlated with increased doxorubicin sensitivity of xenograft RCC tumors (p = 0.017 by Pearson analysis. From patient tumor biopsies we analyzed, CREB3L1 was expressed in 19% of RCC, which is generally resistant to doxorubicin, but in 70% of diffuse large B-cell lymphoma that is sensitive to doxorubicin. Doxorubicin is used as the standard treatment for cancers that express the highest levels of CREB3L1 such as osteosarcoma and malignant fibrous histiocytoma but is not generally used to treat those that express the lowest levels of CREB3L1 such as RCC.Identification of CREB3L1 as the biomarker for doxorubicin sensitivity may markedly improve the doxorubicin response rate by applying doxorubicin only to patients with cancers expressing CREB3L1.

O-GlcNAcylation modulates PKA-CREB signaling in a manner specific to PKA catalytic subunit isoforms.

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Jin, Nana; Ma, Denglei; Gu, Jianlan; Shi, Jianhua; Xu, Xiaotao; Iqbal, Khalid; Gong, Cheng-Xin; Liu, Fei; Chu, Dandan

2018-02-26

O-GlcNAcylation is a post-translational modification of proteins. Protein kinase A (PKA)-cAMP response element binding protein (CREB) signaling plays critical roles in multiple biological processes. Isoforms α and β of PKA catalytic subunit (PKAc) and CREB are modified by O-GlcNAcylation. In the present study, we determined the role of O-GlcNAcylation in PKAc isoform-specific CREB signaling. We found that up-regulation of O-GlcNAcylation enhanced CREB phosphorylation, but suppressed CREB expression in exogenous PKAc isoform-unspecific manner. PKAc isoforms affected exogenous expression of OGT or OGA and protein O-GlcNAcylation differently. Up-regulation of O-GlcNAcylation did not significantly affect net PKAcα-CREB signaling, but enhanced PKAcβ-CREB signaling. The role of O-GlcNAcylation in PKA-CREB signaling was desensitized by insulin treatment. This study suggests a role of O-GlcNAcylation in PKA-CREB signaling by affecting phosphorylation of CREB in a PKAc isoform-specific manner. Copyright © 2018 Elsevier Inc. All rights reserved.

Egr-1 antisense oligodeoxynucleotide administration into the olfactory bulb impairs olfactory learning in the greater short-nosed fruit bat Cynopterus sphinx.

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Ganesh, Ambigapathy; Bogdanowicz, Wieslaw; Balamurugan, Krishnaswamy; Ragu Varman, Durairaj; Rajan, Koilmani Emmanuvel

2012-08-30

Postsynaptic densities (PSDs) contain proteins that regulate synaptic transmission. We examined two important examples of these, calcium/calmodulin-dependent protein kinase II (CaMKII) and PSD-95, in regard to the functional role of early growth response gene-1 (egr-1) in regulation of olfactory learning in the greater short-nosed fruit bat Cynopterus sphinx (family Pteropodidae). To test whether activation of egr-1 in the olfactory bulb (OB) is required for olfactory memory of these bats, bilaterally canulated individuals were infused with antisense (AS) or non-sense (NS)-oligodeoxynucleotides (ODN) of egr-1, or with phosphate buffer saline (PBS), 2h before the olfactory training. Our results showed that behavioral training significantly up-regulates immediate early gene (IEG) EGR-1 and key synaptic proteins Synaptotagmin-1(SYT-1), CaMKII and PSD-95, and phosphorylation of CaMKII in the OB at the protein level per se. Subsequently, we observed that egr-1 antisense-ODN infusion in the OB impaired olfactory memory and down regulates the expression of CaMKII and PSD-95, and the phosphorylation of CaMKII but not SYT-1. In contrast, NS-ODN or PBS had no effect on the expression of the PSDs CaMKII or PSD-95, or on the phosphorylation of CaMKII. When the egr-1 NS-ODN was infused in the OB after training for the novel odor there was no effect on olfactory memory. These findings suggest that egr-1 control the activation of CaMKII and PSD-95 during the process of olfactory memory formation. Copyright © 2012 Elsevier B.V. All rights reserved.

Elevation of endogenous anandamide impairs LTP, learning, and memory through CB1 receptor signaling in mice.

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Basavarajappa, Balapal S; Nagre, Nagaraja N; Xie, Shan; Subbanna, Shivakumar

2014-07-01

In rodents, many exogenous and endogenous cannabinoids, such as anandamide (AEA) and 2-arachidonyl glycerol (2-AG), have been shown to play an important role in certain hippocampal memory processes. However, the mechanisms by which endogenous AEA regulate this processes are not well understood. Here the effects of AEA on long-term potentiation (LTP), hippocampal-dependent learning and memory tasks, pERK1/2, pCaMKIV, and pCREB signaling events in both cannabinoid receptor type 1 (CB1R) wild-type (WT) and knockout (KO) mice were assessed following administration of URB597, an inhibitor of the fatty acid amide hydrolase (FAAH). Acute administration of URB597 enhanced AEA levels without affecting the levels of 2-AG or CB1R in the hippocampus and neocortex as compared to vehicle. In hippocampal slices, URB597 impaired LTP in CB1R WT but not in KO littermates. URB597 impaired object recognition, spontaneous alternation and spatial memory in the Y-maze test in CB1R WT mice but not in KO mice. Furthermore, URB597 enhanced ERK phosphorylation in WT without affecting total ERK levels in WT or KO mice. URB597 impaired CaMKIV and CREB phosphorylation in WT but not in KO mice. CB1R KO mice have a lower pCaMKIV/CaMKIV ratio and higher pCREB/CREB ratio as compared to WT littermates. Our results indicate that pharmacologically elevated AEA impair LTP, learning and memory and inhibit CaMKIV and CREB phosphorylation, via the activation of CB1Rs. Collectively, these findings also suggest that pharmacological elevation of AEA beyond normal concentrations is also detrimental for the underlying physiological responses. © 2014 Wiley Periodicals, Inc.

Golgi Outpost Synthesis Impaired by Toxic Polyglutamine Proteins Contributes to Dendritic Pathology in Neurons

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Chang Geon Chung

2017-07-01

Full Text Available Dendrite aberration is a common feature of neurodegenerative diseases caused by protein toxicity, but the underlying mechanisms remain largely elusive. Here, we show that nuclear polyglutamine (polyQ toxicity resulted in defective terminal dendrite elongation accompanied by a loss of Golgi outposts (GOPs and a decreased supply of plasma membrane (PM in Drosophila class IV dendritic arborization (da (C4 da neurons. mRNA sequencing revealed that genes downregulated by polyQ proteins included many secretory pathway-related genes, including COPII genes regulating GOP synthesis. Transcription factor enrichment analysis identified CREB3L1/CrebA, which regulates COPII gene expression. CrebA overexpression in C4 da neurons restores the dysregulation of COPII genes, GOP synthesis, and PM supply. Chromatin immunoprecipitation (ChIP-PCR revealed that CrebA expression is regulated by CREB-binding protein (CBP, which is sequestered by polyQ proteins. Furthermore, co-overexpression of CrebA and Rac1 synergistically restores the polyQ-induced dendrite pathology. Collectively, our results suggest that GOPs impaired by polyQ proteins contribute to dendrite pathology through the CBP-CrebA-COPII pathway.

Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

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Rodón, Laura; Gonzàlez-Juncà, Alba; Inda, María del Mar; Sala-Hojman, Ada; Martínez-Sáez, Elena; Seoane, Joan

2014-10-01

In advanced cancer, including glioblastoma, the TGFβ pathway acts as an oncogenic factor. Some tumors exhibit aberrantly high TGFβ activity, and the mechanisms underlying this phenomenon are not well understood. We have observed that TGFβ can induce TGFβ2, generating an autocrine loop leading to aberrantly high levels of TGFβ2. We identified cAMP-responsive element-binding protein 1 (CREB1) as the critical mediator of the induction of TGFβ2 by TGFβ. CREB1 binds to the TGFB2 gene promoter in cooperation with SMAD3 and is required for TGFβ to activate transcription. Moreover, the PI3K-AKT and RSK pathways regulate the TGFβ2 autocrine loop through CREB1. The levels of CREB1 and active phosphorylated CREB1 correlate with TGFβ2 in glioblastoma. In addition, using patient-derived in vivo models of glioblastoma, we found that CREB1 levels determine the expression of TGFβ2. Our results show that CREB1 can be considered a biomarker to stratify patients for anti-TGFβ treatments and a therapeutic target in glioblastoma. TGFβ is considered a promising therapeutic target, and several clinical trials using TGFβ inhibitors are generating encouraging results. Here, we discerned the molecular mechanisms responsible for the aberrantly high levels of TGFβ2 found in certain tumors, and we propose biomarkers to predict the clinical response to anti-TGFβ therapies. ©2014 American Association for Cancer Research.

Cooperative interactions between CBP and TORC2 confer selectivity to CREB target gene expression

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Ravnskjær, Kim; Kester, Henri; Liu, Yi

2007-01-01

A number of hormones and growth factors stimulate gene expression by promoting the phosphorylation of CREB (P-CREB), thereby enhancing its association with the histone acetylase paralogs p300 and CBP (CBP/p300). Relative to cAMP, stress signals trigger comparable amounts of CREB phosphorylation...... to stress signals, however; and in its absence, P-CREB is unable to stimulate CRE-dependent transcription, due to a block in CBP recruitment. The effect of TORC2 on CBP/p300 promoter occupancy appears pivotal because a gain of function mutant CREB polypeptide with increased affinity for CBP restored CRE......-mediated transcription in cells exposed to stress signals. Taken together, these results indicate that TORC2 is one of the long sought after cofactors that mediates the differential effects of cAMP and stress pathways on CREB target gene expression....

Aging impairs transcriptional regulation of vascular endothelial growth factor in human microvascular endothelial cells: implications for angiogenesis and cell survival.

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Ahluwalia, A; Jones, M K; Szabo, S; Tarnawski, A S

2014-04-01

In some tissues, aging impairs angiogenesis and reduces expression of vascular endothelial growth factor A (VEGF), a fundamental regulator of angiogenesis. We previously examined angiogenesis in aging and young gastric mucosa in vivo and in vitro and showed that an imbalance between expressions of VEGF (pro-angiogenic factor) and endostatin (anti-angiogenic protein) results in an aging-related impairment of angiogenesis in rats. However, the human relevance of these findings, and whether these mechanisms apply to endothelial cells derived from other tissues, is not clear. Since P-STAT3 and P-CREB are transcription factors that, in association with HIF-1α, can activate VEGF gene expression in some cells (e.g., liver cancer cells, vascular smooth muscle cells), we examined the expression of these two proteins in human dermal microvascular endothelial cells (HMVECs) derived from aging and neonatal individuals. We examined and quantified in vitro angiogenesis, expression of VEGF, P-STAT3, P-CREB and importin-α in HMVECs isolated from neonates (neonatal) and a 66 year old subject (aging). We also examined the effects of treatment with exogenous VEGF and endostatin on in vitro angiogenesis in these cells. Endothelial cells isolated from aging individuals had impaired angiogenesis (vs. neonatal endothelial cells) and reduced expression of VEGF mRNA and protein. Aged HMVECs also had reduced importin-α expression, and reduced expression and nuclear translocation of P-STAT3 and P-CREB. Reduced VEGF gene expression in aged HMVECs strongly correlated with the decreased levels of P-STAT3, P-CREB and importin-α in these cells. Our study clearly demonstrates that endothelial cells from aging individuals have impaired angiogenesis and reduced expression of VEGF likely due to impaired nuclear transport of P-STAT3 and P-CREB transcription factors in these cells.

Involvement of Phosphorylated "Apis Mellifera" CREB in Gating a Honeybee's Behavioral Response to an External Stimulus

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Gehring, Katrin B.; Heufelder, Karin; Feige, Janina; Bauer, Paul; Dyck, Yan; Ehrhardt, Lea; Kühnemund, Johannes; Bergmann, Anja; Göbel, Josefine; Isecke, Marlene; Eisenhardt, Dorothea

2016-01-01

The transcription factor cAMP-response element-binding protein (CREB) is involved in neuronal plasticity. Phosphorylation activates CREB and an increased level of phosphorylated CREB is regarded as an indicator of CREB-dependent transcriptional activation. In honeybees ("Apis mellifera") we recently demonstrated a particular high…

Creb1 regulates late stage mammalian lung development via respiratory epithelial and mesenchymal-independent mechanisms

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Antony, N.; McDougall, A. R.; Mantamadiotis, T.; Cole, T. J.; Bird, A. D.

2016-01-01

During mammalian lung development, the morphological transition from respiratory tree branching morphogenesis to a predominantly saccular architecture, capable of air-breathing at birth, is dependent on physical forces as well as molecular signaling by a range of transcription factors including the cAMP response element binding protein 1 (Creb1). Creb1−/− mutant mice exhibit complete neonatal lethality consistent with a lack of lung maturation beyond the branching phase. To further define its role in the developing mouse lung, we deleted Creb1 separately in the respiratory epithelium and mesenchyme. Surprisingly, we found no evidence of a morphological lung defect nor compromised neonatal survival in either conditional Creb1 mutant. Interestingly however, loss of mesenchymal Creb1 on a genetic background lacking the related Crem protein showed normal lung development but poor neonatal survival. To investigate the underlying requirement for Creb1 for normal lung development, Creb1−/− mice were re-examined for defects in both respiratory muscles and glucocorticoid hormone signaling, which are also required for late stage lung maturation. However, these systems appeared normal in Creb1−/− mice. Together our results suggest that the requirement of Creb1 for normal mammalian lung morphogenesis is not dependent upon its expression in lung epithelium or mesenchyme, nor its role in musculoskeletal development. PMID:27150575

The α and Δ isoforms of CREB1 are required to maintain normal pulmonary vascular resistance.

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Lili Li

Full Text Available Chronic hypoxia causes pulmonary hypertension associated with structural alterations in pulmonary vessels and sustained vasoconstriction. The transcriptional mechanisms responsible for these distinctive changes are unclear. We have previously reported that CREB1 is activated in the lung in response to alveolar hypoxia but not in other organs. To directly investigate the role of α and Δ isoforms of CREB1 in the regulation of pulmonary vascular resistance we examined the responses of mice in which these isoforms of CREB1 had been inactivated by gene mutation, leaving only the β isoform intact (CREB(αΔ mice. Here we report that expression of CREB regulated genes was altered in the lungs of CREB(αΔ mice. CREB(αΔ mice had greater pulmonary vascular resistance than wild types, both basally in normoxia and following exposure to hypoxic conditions for three weeks. There was no difference in rho kinase mediated vasoconstriction between CREB(αΔ and wild type mice. Stereological analysis of pulmonary vascular structure showed characteristic wall thickening and lumen reduction in hypoxic wild-type mice, with similar changes observed in CREB(αΔ. CREB(αΔ mice had larger lungs with reduced epithelial surface density suggesting increased pulmonary compliance. These findings show that α and Δ isoforms of CREB1 regulate homeostatic gene expression in the lung and that normal activity of these isoforms is essential to maintain low pulmonary vascular resistance in both normoxic and hypoxic conditions and to maintain the normal alveolar structure. Interventions that enhance the actions of α and Δ isoforms of CREB1 warrant further investigation in hypoxic lung diseases.

Moringa oleifera Seed Extract Alleviates Scopolamine-Induced Learning and Memory Impairment in Mice

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Juan Zhou

2018-04-01

Full Text Available The extract of Moringa oleifera seeds has been shown to possess various pharmacological properties. In the present study, we assessed the neuropharmacological effects of 70% ethanolic M. oleifera seed extract (MSE on cognitive impairment caused by scopolamine injection in mice using the passive avoidance and Morris water maze (MWM tests. MSE (250 or 500 mg/kg was administered to mice by oral gavage for 7 or 14 days, and cognitive impairment was induced by intraperitoneal injection of scopolamine (4 mg/kg for 1 or 6 days. Mice that received scopolamine alone showed impaired learning and memory retention and considerably decreased cholinergic system reactivity and neurogenesis in the hippocampus. MSE pretreatment significantly ameliorated scopolamine-induced cognitive impairment and enhanced cholinergic system reactivity and neurogenesis in the hippocampus. Additionally, the protein expressions of phosphorylated Akt, ERK1/2, and CREB in the hippocampus were significantly decreased by scopolamine, but these decreases were reversed by MSE treatment. These results suggest that MSE-induced ameliorative cognitive effects are mediated by enhancement of the cholinergic neurotransmission system and neurogenesis via activation of the Akt, ERK1/2, and CREB signaling pathways. These findings suggest that MSE could be a potent neuropharmacological drug against amnesia, and its mechanism might be modulation of cholinergic activity via the Akt, ERK1/2, and CREB signaling pathways.

Epigenetic modification of miR-10a regulates renal damage by targeting CREB1 in type 2 diabetes mellitus

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Shan, Qun, E-mail: shanp@jsnu.edu.cn; Zheng, Guihong, E-mail: ghzhengsd@jsnu.edu.cn; Zhu, Aihua, E-mail: ahzhu@jsnu.edu.cn; Cao, Li, E-mail: 948113717@qq.com; Lu, Jun, E-mail: lu-jun75@163.com; Wu, Dongmei, E-mail: wdm8610@jsnu.edu.cn; Zhang, ZiFeng, E-mail: zhangzifengsuper@jsnu.edu.cn; Fan, Shaohua, E-mail: fshfly@126.com; Sun, Chunhui, E-mail: 306484866@qq.com; Hu, Bin, E-mail: hubin@jsnu.edu.cn; Zheng, Yuanlin, E-mail: ylzheng@jsnu.edu.cn

2016-09-01

Emerging evidence has shown that microRNA-mediated gene expression modulation plays a crucial role in the pathogenesis of type 2 diabetes mellitus, but the novel miRNAs involved in type 2 diabetes and its functional regulatory mechanisms still need to be determined. In this study, we assessed the role of miR-10a in extracellular matrix accumulation in the kidney of diabetic mellitus induced by combining administration of chronic high fat diet (HFD) and low dosage of streptozotocin (STZ, 35 mg/kg). Here, we found that HFD/STZ administration decreased the level of microRNA (miR-10a) expression in ICR strain mice. Overexpression of miR-10a alleviated the increased ratio of urine albumin-to-creatinine (ACR) ratio of HFD/STZ mice. In contrast, knockdown of miR-10a increased the ratio of kidney ACR in naïve mice. Furthermore, cAMP response element binding protein 1 (CREB1) was validated as a target of miR-10a in vitro and in vivo. CREB1 and its downstream fibronectin (FN, extracellular matrix) were increased in HFD/STZ-treated mice, which was reversed by kidney miR-10a overexpression. The content of CREB1 and FN was increased by miR-10a knockdown in kidney of naïve mice. Furthermore, histone deacetylase 3 (HDAC3) was revealed to be increased in kidney of HFD/STZ mice, accompanied with the augmentation of ACR ratio and FN level. Knockdown of HDAC3 with siRNA significantly caused the increase of miR-10a, resulting in the decrease in CREB1 and FN expression in kidney of HFD/STZ mice. Contrarily, HDAC3 overexpression mediated by lentivirus decreased miR-10a content, and enhanced ACR value, CREB1 and FN formation in naïve mice. Collectively, these results elucidate that HDAC3/miR-10a/CREB1 serves as a new mechanism underlying kidney injury, providing potential therapeutic targets in type 2 diabetes. - Highlights: • Diabetes induces the decrease of miR-10a level in the kidney. • MiR-10a overexpression improves kidney damage of diabetes. • MiR-10a targeting CREB1/FN

Epigenetic modification of miR-10a regulates renal damage by targeting CREB1 in type 2 diabetes mellitus

International Nuclear Information System (INIS)

Shan, Qun; Zheng, Guihong; Zhu, Aihua; Cao, Li; Lu, Jun; Wu, Dongmei; Zhang, ZiFeng; Fan, Shaohua; Sun, Chunhui; Hu, Bin; Zheng, Yuanlin

2016-01-01

Emerging evidence has shown that microRNA-mediated gene expression modulation plays a crucial role in the pathogenesis of type 2 diabetes mellitus, but the novel miRNAs involved in type 2 diabetes and its functional regulatory mechanisms still need to be determined. In this study, we assessed the role of miR-10a in extracellular matrix accumulation in the kidney of diabetic mellitus induced by combining administration of chronic high fat diet (HFD) and low dosage of streptozotocin (STZ, 35 mg/kg). Here, we found that HFD/STZ administration decreased the level of microRNA (miR-10a) expression in ICR strain mice. Overexpression of miR-10a alleviated the increased ratio of urine albumin-to-creatinine (ACR) ratio of HFD/STZ mice. In contrast, knockdown of miR-10a increased the ratio of kidney ACR in naïve mice. Furthermore, cAMP response element binding protein 1 (CREB1) was validated as a target of miR-10a in vitro and in vivo. CREB1 and its downstream fibronectin (FN, extracellular matrix) were increased in HFD/STZ-treated mice, which was reversed by kidney miR-10a overexpression. The content of CREB1 and FN was increased by miR-10a knockdown in kidney of naïve mice. Furthermore, histone deacetylase 3 (HDAC3) was revealed to be increased in kidney of HFD/STZ mice, accompanied with the augmentation of ACR ratio and FN level. Knockdown of HDAC3 with siRNA significantly caused the increase of miR-10a, resulting in the decrease in CREB1 and FN expression in kidney of HFD/STZ mice. Contrarily, HDAC3 overexpression mediated by lentivirus decreased miR-10a content, and enhanced ACR value, CREB1 and FN formation in naïve mice. Collectively, these results elucidate that HDAC3/miR-10a/CREB1 serves as a new mechanism underlying kidney injury, providing potential therapeutic targets in type 2 diabetes. - Highlights: • Diabetes induces the decrease of miR-10a level in the kidney. • MiR-10a overexpression improves kidney damage of diabetes. • MiR-10a targeting CREB1/FN

Hepatic CREB3L3 controls whole-body energy homeostasis and improves obesity and diabetes.

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Nakagawa, Yoshimi; Satoh, Aoi; Yabe, Sachiko; Furusawa, Mika; Tokushige, Naoko; Tezuka, Hitomi; Mikami, Motoki; Iwata, Wakiko; Shingyouchi, Akiko; Matsuzaka, Takashi; Kiwata, Shiori; Fujimoto, Yuri; Shimizu, Hidehisa; Danno, Hirosuke; Yamamoto, Takashi; Ishii, Kiyoaki; Karasawa, Tadayoshi; Takeuchi, Yoshinori; Iwasaki, Hitoshi; Shimada, Masako; Kawakami, Yasushi; Urayama, Osamu; Sone, Hirohito; Takekoshi, Kazuhiro; Kobayashi, Kazuto; Yatoh, Shigeru; Takahashi, Akimitsu; Yahagi, Naoya; Suzuki, Hiroaki; Yamada, Nobuhiro; Shimano, Hitoshi

2014-12-01

Transcriptional regulation of metabolic genes in the liver is the key to maintaining systemic energy homeostasis during starvation. The membrane-bound transcription factor cAMP-responsive element-binding protein 3-like 3 (CREB3L3) has been reported to be activated during fasting and to regulate triglyceride metabolism. Here, we show that CREB3L3 confers a wide spectrum of metabolic responses to starvation in vivo. Adenoviral and transgenic overexpression of nuclear CREB3L3 induced systemic lipolysis, hepatic ketogenesis, and insulin sensitivity with increased energy expenditure, leading to marked reduction in body weight, plasma lipid levels, and glucose levels. CREB3L3 overexpression activated gene expression levels and plasma levels of antidiabetic hormones, including fibroblast growth factor 21 and IGF-binding protein 2. Amelioration of diabetes by hepatic activation of CREB3L3 was also observed in several types of diabetic obese mice. Nuclear CREB3L3 mutually activates the peroxisome proliferator-activated receptor (PPAR) α promoter in an autoloop fashion and is crucial for the ligand transactivation of PPARα by interacting with its transcriptional regulator, peroxisome proliferator-activated receptor gamma coactivator-1α. CREB3L3 directly and indirectly controls fibroblast growth factor 21 expression and its plasma level, which contributes at least partially to the catabolic effects of CREB3L3 on systemic energy homeostasis in the entire body. Therefore, CREB3L3 is a therapeutic target for obesity and diabetes.

Central and peripheral administration of antisense oligonucleotide targeting amyloid-β protein precursor improves learning and memory and reduces neuroinflammatory cytokines in Tg2576 (AβPPswe) mice.

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Farr, Susan A; Erickson, Michelle A; Niehoff, Michael L; Banks, William A; Morley, John E

2014-01-01

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Currently, there are no therapies to stop or reverse the symptoms of AD. We have developed an antisense oligonucleotide (OL-1) against the amyloid-β protein precursor (AβPP) that can decrease AβPP expression and amyloid-β protein (Aβ) production. This antisense rapidly crosses the blood-brain barrier, reverses learning and memory impairments, reduces oxidative stress, and restores brain-to-blood efflux of Aβ in SAMP8 mice. Here, we examined the effects of this AβPP antisense in the Tg2576 mouse model of AD. We administered the OL-1 antisense into the lateral ventricle 3 times at 2week intervals. Seventy-two hours after the third injection, we tested learning and memory in T-maze foot shock avoidance. In the second study, we injected the mice with OL-1 antisense 3 times at 2-week intervals via the tail vein. Seventy-two hours later, we tested learning and memory T-maze, novel object recognition, and elevated plus maze. At the end of behavioral testing, brain tissue was collected. OL-1 antisense administered centrally improved acquisition and retention of T-maze foot shock avoidance. OL-1 antisense administered via tail vein improved learning and memory in both T-maze foot shock avoidance and novel object-place recognition. In the elevated plus maze, the mice which received OL-1 antisense spent less time in the open arms and had fewer entries into the open arms indicating reduced disinhibitation. Biochemical analyses reveal significant reduction of AβPP signal and a reduction of measures of neuroinflammation. The current findings support the therapeutic potential of OL-1 AβPP antisense.

Effect of XingPiJieYu decoction on spatial learning and memory and cAMP-PKA-CREB-BDNF pathway in rat model of depression through chronic unpredictable stress.

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Wang, Chunye; Guo, Jianyou; Guo, Rongjuan

2017-01-24

Depression is a mental disorder characterized by a pervasive low mood and loss of pleasure or interest in usual activities, and often results in cognitive dysfunction. The disturbance of cognitive processes associated with depression, especially the impairment of learning and memory, exacerbates illness and increases recurrence of depression. XingPiJieYu (XPJY) is one of the most widely clinical formulas of traditional Chinese medicine (TCM) and can improve the symptoms of depression, including learning and memory. However, its regulatory effects haven't been comprehensively studied so far. Recently, some animal tests have indicated that the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element-binding protein (CREB)-brain derived neurotrophic factor (BDNF) signaling pathway in hippocampus is closely related to depression and the pathogenesis of cognitive function impairments. The present study was performed to investigate the effect and mechanism of XPJY on depression and learning and memory in animal model. The rat model of depression was established by chronic unpredictable stress (CUS) for 21 days. The rats were randomly divided into six groups: control group, CUS group, CUS + XPJY (1.4 g/kg, 0.7 g/kg and 0.35 g/kg) groups, and CUS + sertraline (10 mg/kg) group. The sucrose preference, open field exploration and Morris water maze (MWM) were tested. The expression of cAMP, CREB, PKA and BDNF protein in hippocampus was examined with Elisa and Western Blot. The mRNA level of CREB and BDNF in hippocampus was measured with PCR. The results demonstrated that rats subjected to CUS exhibited decreases in sucrose preference, total ambulation, percentage of central ambulation, rearing in the open field test and spatial performance in the MWM. CUS reduced the expression of cAMP, PKA, CREB and BDNF in hippocampus of model rats. These effects could be reversed by XPJY. The results indicated that XPJY can improve depression and

Peripheral administration of antisense oligonucleotides targeting the amyloid-β protein precursor reverses AβPP and LRP-1 overexpression in the aged SAMP8 mouse brain.

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Erickson, Michelle A; Niehoff, Michael L; Farr, Susan A; Morley, John E; Dillman, Lucy A; Lynch, Kristin M; Banks, William A

2012-01-01

The senescence accelerated mouse-prone 8 (SAMP8) mouse model of Alzheimer's disease has a natural mutation leading to age-related increases in the amyloid-β protein precursor (AβPP) and amyloid-β (Aβ) in the brain, memory impairment, and deficits in Aβ removal from the brain. Previous studies show that centrally administered antisense oligonucleotide directed against AβPP can decrease AβPP expression and Aβ production in the brains of aged SAMP8 mice, and improve memory. The same antisense crosses the blood-brain barrier and reverses memory deficits when injected intravenously. Here, we give 6 μg of AβPP or control antisense 3 times over 2 week intervals to 12 month old SAMP8 mice. Object recognition test was done 48 hours later, followed by removal of whole brains for immunoblot analysis of AβPP, low-density lipoprotein-related protein-1 (LRP-1), p-glycoprotein (Pgp), receptor for advanced glycation endproducts (RAGE), or ELISA of soluble Aβ(40). Our results show that AβPP antisense completely reverses a 30% age-associated increase in AβPP signal (p < 0.05 versus untreated 4 month old SAMP8). Soluble Aβ(40) increased with age, but was not reversed by antisense. LRP-1 large and small subunits increased significantly with age (147.7%, p < 0.01 and 123.7%, p < 0.05 respectively), and AβPP antisense completely reversed these increases (p < 0.05). Pgp and RAGE were not significantly altered with age or antisense. Antisense also caused improvements in memory (p < 0.001). Together, these data support the therapeutic potential of AβPP antisense and show a unique association between AβPP and LRP-1 expression in the SAMP8 mouse.

Effects of thioperamide on seizure development and memory impairment induced by pentylenetetrazole-kindling epilepsy in rats

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ZHANG Li-san; CHEN Jie-fang; CHEN Guan-feng; HU Xing-yue; DING Mei-ping

2013-01-01

Background Histamine H3 receptor antagonists have been considered as potential drugs to treat central nervous system diseases.However,whether these drugs can inhibit epileptogenesis remains unclear.This study aimed to investigate the effects of thioperamide,a selective and potent histamine H3 receptor antagonist,on the seizure development and memory impairment induced by pentylenetetrazole (PTZ)-kindling epilepsy in rats.Methods Chemical kindling was elicited by repeated intraperitoneal (ip) injections of a subconvulsant dose of PTZ (35 mg/kg) once every 48 hours for 12 times,and seizure activity of kindling was recorded for 30 minutes.Control rats were ip injected with saline instead of PTZ.Morris water maze was used to evaluate the spatial memory.Phosphorylated cyclic adenosine monophosphate response element binding protein (p-CREB) was tested by Western blotting in hippocampus.Results Intracerebroventricular (icv) injections with thioperamide (10 μg,20 μg) 30 minutes before every PTZ injections,significantly prolonged the onset of PTZ-kindling and inhibited the seizure stages.PTZ-kindling seizures led to the impairment of spatial memory in rats,and thioperamide ameliorated the impairment of spatial learning and memory.Compared to non-kindling rats,there was a significant decrease in p-CREB level in hippocampus of the PTZ-kindling rats,which was reversed by thioperamide.Conclusions Thioperamide plays a protective role in seizure development and cognitive impairment of PTZ-induced kindling in rats.The protection of thioperamide in cognitive impairment is possibly associated with the enhancement of CREB-dependent transcription.

Antisense downregulation of mutant huntingtin in a cell model

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Hasholt, L.; Abell, K.; Norremolle, A.

2003-01-01

or by addition to the culture medium. Results Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2-N neurons. We obtained antisense sequence-specific inhibition of expression...... of the fusion protein and/or suppression of the aggregate formation in both cell types. In the NT2 cells the antisense effect was dependent on the way of administration of the oligo. Conclusions The PS-antisense oligo is effective in downregulation of mutant huntingtin, and the reduction of aggregate formation...... is a sensitive biological marker. The findings suggest that antisense knockdown of huntingtin could be a useful strategy for treatment of HD, and could also be suitable for studies of the normal and pathological function of huntingtin in different cellular model systems....

A role for neuronal cAMP responsive-element binding (CREB)-1 in brain responses to calorie restriction

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Fusco, Salvatore; Ripoli, Cristian; Podda, Maria Vittoria; Ranieri, Sofia Chiatamone; Leone, Lucia; Toietta, Gabriele; McBurney, Michael W.; Schütz, Günther; Riccio, Antonella; Grassi, Claudio; Galeotti, Tommaso; Pani, Giovambattista

2012-01-01

Calorie restriction delays brain senescence and prevents neurodegeneration, but critical regulators of these beneficial responses other than the NAD+-dependent histone deacetylase Sirtuin-1 (Sirt-1) are unknown. We report that effects of calorie restriction on neuronal plasticity, memory and social behavior are abolished in mice lacking cAMP responsive-element binding (CREB)-1 in the forebrain. Moreover, CREB deficiency drastically reduces the expression of Sirt-1 and the induction of genes relevant to neuronal metabolism and survival in the cortex and hippocampus of dietary-restricted animals. Biochemical studies reveal a complex interplay between CREB and Sirt-1: CREB directly regulates the transcription of the sirtuin in neuronal cells by binding to Sirt-1 chromatin; Sirt-1, in turn, is recruited by CREB to DNA and promotes CREB-dependent expression of target gene peroxisome proliferator-activated receptor-γ coactivator-1α and neuronal NO Synthase. Accordingly, expression of these CREB targets is markedly reduced in the brain of Sirt KO mice that are, like CREB-deficient mice, poorly responsive to calorie restriction. Thus, the above circuitry, modulated by nutrient availability, links energy metabolism with neurotrophin signaling, participates in brain adaptation to nutrient restriction, and is potentially relevant to accelerated brain aging by overnutrition and diabetes. PMID:22190495

Role of hippocampal and prefrontal cortical signaling pathways in dextromethorphan effect on morphine-induced memory impairment in rats.

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Ghasemzadeh, Zahra; Rezayof, Ameneh

2016-02-01

Evidence suggests that dextromethorphan (DM), an NMDA receptor antagonist, induces memory impairment. Considering that DM is widely used in cough-treating medications, and the co-abuse of DM with morphine has recently been reported, the aims of the present study was (1) to investigate whether there is a functional interaction between morphine and DM in passive avoidance learning and (2) to assess the possible role of the hippocampal and prefrontal cortical (PFC) signaling pathways in the effects of the drugs on memory formation. Our findings indicated that post-training or pre-test administration of morphine (2 and 6 mg/kg) or DM (10-30 mg/kg) impaired memory consolidation and retrieval which was associated with the attenuation of the levels of phosphorylated Ca(2+)/calmodulin-dependent protein kinase II (p-CAMKII) and cAMP responsive element-binding protein (p-CREB) in the targeted sites. Moreover, the memory impairment induced by post-training administration of morphine was reversed by pre-test administration of the same dose of morphine or DM (30 mg/kg), indicating state-dependent learning (SDL) and a cross-SDL between the drugs. It is important to note that the levels of p-CAMKII/CAMKII and p-CREB/CREB in the hippocampus and the PFC increased in drugs-induced SDL. In addition, DM administration potentiated morphine-induced SDL which was related to the enhanced levels of hippocampal and PFC CAMKII-CREB signaling pathways. It can be concluded that there is a relationship between the hippocampus and the PFC in the effect of DM and/or morphine on memory retrieval. Moreover, a cross SDL can be induced between the co-administration of DM and morphine. Interestingly, CAMKII-CREB signaling pathways also mediate the drugs-induced SDL. Copyright © 2015 Elsevier Inc. All rights reserved.

Deletion of creB in Aspergillus oryzae increases secreted hydrolytic enzyme activity.

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Hunter, A J; Morris, T A; Jin, B; Saint, C P; Kelly, J M

2013-09-01

Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes.

Peptide nucleic acid (PNA) antisense effects in Escherichia coli

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Good, L; Nielsen, P E

1999-01-01

Antisense peptide nucleic acid (PNA) can be used to control cell growth, gene expression and growth phenotypes in the bacteria Escherichia coli. PNAs targeted to the RNA components of the ribosome can inhibit translation and cell growth, and PNAs targeted to mRNA can limit gene expression with gene...... and sequence specificity. In an E. coli cell extract, efficient inhibition is observed when using PNA concentrations in the nanomolar range, whereas micromolar concentrations are required for inhibition in growing cells. A mutant strain of E. coli that is more permeable to antibiotics also is more susceptible...... to antisense PNAs than the wild type. This chapter details methods for testing the antisense activities of PNA in E. coli. As an example of the specific antisense inhibition possible, we show the effects of an anti-beta-galactosidase PNA in comparison to control PNAs. With improvements in cell uptake...

Advancements of antisense oligonucleotides in treatment of breast cancer

Institute of Scientific and Technical Information of China (English)

YANGShuan-Ping; SONGSan-Tai; 等

2003-01-01

Breast cancer is one kind of multi-gene related malignancy.Overexpression of some oncogenes such as HER-2(c-erbB-2,Neu),bcl-2/bcl-xL,protein kinase A(PKA),and transferrin receptor gene(TfR gene),etc significantly affect the prognosis of breast cancer.It was shown that specific suppression of the overexpressed genes above resulted in the improvement of the therapy of breast cancer.Antisense interference.one of useful tools for inhibiting the overexpression of specific oncogenes,was involved in the therapy of breast cancer in recent years. Data indicated that antisense oligonucleotides(ON)could inhibit specially the expression of the target genes on mRNA or protein levels in most of cases;some ON candidates showed encouraging therapeutic effects in vitro and in vivo on breast cancer cell lines or xenografts.Furthermore,the combination use of the antisense ON and normal chemotherapeutic agents indicated synergistic antitumor effects,which was probably the best utilization of antisense ON in the treatment of breast cancer.

Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters.

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Christopher A Lavender

2016-08-01

Full Text Available Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment.

Antiviral effects of herpes simplex virus specific anti-sense nucleic acids.

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Cantin, E M; Podsakoff, G; Willey, D E; Openshaw, H

1992-01-01

We have targeted mRNA sequences encompassing the translation initiation codon of the essential herpes simplex virus type 1 (HSV-1) IE3 gene with three kinds of anti-sense molecule. Addition of a 15mer oligodeoxyribonucleoside methylphosphonate to tissue culture cells resulted in suppression of viral replication. HSV-1 replication was also inhibited in cultured cells containing anti-sense vectors expressing transcripts complementary to the IE3 mRNA. We have also constructed a ribozyme which upon base pairing with the target IE3 mRNA induces cleavage at the predicted GUC site. A major obstacle to anti-sense studies in animals is drug delivery of preformed antisense molecules to ganglionic neurons, the site of HSV latency and reactivation. We speculate as to how this may be accomplished through carrier compounds which are taken up by nerve terminals and transported by retrograde axoplasmic flow. By the same route, HSV itself may be used as an anti-sense vector.

CREB mediates the insulinotropic and anti-apoptotic effects of GLP-1 signaling in adult mouse β-cells

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Soona Shin

2014-11-01

Conclusions: In sum, our studies using conditional gene deletion put into question current notions about the importance of CREB in regulating β-cell function and mass. However, we reveal an important role for CREB in the β-cell response to GLP-1 receptor signaling, further validating CREB as a therapeutic target for diabetes.

Inducible forebrain-specific ablation of the transcription factor Creb during adulthood induces anxiety but no spatial/contextual learning deficits

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Miriam Annika Vogt

2014-11-01

Full Text Available The cyclic AMP (cAMP-response element binding protein (CREB is an activity-dependent transcription factor playing a role in synaptic plasticity, learning and memory, and emotional behavior. However, the impact of Creb ablation on rodent behavior is vague as e.g. memory performance of different Creb mutant mice depends on the specific type of mutation per se but additionally on the background and learning protocol differences. Here we present the first targeted ablation of CREB induced during adulthood selectively in principal forebrain neurons in a pure background strain of C57BL/6 mice. All hippocampal principal neurons exhibited lack of CREB expression. Mutant mice showed a severe anxiety phenotype in the openfield and novel object exploration test as well as in the Dark-Light Box Test, but unaltered hippocampus-dependent long-term memory in the Morris water maze and in context dependent fear conditioning. On the molecular level, CREB ablation led to CREM up regulation in the hippocampus and frontal cortex which may at least in part compensate for the loss of CREB. BDNF, a postulated CREB target gene, was down regulated in the frontal lobe but not in the hippocampus; neurogenesis remained unaltered. Our data indicate that in the adult mouse forebrain the late onset of CREB ablation can, in case of memory functionality, be compensated for and is not essential for memory consolidation and retrieval during adulthood. In contrast, the presence of CREB protein during adulthood seems to be pivotal for the regulation of emotional behavior.

Impairment of the spatial learning and memory induced by learned helplessness and chronic mild stress.

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Song, Li; Che, Wang; Min-Wei, Wang; Murakami, Yukihisa; Matsumoto, Kinzo

2006-02-01

Increasing evidences indicate the concurrence and interrelationship of depression and cognitive impairments. The present study was undertaken to investigate the effects of two depressive animal models, learned helplessness (LH) and chronic mild stress (CMS), on the cognitive functions of mice in the Morris water maze task. Our results demonstrated that both LH and CMS significantly decreased the cognitive performance of stressed mice in the water maze task. The escaping latency to the platform was prolonged and the probe test percentage in the platform quadrant was reduced. These two models also increased the plasma corticosterone concentration and decreased the brain derived neurotrophic factor (BDNF) and cAMP-response element-biding protein (CREB) messenger ribonucleic acid (mRNA) levels in hippocampus, which might cause the spatial cognition deficits. Repeated treatment with antidepressant drugs, imipramine (Imi) and fluoxetine (Flu), significantly reduced the plasma corticosterone concentration and enhanced the BDNF and CREB levels. Furthermore, antidepressant treated animals showed an ameliorated cognitive performance compared with the vehicle treated stressed animals. These data suggest that both LH and CMS impair the spatial cognitive function and repeated treatment with antidepressant drugs decreases the prevalence of cognitive impairments induced by these two animal models. Those might in part be attributed to the reduced plasma corticosterone and enhanced hippocampal BDNF and CREB expressions. This study provided a better understanding of molecular mechanisms underlying interactions of depression and cognitive impairments, although animal models used in this study can mimic only some aspects of depression or cognition of human.

Involvement of HTLV-I Tax and CREB in aneuploidy: a bioinformatics approach

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Pumfery Anne

2006-07-01

Full Text Available Abstract Background Adult T-cell leukemia (ATL is a complex and multifaceted disease associated with human T-cell leukemia virus type 1 (HTLV-I infection. Tax, the viral oncoprotein, is considered a major contributor to cell cycle deregulation in HTLV-I transformed cells by either directly disrupting cellular factors (protein-protein interactions or altering their transcription profile. Tax transactivates these cellular promoters by interacting with transcription factors such as CREB/ATF, NF-κB, and SRF. Therefore by examining which factors upregulate a particular set of promoters we may begin to understand how Tax orchestrates leukemia development. Results We observed that CTLL cells stably expressing wild-type Tax (CTLL/WT exhibited aneuploidy as compared to a Tax clone deficient for CREB transactivation (CTLL/703. To better understand the contribution of Tax transactivation through the CREB/ATF pathway to the aneuploid phenotype, we performed microarray analysis comparing CTLL/WT to CTLL/703 cells. Promoter analysis of altered genes revealed that a subset of these genes contain CREB/ATF consensus sequences. While these genes had diverse functions, smaller subsets of genes were found to be involved in G2/M phase regulation, in particular kinetochore assembly. Furthermore, we confirmed the presence of CREB, Tax and RNA Polymerase II at the p97Vcp and Sgt1 promoters in vivo through chromatin immunoprecipitation in CTLL/WT cells. Conclusion These results indicate that the development of aneuploidy in Tax-expressing cells may occur in response to an alteration in the transcription profile, in addition to direct protein interactions.

Activation of the CREB/c-Fos Pathway during Long-Term Synaptic Plasticity in the Cerebellum Granular Layer

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Daniela Gandolfi

2017-06-01

Full Text Available The induction of long-term potentiation and depression (LTP and LTD is thought to trigger gene expression and protein synthesis, leading to consolidation of synaptic and neuronal changes. However, while LTP and LTD have been proposed to play important roles for sensori-motor learning in the cerebellum granular layer, their association with these mechanisms remained unclear. Here, we have investigated phosphorylation of the cAMP-responsive element binding protein (CREB and activation of the immediate early gene c-Fos pathway following the induction of synaptic plasticity by theta-burst stimulation (TBS in acute cerebellar slices. LTP and LTD were localized using voltage-sensitive dye imaging (VSDi. At two time points following TBS (15 min and 120 min, corresponding to the early and late phases of plasticity, slices were fixed and processed to evaluate CREB phosphorylation (P-CREB and c-FOS protein levels, as well as Creb and c-Fos mRNA expression. High levels of P-CREB and Creb/c-Fos were detected before those of c-FOS, as expected if CREB phosphorylation triggered gene expression followed by protein synthesis. No differences between control slices and slices stimulated with TBS were observed in the presence of an N-methyl-D-aspartate receptor (NMDAR antagonist. Interestingly, activation of the CREB/c-Fos system showed a relevant degree of colocalization with long-term synaptic plasticity. These results show that NMDAR-dependent plasticity at the cerebellum input stage bears about transcriptional and post-transcriptional processes potentially contributing to cerebellar learning and memory consolidation.

Antisense Treatments for Biothreat Agents

National Research Council Canada - National Science Library

Warfield, Kelly L; Panchal, Rekha G; Aman, M J; Bavari, Sina

2006-01-01

... a variety of pathogens in cell culture studies and nonhuman primate models of infection. For these reasons, antisense technologies are being pursued as treatments against biothreat agents such as Ebola virus, dengue virus and Bacillus anthracis...

Transcription factor CREB is involved in CaSR-mediated cytoskeleton gene expression.

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Huang, Shuaishuai; Ren, Yu; Wang, Ping; Li, Yanyuan; Wang, Xue; Zhuang, Haihui; Fang, Rong; Wang, Yuduo; Liu, Ningsheng; Hehir, Michael; Zhou, Jeff X

2015-03-01

Our previous studies illustrated that a steady increase of intracellular calcium concentration ([Ca2+]i) was important for maintaining microtubules (MTs) rearrangement in apoptotic cells. However, little is known about the effect of calcium sensing receptor (CaSR)-mediated increase in [Ca2+]i on cytoskeleton gene expression. We examined the impact of taxol or CaSR agonist/antagonist on the regulation of [Ca2+]i concentration, cytoskeleton arrangement, phosphorylated CREB and cytoskeleton gene expressions in HeLa cells with dominant negative plasmid of CREB (PM). This study demonstrated that Gdcl3 (a specific CaSR agonist) evoked a rapid increase of [Ca2+]i, formed a rigid bundle of MTs which surrounded the nucleus and decreased the cytoskeleton gene expressions in HeLa cells. These effects were rescued by addition of NPS2390 (a specific CaSR antagonist). Moreover, CaSR activity affected cytoskeleton gene expression through transcription factor CREB. Histoscores of pCREB immunoreactivity in tissues of cervical adenocarcinoma, renal clear cell carcinoma, and diffuse large B-cell lymphoma were markedly increased compared with non malignant tissue. These data demonstrate, for the first time, that CaSR-mediated increase in [Ca2+]i probably modulate cytoskeleton organization and gene expression via transcription factor. © 2014 Wiley Periodicals, Inc.

Time of Day Influences Memory Formation and dCREB2 Proteins in Drosophila

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Robin eFropf

2014-03-01

Full Text Available Many biological phenomena oscillate under the control of the circadian system, exhibiting peaks and troughs of activity across the day/night cycle. In most animal models, memory formation also exhibits this property, but the underlying neuronal and molecular mechanisms remain unclear. The dCREB2 transcription factor shows circadian regulated oscillations in its activity, and has been shown to be important for both circadian biology and memory formation. We show that the time-of-day (TOD of behavioral training affects Drosophila memory formation. dCREB2 exhibits complex changes in protein levels across the daytime and nighttime, and these changes in protein abundance are likely to contribute to oscillations in dCREB2 activity and TOD effects on memory formation.

Caffeine and diphenyl diselenide improve long-term memory impaired in middle-aged rats.

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Leite, Marlon R; Marcondes Sari, Marcel Henrique; de Freitas, Mayara L; Oliveira, Lia P; Dalmolin, Laíza; Brandão, Ricardo; Zeni, Gilson

2014-05-01

The aim of the present study was to evaluate the effects of diphenyl diselenide (PhSe)2 supplemented diet (10ppm) associated to the administration of caffeine (15mg/kg; i.g.) for 30days on the novel object recognition memory in middle-aged rats. The present findings showed that (PhSe)2-supplemented diet enhanced short-term memory, but not long-term memory, of middle-aged rats in the novel object recognition task. The (PhSe)2 supplemented diet associated with caffeine administration improved long-term memory, but did not alter short-term memory, impaired in middle-aged rats. Daily caffeine administration to middle-aged rats had no effect on the memory tasks. Diet supplemented with (PhSe)2 plus caffeine administration increased the number of crossings and rearings reduced in middle-aged rats. Caffeine administration plus (PhSe)2 diets were effective in increasing the number of rearings and crossings, respectively, in middle-aged rats, [(3)H] glutamate uptake was reduced in hippocampal slices of rats from (PhSe)2 and caffeine plus (PhSe)2 groups. In addition, animals supplemented with (PhSe)2 showed an increase in the pCREB/CREB ratio whereas pAkt/Akt ratio was not modified. These results suggest that the effects of (PhSe)2 on the short-term memory may be related to its ability to decrease the uptake of glutamate, influencing the increase of CREB phosphorylation. (PhSe)2-supplemented diet associated to the administration of caffeine improved long-term memory impaired in middle-aged rats, an effect independent of CREB and Akt phosphorylation. Copyright © 2014 Elsevier Inc. All rights reserved.

Curcumin confers neuroprotection against alcohol-induced hippocampal neurodegeneration via CREB-BDNF pathway in rats.

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Motaghinejad, Majid; Motevalian, Manijeh; Fatima, Sulail; Hashemi, Hajar; Gholami, Mina

2017-03-01

Alcohol abuse causes severe damage to the brain neurons. Studies have reported the neuroprotective effects of curcumin against alcohol-induced neurodegeneration. However, the precise mechanism of action remains unclear. Seventy rats were equally divided into 7 groups (10 rats per group). Group 1 received normal saline (0.7ml/rat) and group 2 received alcohol (2g/kg/day) for 21days. Groups 3, 4, 5 and 6 concurrently received alcohol (2g/kg/day) and curcumin (10, 20, 40 and 60mg/kg, respectively) for 21days. Animals in group 7 self- administered alcohol for 21days. Group 8 treated with curcumin (60mg/kg, i.p.) alone for 21days. Open Field Test (OFT) was used to investigate motor activity in rats. Hippocampal oxidative, antioxidative and inflammatory factors were evaluated. Furthermore, brain cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) and brain derived neurotrophic factor (BDNF) levels were studied at gene level by reverse transcriptase polymerase chain reaction (RT-PCR). In addition, protein expression for BDNF, CREB, phosphorylated CREB (CREB-P), Bax and Bcl-2 was determined by western blotting. Voluntary and involuntary administration of alcohol altered motor activity in OFT, and curcumin treatment inhibited this alcohol-induced motor disturbance. Also, alcohol administration augmented lipid peroxidation, mitochondrial oxidized glutathione (GSSG), interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and Bax levels in isolated hippocampal tissues. Furthermore, alcohol-induced significant reduction were observed in reduced form of glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) activities and CREB, BDNF and Bcl-2 levels. Also curcumin alone did not change the behavior and biochemical and molecular parameters. Curcumin can act as a neuroprotective agent against neurodegenerative effects of alcohol abuse, probably via activation of CREB-BDNF signaling pathway

Dyslipidemia, sense, antisense or nonsense?

NARCIS (Netherlands)

Visser, M.E.

2011-01-01

Maartje Visser onderzocht het remmen van de synthese van apoB met behulp van antisense - een nieuwe farmacologische techniek. Dit blijkt het slechte LDL-cholesterol op een effectieve manier te verlagen. Bij sommige proefpersonen resulteerde dit in leververvetting. Of dit op de lange termijn

Identification of antisense long noncoding RNAs that function as SINEUPs in human cells.

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Schein, Aleks; Zucchelli, Silvia; Kauppinen, Sakari; Gustincich, Stefano; Carninci, Piero

2016-09-20

Mammalian genomes encode numerous natural antisense long noncoding RNAs (lncRNAs) that regulate gene expression. Recently, an antisense lncRNA to mouse Ubiquitin carboxyl-terminal hydrolase L1 (Uchl1) was reported to increase UCHL1 protein synthesis, representing a new functional class of lncRNAs, designated as SINEUPs, for SINE element-containing translation UP-regulators. Here, we show that an antisense lncRNA to the human protein phosphatase 1 regulatory subunit 12A (PPP1R12A), named as R12A-AS1, which overlaps with the 5' UTR and first coding exon of the PPP1R12A mRNA, functions as a SINEUP, increasing PPP1R12A protein translation in human cells. The SINEUP activity depends on the aforementioned sense-antisense interaction and a free right Alu monomer repeat element at the 3' end of R12A-AS1. In addition, we identify another human antisense lncRNA with SINEUP activity. Our results demonstrate for the first time that human natural antisense lncRNAs can up-regulate protein translation, suggesting that endogenous SINEUPs may be widespread and present in many mammalian species.

cAMP response element binding protein (CREB activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

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Stefano Luisa

2005-01-01

Full Text Available Abstract Background The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE. Results The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2, known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Conclusions Using a constitutively active CREB2

DPPC regulates COX-2 expression in monocytes via phosphorylation of CREB

International Nuclear Information System (INIS)

Morris, R.H.K.; Tonks, A.J.; Jones, K.P.; Ahluwalia, M.K.; Thomas, A.W.; Tonks, A.; Jackson, S.K.

2008-01-01

The major phospholipid in pulmonary surfactant dipalmitoyl phosphatidylcholine (DPPC) has been shown to modulate inflammatory responses. Using human monocytes, this study demonstrates that DPPC significantly increased PGE 2 (P < 0.05) production by 2.5-fold when compared to untreated monocyte controls. Mechanistically, this effect was concomitant with an increase in COX-2 expression which was abrogated in the presence of a COX-2 inhibitor. The regulation of COX-2 expression was independent of NF-κB activity. Further, DPPC increased the phosphorylation of the cyclic AMP response element binding protein (CREB; an important nuclear transcription factor important in regulating COX-2 expression). In addition, we also show that changing the fatty acid groups of PC (e.g. using L-α-phosphatidylcholine β-arachidonoyl-γ-palmitoyl (PAPC)) has a profound effect on the regulation of COX-2 expression and CREB activation. This study provides new evidence for the anti-inflammatory activity of DPPC and that this activity is at least in part mediated via CREB activation of COX-2

Scintigraphic imaging of oncogenes with antisense probes: does it make sense?

International Nuclear Information System (INIS)

Urbain, J.L.C.; Shore, S.K.; Vekemans, M.C.; Cosenza, S.C.; DeRiel, K.; Patel, G.V.; Charkes, N.D.; Malmud, L.S.; Reddy, E.P.

1995-01-01

The aim of this study was to demonstrate that cells which are expressing a particular mRNA transcript do preferentially and specifically retain the antisense probe targeting that mRNA. Using a mouse plasmacytoma cell line (MOPC315) which produces high levels of IgA heavy chain mRNA, a control mouse pre B cell line (7OZ/3B), a human mammary cell line (MCF7) which expresses the erbB2 or neu oncogene, MOPC315 cells as neu-negative controls, and antisense DNA oligonucleotides complementary to the 5' region of the mRNAs and the sense sequence, we have shown that there is a preferential, specific retention of the IgA and neu antisense sequence in MOPC315 and MCF7 cells, respectively. We have further demonstrated that this retention is time and concentration dependent with a maximum at 24 h. We conclude that cancer cells which express a particular oncogene are suitable targets for radiolabeled antisense deoxyoligonucleotides directed toward the oncogene transcript. (orig.)

Novel CREB3L3 Nonsense Mutation in a Family With Dominant Hypertriglyceridemia.

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Cefalù, Angelo B; Spina, Rossella; Noto, Davide; Valenti, Vincenza; Ingrassia, Valeria; Giammanco, Antonina; Panno, Maria D; Ganci, Antonina; Barbagallo, Carlo M; Averna, Maurizio R

2015-12-01

Cyclic AMP responsive element-binding protein 3-like 3 (CREB3L3) is a novel candidate gene for dominant hypertriglyceridemia. To date, only 4 kindred with dominant hypertriglyceridemia have been found to be carriers of 2 nonsense mutations in CREB3L3 gene (245fs and W46X). We investigated a family in which hypertriglyceridemia displayed an autosomal dominant pattern of inheritance. The proband was a 49-year-old woman with high plasma triglycerides (≤1300 mg/dL; 14.68 mmol/L). Her father had a history of moderate hypertriglyceridemia, and her 51-year-old brother had triglycerides levels as high as 1600 mg/dL (18.06 mmol/L). To identify the causal mutation in this family, we analyzed the candidate genes of recessive and dominant forms of primary hypertriglyceridemia by direct sequencing. The sequencing of CREB3L3 gene led to the discovery of a novel minute frame shift mutation in exon 3 of CREB3L3 gene, predicted to result in the formation of a truncated protein devoid of function (c.359delG-p.K120fsX20). Heterozygosity for the c.359delG mutation resulted in a severe phenotype occurring later in life in the proband and her brother and a good response to diet and a hypotriglyceridemic treatment. The same mutation was detected in a 13-year-old daughter who to date is normotriglyceridemic. We have identified a novel pathogenic mutation in CREB3L3 gene in a family with dominant hypertriglyceridemia with a variable pattern of penetrance. © 2015 American Heart Association, Inc.

Bcl-2 antisense therapy in B-cell malignancies.

Science.gov (United States)

Chanan-Khan, Asher

2005-07-01

Bcl-2 is an apoptosis regulating protein, overexpression of which is associated with chemotherapy resistant disease, aggressive clinical course, and poor survival in patients with B-cell lymphoproliferative disorders. Overexpression of Bcl-2 protein results in an aberrant intrinsic apoptotic pathway that confers a protective effect on malignant cells against a death signal (e.g., chemotherapy or radiotherapy). Downregulation of this oncoprotein, thus, represents a possible new way to target clinically aggressive disease. Preclinical studies have shown that this oncoprotein can be effectively decreased by Bcl-2 antisense in malignant lymphoid cells and can reverse chemotherapy resistance, as well as enhance the anti-apoptotic potential of both chemotherapeutic and biologic agents. Ongoing clinical trials are exploring the role of Bcl-2 downregulation with oblimersen (Bcl-2 antisense) in patients with non-Hodgkin's lymphoma, chronic lymphocytic leukemia and multiple myeloma. Early results from these studies are promising and support the proof of the principle. As these studies are completed and mature data emerges, the role of Bcl-2 antisense therapy in the treatment of B-cell malignancies will become clearer.

GPR39 (zinc receptor) knockout mice exhibit depression-like behavior and CREB/BDNF down-regulation in the hippocampus.

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Młyniec, Katarzyna; Budziszewska, Bogusława; Holst, Birgitte; Ostachowicz, Beata; Nowak, Gabriel

2014-10-31

Zinc may act as a neurotransmitter in the central nervous system by activation of the GPR39 metabotropic receptors. In the present study, we investigated whether GPR39 knockout would cause depressive-like and/or anxiety-like behavior, as measured by the forced swim test, tail suspension test, and light/dark test. We also investigated whether lack of GPR39 would change levels of cAMP response element-binding protein (CREB),brain-derived neurotrophic factor (BDNF) and tropomyosin related kinase B (TrkB) protein in the hippocampus and frontal cortex of GPR39 knockout mice subjected to the forced swim test, as measured by Western-blot analysis. In this study, GPR39 knockout mice showed an increased immobility time in both the forced swim test and tail suspension test, indicating depressive-like behavior and displayed anxiety-like phenotype. GPR39 knockout mice had lower CREB and BDNF levels in the hippocampus, but not in the frontal cortex, which indicates region specificity for the impaired CREB/BDNF pathway (which is important in antidepressant response) in the absence of GPR39. There were no changes in TrkB protein in either structure. In the present study, we also investigated activity in the hypothalamus-pituitary-adrenal axis under both zinc- and GPR39-deficient conditions. Zinc-deficient mice had higher serum corticosterone levels and lower glucocorticoid receptor levels in the hippocampus and frontal cortex. There were no changes in the GPR39 knockout mice in comparison with the wild-type control mice, which does not support a role of GPR39 in hypothalamus-pituitary-adrenal axis regulation. The results of this study indicate the involvement of the GPR39 Zn(2+)-sensing receptor in the pathophysiology of depression with component of anxiety. © The Author 2015. Published by Oxford University Press on behalf of CINP.

Propofol prevents electroconvulsive-shock-induced memory impairment through regulation of hippocampal synaptic plasticity in a rat model of depression

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Luo J

2014-09-01

Full Text Available Jie Luo, Su Min, Ke Wei, Jun Cao, Bin Wang, Ping Li, Jun Dong, Yuanyuan Liu Department of Anesthesiology, the First Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China Background: Although a rapid and efficient psychiatric treatment, electroconvulsive therapy (ECT induces memory impairment. Modified ECT requires anesthesia for safety purposes. Although traditionally found to exert amnesic effects in general anesthesia, which is an inherent part of modified ECT, some anesthetics have been found to protect against ECT-induced cognitive impairment. However, the mechanisms remain unclear. We investigated the effects of propofol (2,6-diisopropylphenol on memory in depressed rats undergoing electroconvulsive shock (ECS, the analog of ECT in animals, under anesthesia as well as its mechanisms.Methods: Chronic unpredictable mild stresses were adopted to reproduce depression in a rodent model. Rats underwent ECS (or sham ECS with anesthesia with propofol or normal saline. Behavior was assessed in sucrose preference, open field and Morris water maze tests. Hippocampal long-term potentiation (LTP was measured using electrophysiological techniques. PSD-95, CREB, and p-CREB protein expression was assayed with western blotting.Results: Depression induced memory damage, and downregulated LTP, PSD-95, CREB, and p-CREB; these effects were exacerbated in depressed rats by ECS; propofol did not reverse the depression-induced changes, but when administered in modified ECS, propofol improved memory and reversed the downregulation of LTP and the proteins. Conclusion: These findings suggest that propofol prevents ECS-induced memory impairment, and modified ECS under anesthesia with propofol improves memory in depressed rats, possibly by reversing the excessive changes in hippocampal synaptic plasticity. These observations provide a novel insight into potential targets for optimizing the clinical use of ECT for psychiatric

Developmental transitions in Arabidopsis are regulated by antisense RNAs resulting from bidirectionally transcribed genes.

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Krzyczmonik, Katarzyna; Wroblewska-Swiniarska, Agata; Swiezewski, Szymon

2017-07-03

Transcription terminators are DNA elements located at the 3' end of genes that ensure efficient cleavage of nascent RNA generating the 3' end of mRNA, as well as facilitating disengagement of elongating DNA-dependent RNA polymerase II. Surprisingly, terminators are also a potent source of antisense transcription. We have recently described an Arabidopsis antisense transcript originating from the 3' end of a master regulator of Arabidopsis thaliana seed dormancy DOG1. In this review, we discuss the broader implications of our discovery in light of recent developments in yeast and Arabidopsis. We show that, surprisingly, the key features of terminators that give rise to antisense transcription are preserved between Arabidopsis and yeast, suggesting a conserved mechanism. We also compare our discovery to known antisense-based regulatory mechanisms, highlighting the link between antisense-based gene expression regulation and major developmental transitions in plants.

Identification of sumoylation sites in CCDC6, the first identified RET partner gene in papillary thyroid carcinoma, uncovers a mode of regulating CCDC6 function on CREB1 transcriptional activity.

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Chiara Luise

Full Text Available CCDC6 was originally identified in chimeric genes as caused by chromosomal translocation involving the RET protooncogene in some thyroid tumors. Recognised as a 65 kDa pro-apoptotic phosphoprotein, CCDC6 has been enrolled as an ATM substrate that contribute to protect genome integrity by modulating PP4c activity in response to genotoxic stress. Recently, CCDC6 has been identified as a repressor of CREB1-dependent transcription. Sumoylation has emerged as an important mechanism in transcriptional control. Here, we report the identification and characterization of three sites of sumoylation in CCDC6 (K74, K266 and K424 which are highly conserved in vertebrates. We demonstrate that the post-translational modifications by SUMO2 constrain most of the CCDC6 protein in the cytosol and affect its functional interaction with CREB1 with a decrease of CCDC6 repressive function on CREB1 transcriptional activity. Indeed, the impairment of functional outcome of sumoylated CCDC6 is obtained knocking down all three the sumoylation sites. Interestingly, in thyroid cells the SUMO2-mediated CCDC6 post-translational modifications are induced by Forskolin, a cAMP analog. Signal transduction via the cAMP pathway is known to be ubiquitous and represents a major line of communication between many organisms and their environment. We believe that CCDC6 could be an important player in the dynamics of cAMP signaling by fine regulating CREB1 transcriptional activity in normal and transformed thyroid cells.

CREB activity in dopamine D1 receptor expressing neurons regulates cocaine-induced behavioral effects

Science.gov (United States)

Bilbao, Ainhoa; Rieker, Claus; Cannella, Nazzareno; Parlato, Rosanna; Golda, Slawomir; Piechota, Marcin; Korostynski, Michal; Engblom, David; Przewlocki, Ryszard; Schütz, Günther; Spanagel, Rainer; Parkitna, Jan R.

2014-01-01

It is suggested that striatal cAMP responsive element binding protein (CREB) regulates sensitivity to psychostimulants. To test the cell-specificity of this hypothesis we examined the effects of a dominant-negative CREB protein variant expressed in dopamine receptor D1 (D1R) neurons on cocaine-induced behaviors. A transgenic mouse strain was generated by pronuclear injection of a BAC-derived transgene harboring the A-CREB sequence under the control of the D1R gene promoter. Compared to wild-type, drug-naïve mutants showed moderate alterations in gene expression, especially a reduction in basal levels of activity-regulated transcripts such as Arc and Egr2. The behavioral responses to cocaine were elevated in mutant mice. Locomotor activity after acute treatment, psychomotor sensitization after intermittent drug injections and the conditioned locomotion after saline treatment were increased compared to wild-type littermates. Transgenic mice had significantly higher cocaine conditioned place preference, displayed normal extinction of the conditioned preference, but showed an augmented cocaine-seeking response following priming-induced reinstatement. This enhanced cocaine-seeking response was associated with increased levels of activity-regulated transcripts and prodynorphin. The primary reinforcing effects of cocaine were not altered in the mutant mice as they did not differ from wild-type in cocaine self-administration under a fixed ratio schedule at the training dose. Collectively, our data indicate that expression of a dominant-negative CREB variant exclusively in neurons expressing D1R is sufficient to recapitulate the previously reported behavioral phenotypes associated with virally expressed dominant-negative CREB. PMID:24966820

Specific rescue by ortho-hydroxy atorvastatin of cortical GABAergic neurons from previous oxygen/glucose deprivation: role of pCREB.

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Guirao, Verónica; Martí-Sistac, Octavi; DeGregorio-Rocasolano, Núria; Ponce, Jovita; Dávalos, Antoni; Gasull, Teresa

2017-11-01

The statin atorvastatin (ATV) given as a post-treatment has been reported beneficial in stroke, although the mechanisms involved are not well understood so far. Here, we investigated in vitro the effect of post-treatment with ATV and its main bioactive metabolite ortho-hydroxy ATV (o-ATV) on neuroprotection after oxygen and glucose deprivation (OGD), and the role of the pro-survival cAMP response element-binding protein (CREB). Post-OGD treatment of primary cultures of rat cortical neurons with o-ATV, but not ATV, provided neuroprotection to a specific subset of cortical neurons that were large and positive for glutamic acid decarboxylase (large-GAD (+) neurons, GABAergic). Significantly, only these GABAergic neurons showed an increase in phosphorylated CREB (pCREB) early after neuronal cultures were treated post-OGD with o-ATV. We found that o-ATV, but not ATV, increased the neuronal uptake of glutamate from the medium; this provides a rationale for the specific effect of o-ATV on pCREB in large-GABAergic neurons, which have a higher ratio of synaptic (pCREB-promoting) vs extrasynaptic (pCREB-reducing) N-methyl-D-aspartate (NMDA) receptors (NMDAR) than that of small-non-GABAergic neurons. When we pharmacologically increased pCREB levels post-OGD in non-GABAergic neurons, through the selective activation of synaptic NMDAR, we observed as well long-lasting neuronal survival. We propose that the statin metabolite o-ATV given post-OGD boosts the intrinsic pro-survival factor pCREB in large-GABAergic cortical neurons in vitro, this contributing to protect them from OGD. © 2017 International Society for Neurochemistry.

Effective intracellular delivery of oligonucleotides in order to make sense of antisense

NARCIS (Netherlands)

Shi, FX; Hoekstra, D

2004-01-01

For more than two decades, antisense oligonucleotides (ODNs) have been used to modulate gene expression for the purpose of applications in cell biology and for development of novel sophisticated medical therapeutics. Conceptually, the antisense approach represents an elegant strategy, involving the

CREB binding protein is required for both short-term and long-term memory formation.

Science.gov (United States)

Chen, Guiquan; Zou, Xiaoyan; Watanabe, Hirotaka; van Deursen, Jan M; Shen, Jie

2010-09-29

CREB binding protein (CBP) is a transcriptional coactivator with histone acetyltransferase activity. Our prior study suggested that CBP might be a key target of presenilins in the regulation of memory formation and neuronal survival. To elucidate the role of CBP in the adult brain, we generated conditional knock-out (cKO) mice in which CBP is completely inactivated in excitatory neurons of the postnatal forebrain. Histological analysis revealed normal neuronal morphology and absence of age-dependent neuronal degeneration in the CBP cKO cerebral cortex. CBP cKO mice exhibited robust impairment in the formation of spatial, associative, and object-recognition memory. In addition to impaired long-term memory, CBP cKO mice also displayed deficits in short-term associative and object-recognition memory. Administration of a histone deacetylase inhibitor, trichostatin A, rescued the reduction of acetylated histones in the CBP cKO cortex but failed to rescue either short- or long-term memory deficits, suggesting that the memory impairment may not be caused by general reduction of histone acetyltransferase activity in CBP cKO mice. Further microarray and Western analysis showed decreased expression of calcium-calmodulin-dependent kinase isoforms and NMDA and AMPA receptor subunits in the cerebral cortex of CBP cKO mice. Collectively, these findings suggest a crucial role for CBP in the formation of both short- and long-term memory.

The requirement for enhanced CREB1 expression in consolidation of long-term synaptic facilitation and long-term excitability in sensory neurons of Aplysia

Science.gov (United States)

Liu, Rong-Yu; Cleary, Leonard J.; Byrne, John H.

2011-01-01

Accumulating evidence suggests that the transcriptional activator CREB1 is important for serotonin (5-HT)-induced long-term facilitation (LTF) of the sensorimotor synapse in Aplysia. Moreover, creb1 is among the genes activated by CREB1, suggesting a role for this protein beyond the induction phase of LTF. The time course of the requirement for CREB1 synthesis in the consolidation of long-term facilitation was examined using RNA interference (RNAi) techniques in sensorimotor co-cultures. Injection of CREB1 small-interfering RNA (siRNA) immediately or 10 h after 5-HT treatment blocked LTF when measured at 24 h and 48 h after treatment. In contrast, CREB1 siRNA did not block LTF when injected 16 h after 5-HT treatment. These results demonstrate that creb1 expression must be sustained for a relatively long time in order to support the consolidation of LTF. In addition, LTF is also accompanied by a long-term increase in the excitability (LTE) of sensory neurons (SNs). Because LTE was observed in the isolated SN after 5-HT treatment, this long-term change was intrinsic to that element of the circuit. LTE was blocked when CREB1 siRNA was injected into isolated SNs immediately after 5-HT treatment. These data suggest that 5-HT-induced CREB1 synthesis is required for consolidation of both LTF and LTE. PMID:21543617

Large-scale analysis of antisense transcription in wheat using the Affymetrix GeneChip Wheat Genome Array

Directory of Open Access Journals (Sweden)

Settles Matthew L

2009-05-01

Full Text Available Abstract Background Natural antisense transcripts (NATs are transcripts of the opposite DNA strand to the sense-strand either at the same locus (cis-encoded or a different locus (trans-encoded. They can affect gene expression at multiple stages including transcription, RNA processing and transport, and translation. NATs give rise to sense-antisense transcript pairs and the number of these identified has escalated greatly with the availability of DNA sequencing resources and public databases. Traditionally, NATs were identified by the alignment of full-length cDNAs or expressed sequence tags to genome sequences, but an alternative method for large-scale detection of sense-antisense transcript pairs involves the use of microarrays. In this study we developed a novel protocol to assay sense- and antisense-strand transcription on the 55 K Affymetrix GeneChip Wheat Genome Array, which is a 3' in vitro transcription (3'IVT expression array. We selected five different tissue types for assay to enable maximum discovery, and used the 'Chinese Spring' wheat genotype because most of the wheat GeneChip probe sequences were based on its genomic sequence. This study is the first report of using a 3'IVT expression array to discover the expression of natural sense-antisense transcript pairs, and may be considered as proof-of-concept. Results By using alternative target preparation schemes, both the sense- and antisense-strand derived transcripts were labeled and hybridized to the Wheat GeneChip. Quality assurance verified that successful hybridization did occur in the antisense-strand assay. A stringent threshold for positive hybridization was applied, which resulted in the identification of 110 sense-antisense transcript pairs, as well as 80 potentially antisense-specific transcripts. Strand-specific RT-PCR validated the microarray observations, and showed that antisense transcription is likely to be tissue specific. For the annotated sense-antisense

Protein domains involved in both in vivo and in vitro interactions between human T-cell leukemia virus type I tax and CREB.

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Yin, M J; Paulssen, E J; Seeler, J S; Gaynor, R B

1995-06-01

Gene expression from the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) is mediated by three cis-acting regulatory elements known as 21-bp repeats and the transactivator protein Tax. The 21-bp repeats can be subdivided into three motifs known as A, B, and C, each of which is important for maximal gene expression in response to Tax. The B motif contains nucleotide sequences known as a cyclic AMP response element (CRE) or tax-response element which binds members of the ATF/CREB family of transcription factors. Though mutations of this element in the HTLV-I LTR eliminate tax activation, Tax will not activate most other promoters containing these CRE sites. In this study, we investigated the mechanism by which Tax activates gene expression in conjunction with members of the ATF/CREB family. We found that Tax enhanced the binding of one member of the ATF/CREB family, CREB 1, to each of the three HTLV-I LTR 21-bp repeats but not another member designated CRE-BP1 or CREB2. Tax enhanced the binding of CREB1 to nonpalindromic CRE binding sites such as those found in the HTLV-I LTR, but Tax did not enhance the binding of CREB1 to palindromic CRE binding sites such as found in the somatostatin promoter. This finding may help explain the failure of Tax to activate promoters containing consensus CRE sites. These studies were extended by use of the mammalian two-hybrid system. Tax was demonstrated to interact directly with CREB1 but not with other bZIP proteins, including CREB2 and Jun. Site-directed mutagenesis of both Tax and CREB1 demonstrated that the amino terminus of Tax and both the basic and the leucine zipper regions of CREB1 were required for direct interactions between these proteins both in vivo and in vitro. This interaction occurred in vivo and thus did not require the presence of the HTLV-I 21-bp repeats, as previously suggested. These results define the domains required for interaction between Tax and CREB that are likely critical for the

A CREB-MPP7-AMOT Regulatory Axis Controls Muscle Stem Cell Expansion and Self-Renewal Competence

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Lydia Li

2017-10-01

Full Text Available Summary: Skeletal muscle regeneration requires resident muscle stem cells, termed satellite cells (SCs. SCs are largely quiescent during homeostasis yet become activated upon injury to supply myonuclei and self-renewed SCs. Molecular mechanisms underlying the competence of SCs to proliferate and self-renew in response to injury remain unclear. Here, we show that CREB activity establishes proliferative potential during SC quiescence. SCs with inhibited CREB activity remain quiescent and positioned in their niche, but upon injury, they cannot enter or maintain a proliferative state for expansion and self-renewal. We demonstrate mechanistically that Mpp7 is a CREB target and its functional mediator. MPP7 loss affects the level and sub-cellular localization of AMOT and YAP1 in quiescent SCs. Furthermore, MPP7 and AMOT are required for YAP1 nuclear accumulation, and the three are individually required for a proliferative state in myoblasts. We propose that the CREB-MPP7-AMOT-YAP1 axis establishes the competence of quiescent SCs to expand and self-renew, thereby preserving stem cell function. : Satellite cells are quiescent muscle stem cells that have the ability to regenerate muscles after injury. Li and Fan reveal an MPP7-AMOT-YAP1 regulatory axis that acts downstream of CREB to instill satellite cell competence. They also show how this regulatory axis prepares satellite cells for robust muscle regeneration after injury.

Up-regulation of Ca2+/CaMKII/CREB signaling in salicylate-induced tinnitus in rats.

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Zhao, Jiuhan; Wang, Biao; Wang, Xiaohong; Shang, Xiuli

2018-02-09

The purpose of the study was to investigate the changes of Ca 2+ /calmodulin-dependent protein kinases II (CaMKII)/cAMP response element-binding protein (CREB) signaling pathway in a rat tinnitus model. Eighteen Wistar rats were randomly divided into three groups: normal control (NC), normal saline (NS), and tinnitus model (TM) groups. Tinnitus model was induced by intraperitoneal injection of salicylate. The concentration of intracellular calcium level in auditory cortex cells was determined using Fura-2 acetoxymethyl ester (Fura-2 AM) method with fluorospectrophotometer. Expressions of calmodulin (CaM), N-methyl-D-aspartate receptor 2B subunit (NR2B), calcium-calmodulin kinase II (CaMKII), and cAMP response element-binding protein (CREB) were detected with Western blot. Tinnitus model was successfully established by the intraperitoneal administration of salicylate in rats. Compared with rats in NC and NS groups, salicylate administration significantly elevated CaM, NR2B, phospho-CaMKII and phospho-CREB expression in auditory cortex from tinnitus model group (p salicylate administration causes tinnitus symptoms and elevates Ca 2+ /CaMKII/CREB signaling pathway in auditory cortex cells. Our study likely provides a new understanding of the development of tinnitus.

Expression of phosphorylated cAMP response element binding protein (p-CREB) in bladder afferent pathways in VIP-/- mice with cyclophosphamide (CYP)-induced cystitis

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Jensen, Dorthe G; Studeny, Simon; May, Victor

2008-01-01

The expression of phosphorylated cAMP response element binding protein (p-CREB) in dorsal root ganglia (DRG) with and without cyclophosphamide (CYP)-induced cystitis (150 mg/kg, i.p; 48 h) was determined in VIP(-/-) and wild-type (WT) mice. p-CREB immunoreactivity (IR) was determined in bladder...... (Fast blue) afferent cells. Nerve growth factor (NGF) bladder content was determined by enzyme-linked immunosorbent assays. Basal expression of p-CREB-IR in DRG of VIP(-/-) mice was (p DRG compared to WT mice. CYP treatment in WT mice increased (p ...-CREB-IR in L1, L2, L5-S1 DRG. CYP treatment in VIP(-/-) mice (p DRG compared to WT with CYP. In WT mice, bladder afferent cells (20-38%) in DRG expressed p-CREB-IR under basal conditions. With CYP, p-CREB-IR increased in bladder afferent cells (60...

Fluoxetine increases the activity of the ERK-CREB signal system and alleviates the depressive-like behavior in rats exposed to chronic forced swim stress.

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Qi, Xiaoli; Lin, Wenjuan; Li, Junfa; Li, Huanhuan; Wang, Weiwen; Wang, Donglin; Sun, Meng

2008-08-01

Our previous research indicates that the extracellular signal-regulated kinase (ERK)-cyclic AMP-responsive-element-binding protein (CREB) signal system may be involved in the molecular mechanism of depression. The present study further investigated the effect of antidepressant fluoxetine on the ERK-CREB signal system and the depressive-like behaviors in rats. Fluoxetine was administrated to either naive rats or stressed rats for 21 days. The results showed that chronic forced swim stress induced depressive-like behaviors and decreased the levels of P-ERK2, P-CREB, ERK1/2 and CREB in hippocampus and prefrontal cortex. Fluoxetine alleviated the depressive-like behaviors and reversed the disruptions of the P-ERK2 and P-CREB in stressed rats. Fluoxetine also exerted mood-elevating effect and increased the levels of the P-ERK2 and P-CREB in naive rats. These results suggest that the ERK-CREB signal system may be the targets of the antidepressant action of fluoxetine and participate in the neuronal mechanism of depression.

A CREB1 Gene Polymorphism (rs2253206) Is Associated with Prospective Memory in a Healthy Cohort.

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Avgan, Nesli; Sutherland, Heidi G; Lea, Rodney A; Spriggens, Lauren K; Haupt, Larisa M; Shum, David H K; Griffiths, Lyn R

2017-01-01

Prospective memory (PM) is generally defined as remembering to perform intended actions in the future and is important for functioning in daily life. Cyclic adenosine monophosphate (cAMP) responsive element binding protein 1 (CREB1) plays an important role in cognitive functions. In this study, we hypothesized that genetic variation in the CREB1 gene is associated with PM. We genotyped a CREB1 promoter polymorphism rs2253206 and tested it for association with PM in 619 healthy subjects. PM performance was measured using the Prospective and Retrospective Memory Questionnaire (PRMQ), the Comprehensive Assessment of Prospective Memory (CAPM), and the Memory for Intentions Screening Test (MIST). Generalized linear model analysis was conducted for each PM test independently using different inheritance models to identify any associations ( p CAPM instrumental activities of daily living measure ( p = 0.016). These results suggest that the rs2253206 polymorphism in the CREB1 gene locus is associated with PM in healthy individuals and contributes to knowledge on the genetics of this particular type of memory.

Distinct changes in CREB phosphorylation in frontal cortex and striatum during contingent and non-contingent performance of a visual attention task

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Mirjana eCarli

2011-10-01

Full Text Available The cyclic-AMP response element binding protein (CREB family of transcription factors has been implicated in numerous forms of behavioural plasticity. We investigated CREB phosphorylation along some nodes of corticostriatal circuitry such as frontal cortex (FC and dorsal (caudate putamen, CPu and ventral (nucleus accumbens, NAC striatum in response to the contingent or non-contingent performance of the five-choice serial reaction time task (5-CSRTT used to assess visuospatial attention. Three experimental manipulations were used; an attentional performance group (contingent, master, a group trained previously on the task but for whom the instrumental contingency coupling responding with stimulus detection and reward was abolished (non-contingent, yoked and a control group matched for food deprivation and exposure to the test apparatus (untrained. Rats trained on the 5-CSRTT (both master and yoked had higher levels of CREB protein in the FC, CPu and NAC compared to untrained controls. Despite the divergent behaviour of master and yoked rats CREB activity in the FC was not substantially different. In rats performing the 5-CSRTT (master, CREB activity was completely abolished in the CPu whereas in the NAC it remained unchanged. In contrast, CREB phosphorylation in CPu and NAC increased only when the contingency changed from goal-dependent to goal-independent reinforcement (yoked. The present results indicate that up-regulation of CREB protein expression across cortical and striatal regions possibly reflects the extensive instrumental learning and performance whereas increased CREB activity in striatal regions may signal the unexpected change in the relationship between instrumental action and reinforcement.

Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products

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Hsiao Chiu-Bin

2006-11-01

Full Text Available Abstract Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1 long terminal repeat (LTR. Results Inspection of published sequences revealed a potential transcription initiator element (INR situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The

Forced running exercise attenuates hippocampal neurogenesis impairment and the neurocognitive deficits induced by whole-brain irradiation via the BDNF-mediated pathway

International Nuclear Information System (INIS)

Ji, Jian-feng; Ji, Sheng-jun; Sun, Rui; Li, Kun; Zhang, Yuan; Zhang, Li-yuan; Tian, Ye

2014-01-01

Highlights: •Forced exercise can ameliorate WBI induced cognitive impairment in our rat model. •Mature BDNF plays an important role in the effects of forced exercise. •Exercise may be a possible treatment of the radiation-induced cognitive impairment. -- Abstract: Cranial radiotherapy induces progressive and debilitating cognitive deficits, particularly in long-term cancer survivors, which may in part be caused by the reduction of hippocampal neurogenesis. Previous studies suggested that voluntary exercise can reduce the cognitive impairment caused by radiation therapy. However, there is no study on the effect of forced wheel exercise and little is known about the molecular mechanisms mediating the effect of exercise. In the present study, we investigated whether the forced running exercise after irradiation had the protective effects of the radiation-induced cognitive impairment. Sixty-four Male Sprague–Dawley rats received a single dose of 20 Gy or sham whole-brain irradiation (WBI), behavioral test was evaluated using open field test and Morris water maze at 2 months after irradiation. Half of the rats accepted a 3-week forced running exercise before the behavior detection. Immunofluorescence was used to evaluate the changes in hippocampal neurogenesis and Western blotting was used to assess changes in the levels of mature brain-derived neurotrophic factor (BDNF), phosphorylated tyrosine receptor kinase B (TrkB) receptor, protein kinase B (Akt), extracellular signal-regulated kinase (ERK), calcium-calmodulin dependent kinase (CaMKII), cAMP-calcium response element binding protein (CREB) in the BDNF–pCREB signaling. We found forced running exercise significantly prevented radiation-induced cognitive deficits, ameliorated the impairment of hippocampal neurogenesis and attenuated the down-regulation of these proteins. Moreover, exercise also increased behavioral performance, hippocampal neurogenesis and elevated BDNF–pCREB signaling in non

Forced running exercise attenuates hippocampal neurogenesis impairment and the neurocognitive deficits induced by whole-brain irradiation via the BDNF-mediated pathway

Energy Technology Data Exchange (ETDEWEB)

Ji, Jian-feng; Ji, Sheng-jun; Sun, Rui; Li, Kun; Zhang, Yuan; Zhang, Li-yuan; Tian, Ye, E-mail: dryetian@hotmail.com

2014-01-10

Highlights: •Forced exercise can ameliorate WBI induced cognitive impairment in our rat model. •Mature BDNF plays an important role in the effects of forced exercise. •Exercise may be a possible treatment of the radiation-induced cognitive impairment. -- Abstract: Cranial radiotherapy induces progressive and debilitating cognitive deficits, particularly in long-term cancer survivors, which may in part be caused by the reduction of hippocampal neurogenesis. Previous studies suggested that voluntary exercise can reduce the cognitive impairment caused by radiation therapy. However, there is no study on the effect of forced wheel exercise and little is known about the molecular mechanisms mediating the effect of exercise. In the present study, we investigated whether the forced running exercise after irradiation had the protective effects of the radiation-induced cognitive impairment. Sixty-four Male Sprague–Dawley rats received a single dose of 20 Gy or sham whole-brain irradiation (WBI), behavioral test was evaluated using open field test and Morris water maze at 2 months after irradiation. Half of the rats accepted a 3-week forced running exercise before the behavior detection. Immunofluorescence was used to evaluate the changes in hippocampal neurogenesis and Western blotting was used to assess changes in the levels of mature brain-derived neurotrophic factor (BDNF), phosphorylated tyrosine receptor kinase B (TrkB) receptor, protein kinase B (Akt), extracellular signal-regulated kinase (ERK), calcium-calmodulin dependent kinase (CaMKII), cAMP-calcium response element binding protein (CREB) in the BDNF–pCREB signaling. We found forced running exercise significantly prevented radiation-induced cognitive deficits, ameliorated the impairment of hippocampal neurogenesis and attenuated the down-regulation of these proteins. Moreover, exercise also increased behavioral performance, hippocampal neurogenesis and elevated BDNF–pCREB signaling in non

Directional gene expression and antisense transcripts in sexual and asexual stages of Plasmodium falciparum

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López-Barragán María J

2011-11-01

Full Text Available Abstract Background It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent and pattern of antisense transcripts in different developmental stages remain largely unknown. Results We have sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 parasites. Alignment of the cDNA sequences to the 3D7 reference genome revealed stage-specific antisense transcripts and novel intron-exon splicing junctions. Sequencing of strand-specific cDNA libraries suggested that more genes are expressed in one direction in gametocyte than in schizont. Alternatively spliced genes, antisense transcripts, and stage-specific expressed genes were also characterized. Conclusions It is necessary to continue to sequence cDNA from different developmental stages, particularly those of non-erythrocytic stages. The presence of antisense transcripts in some gametocyte and ookinete genes suggests that these antisense RNA may play an important role in gene expression regulation and parasite development. Future gene expression studies should make use of directional cDNA libraries. Antisense transcripts may partly explain the observed discrepancy between levels of mRNA and protein expression.

Molecular interactions involved in the transactivation of the human T-cell leukemia virus type 1 promoter mediated by Tax and CREB-2 (ATF-4).

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Gachon, F; Thebault, S; Peleraux, A; Devaux, C; Mesnard, J M

2000-05-01

The human T-cell leukemia virus type 1 (HTLV-1) Tax protein activates viral transcription through three 21-bp repeats located in the U3 region of the HTLV-1 long terminal repeat and called Tax-responsive elements (TxREs). Each TxRE contains nucleotide sequences corresponding to imperfect cyclic AMP response elements (CRE). In this study, we demonstrate that the bZIP transcriptional factor CREB-2 is able to bind in vitro to the TxREs and that CREB-2 binding to each of the 21-bp motifs is enhanced by Tax. We also demonstrate that Tax can weakly interact with CREB-2 bound to a cellular palindromic CRE motif such as that found in the somatostatin promoter. Mutagenesis of Tax and CREB-2 demonstrates that both N- and C-terminal domains of Tax and the C-terminal region of CREB-2 are required for direct interaction between the two proteins. In addition, the Tax mutant M47, defective for HTLV-1 activation, is unable to form in vitro a ternary complex with CREB-2 and TxRE. In agreement with recent results suggesting that Tax can recruit the coactivator CREB-binding protein (CBP) on the HTLV-1 promoter, we provide evidence that Tax, CREB-2, and CBP are capable of cooperating to stimulate viral transcription. Taken together, our data highlight the major role played by CREB-2 in Tax-mediated transactivation.

Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus

DEFF Research Database (Denmark)

tang, T. H.; Polacek, N.; Zywicki, M.

2005-01-01

By generating a specialized cDNA library from the archaeon Sulfolobus solfataricus, we have identified 57 novel small non-coding RNA (ncRNA) candidates and confirmed their expression by Northern blot analysis. The majority was found to belong to one of two classes, either antisense or antisense...... elements by inhibiting expression of the transposase mRNA. Surprisingly, the class of antisense RNAs also contained RNAs complementary to tRNAs or sRNAs (small-nucleolar-like RNAs). For the antisense-box ncRNAs, the majority could be assigned to the class of C/D sRNAs, which specify 2'-O-methylation sites...... on rRNAs or tRNAs. Five C/D sRNAs of this group are predicted to target methylation at six sites in 13 different tRNAs, thus pointing to the widespread role of these sRNA species in tRNA modification in Archaea. Another group of antisense-box RNAs, lacking typical C/D sRNA motifs, was predicted...

Bcl-2 antisense therapy in B-cell malignant proliferative disorders.

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Chanan-Khan, Asher; Czuczman, Myron S

2004-08-01

Overexpression of Bcl-2 oncogene has been clinically associated with an aggressive clinical course, chemotherapy and radiotherapy resistance, and poor survival in patients with malignant B-cell disorders. Patients with relapsed or refractory chronic lymphocytic leukemia, multiple myeloma, or non-Hodgkin's lymphoma have limited therapeutic options. Preclinical and early clinical data have shown that Bcl-2 oncoprotein can be decreased by Bcl-2 antisense therapy. Also, downregulation of Bcl-2 protein can result in reversal of chemotherapy resistance and improved antitumor activity of biologic agents. Various clinical trials are evaluating the role of targeting Bcl-2 as a mechanism to enhance the antitumor potential of chemotherapy and immunotherapy. Early results from these clinical studies are encouraging and confirm the proof of principle for antisense therapy. As current data mature, these trials will hopefully validate preliminary results and establish Bcl-2 antisense as an important addition to the current armamentarium used in the treatment of patients with B-cell neoplasms.

The integral membrane protein ITM2A, a transcriptional target of PKA-CREB, regulates autophagic flux via interaction with the vacuolar ATPase.

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Namkoong, Sim; Lee, Kang Il; Lee, Jin I; Park, Rackhyun; Lee, Eun-Ju; Jang, Ik-Soon; Park, Junsoo

2015-01-01

The PKA-CREB signaling pathway is involved in many cellular processes including autophagy. Recent studies demonstrated that PKA-CREB inhibits autophagy in yeast; however, the role of PKA-CREB signaling in mammalian cell autophagy has not been fully characterized. Here, we report that the integral membrane protein ITM2A expression is positively regulated by PKA-CREB signaling and ITM2A expression interferes with autophagic flux by interacting with vacuolar ATPase (v-ATPase). The ITM2A promoter contains a CRE element, and mutation at the CRE consensus site decreases the promoter activity. Forskolin treatment and PKA expression activate the ITM2A promoter confirming that ITM2A expression is dependent on the PKA-CREB pathway. ITM2A expression results in the accumulation of autophagosomes and interferes with autolysosome formation by blocking autophagic flux. We demonstrated that ITM2A physically interacts with v-ATPase and inhibits lysosomal function. These results support the notion that PKA-CREB signaling pathway regulates ITM2A expression, which negatively regulates autophagic flux by interfering with the function of v-ATPase.

The Inhibitory Effect of Somatostatin Receptor Activation on Bee Venom-Evoked Nociceptive Behavior and pCREB Expression in Rats

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Li Li

2014-01-01

Full Text Available The present study examined nociceptive behaviors and the expression of phosphorylated cAMP response element-binding protein (pCREB in the dorsal horn of the lumbar spinal cord and the dorsal root ganglion (DRG evoked by bee venom (BV. The effect of intraplantar preapplication of the somatostatin analog octreotide on nociceptive behaviors and pCREB expression was also examined. Subcutaneous injection of BV into the rat unilateral hindpaw pad induced significant spontaneous nociceptive behaviors, primary mechanical allodynia, primary thermal hyperalgesia, and mirror-thermal hyperalgesia, as well as an increase in pCREB expression in the lumbar spinal dorsal horn and DRG. Octreotide pretreatment significantly attenuated the BV-induced lifting/licking response and mechanical allodynia. Local injection of octreotide also significantly reduced pCREB expression in the lumbar spinal dorsal horn and DRG. Furthermore, pretreatment with cyclosomatostatin, a somatostatin receptor antagonist, reversed the octreotide-induced inhibition of the lifting/licking response, mechanical allodynia, and the expression of pCREB. These results suggest that BV can induce nociceptive responses and somatostatin receptors are involved in mediating the antinociception, which provides new evidence for peripheral analgesic action of somatostatin in an inflammatory pain state.

CREB Selectively Controls Learning-Induced Structural Remodeling of Neurons

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Middei, Silvia; Spalloni, Alida; Longone, Patrizia; Pittenger, Christopher; O'Mara, Shane M.; Marie, Helene; Ammassari-Teule, Martine

2012-01-01

The modulation of synaptic strength associated with learning is post-synaptically regulated by changes in density and shape of dendritic spines. The transcription factor CREB (cAMP response element binding protein) is required for memory formation and in vitro dendritic spine rearrangements, but its role in learning-induced remodeling of neurons…

Morphine Reward Promotes Cue-Sensitive Learning: Implication of Dorsal Striatal CREB Activity

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Mathieu Baudonnat

2017-05-01

Full Text Available Different parallel neural circuits interact and may even compete to process and store information: whereas stimulus–response (S–R learning critically depends on the dorsal striatum (DS, spatial memory relies on the hippocampus (HPC. Strikingly, despite its potential importance for our understanding of addictive behaviors, the impact of drug rewards on memory systems dynamics has not been extensively studied. Here, we assessed long-term effects of drug- vs food reinforcement on the subsequent use of S–R vs spatial learning strategies and their neural substrates. Mice were trained in a Y-maze cue-guided task, during which either food or morphine injections into the ventral tegmental area (VTA were used as rewards. Although drug- and food-reinforced mice learned the Y-maze task equally well, drug-reinforced mice exhibited a preferential use of an S–R learning strategy when tested in a water-maze competition task designed to dissociate cue-based and spatial learning. This cognitive bias was associated with a persistent increase in the phosphorylated form of cAMP response element-binding protein phosphorylation (pCREB within the DS, and a decrease of pCREB expression in the HPC. Pharmacological inhibition of striatal PKA pathway in drug-rewarded mice limited the morphine-induced increase in levels of pCREB in DS and restored a balanced use of spatial vs cue-based learning. Our findings suggest that drug (opiate reward biases the engagement of separate memory systems toward a predominant use of the cue-dependent system via an increase in learning-related striatal pCREB activity. Persistent functional imbalance between striatal and hippocampal activity could contribute to the persistence of addictive behaviors, or counteract the efficiency of pharmacological or psychotherapeutic treatments.

Steamed and Fermented Ethanolic Extract from Codonopsis lanceolata Attenuates Amyloid-β-Induced Memory Impairment in Mice

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Jin Bae Weon

2016-01-01

Full Text Available Codonopsis lanceolata (C. lanceolata is a traditional medicinal plant used for the treatment of certain inflammatory diseases such as asthma, tonsillitis, and pharyngitis. We evaluated whether steamed and fermented C. lanceolata (SFC extract improves amyloid-β- (Aβ- induced learning and memory impairment in mice. The Morris water maze and passive avoidance tests were used to evaluate the effect of SFC extract. Moreover, we investigated acetylcholinesterase (AChE activity and brain-derived neurotrophic factor (BDNF, cyclic AMP response element-binding protein (CREB, and extracellular signal-regulated kinase (ERK signaling in the hippocampus of mice to determine a possible mechanism for the cognitive-enhancing effect. Saponin compounds in SFC were identified by Ultra Performance Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry (UPLC-Q-TOF-MS. SFC extract ameliorated amyloid-β-induced memory impairment in the Morris water maze and passive avoidance tests. SFC extract inhibited AChE activity and also significantly increased the level of CREB phosphorylation, BDNF expression, and ERK activation in hippocampal tissue of amyloid-β-treated mice. Lancemasides A, B, C, D, E, and G and foetidissimoside A compounds present in SFC were determined by UPLC-Q-TOF-MS. These results indicate that SFC extract improves Aβ-induced memory deficits and that AChE inhibition and CREB/BDNF/ERK expression is important for the effect of the SFC extract. In addition, lancemaside A specifically may be responsible for efficacious effect of SFC.

Efficacy and Safety Profile of Tricyclo-DNA Antisense Oligonucleotides in Duchenne Muscular Dystrophy Mouse Model

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Karima Relizani

2017-09-01

Full Text Available Antisense oligonucleotides (AONs hold promise for therapeutic splice-switching correction in many genetic diseases. However, despite advances in AON chemistry and design, systemic use of AONs is limited due to poor tissue uptake and sufficient therapeutic efficacy is still difficult to achieve. A novel class of AONs made of tricyclo-DNA (tcDNA is considered very promising for the treatment of Duchenne muscular dystrophy (DMD, a neuromuscular disease typically caused by frameshifting deletions or nonsense mutations in the gene-encoding dystrophin and characterized by progressive muscle weakness, cardiomyopathy, and respiratory failure in addition to cognitive impairment. Herein, we report the efficacy and toxicology profile of a 13-mer tcDNA in mdx mice. We show that systemic delivery of 13-mer tcDNA allows restoration of dystrophin in skeletal muscles and to a lower extent in the brain, leading to muscle function improvement and correction of behavioral features linked to the emotional/cognitive deficiency. More importantly, tcDNA treatment was generally limited to minimal glomerular changes and few cell necroses in proximal tubules, with only slight variation in serum and urinary kidney toxicity biomarker levels. These results demonstrate an encouraging safety profile for tcDNA, albeit typical of phosphorothiate AONs, and confirm its therapeutic potential for the systemic treatment of DMD patients. Keywords: antisense oligonucleotides, Duchenne muscular dystrophy, preclinical, splice switching, tcDNA-AONs

The Neuroprotective Effect of Curcumin Against Nicotine-Induced Neurotoxicity is Mediated by CREB-BDNF Signaling Pathway.

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Motaghinejad, Majid; Motevalian, Manijeh; Fatima, Sulail; Faraji, Fahimeh; Mozaffari, Shiva

2017-10-01

Nicotine abuse adversely affects brain and causes apoptotic neurodegeneration. Curcumin- a bright yellow chemical compound found in turmeric is associated with neuroprotective properties. The current study was designed to evaluate the role of CREB-BDNF signaling in mediating the neuroprotective effects of curcumin against nicotine-induced apoptosis, oxidative stress and inflammation in rats. Sixty adult male rats were divided randomly into six groups. Group 1 received 0.7 ml/rat normal saline, group 2 received 6 mg/kg nicotine. Groups 3, 4, 5 and 6 were treated concurrently with nicotine (6 mg/kg) and curcumin (10, 20, 40 and 60 mg/kg i.p. respectively) for 21 days. Open Field Test (OFT) was used to evaluate the motor activity. Hippocampal oxidative, anti-oxidant, inflammatory and apoptotic factors were evaluated. Furthermore, phosphorylated brain cyclic adenosine monophosphate (cAMP) response element binding protein (P-CREB) and brain derived neurotrophic factor (BDNF) levels were studied at gene and protein levels. We found that nicotine disturbed the motor activity in OFT and simultaneous treatment with curcumin (40 and 60 mg/kg) reduced the nicotine-induced motor activity disturbances. In addition, nicotine treatment increased lipid peroxidation and the levels of GSH, IL-1β, TNF-α and Bax, while reducing Bcl-2, P-CREB and BDNF levels in the hippocampus. Nicotine also reduced the activity of superoxide dismutase, glutathione peroxidase and glutathione reductase in hippocampus. In contrast, various doses of curcumin attenuated nicotine-induced apoptosis, oxidative stress and inflammation; while elevating P-CREB and BDNF levels. Thus, curcumin via activation of P-CREB/BDNF signaling pathway, confers neuroprotection against nicotine-induced inflammation, apoptosis and oxidative stress.

The Antidepressant Agomelatine Improves Memory Deterioration and Upregulates CREB and BDNF Gene Expression Levels in Unpredictable Chronic Mild Stress (UCMS-Exposed Mice

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Esen Gumuslu

2014-01-01

Full Text Available Agomelatine, a novel antidepressant with established clinical efficacy, acts as an agonist of melatonergic MT 1 and MT 2 receptors and as an antagonist of 5-HT 2C receptors. The present study was undertaken to investigate whether chronic treatment with agomelatine would block unpredictable chronic mild stress (UCMS-induced cognitive deterioration in mice in passive avoidance (PA, modified elevated plus maze (mEPM, novel object recognition (NOR, and Morris water maze (MWM tests. Moreover, the effects of stress and agomelatine on brain-derived neurotrophic factor (BDNF and cyclic adenosine monophosphate (cAMP response element binding protein (CREB messenger ribonucleic acid (mRNA levels in the hippocampus was also determined using quantitative real-time polymerase chain reaction (RT-PCR. Male inbred BALB/c mice were treated with agomelatine (10 mg/kg, i.p., melatonin (10 mg/kg, or vehicle daily for five weeks. The results of this study revealed that UCMS-exposed animals exhibited memory deterioration in the PA, mEPM, NOR, and MWM tests. The chronic administration of melatonin had a positive effect in the PA and +mEPM tests, whereas agomelatine had a partial effect. Both agomelatine and melatonin blocked stress-induced impairment in visual memory in the NOR test and reversed spatial learning and memory impairment in the stressed group in the MWM test. Quantitative RT-PCR revealed that CREB and BDNF gene expression levels were downregulated in UCMS-exposed mice, and these alterations were reversed by chronic agomelatine or melatonin treatment. Thus, agomelatine plays an important role in blocking stress-induced hippocampal memory deterioration and activates molecular mechanisms of memory storage in response to a learning experience.

Monosialotetrahexosylganglioside Inhibits the Expression of p-CREB and NR2B in the Auditory Cortex in Rats with Salicylate-Induced Tinnitus.

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Song, Rui-Biao; Lou, Wei-Hua

2015-01-01

This study investigated the effects of monosialotetrahexosylganglioside (GM1) on the expression of N-methyl-D-aspartate receptor subunit 2B (NR2B) and phosphorylated (p)-cyclic AMP response element-binding protein (CREB) in the auditory cortex of rats with tinnitus. Tinnitus-like behavior in rats was tested with the gap prepulse inhibition of acoustic startle paradigm. We then investigated the NR2B mRNA and protein and p-CREB protein levels in the auditory cortex of tinnitus rats compared with normal rats. Rats treated for 4 days with salicylate exhibited tinnitus. NR2B mRNA and protein and p-CREB protein levels were upregulated in these animals, with expression returning to normal levels 14 days after cessation of treatment; baseline levels of NR2B and p-CREB were also restored by GM1 administration. These data suggest that chronic salicylate administration induces tinnitus via upregulation of p-CREB and NR2B expression, and that GM1 can potentially be used to treat tinnitus.

Curcumin reverses the effects of chronic stress on behavior, the HPA axis, BDNF expression and phosphorylation of CREB.

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Xu, Ying; Ku, Baoshan; Tie, Lu; Yao, Haiyan; Jiang, Wengao; Ma, Xing; Li, Xuejun

2006-11-29

Curcuma longa is a major constituent of the traditional Chinese medicine Xiaoyao-san, which has been used to effectively manage stress and depression-related disorders in China. Curcumin is the active component of curcuma longa, and its antidepressant effects were described in our prior studies in mouse models of behavioral despair. We hypothesized that curcumin may also alleviate stress-induced depressive-like behaviors and hypothalamic-pituitary-adrenal (HPA) axis dysfunction. Thus in present study we assessed whether curcumin treatment (2.5, 5 and 10 mg/kg, p.o.) affects behavior in a chronic unpredictable stress model of depression in rats and examined what its molecular targets may be. We found that subjecting animals to the chronic stress protocol for 20days resulted in performance deficits in the shuttle-box task and several physiological effects, such as an abnormal adrenal gland weight to body weight (AG/B) ratio and increased thickness of the adrenal cortex as well as elevated serum corticosterone levels and reduced glucocorticoid receptor (GR) mRNA expression. These changes were reversed by chronic curcumin administration (5 or 10 mg/kg, p.o.). In addition, we also found that the chronic stress procedure induced a down-regulation of brain-derived neurotrophic factor (BDNF) protein levels and reduced the ratio of phosphorylated cAMP response element-binding protein (pCREB) to CREB levels (pCREB/CREB) in the hippocampus and frontal cortex of stressed rats. Furthermore, these stress-induced decreases in BDNF and pCREB/CREB were also blocked by chronic curcumin administration (5 or 10 mg/kg, p.o.). These results provide compelling evidence that the behavioral effects of curcumin in chronically stressed animals, and by extension humans, may be related to their modulating effects on the HPA axis and neurotrophin factor expressions.

Antisense oligodeoxynucleotide inhibition as a potent diagnostic tool for gene function in plant biology

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Jansson, Christer; Sun, Chuanxin; Ghebramedhin, Haile; Hoglund, Anna-Stina; Jansson, Christer

2008-01-15

Antisense oligodeoxynucleotide (ODN) inhibition emerges as an effective means for probing gene function in plant cells. Employing this method we have established the importance of the SUSIBA2 transcription factor for regulation of starch synthesis in barley endosperm, and arrived at a model for the role of the SUSIBAs in sugar signaling and source-sink commutation during cereal endosperm development. In this addendum we provide additional data demonstrating the suitability of the antisense ODN technology in studies on starch branching enzyme activities in barley leaves. We also comment on the mechanism for ODN uptake in plant cells. Antisense ODNs are short (12-25 nt-long) stretches of single-stranded ODNs that hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. They are naturally occurring in both prokaryotes and eukaryotes where they partake in gene regulation and defense against viral infection. The mechanisms for antisense ODN inhibition are not fully understood but it is generally considered that the ODN either sterically interferes with translation or promotes transcript degradation by RNase H activation. The earliest indication of the usefulness of antisense ODN technology for the purposes of molecular biology and medical therapy was the demonstration in 1978 that synthetic ODNs complementary to Raos sarcoma virus could inhibit virus replication in tissue cultures of chick embryo fibroblasts. Since then the antisense ODN technology has been widely used in animal sciences and as an important emerging therapeutic approach in clinical medicine. However, antisense ODN inhibition has been an under-exploited strategy for plant tissues, although the prospects for plant cells in suspension cultures to take up single-stranded ODNs was reported over a decade ago. In 2001, two reports from Malho and coworker demonstrated the use of cationic-complexed antisense ODNs to suppress expression of genes encoding pollen

Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe

OpenAIRE

Chen, Huei-Mei; Rosebrock, Adam P.; Khan, Sohail R.; Futcher, Bruce; Leatherwood, Janet K.

2012-01-01

In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription ...

Modulation of lipoprotein metabolism by antisense technology: preclinical drug discovery methodology.

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Crooke, Rosanne M; Graham, Mark J

2013-01-01

Antisense oligonucleotides (ASOs) are a new class of specific therapeutic agents that alter the intermediary metabolism of mRNA, resulting in the suppression of disease-associated gene products. ASOs exert their pharmacological effects after hybridizing, via Watson-Crick base pairing, to a specific target RNA. If appropriately designed, this event results in the recruitment of RNase H, the degradation of targeted mRNA or pre-mRNA, and subsequent inhibition of the synthesis of a specific protein. A key advantage of the technology is the ability to selectively inhibit targets that cannot be modulated by traditional therapeutics such as structural proteins, transcription factors, and, of topical interest, lipoproteins. In this chapter, we will first provide an overview of antisense technology, then more specifically describe the status of lipoprotein-related genes that have been studied using the antisense platform, and finally, outline the general methodology required to design and evaluate the in vitro and in vivo efficacy of those drugs.

Nanoparticle for delivery of antisense γPNA oligomers targeting CCR5.

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Bahal, Raman; McNeer, Nicole Ali; Ly, Danith H; Saltzman, W Mark; Glazer, Peter M

2013-01-01

The development of a new class of peptide nucleic acids (PNAs), i.e., gamma PNAs (γPNAs), creates the need for a general and effective method for its delivery into cells for regulating gene expression in mammalian cells. Here we report the antisense activity of a recently developed hydrophilic and biocompatible diethylene glycol (miniPEG)-based gamma peptide nucleic acid called MPγPNAs via its delivery by poly(lactide-co-glycolide) (PLGA)-based nanoparticle system. We show that MPγPNA oligomers designed to bind to the selective region of chemokine receptor 5 (CC R5) transcript, induce potent and sequence-specific antisense effects as compared with regular PNA oligomers. In addition, PLGA nanoparticle delivery of MPγPNAs is not toxic to the cells. The findings reported in this study provide a combination of γPNA technology and PLGA-based nanoparticle delivery method for regulating gene expression in live cells via the antisense mechanism.

A Simple Three-Step Method for Design and Affinity Testing of New Antisense Peptides: An Example of Erythropoietin

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Nikola Štambuk

2014-05-01

Full Text Available Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide–receptor modulation. It is based on the fact that peptides specified by the complementary (antisense nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense–antisense (epitope–paratope peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.

Small molecule kaempferol modulates PDX-1 protein expression and subsequently promotes pancreatic β-cell survival and function via CREB

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Zhang, Yanling.; Zhen, Wei.; Maechler, Pierre; Liu, Dongmin

2013-01-01

Chronic hyperlipidemia causes β-cell apoptosis and dysfunction, thereby contributing to the pathogenesis of T2D. Thus, searching for agents to promote pancreatic β-cell survival and improve its function could be a promising strategy to prevent and treat T2D. We investigated the effects of kaempferol, a small molecule isolated from ginkgo biloba, on apoptosis and function of β-cells and further determined the mechanism underlying its actions. Kaempferol treatment promoted viability, inhibited apoptosis, and reduced caspase-3 activity in INS-1E cells and human islets chronically exposed to palmitate. In addition, kaempferol prevented the lipotoxicity-induced down-regulation of anti-apoptotic proteins Akt and Bcl-2. The cytoprotective effects of kaempferol were associated with improved insulin secretion, synthesis, and PDX-1 expression. Chronic hyperlipidemia significantly diminished cAMP production, PKA activation, and CREB phosphorylation and its regulated transcriptional activity in β-cells, all of which were restored by kaempferol treatment. Disruption of CREB expression by transfection of CREB siRNA in INS-1E cells or adenoviral transfer of dominant-negative forms of CREB in human islets ablated kaempferol protection of β-cell apoptosis and dysfunction caused by palmitate. Incubation of INS-1E cells or human islets with kaempferol for 48 h induced PDX-1 expression. This effect of kaempferol on PDX-1 expression was not shared by a host of structurally related flavonoid compounds. PDX-1 gene knockdown reduced kaempferol–stimulated cAMP generation and CREB activation in INS-1E cells. These findings demonstrate that kaempferol is a novel survivor factor for pancreatic β-cells via up-regulating the PDX-1/cAMP/PKA/CREB signaling cascade. PMID:22819546

The Role of CREB, SRF, and MEF2 in Activity-Dependent Neuronal Plasticity in the Visual Cortex.

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Pulimood, Nisha S; Rodrigues, Wandilson Dos Santos; Atkinson, Devon A; Mooney, Sandra M; Medina, Alexandre E

2017-07-12

The transcription factors CREB (cAMP response element binding factor), SRF (serum response factor), and MEF2 (myocyte enhancer factor 2) play critical roles in the mechanisms underlying neuronal plasticity. However, the role of the activation of these transcription factors in the different components of plasticity in vivo is not well known. In this study, we tested the role of CREB, SRF, and MEF2 in ocular dominance plasticity (ODP), a paradigm of activity-dependent neuronal plasticity in the visual cortex. These three proteins bind to the synaptic activity response element (SARE), an enhancer sequence found upstream of many plasticity-related genes (Kawashima et al., 2009; Rodríguez-Tornos et al., 2013), and can act cooperatively to express Arc , a gene required for ODP (McCurry et al., 2010). We used viral-mediated gene transfer to block the transcription function of CREB, SRF, and MEF2 in the visual cortex, and measured visually evoked potentials in awake male and female mice before and after a 7 d monocular deprivation, which allowed us to examine both the depression component (Dc-ODP) and potentiation component (Pc-ODP) of plasticity independently. We found that CREB, SRF, and MEF2 are all required for ODP, but have differential effects on Dc-ODP and Pc-ODP. CREB is necessary for both Dc-ODP and Pc-ODP, whereas SRF and MEF2 are only needed for Dc-ODP. This finding supports previous reports implicating SRF and MEF2 in long-term depression (required for Dc-ODP), and CREB in long-term potentiation (required for Pc-ODP). SIGNIFICANCE STATEMENT Activity-dependent neuronal plasticity is the cellular basis for learning and memory, and it is crucial for the refinement of neuronal circuits during development. Identifying the mechanisms of activity-dependent neuronal plasticity is crucial to finding therapeutic interventions in the myriad of disorders where it is disrupted, such as Fragile X syndrome, Rett syndrome, epilepsy, major depressive disorder, and autism

Neurons efficiently repair glutamate-induced oxidative DNA damage by a process involving CREB-mediated up-regulation of apurinic endonuclease 1

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Yang, Jenq-Lin; Tadokoro, Takashi; Keijzers, Guido

2010-01-01

inhibitor (KN-93) blocked the ability of glutamate to induce CREB phosphorylation and APE1 expression. Selective depletion of CREB using RNA interference prevented glutamate-induced up-regulation of APE1. Thus, glutamate receptor stimulation triggers Ca(2+)- and mitochondrial reactive oxygen species...

Mismatched single stranded antisense oligonucleotides can induce efficient dystrophin splice switching

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Kole Ryszard

2011-10-01

Full Text Available Abstract Background Antisense oligomer induced exon skipping aims to reduce the severity of Duchenne muscular dystrophy by redirecting splicing during pre-RNA processing such that the causative mutation is by-passed and a shorter but partially functional Becker muscular dystrophy-like dystrophin isoform is produced. Normal exons are generally targeted to restore the dystrophin reading frame however, an appreciable subset of dystrophin mutations are intra-exonic and therefore have the potential to compromise oligomer efficiency, necessitating personalised oligomer design for some patients. Although antisense oligomers are easily personalised, it remains unclear whether all patient polymorphisms within antisense oligomer target sequences will require the costly process of producing and validating patient specific compounds. Methods Here we report preclinical testing of a panel of splice switching antisense oligomers, designed to excise exon 25 from the dystrophin transcript, in normal and dystrophic patient cells. These patient cells harbour a single base insertion in exon 25 that lies within the target sequence of an oligomer shown to be effective at removing exon 25. Results It was anticipated that such a mutation would compromise oligomer binding and efficiency. However, we show that, despite the mismatch an oligomer, designed and optimised to excise exon 25 from the normal dystrophin mRNA, removes the mutated exon 25 more efficiently than the mutation-specific oligomer. Conclusion This raises the possibility that mismatched AOs could still be therapeutically applicable in some cases, negating the necessity to produce patient-specific compounds.

Tumor delivery of antisense oligomer using trastuzumab within a streptavidin nanoparticle

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Wang, Yi [University of Massachusetts Medical School, Division of Nuclear Medicine, Department of Radiology, Worcester, MA (United States); Yale University, Yale PET Center, Department of Diagnostic Radiology, New Haven, CT (United States); Liu, Xinrong; Chen, Ling; Cheng, Dengfeng; Rusckowski, Mary [University of Massachusetts Medical School, Division of Nuclear Medicine, Department of Radiology, Worcester, MA (United States); Hnatowich, Donald J. [University of Massachusetts Medical School, Division of Nuclear Medicine, Department of Radiology, Worcester, MA (United States); Umass Medical School, Department of Radiology, Worcester, MA (United States)

2009-12-15

Trastuzumab (Herceptin trademark) is often internalized following binding to Her2+ tumor cells. The objective of this study was to investigate whether trastuzumab can be used as a specific carrier to deliver antisense oligomers into Her2+ tumor cells both in vitro and in vivo. A biotinylated MORF oligomer antisense to RhoC mRNA and its biotinylated sense control were labeled with either lissamine for fluorescence detection or {sup 99m}Tc for radioactivity detection and were linked to biotinylated trastuzumab via streptavidin. The nanoparticles were studied in SUM190 (RhoC+, Her2+) study and SUM149 (RhoC+, Her2-) control cells in culture and as xenografts in mice. As evidence of unimpaired Her2+ binding of trastuzumab within the nanoparticle, accumulations were clearly higher in SUM190 compared to SUM149 cells and, by whole-body imaging, targeting of SUM190 tumor was similar to that expected for a radiolabeled trastuzumab. As evidence of internalization, fluorescence microscopy images of cells grown in culture and obtained from xenografts showed uniform cytoplasm distribution of the lissamine-MORF. An invasion assay showed decreased RhoC expression in SUM190 cells when incubated with the antisense MORF nanoparticles at only 100 nM. Both in cell culture and in animals, the nanoparticle with trastuzumab as specific carrier greatly improved tumor delivery of the antisense oligomer against RhoC mRNA into tumor cells overexpressing Her2 and may be of general utility. (orig.)

Repression of meiotic genes by antisense transcription and by Fkh2 transcription factor in Schizosaccharomyces pombe.

Science.gov (United States)

Chen, Huei-Mei; Rosebrock, Adam P; Khan, Sohail R; Futcher, Bruce; Leatherwood, Janet K

2012-01-01

In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the "unspliced" signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.

Cross-Sectional and Longitudinal Effects of CREB1 Genotypes on Individual Differences in Memory and Executive Function: Findings from the BLSA

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Claudia Wolf

2017-05-01

Full Text Available Purpose: Previously, we have shown that the SNP rs10932201 genotype of the cyclic AMP responsive element binding protein 1 gene (CREB1 contributes to individual differences in executive and memory function at the neural system and behavioral levels in healthy, young adults. However, longitudinal effects of CREB1 genotypes on cognition have not yet been addressed. Furthermore we were interested in replicating associations between CREB1 genotypes and human cognition in previous cross-sectional studies and explore whether APOE4 status might modify these relations.Materials and Methods: We investigated whether common, independent tag SNPs within CREB1 (rs2253206, rs10932201, rs6785 influence individual differences in age-related longitudinal change and level of executive function and memory performance independent of baseline age, sex, APOE4 status, and education. Our analysis included data from cognitively unimpaired older adults participating in the Baltimore Longitudinal Study of Aging. Eleven measures from six cognitive tests (sample sizes range 617–786 were analyzed using linear mixed effects and generalized estimating equations models. Mean baseline age ranged from 50 to 69 years and mean time of follow-up (interval ranged from 8 to 22 years.Results: We found significant effects of all three CREB1 SNPs on performance level and/or longitudinal change in performance based on eight measures assessing semantic memory, episodic memory, or both executive function and semantic memory. SNP rs10932201 showed the most significant and largest effect (Cohen’s d = -0.70, p < 0.01 on age-related longitudinal decline of semantic memory. Additionally, we show interactions between all three CREB1 SNPs and APOE4 status on age-related longitudinal declines and levels of memory and executive function.Conclusion: Our results suggest that CREB1 genotypes independently and by interactions with APOE4 status contribute to individual differences in cognitive aging.

Extracellular visfatin activates gluconeogenesis in HepG2 cells through the classical PKA/CREB-dependent pathway.

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Choi, Y J; Choi, S-E; Ha, E S; Kang, Y; Han, S J; Kim, D J; Lee, K W; Kim, H J

2014-04-01

Adipokines reportedly affect hepatic gluconeogenesis, and the adipokine visfatin is known to be related to insulin resistance and type 2 diabetes. However, whether visfatin contributes to hepatic gluconeogenesis remains unclear. Visfatin, also known as nicotinamide phosphoribosyltransferase (NAMPT), modulates sirtuin1 (SIRT1) through the regulation of nicotinamide adenine dinucleotide (NAD). Therefore, we investigated the effect of extracellular visfatin on glucose production in HepG2 cells, and evaluated whether extracellular visfatin affects hepatic gluconeogenesis via an NAD+-SIRT1-dependent pathway. Treatment with visfatin significantly increased glucose production and the mRNA expression and protein levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) in HepG2 cells in a time- and concentration-dependent manner. Knockdown of SIRT1 had no remarkable effect on the induction of gluconeogenesis by visfatin. Subsequently, we evaluated if extracellular visfatin stimulates the production of gluconeogenic enzymes through the classical protein kinase A (PKA)/cyclic AMP-responsive element (CRE)-binding protein (CREB)-dependent process. The phosphorylation of CREB and PKA increased significantly in HepG2 cells treated with visfatin. Additionally, knockdown of CREB and PKA inhibited visfatin-induced gluconeogenesis in HepG2 cells. In summary, extracellular visfatin modulates glucose production in HepG2 cells through the PKA/CREB pathway, rather than via SIRT1 signaling. © Georg Thieme Verlag KG Stuttgart · New York.

Sense-antisense (complementary) peptide interactions and the proteomic code; potential opportunities in biology and pharmaceutical science.

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Miller, Andrew D

2015-02-01

A sense peptide can be defined as a peptide whose sequence is coded by the nucleotide sequence (read 5' → 3') of the sense (positive) strand of DNA. Conversely, an antisense (complementary) peptide is coded by the corresponding nucleotide sequence (read 5' → 3') of the antisense (negative) strand of DNA. Research has been accumulating steadily to suggest that sense peptides are capable of specific interactions with their corresponding antisense peptides. Unfortunately, although more and more examples of specific sense-antisense peptide interactions are emerging, the very idea of such interactions does not conform to standard biology dogma and so there remains a sizeable challenge to lift this concept from being perceived as a peripheral phenomenon if not worse, into becoming part of the scientific mainstream. Specific interactions have now been exploited for the inhibition of number of widely different protein-protein and protein-receptor interactions in vitro and in vivo. Further, antisense peptides have also been used to induce the production of antibodies targeted to specific receptors or else the production of anti-idiotypic antibodies targeted against auto-antibodies. Such illustrations of utility would seem to suggest that observed sense-antisense peptide interactions are not just the consequence of a sequence of coincidental 'lucky-hits'. Indeed, at the very least, one might conclude that sense-antisense peptide interactions represent a potentially new and different source of leads for drug discovery. But could there be more to come from studies in this area? Studies on the potential mechanism of sense-antisense peptide interactions suggest that interactions may be driven by amino acid residue interactions specified from the genetic code. If so, such specified amino acid residue interactions could form the basis for an even wider amino acid residue interaction code (proteomic code) that links gene sequences to actual protein structure and function, even

Targeted disruption of the CREB coactivator Crtc2 increases insulin sensitivity

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Wang, Yiguo; Inoue, Hiroshi; Ravnskjær, Kim

2010-01-01

Under fasting conditions, increases in circulating concentrations of pancreatic glucagon maintain glucose homeostasis through induction of gluconeogenic genes by the CREB coactivator CRTC2. Hepatic CRTC2 activity is elevated in obesity, although the extent to which this cofactor contributes to at...

Regulation of cAMP Responsive Element Binding Protein 3-Like 1 (Creb3l1 Expression by Orphan Nuclear Receptor Nr4a1

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Michael P. Greenwood

2017-12-01

Full Text Available Cyclic AMP (cAMP inducible transcription factor cAMP responsive element binding protein 3 like 1 (Creb3l1 is strongly activated in the hypothalamus in response to hyperosmotic cues such as dehydration (DH. We have recently shown that Creb3l1 expression is upregulated by cAMP pathways in vitro, however the exact mechanisms are not known. Here we show that increasing Creb3l1 transcription by raising cAMP levels in mouse pituitary AtT20 cells automatically initiates cleavage of Creb3l1, leading to a greater abundance of the transcriptionally active N-terminal portion. Inhibiting protein synthesis indicated that de novo protein synthesis of an intermediary transcription factor was required for Creb3l1 induction. Strategic mining of our microarray data from dehydrated rodent hypothalamus revealed four candidates, reduced to two by analysis of acute hyperosmotic-induced transcriptional activation profiles in the hypothalamus, and one, orphan nuclear receptor Nr4a1, by direct shRNA mediated silencing in AtT20 cells. We show that activation of Creb3l1 transcription by Nr4a1 involves interaction with a single NBRE site in the promoter region. The ability to activate Creb3l1 transcription by this pathway in vitro is dictated by the level of methylation of a CpG island within the proximal promoter/5′UTR of this gene. We thus identify a novel cAMP-Nr4a1-Creb3l1 transcriptional pathway in AtT20 cells and also, our evidence would suggest, in the hypothalamus.

The transcription factors CREB and c-Fos play key roles in NCAM-mediated neuritogenesis in PC12-E2 cells

DEFF Research Database (Denmark)

Jessen, U; Novitskaya, V; Pedersen, N

2001-01-01

The neural cell adhesion molecule (NCAM) stimulates axonal outgrowth by activation of the Ras-mitogen activated protein kinase (MAPK) pathway and by generation of arachidonic acid. We investigated whether the transcription factors, cyclic-AMP response-element binding protein (CREB) and c-Fos play...... roles in this process by estimating NCAM-dependent neurite outgrowth from PC12-E2 cells grown in co-culture with NCAM-negative or NCAM-positive fibroblasts. PC12-E2 cells were transiently transfected with expression plasmids encoding wild-type or dominant negative forms of CREB and c-Fos or an activated...... form of the MAPK kinase, MEK2. Alternatively, PC12-E2 cells were treated with arachidonic acid, the cAMP analogue dBcAMP, or protein kinase A (PKA) inhibitors. The negative forms of CREB and c-Fos inhibited neurite outgrowth mediated by NCAM, arachidonic acid, dBcAMP, or MEK2. Neither CREB nor c...

Re-sensitizing drug-resistant bacteria to antibiotics by designing Antisense Therapeutics

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Courtney, Colleen; Chatterjee, Anushree

2014-03-01

``Super-bugs'' or ``multi-drug resistant organisms'' are a serious international health problem, with devastating consequences to patient health care. The Center for Disease Control has identified antibiotic resistance as one of the world's most pressing public health problems as a significant fraction of bacterial infections contracted are drug resistant. Typically, antibiotic resistance is encoded by ``resistance-genes'' which express proteins that carryout the resistance causing functions inside the bacterium. We present a RNA based therapeutic strategy for designing antimicrobials capable of re-sensitizing resistant bacteria to antibiotics by targeting labile regions of messenger RNAs encoding for resistance-causing proteins. We perform in silico RNA secondary structure modeling to identify labile target regions in an mRNA of interest. A synthetic biology approach is then used to administer antisense nucleic acids to our model system of ampicillin resistant Escherichia coli. Our results show a prolonged lag phase and decrease in viability of drug-resistant E. colitreated with antisense molecules. The antisense strategy can be applied to alter expression of other genes in antibiotic resistance pathways or other pathways of interest.

Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe

Science.gov (United States)

Chen, Huei-Mei; Rosebrock, Adam P.; Khan, Sohail R.; Futcher, Bruce; Leatherwood, Janet K.

2012-01-01

In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the “unspliced” signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression. PMID:22238674

Repression of meiotic genes by antisense transcription and by Fkh2 transcription factor in Schizosaccharomyces pombe.

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Huei-Mei Chen

Full Text Available In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the "unspliced" signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.

Lysine metabolism in antisense C-hordein barley grains

DEFF Research Database (Denmark)

Schmidt, Daiana; Rizzi, Vanessa; Gaziola, Salete A

2015-01-01

The grain proteins of barley are deficient in lysine and threonine due to their low concentrations in the major storage protein class, the hordeins, especially in the C-hordein subgroup. Previously produced antisense C-hordein transgenic barley lines have an improved amino acid composition, with ...

Gene network inference and biochemical assessment delineates GPCR pathways and CREB targets in small intestinal neuroendocrine neoplasia.

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Ignat Drozdov

Full Text Available Small intestinal (SI neuroendocrine tumors (NET are increasing in incidence, however little is known about their biology. High throughput techniques such as inference of gene regulatory networks from microarray experiments can objectively define signaling machinery in this disease. Genome-wide co-expression analysis was used to infer gene relevance network in SI-NETs. The network was confirmed to be non-random, scale-free, and highly modular. Functional analysis of gene co-expression modules revealed processes including 'Nervous system development', 'Immune response', and 'Cell-cycle'. Importantly, gene network topology and differential expression analysis identified over-expression of the GPCR signaling regulators, the cAMP synthetase, ADCY2, and the protein kinase A, PRKAR1A. Seven CREB response element (CRE transcripts associated with proliferation and secretion: BEX1, BICD1, CHGB, CPE, GABRB3, SCG2 and SCG3 as well as ADCY2 and PRKAR1A were measured in an independent SI dataset (n = 10 NETs; n = 8 normal preparations. All were up-regulated (p<0.035 with the exception of SCG3 which was not differently expressed. Forskolin (a direct cAMP activator, 10(-5 M significantly stimulated transcription of pCREB and 3/7 CREB targets, isoproterenol (a selective ß-adrenergic receptor agonist and cAMP activator, 10(-5 M stimulated pCREB and 4/7 targets while BIM-53061 (a dopamine D(2 and Serotonin [5-HT(2] receptor agonist, 10(-6 M stimulated 100% of targets as well as pCREB; CRE transcription correlated with the levels of cAMP accumulation and PKA activity; BIM-53061 stimulated the highest levels of cAMP and PKA (2.8-fold and 2.5-fold vs. 1.8-2-fold for isoproterenol and forskolin. Gene network inference and graph topology analysis in SI NETs suggests that SI NETs express neural GPCRs that activate different CRE targets associated with proliferation and secretion. In vitro studies, in a model NET cell system, confirmed that transcriptional

Pharmacological Activators of the NR4A Nuclear Receptors Enhance LTP in a CREB/CBP-Dependent Manner.

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Bridi, Morgan S; Hawk, Joshua D; Chatterjee, Snehajyoti; Safe, Stephen; Abel, Ted

2017-05-01

Nr4a nuclear receptors contribute to long-term memory formation and are required for long-term memory enhancement by a class of broad-acting drugs known as histone deacetylase (HDAC) inhibitors. Understanding the molecular mechanisms that regulate these genes and identifying ways to increase their activity may provide novel therapeutic approaches for ameliorating cognitive dysfunction. In the present study, we find that Nr4a gene expression after learning requires the cAMP-response element binding (CREB) interaction domain of the histone acetyltransferase CREB-binding protein (CBP). These gene expression deficits emerge at a time after learning marked by promoter histone acetylation in wild-type mice. Further, mutation of the CREB-CBP interaction domain reduces Nr4a promoter acetylation after learning. As memory enhancement by HDAC inhibitors requires CREB-CBP interaction and Nr4a gene function, these data support the notion that the balance of histone acetylation at the Nr4a promoters is critical for memory formation. NR4A ligands have recently been described, but the effect of these drugs on synaptic plasticity or memory has not been investigated. We find that the 'C-DIM' NR4A ligands, para-phenyl substituted di-indolylmethane compounds, enhance long-term contextual fear memory and increase the duration of long-term potentiation (LTP), a form of hippocampal synaptic plasticity. LTP enhancement by these drugs is eliminated in mice expressing a dominant negative form of NR4A and attenuated in mice with mutation of the CREB-CBP interaction domain. These data define the molecular connection between histone acetylation and Nr4a gene expression after learning. In addition, they suggest that NR4A-activating C-DIM compounds may serve as a potent and selective means to enhance memory and synaptic plasticity.

Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress.

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Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

2012-07-01

Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages.

Antisense PMO found in dystrophic dog model was effective in cells from exon 7-deleted DMD patient.

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Takashi Saito

Full Text Available BACKGROUND: Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD. We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD(J lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells. METHODOLOGY/PRINCIPAL FINDINGS: We converted fibroblasts of CXMD(J and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD(J and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species. CONCLUSION/SIGNIFICANCE: Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.

Ginsenosides Rb1 and Rg1 Stimulate Melanogenesis in Human Epidermal Melanocytes via PKA/CREB/MITF Signaling

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Mao Lin

2014-01-01

Full Text Available Reduced or defective melanin skin pigmentation may cause many hypopigmentation disorders and increase the risk of damage to the skin triggered by UV irradiation. Ginsenosides Rb1 and Rg1 have many molecular targets including the cAMP-response element-binding protein (CREB, which is involved in melanogenesis. This study aimed to investigate the effects of ginsenosides Rb1 and Rg1 on melanogenesis in human melanocytes and their related mechanisms. The effects of Rb1 and Rg1 on cell viability, tyrosinase activity, cellular melanin content and protein levels of tyrosinase, microphthalmia-associated transcription factor (MITF, and activation of CREB in melanocytes were assessed. Results showed that Rb1 or Rg1 significantly increased cellular melanin content and tyrosinase activity in a dose-dependent manner. By contrast, the cell viability of melanocytes remained unchanged. After exposure to Rb1 or Rg1, the protein levels of tyrosinase, MITF, and phosphorylated CREB were significantly increased. Furthermore, pretreatment with the selective PKA inhibitor H-89 significantly blocked the Rb1- or Rg1-induced increase of melanin content. These findings indicated that Rb1 and Rg1 increased melanogenesis and tyrosinase activity in human melanocytes, which was associated with activation of PKA/CREB/MITF signaling. The effects and mechanisms of Rb1 or Rg1 on skin pigmentation deserve further study.

Aqueous extracts from asparagus stems prevent memory impairments in scopolamine-treated mice.

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Sui, Zifang; Qi, Ce; Huang, Yunxiang; Ma, Shufeng; Wang, Xinguo; Le, Guowei; Sun, Jin

2017-04-19

Aqueous extracts from Asparagus officinalis L. stems (AEAS) are rich in polysaccharides, gamma-amino butyric acid (GABA), and steroidal saponin. This study was designed to investigate the effects of AEAS on learning, memory, and acetylcholinesterase-related activity in a scopolamine-induced model of amnesia. Sixty ICR mice were randomly divided into 6 groups (n = 10) including the control group (CT), scopolamine group (SC), donepezil group (DON), low, medium, and high dose groups of AEAS (LS, MS, HS; 1.6 mL kg -1 , 8 mL kg -1 , 16 mL kg -1 ). The results showed that 8 mL kg -1 of AEAS used in this study significantly reversed scopolamine-induced cognitive impairments in mice in the novel object recognition test (P < 0.05) and the Y-maze test (P < 0.05), and also improved the latency to escape in the Morris water maze test (P < 0.05). Moreover, it significantly increased acetylcholine and inhibited acetylcholinesterase activity in the hippocampus, which was directly related to the reduction in learning and memory impairments. It also reversed scopolamine-induced reduction in the hippocampal brain-derived neurotrophic factor (BDNF) and the cAMP response element-binding protein (CREB) mRNA expression. AEAS protected against scopolamine-induced memory deficits. In conclusion, AEAS protected learning and memory function in mice by enhancing the activity of the cholinergic nervous system, and increasing BDNF and CREB expression. This suggests that AEAS has the potential to prevent cognitive impairments in age-related diseases, such as Alzheimer's disease.

Aripiprazole Increases the PKA Signalling and Expression of the GABAA Receptor and CREB1 in the Nucleus Accumbens of Rats.

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Pan, Bo; Lian, Jiamei; Huang, Xu-Feng; Deng, Chao

2016-05-01

The GABAA receptor is implicated in the pathophysiology of schizophrenia and regulated by PKA signalling. Current antipsychotics bind with D2-like receptors, but not the GABAA receptor. The cAMP-responsive element-binding protein 1 (CREB1) is also associated with PKA signalling and may be related to the positive symptoms of schizophrenia. This study investigated the effects of antipsychotics in modulating D2-mediated PKA signalling and its downstream GABAA receptors and CREB1. Rats were treated orally with aripiprazole (0.75 mg/kg, ter in die (t.i.d.)), bifeprunox (0.8 mg/kg, t.i.d.), haloperidol (0.1 mg/kg, t.i.d.) or vehicle for 1 week. The levels of PKA-Cα and p-PKA in the prefrontal cortex (PFC), nucleus accumbens (NAc) and caudate putamen (CPu) were detected by Western blots. The mRNA levels of Gabrb1, Gabrb2, Gabrb3 and Creb1, and their protein expression were measured by qRT-PCR and Western blots, respectively. Aripiprazole elevated the levels of p-PKA and the ratio of p-PKA/PKA in the NAc, but not the PFC and CPu. Correlated with this elevated PKA signalling, aripiprazole elevated the mRNA and protein expression of the GABAA (β-1) receptor and CREB1 in the NAc. While haloperidol elevated the levels of p-PKA and the ratio of p-PKA/PKA in both NAc and CPu, it only tended to increase the expression of the GABAA (β-1) receptor and CREB1 in the NAc, but not the CPu. Bifeprunox had no effects on PKA signalling in these brain regions. These results suggest that aripiprazole has selective effects on upregulating the GABAA (β-1) receptor and CREB1 in the NAc, probably via activating PKA signalling.

Membrane-Associated Effects of Glucocorticoid on BACE1 Upregulation and Aβ Generation: Involvement of Lipid Raft-Mediated CREB Activation.

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Choi, Gee Euhn; Lee, Sei-Jung; Lee, Hyun Jik; Ko, So Hee; Chae, Chang Woo; Han, Ho Jae

2017-08-30

Glucocorticoid has been widely accepted to induce Alzheimer's disease, but the nongenomic effect of glucocorticoid on amyloid β (Aβ) generation has yet to be studied. Here, we investigated the effect of the nongenomic pathway induced by glucocorticoid on amyloid precursor protein processing enzymes as well as Aβ production using male ICR mice and human neuroblastoma SK-N-MC cells. Mice groups exposed to restraint stress or intracerebroventricular injection of Aβ showed impaired cognition, decreased intracellular glucocorticoid receptor (GR) level, but elevated level of membrane GR (mGR). In this respect, we identified the mGR-dependent pathway evoked by glucocorticoid using impermeable cortisol conjugated to BSA (cortisol-BSA) on SK-N-MC cells. Cortisol-BSA augmented the expression of β-site amyloid precursor protein cleaving enzyme 1 (BACE1), the level of C-terminal fragment β of amyloid precursor protein (C99) and Aβ production, which were maintained even after blocking intracellular GR. We also found that cortisol-BSA enhanced the interaction between mGR and Gαs, which colocalized in the lipid raft. The subsequently activated CREB by cortisol-BSA bound to the CRE site of the BACE1 promoter increasing its expression, which was downregulated by inhibiting CBP. Consistently, blocking CBP attenuated cognitive impairment and Aβ production induced by corticosterone treatment or intracerebroventricular injection of Aβ more efficiently than inhibiting intracellular GR in mice. In conclusion, glucocorticoid couples mGR with Gαs and triggers cAMP-PKA-CREB axis dependent on the lipid raft to stimulate BACE1 upregulation and Aβ generation. SIGNIFICANCE STATEMENT Patients with Alzheimer's disease (AD) have been growing sharply and stress is considered as the major environment factor of AD. Glucocorticoid is the primarily responsive factor to stress and is widely known to induce AD. However, most AD patients usually have impaired genomic pathway of glucocorticoid

PKC/CREB pathway mediates the expressions of GABAA receptor subunits in cultured hippocampal neurons after low-Mg2+ solution treatment.

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Wu, Guofeng; Yu, Jinpeng; Wang, Likun; Ren, Siying; Zhang, Yixia

2018-02-01

To investigate the potential effects of the PKC/CREB pathway on the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons using a model of epilepsy that employed conditions of low magnesium (Mg 2+ ). A total of 108 embryonic rats at the age of 18 embryonic days (E18)prepared from adult female SD rats were used as experimental subjects. Primary rat hippocampal cultures were prepared from the embryonic 18 days rats. The cultured hippocampal neurons were then treated with artificial cerebrospinal fluid containing low Mg 2+ solutions to generate a low Mg 2+ model of epilepsy. The low Mg 2+ stimulation lasted for 3 h and then returned to in maintenance medium for 20 h. The changes of the GABA A receptor subunit α1, γ2, δ were observed by blocking or activating the function of the CREB. The quantification of the GABA A receptor subunit α1, γ2, δ and the CREB were determined by a qRT-PCR and a Western blot method. After the neurons were exposed to a low-Mg 2+ solution for 3 h, GABA A receptor mRNA expression markedly increased compared to the control, and then gradually decreased. In contrast, CREB mRNA levels exhibited a dramatic down-regulation 3 h after terminating low-Mg 2+ treatment, and then peaked at 9 h. Western blot analyses verified that staurosporine suppressed CREB phosphorylation (p-CREB). The mRNA expression of GABA A receptor subunit α1 increased only in the presence of staurosporine, whereas the expressions of subunits γ2 and δ significantly increased in the presence of either KG-501 or staurosporine. Furthermore, phorbol 12-myristate 13-acetate (PMA) decreased the expressions of GABA A subunits α1, γ2, and δ when administered alone. However, the administration of either KG-501 or staurosporine reversed the inhibitory effects of PMA. The PKC/CREB pathway may negatively regulate the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons in low Mg 2+ model of

YC-1 potentiates cAMP-induced CREB activation and nitric oxide production in alveolar macrophages

Energy Technology Data Exchange (ETDEWEB)

Hwang, Tsong-Long, E-mail: htl@mail.cgu.edu.tw [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan, Taiwan (China); Tang, Ming-Chi [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China); Kuo, Liang-Mou [Department of General Surgery, Chang Gung Memorial Hospital at Chia-Yi, Taiwan (China); Chang, Wen-De; Chung, Pei-Jen; Chang, Ya-Wen; Fang, Yao-Ching [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan, Taiwan (China)

2012-04-15

Alveolar macrophages play significant roles in the pathogenesis of several inflammatory lung diseases. Increases in exhaled nitric oxide (NO) are well documented to reflect disease severity in the airway. In this study, we investigated the effect of 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1), a known activator of soluble guanylyl cyclase, on prostaglandin (PG)E{sub 1} (a stable PGE{sub 2} analogue) and forskolin (a adenylate cyclase activator) induced NO production and inducible NO synthase (iNOS) expression in rat alveolar macrophages (NR8383). YC-1 did not directly cause NO production or iNOS expression, but drastically potentiated PGE{sub 1}- or forskolin-induced NO production and iNOS expression in NR8383 alveolar macrophages. Combination treatment with YC-1 and PGE{sub 1} significantly increased phosphorylation of the cAMP response element-binding protein (CREB), but not nuclear factor (NF)-κB activation. The combined effect on NO production, iNOS expression, and CREB phosphorylation was reversed by a protein kinase (PK)A inhibitor (H89), suggesting that the potentiating functions were mediated through a cAMP/PKA signaling pathway. Consistent with this, cAMP analogues, but not the cGMP analogue, caused NO release, iNOS expression, and CREB activation. YC-1 treatment induced an increase in PGE{sub 1}-induced cAMP formation, which occurred through the inhibition of cAMP-specific phosphodiesterase (PDE) activity. Furthermore, the combination of rolipram (an inhibitor of PDE4), but not milronone (an inhibitor of PDE3), and PGE{sub 1} also triggered NO production and iNOS expression. In summary, YC-1 potentiates PGE{sub 1}-induced NO production and iNOS expression in alveolar macrophages through inhibition of cAMP PDE activity and activation of the cAMP/PKA/CREB signaling pathway. Highlights: ► YC-1 potentiated PGE1-induced iNOS expression in alveolar macrophages. ► The combination of YC-1 and PGE1 increased CREB but not NFκB activation. �

Epigenetic modification of miR-10a regulates renal damage by targeting CREB1 in type 2 diabetes mellitus.

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Shan, Qun; Zheng, Guihong; Zhu, Aihua; Cao, Li; Lu, Jun; Wu, Dongmei; Zhang, ZiFeng; Fan, Shaohua; Sun, Chunhui; Hu, Bin; Zheng, Yuanlin

2016-09-01

Emerging evidence has shown that microRNA-mediated gene expression modulation plays a crucial role in the pathogenesis of type 2 diabetes mellitus, but the novel miRNAs involved in type 2 diabetes and its functional regulatory mechanisms still need to be determined. In this study, we assessed the role of miR-10a in extracellular matrix accumulation in the kidney of diabetic mellitus induced by combining administration of chronic high fat diet (HFD) and low dosage of streptozotocin (STZ, 35mg/kg). Here, we found that HFD/STZ administration decreased the level of microRNA (miR-10a) expression in ICR strain mice. Overexpression of miR-10a alleviated the increased ratio of urine albumin-to-creatinine (ACR) ratio of HFD/STZ mice. In contrast, knockdown of miR-10a increased the ratio of kidney ACR in naïve mice. Furthermore, cAMP response element binding protein 1 (CREB1) was validated as a target of miR-10a in vitro and in vivo. CREB1 and its downstream fibronectin (FN, extracellular matrix) were increased in HFD/STZ-treated mice, which was reversed by kidney miR-10a overexpression. The content of CREB1 and FN was increased by miR-10a knockdown in kidney of naïve mice. Furthermore, histone deacetylase 3 (HDAC3) was revealed to be increased in kidney of HFD/STZ mice, accompanied with the augmentation of ACR ratio and FN level. Knockdown of HDAC3 with siRNA significantly caused the increase of miR-10a, resulting in the decrease in CREB1 and FN expression in kidney of HFD/STZ mice. Contrarily, HDAC3 overexpression mediated by lentivirus decreased miR-10a content, and enhanced ACR value, CREB1 and FN formation in naïve mice. Collectively, these results elucidate that HDAC3/miR-10a/CREB1 serves as a new mechanism underlying kidney injury, providing potential therapeutic targets in type 2 diabetes. Copyright © 2016. Published by Elsevier Inc.

Peptide nucleic acid (PNA) cell penetrating peptide (CPP) conjugates as carriers for cellular delivery of antisense oligomers

DEFF Research Database (Denmark)

Shiraishi, Takehiko; Nielsen, Peter E

2012-01-01

We have explored the merits of a novel delivery strategy for the antisense oligomers based on cell penetrating peptide (CPP) conjugated to a carrier PNA with sequence complementary to part of the antisense oligomer. The effect of these carrier CPP-PNAs was evaluated by using antisense PNA targeting......-PNA (cPNA1(7)-(D-Arg)8) and hexamer carrier decanoyl-CPP-PNA (Deca-cPNA1(6)-(D-Arg)8), respectively, without showing significant additional cellular toxicity. Most interestingly, the activity reached the same level obtained by enhancement with endosomolytic chloroquine (CQ) treatment, suggesting...... that the carrier might facilitate endosomal escape. Furthermore, 50% downregulation of luciferase expression at 60 nM siRNA was obtained using this carrier CPP-PNA delivery strategy (with CQ co-treatment) for a single stranded antisense RNA targeting normal luciferase mRNA. These results indicated that CPP...

Transfection of mouse cytotoxic T lymphocyte with an antisense granzyme A vector reduces lytic activity.

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Talento, A; Nguyen, M; Law, S; Wu, J K; Poe, M; Blake, J T; Patel, M; Wu, T J; Manyak, C L; Silberklang, M

1992-12-15

Murine CTL have seven serine proteases, known as granzymes, in their lytic granules. Despite considerable effort, convincing evidence that these enzymes play an obligatory role in the lytic process has not been presented. To investigate the function of one of these proteases, granzyme A (GA), we utilized an antisense expression vector to lower the level of the enzyme in the cells. An expression vector containing antisense cDNA for GA and the gene for hygromycin B resistance was constructed and electroporated into the murine CTL line, AR1. Transfectants were selected based on resistance to hygromycin B, and a number of stable lines were developed. One of the antisense lines had greatly reduced levels of GA mRNA, when compared to the parental cells or to control lines transfected with the vector lacking the antisense DNA. The message levels for two other CTL granule proteins, granzyme B and perforin, were unaffected by the antisense vector. The amount of GA, as measured by enzymatic activity, was 3- to 10-fold lower in the transfectant. Most significantly, this line also consistently showed 50 to 70% lower ability to lyse nucleated target cells and to degrade their DNA. Furthermore, it exhibited 90 to 95% lower lytic activity to anti-CD3-coated SRBC. Conjugate formation with target cells, however, was normal. These data provide strong evidence that GA plays an important role in the cytolytic cycle, and that the quantity of enzyme is a limiting factor in these cytolytic cells.

Review on investigations of antisense oligonucleotides with the use of mass spectrometry.

Science.gov (United States)

Studzińska, Sylwia

2018-01-01

Antisense oligonucleotides have been investigated as potential drugs for years. They inhibit target gene or protein expression. The present review summarizes their modifications, modes of action, and applications of liquid chromatography coupled with mass spectrometry for qualitative and quantitative analysis of these compounds. The most recent reports on a given topic were given prominence, while some early studies were reviewed in order to provide a theoretical background. The present review covers the issues of using ion-exchange chromatography, ion-pair reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography for the separation of antisense oligonucleotides. The application of mass spectrometry was described with regard to the ionization type used for the determination of these potential therapeutics. Moreover, the current approaches and applications of mass spectrometry for quantitative analysis of antisense oligonucleotides and their metabolites as well as their impurities during in vitro and in vivo studies were discussed. Finally, certain conclusions and perspectives on the determination of therapeutic oligonucleotides in various samples were briefly described. Copyright © 2017 Elsevier B.V. All rights reserved.

Dopamine-induced apoptosis in human neuronal cells: inhibition by nucleic acides antisense to the dopamine transporter

International Nuclear Information System (INIS)

Porat, S.; Gabbay, M.; Tauber, M.; Ratovitski, T.; Blinder, E.; Simantov, R.

1996-01-01

Human neuroblastoma NMB cells take up [ 3 H]dopamine in a selective manner indicating that dopamine transporters are responsible for this uptake. These cells were therefore used as a model to study dopamine neurotoxicity, and to elucidate the role of dopamine transporters in controlling cell death. Treatment with 0.05-0.4 mM dopamine changed cells' morphology within 4 h, accompanied by retraction of processes, shrinkage, apoptosis-like atrophy, accumulation of apoptotic particles, DNA fragmentation and cell death. Cycloheximide inhibited dopamine's effect, suggesting that induction of apoptosis by dopamine was dependent upon protein synthesis. Dopamine cytotoxicity, monitored morphologically by flow cytometric analysis, and by lactate dehydrogenase released, was blocked by cocaine but not by the noradrenaline and serotonin uptake blockers desimipramine and imipramine, respectively. Attempting to inhibit dopamine transport and toxicity in a drug-free and highly selective way, three 18-mer dopamine transporter antisense phosphorothioate oligonucleotides (numbers 1, 2 and 3) and a new plasmid vector expressing the entire rat dopamine transporter complementary DNA in the antisense orientation were prepared and tested. Antisense phosphorothioate oligonucleotide 3 inhibited [ 3 H]dopamine uptake in a time- and dose-dependent manner. Likewise, transient transfection of NMB cells with the plasmid expressing dopamine transporter complementary DNA in the antisense orientation partially blocked [ 3 H]dopamine uptake. Antisense phosphorothioate oligonucleotide 3 also decreased, dose-dependently, the toxic effect of dopamine and 6-hydroxydopamine. Western blot analysis with newly prepared anti-human dopamine transporter antibodies showed that antisense phosphorothioate oligonucleotide 3 decreased the transporter protein level. These studies contribute to better understand the mechanism of dopamine-induced apoptosis and neurotoxicity. (Copyright (c) 1996 Elsevier Science B

Technetium-99m labeled antisense oligonucleotide-noninvasive tumor imaging in mice

International Nuclear Information System (INIS)

Qin, G.M.; Zhang, Y.X.; An, R.; Gao, Z.R.; Cao, W.; Cao, G.X.; Hnatowich, D.J.

2002-01-01

Single-stranded RNA and DNA oligonucleotides may be useful as radiopharmaceuticals for antisense and other in vivo applications if convenient methods for stably attaching radionuclides such as 99m Tc can be developed. The c-myc oncogene works in cooperation with other oncogenes in a variety of malignant tumors. The concentration of c-myc messenger RNA increases rapidly 30 to 50 fold during DNA synthesis, thus making it a suitable target for following the progression of malignancy by noninvasive imaging with radiolabeled antisense oligonucleotide probes. Methods: 1 Oligonucleotide Conjugation: A solution of single stranded amine-derivatized DNA (100-1000μg) was prepared at a concentration of 2 mg/ml in 0.25M sodium bicarbonate, 1 M sodium chloride, 1mM EDTA, pH8.5. 2 Oligonucleotide Labeling: A fresh 50mg/ml solution of sodium tartrate was prepared in sterile 0.5 M ammonium The ability of the labeled DNA to hybridize to its complement was analyzed by Sep-Pak column chromatography before and after the addition of the complementary DNA. 3 Biodistribution and Tumor Imaging Studies: A colony of KM mice (15-20g) were inoculated with 1x10 6 Ehrlich carcinoma tumor cells in the right thigh, and the tumors were allowed to grow for 6-7 days to a size of 1.0-1.5 cm in diameter. Biodistribution studies were performed in 32 KM mice after 50 μCi per mouse of 99m Tc-labeled oncogene probes were injected intravenously. A total of 8 mice were injected intravenously in the tail vein with 1-2 mCi of 99m Tc-labeled sense or antisense probes, immobilized with ketamine hydrochloride and imaged periodically from 0.5hr to 24hr with a gamma camera. Results: Essentially complete conjugation was achieved by reverse-phase Sep-Pak C18 chromatography analysis. The labeled antisense DNA still remained the ability to hybridize with its complementary DNA. The highest accumulation of label was in the liver first, with the kidney and small bowel next. The injected activity localized in the lesion

Factor XI Antisense Oligonucleotide for Prevention of Venous Thrombosis

NARCIS (Netherlands)

Büller, Harry R.; Bethune, Claudette; Bhanot, Sanjay; Gailani, David; Monia, Brett P.; Raskob, Gary E.; Segers, Annelise; Verhamme, Peter; Weitz, Jeffrey I.; Weitz, Jeffrey; Prins, Martin; Beenen, Ludo; Otten, Hans-Martin; Roos, Yvo; Slagboom, Ton; Vandenbriele, Christophe; Vanassche, Thomas; Dani, Vidhi; Schulz, Dan; Shapiro, Cara; Kwoh, Katherine; Jung, Bill; Gawinek-Samelczak, Agata; Kaemmer, Christina; Angelov, S.; Stavrev, V.; Kinov, P.; Dessouki, E.; Abuzgaya, F.; Baurovskis, A.; Peredistijs, A.; Petronis, S.; Danilyak, V.; Driagin, V.; Kuropatkin, G.; Parfeev, S.; Safronov, A.; Ankin, M.; Korzh, M.; Olinichenko, G.; Polivoda, A.; Shevchenko, V.; Sulyma, V.

2015-01-01

Background Experimental data indicate that reducing factor XI levels attenuates thrombosis without causing bleeding, but the role of factor XI in the prevention of postoperative venous thrombosis in humans is unknown. FXI-ASO (ISIS 416858) is a second-generation antisense oligonucleotide that

Presenilins Regulate Neurotrypsin Gene Expression and Neurotrypsin-dependent Agrin Cleavage via Cyclic AMP Response Element-binding Protein (CREB) Modulation*

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Almenar-Queralt, Angels; Kim, Sonia N.; Benner, Christopher; Herrera, Cheryl M.; Kang, David E.; Garcia-Bassets, Ivan; Goldstein, Lawrence S. B.

2013-01-01

Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment. PMID:24145027

Presenilins regulate neurotrypsin gene expression and neurotrypsin-dependent agrin cleavage via cyclic AMP response element-binding protein (CREB) modulation.

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Almenar-Queralt, Angels; Kim, Sonia N; Benner, Christopher; Herrera, Cheryl M; Kang, David E; Garcia-Bassets, Ivan; Goldstein, Lawrence S B

2013-12-06

Presenilins, the catalytic components of the γ-secretase complex, are upstream regulators of multiple cellular pathways via regulation of gene transcription. However, the underlying mechanisms and the genes regulated by these pathways are poorly characterized. In this study, we identify Tequila and its mammalian ortholog Prss12 as genes negatively regulated by presenilins in Drosophila larval brains and mouse embryonic fibroblasts, respectively. Prss12 encodes the serine protease neurotrypsin, which cleaves the heparan sulfate proteoglycan agrin. Altered neurotrypsin activity causes serious synaptic and cognitive defects; despite this, the molecular processes regulating neurotrypsin expression and activity are poorly understood. Using γ-secretase drug inhibitors and presenilin mutants in mouse embryonic fibroblasts, we found that a mature γ-secretase complex was required to repress neurotrypsin expression and agrin cleavage. We also determined that PSEN1 endoproteolysis or processing of well known γ-secretase substrates was not essential for this process. At the transcriptional level, PSEN1/2 removal induced cyclic AMP response element-binding protein (CREB)/CREB-binding protein binding, accumulation of activating histone marks at the neurotrypsin promoter, and neurotrypsin transcriptional and functional up-regulation that was dependent on GSK3 activity. Upon PSEN1/2 reintroduction, this active epigenetic state was replaced by a methyl CpG-binding protein 2 (MeCP2)-containing repressive state and reduced neurotrypsin expression. Genome-wide analysis revealed hundreds of other mouse promoters in which CREB binding is similarly modulated by the presence/absence of presenilins. Our study thus identifies Tequila and neurotrypsin as new genes repressed by presenilins and reveals a novel mechanism used by presenilins to modulate CREB signaling based on controlling CREB recruitment.

Chronic copper exposure causes spatial memory impairment, selective loss of hippocampal synaptic proteins, and activation of PKR/eIF2α pathway in mice.

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Ma, Quan; Ying, Ming; Sui, Xiaojing; Zhang, Huimin; Huang, Haiyan; Yang, Linqing; Huang, Xinfeng; Zhuang, Zhixiong; Liu, Jianjun; Yang, Xifei

2015-01-01

Copper is an essential element for human growth and development; however, excessive intake of copper could contribute to neurotoxicity. Here we show that chronic exposure to copper in drinking water impaired spatial memory with simultaneous selective loss of hippocampal pre-synaptic protein synapsin 1, and post-synaptic density protein (PSD)-93/95 in mice. Copper exposure was shown to elevate the levels of nitrotyrosine and 8-hydroxydeoxyguanosine (8-OHdG) in hippocampus, two markers of oxidative stress. Concurrently, we also found that copper exposure activated double stranded RNA-dependent protein kinase (PKR) as evidenced by increased ratio of phosphorylated PKR at Thr451 and total PKR and increased the phosphorylation of its downstream signaling molecule eukaryotic initiation factor 2α (eIF2α) at Ser51 in hippocampus. Consistent with activation of PKR/eIF2α signaling pathway which was shown to mediate synaptic deficit and cognitive impairment, the levels of activating transcription factor 4 (ATF-4), a downstream signaling molecule of eIF2α and a repressor of CREB-mediated gene expression, were significantly increased, while the activity of cAMP response elements binding protein (CREB) was inactivated as suggested by decreased phosphorylation of CREB at Ser133 by copper exposure. In addition, the expression of the pro-apoptotic target molecule C/EBP homology protein (CHOP) of ATF-4 was upregulated and hippocampal neuronal apoptosis was induced by copper exposure. Taken together, we propose that chronic copper exposure might cause spatial memory impairment, selective loss of synaptic proteins, and neuronal apoptosis through the mechanisms involving activation of PKR/eIF2α signaling pathway.

DA-9801 promotes neurite outgrowth via ERK1/2-CREB pathway in PC12 cells.

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Won, Jong Hoon; Ahn, Kyong Hoon; Back, Moon Jung; Ha, Hae Chan; Jang, Ji Min; Kim, Ha Hyung; Choi, Sang-Zin; Son, Miwon; Kim, Dae Kyong

2015-01-01

In the present study, we examined the mechanisms underlying the effect of DA-9801 on neurite outgrowth. We found that DA-9801 elicits its effects via the mitogen-activated protein kinase (MEK) extracellular signal-regulated kinase (ERK)1/2-cAMP response element-binding protein (CREB) pathway. DA-9801, an extract from a mixture of Dioscorea japonica and Dioscorea nipponica, was reported to promote neurite outgrowth in PC12 cells. The effects of DA-9801 on cell viability and expression of neuronal markers were evaluated in PC12 cells. To investigate DA-9801 action, specific inhibitors targeting the ERK signaling cascade were used. No cytotoxicity was observed in PC12 cells at DA-9801 concentrations of less than 30 µg/mL. In the presence of nerve growth factor (NGF, 2 ng/mL), DA-9801 promoted neurite outgrowth and increased the relative mRNA levels of neurofilament-L (NF-L), a marker of neuronal differentiation. The Raf-1 inhibitor GW5074 and MEK inhibitor PD98059 significantly attenuated DA-9801-induced neurite outgrowth. Additionally, the MEK1 and MEK2 inhibitor SL327 significantly attenuated the increase in the percentage of neurite-bearing PC12 cells induced by DA-9801 treatment. Conversely, the selective p38 mitogen-activated protein kinase inhibitor SB203580 did not attenuate the DA-9801 treatment-induced increase in the percentage of neurite-bearing PC12 cells. DA-9801 enhanced the phosphorylation of ERK1/2 and CREB in PC12 cells incubated with and without NGF. Pretreatment with PD98059 blocked the DA-9801-induced phosphorylation of ERK1/2 and CREB. In conclusion, DA-9801 induces neurite outgrowth by affecting the ERK1/2-CREB signaling pathway. Insights into the mechanism underlying this effect of DA-9801 may suggest novel potential strategies for the treatment of peripheral neuropathy.

Analysis of antisense expression by whole genome tiling microarrays and siRNAs suggests mis-annotation of Arabidopsis orphan protein-coding genes.

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Casey R Richardson

2010-05-01

Full Text Available MicroRNAs (miRNAs and trans-acting small-interfering RNAs (tasi-RNAs are small (20-22 nt long RNAs (smRNAs generated from hairpin secondary structures or antisense transcripts, respectively, that regulate gene expression by Watson-Crick pairing to a target mRNA and altering expression by mechanisms related to RNA interference. The high sequence homology of plant miRNAs to their targets has been the mainstay of miRNA prediction algorithms, which are limited in their predictive power for other kingdoms because miRNA complementarity is less conserved yet transitive processes (production of antisense smRNAs are active in eukaryotes. We hypothesize that antisense transcription and associated smRNAs are biomarkers which can be computationally modeled for gene discovery.We explored rice (Oryza sativa sense and antisense gene expression in publicly available whole genome tiling array transcriptome data and sequenced smRNA libraries (as well as C. elegans and found evidence of transitivity of MIRNA genes similar to that found in Arabidopsis. Statistical analysis of antisense transcript abundances, presence of antisense ESTs, and association with smRNAs suggests several hundred Arabidopsis 'orphan' hypothetical genes are non-coding RNAs. Consistent with this hypothesis, we found novel Arabidopsis homologues of some MIRNA genes on the antisense strand of previously annotated protein-coding genes. A Support Vector Machine (SVM was applied using thermodynamic energy of binding plus novel expression features of sense/antisense transcription topology and siRNA abundances to build a prediction model of miRNA targets. The SVM when trained on targets could predict the "ancient" (deeply conserved class of validated Arabidopsis MIRNA genes with an accuracy of 84%, and 76% for "new" rapidly-evolving MIRNA genes.Antisense and smRNA expression features and computational methods may identify novel MIRNA genes and other non-coding RNAs in plants and potentially other

Single nucleotide polymorphism near CREB1, rs7591784, is associated with pretreatment methamphetamine use frequency and outcome of outpatient treatment for methamphetamine use disorder.

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Heinzerling, Keith G; Demirdjian, Levon; Wu, Yingnian; Shoptaw, Steven

2016-03-01

Although stimulant dependence is highly heritable, few studies have examined genetic influences on methamphetamine dependence. We performed a candidate gene study of 52 SNPs and pretreatment methamphetamine use frequency among 263 methamphetamine dependent Hispanic and Non-Hispanic White participants of several methamphetamine outpatient clinical trials in Los Angeles. One SNP, rs7591784 was significantly associated with pretreatment methamphetamine use frequency following Bonferroni correction (p methamphetamine urine drug screen results during 12 weeks of outpatient treatment among males with treatment outcome data available (N = 94) and found rs7591784 was significantly associated with methamphetamine use during treatment controlling for pretreatment methamphetamine use. rs7591784 is near CREB1 and in a linkage disequilibrium block with rs2952768, previously shown to influence CREB1 expression. The CREB signaling pathway is involved in gene expression changes related to chronic use of multiple drugs of abuse including methamphetamine and these results suggest that variability in CREB signaling may influence pretreatment frequency of methamphetamine use as well as outcomes of outpatient treatment. Medications targeting the CREB pathway, including phosphodiesterase inhibitors, warrant investigation as pharmacotherapies for methamphetamine use disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

Single nucleotide polymorphism near CREB1, rs7591784, is associated with pretreatment methamphetamine use frequency and outcome of outpatient treatment for methamphetamine use disorder

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Heinzerling, Keith G.; Demirdjian, Levon; Wu, Yingnian; Shoptaw, Steven

2016-01-01

Although stimulant dependence is highly heritable, few studies have examined genetic influences on methamphetamine dependence. We performed a candidate gene study of 52 SNPs and pretreatment methamphetamine use frequency among 263 methamphetamine dependent Hispanic and Non-Hispanic White participants of several methamphetamine outpatient clinical trials in Los Angeles. One SNP, rs7591784 was significantly associated with pretreatment methamphetamine use frequency following Bonferroni correction (p methamphetamine urine drug screen results during 12 weeks of outpatient treatment among males with treatment outcome data available (N = 94) and found rs7591784 was significantly associated with methamphetamine use during treatment controlling for pretreatment methamphetamine use. rs7591784 is near CREB1 and in a linkage disequilibrium block with rs2952768, previously shown to influence CREB1 expression. The CREB signaling pathway is involved in gene expression changes related to chronic use of multiple drugs of abuse including methamphetamine and these results suggest that variability in CREB signaling may influence pretreatment frequency of methamphetamine use as well as outcomes of outpatient treatment. Medications targeting the CREB pathway, including phosphodiesterase inhibitors, warrant investigation as pharmacotherapies for methamphetamine use disorders. PMID:26736037

Glutamatergic induction of CREB phosphorylation and Fos expression in primary cultures of the suprachiasmatic hypothalamus in vitro is mediated by co-ordinate activity of NMDA and non-NMDA receptors.

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Schurov, I L; McNulty, S; Best, J D; Sloper, P J; Hastings, M H

1999-01-01

Exposure of Syrian hamsters to light 1 h after lights-off rapidly (10 min) induced nuclear immunoreactivity (-ir) to the phospho-Ser133 form of the Ca2+/cAMP response element (CRE) binding protein (pCREB) in the retinorecipient zone of the suprachiasmatic nuclei (SCN). Light also induced nuclear Fos-ir in the same region of the SCN after 1 h. The glutamatergic N-methyl-D-aspartate (NMDA) receptor blocker MK801 attenuated the photic induction of both factors. To investigate glutamatergic regulation of pCREB and Fos further, tissue blocks and primary cultures of neonatal hamster SCN were examined by Western blotting and immunocytochemistry in vitro. On Western blots of SCN tissue, the pCREB-ir signal at 45 kDa was enhanced by glutamate or a mixture of glutamatergic agonists (NMDA, amino-methyl proprionic acid (AMPA), and Kainate (KA)), whereas total CREB did not change. Glutamate or the mixture of agonists also induced a 56 kDa band identified as Fos protein in SCN tissue. In dissociated cultures of SCN, glutamate caused a rapid (15 min) induction of nuclear pCREB-ir and Fos-ir (after 60 min) exclusively in neurones, both GABA-ir and others. Treatment with NMDA alone had no effect on pCREB-ir. AMPA alone caused a slight increase in pCREB-ir. However, kainate alone or in combination with NMDA and AMPA induced nuclear pCREB-ir equal to that induced by glutamate. The effects of glutamate on pCREB-ir and Fos-ir were blocked by antagonists of both NMDA (MK801) and AMPA/KA (NBQX) receptors. In the absence of extracellular Mg2+, MK801 blocked glutamatergic induction of Fos-ir. However, the AMPA/KA receptor antagonist was no longer effective at blocking glutamatergic induction of either Fos-ir or pCREB-ir, consistent with the model that glutamate regulates gene expression in the SCN by a co-ordinate action through both NMDA and AMPA/KA receptors. Glutamatergic induction of nuclear pCREB-ir in GABA-ir neurones was blocked by KN-62 an inhibitor of Ca2+/Calmodulin (Ca

Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.

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Duchrow, M; Schmidt, M H; Zingler, M; Anemüller, S; Bruch, H P; Broll, R

2001-01-01

The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements. Copyright 2001 S. Karger AG, Basel

Atorvastatin inhibits insulin synthesis by inhibiting the Ras/Raf/ERK/CREB pathway in INS-1 cells

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Sun, Hongxi; Li, Yu; Sun, Bei; Hou, Ningning; Yang, Juhong; Zheng, Miaoyan; Xu, Jie; Wang, Jingyu; Zhang, Yi; Zeng, Xianwei; Shan, Chunyan; Chang, Bai; Chen, Liming; Chang, Baocheng

2016-01-01

Abstract Backround: Type 2 diabetes has become a global epidemic disease. Atorvastatin has become a cornerstone in the prevention and treatment of atherosclerosis. However, increasing evidence showed that statins can dose-dependently increase the risk of diabetes mellitus. The mechanism is not clear. Objective: The Ras complex pathway (Ras/Raf/extracellular signal-regulated kinase [ERK]/cAMP response element-binding protein [CREB]) is the major pathway that regulates the gene transcription. Except for the inhibition of cholesterol synthesis by inhibiting the 3-hydroxy-3-methyl glutaryl coenzyme A (HMG-COA) reductase, statins can also downregulate the phosphorylation of a series of downstream substrates including the key proteins of the Ras complex pathway, therefore may inhibit the insulin syntheses in pancreatic beta cells. In our study, we investigated the inhibitory effect and the underlying mechanism of atorvastatin on insulin synthesis in rat islets. Methods: Islets were isolated from Wistar rats and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium. The insulin content in the medium was measured by radioimmunoassay before and after the treatment of 50 μM atorvastatin. Effect of atorvastatin on the expression of insulin message Ribonucleic acid (mRNA) in pancreatic islet beta cells was also detected using quantitative real-time polymerase chain reaction. Western blotting was used to explore the possible role of the Ras complex pathway (Ras/Raf/ERK/CREB) in atorvastatin-inhibited insulin synthesis. The effects of atorvastatin on the binding of nuclear transcription factor p-CREB with CRE in INS-1 cells were examined via chromatin immunoprecipitation assay. Results: Compared with the control group, the insulin level decreased by 27.1% at 24 hours after atorvastatin treatment. Atorvastatin inhibited insulin synthesis by decreasing insulin mRNA expression of pancreatic islet beta cells. The activities of Ras, Raf-1, and p-CREB in the Ras complex

Two Alkaloids from Bulbs of Lycoris sanguinea MAXIM. Suppress PEPCK Expression by Inhibiting the Phosphorylation of CREB.

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Yun, Young Sook; Tajima, Miki; Takahashi, Shigeru; Takahashi, Yuji; Umemura, Mariko; Nakano, Haruo; Park, Hyun Sun; Inoue, Hideshi

2016-10-01

In the fasting state, gluconeogenesis is upregulated by glucagon. Glucagon stimulates cyclic adenosine monophosphate production, which induces the expression of key enzymes for gluconeogenesis, such as cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), which are involved in gluconeogenesis through the protein kinase A/cAMP response element-binding protein (CREB) pathway. Using a luciferase reporter gene assay, a methanol extract of the bulbs of Lycoris sanguinea M AXIM. var. kiushiana Makino was found to suppress cAMP-enhanced PEPCK-C promoter activity. In addition, two alkaloids, lycoricidine and lycoricidinol, in the extract were identified as active constituents. In forskolin-stimulated human hepatoma cells, these alkaloids suppressed the expression of a reporter gene under the control of cAMP response element and also prevented increases in the endogenous levels of phosphorylated CREB and PEPCK mRNA expression. These results suggest that lycoricidine and lycoricidinol suppress PEPCK-C expression by inhibiting the phosphorylation of CREB and may thus have the potential to prevent excessive gluconeogenesis in type 2 diabetes. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Human T-cell leukemia virus type 1 Tax requires direct access to DNA for recruitment of CREB binding protein to the viral promoter.

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Lenzmeier, B A; Giebler, H A; Nyborg, J K

1998-02-01

Efficient human T-cell leukemia virus type 1 (HTLV-1) replication and viral gene expression are dependent upon the virally encoded oncoprotein Tax. To activate HTLV-1 transcription, Tax interacts with the cellular DNA binding protein cyclic AMP-responsive element binding protein (CREB) and recruits the coactivator CREB binding protein (CBP), forming a nucleoprotein complex on the three viral cyclic AMP-responsive elements (CREs) in the HTLV-1 promoter. Short stretches of dG-dC-rich (GC-rich) DNA, immediately flanking each of the viral CREs, are essential for Tax recruitment of CBP in vitro and Tax transactivation in vivo. Although the importance of the viral CRE-flanking sequences is well established, several studies have failed to identify an interaction between Tax and the DNA. The mechanistic role of the viral CRE-flanking sequences has therefore remained enigmatic. In this study, we used high resolution methidiumpropyl-EDTA iron(II) footprinting to show that Tax extended the CREB footprint into the GC-rich DNA flanking sequences of the viral CRE. The Tax-CREB footprint was enhanced but not extended by the KIX domain of CBP, suggesting that the coactivator increased the stability of the nucleoprotein complex. Conversely, the footprint pattern of CREB on a cellular CRE lacking GC-rich flanking sequences did not change in the presence of Tax or Tax plus KIX. The minor-groove DNA binding drug chromomycin A3 bound to the GC-rich flanking sequences and inhibited the association of Tax and the Tax-CBP complex without affecting CREB binding. Tax specifically cross-linked to the viral CRE in the 5'-flanking sequence, and this cross-link was blocked by chromomycin A3. Together, these data support a model where Tax interacts directly with both CREB and the minor-groove viral CRE-flanking sequences to form a high-affinity binding site for the recruitment of CBP to the HTLV-1 promoter.

Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum.

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Amit-Avraham, Inbar; Pozner, Guy; Eshar, Shiri; Fastman, Yair; Kolevzon, Netanel; Yavin, Eylon; Dzikowski, Ron

2015-03-03

The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression.

Rolipram improves facilitation of contextual fear extinction in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse model of Parkinson's disease

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Ken-ichi Kinoshita

2017-05-01

Full Text Available Cognitive impairment often occurs in Parkinson's disease (PD, but the mechanism of onset remains unknown. Recently, we reported that PD model mice produced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP show facilitation of hippocampal memory extinction, which may be the cause of cognitive impairment in PD. When we examined the cAMP/CREB signaling in the hippocampus, decreased levels of cAMP and phosphorylated CREB were observed in the dentate gyrus (DG of MPTP-treated mice. Administration of rolipram improved the memory deficits with concomitant recovery of cAMP and phosphorylated CREB levels, suggesting that reduced cAMP/CREB signaling in the DG leads to cognitive impairment in MPTP-treated mice.

Alteration of rice growth and development via antisense expression ...

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OsGA20ox2 in regulating plant growth and development, we used reverse genomic approach to ... pathways. Similarly, Carmen et al. (2007) suggested that. Carrizo citrange plants have produced antisense ... universal SP6 and T7 primers to conform their reality (Sangon, ..... Optimising the tissue culture conditions for.

CREB, NF-Y and MEIS1 conserved binding sites are essential to balance Myostatin promoter/enhancer activity during early myogenesis.

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Grade, Carla Vermeulen Carvalho; Mantovani, Carolina Stefano; Fontoura, Marina Alves; Yusuf, Faisal; Brand-Saberi, Beate; Alvares, Lúcia Elvira

2017-10-01

Myostatin (MSTN) is a strong inhibitor of skeletal muscle growth in human and other vertebrates. Its transcription is controlled by a proximal promoter/enhancer (Mstn P/E) containing a TATA box besides CREB, NF-Y, MEIS1 and FXR transcription factor binding sites (TFBSs), which are conserved throughout evolution. The aim of this work was to investigate the role of these TFBSs on Mstn P/E activity and evaluate the potential of their putative ligands as Mstn trans regulators. Mstn P/E mutant constructs were used to establish the role of conserved TFBSs using dual-luciferase assays. Expression analyses were performed by RT-PCR and in situ hybridization in C2C12 myoblasts and E10.5 mouse embryos, respectively. Our results revealed that CREB, NF-Y and MEIS1 sites are required to balance Mstn P/E activity, keeping Mstn transcription within basal levels during myoblast proliferation. Furthermore, our data showed that NF-Y site is essential, although not sufficient, to mediate Mstn P/E transcriptional activity. In turn, CREB and MEIS1 binding sites seem to depend on the presence of NF-Y site to induce Mstn P/E. FXR appears not to confer any effect on Mstn P/E activity, except in the absence of all other conserved TFBS. Accordingly, expression studies pointed to CREB, NF-Y and MEIS1 but not to FXR factors as possible regulators of Mstn transcription in the myogenic context. Altogether, our findings indicated that CREB, NF-Y and MEIS1 conserved sites are essential to control basal Mstn transcription during early myogenesis, possibly by interacting with these or other related factors.

Period1 gates the circadian modulation of memory-relevant signaling in mouse hippocampus by regulating the nuclear shuttling of the CREB kinase pP90RSK.

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Rawashdeh, Oliver; Jilg, Antje; Maronde, Erik; Fahrenkrug, Jan; Stehle, Jörg H

2016-09-01

Memory performance varies over a 24-h day/night cycle. While the detailed underlying mechanisms are yet unknown, recent evidence suggests that in the mouse hippocampus, rhythmic phosphorylation of mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate response element-binding protein (CREB) are central to the circadian (~ 24 h) regulation of learning and memory. We recently identified the clock protein PERIOD1 (PER1) as a vehicle that translates information encoding time of day to hippocampal plasticity. We here elaborate how PER1 may gate the sensitivity of memory-relevant hippocampal signaling pathways. We found that in wild-type mice (WT), spatial learning triggers CREB phosphorylation only during the daytime, and that this effect depends on the presence of PER1. The time-of-day-dependent induction of CREB phosphorylation can be reproduced pharmacologically in acute hippocampal slices prepared from WT mice, but is absent in preparations made from Per1-knockout (Per1(-/-) ) mice. We showed that the PER1-dependent CREB phosphorylation is regulated downstream of MAPK. Stimulation of WT hippocampal neurons triggered the co-translocation of PER1 and the CREB kinase pP90RSK (pMAPK-activated ribosomal S6 kinase) into the nucleus. In hippocampal neurons from Per1(-/-) mice, however, pP90RSK remained perinuclear. A co-immunoprecipitation assay confirmed a high-affinity interaction between PER1 and pP90RSK. Knocking down endogenous PER1 in hippocampal cells inhibited adenylyl cyclase-dependent CREB activation. Taken together, the PER1-dependent modulation of cytoplasmic-to-nuclear signaling in the murine hippocampus provides a molecular explanation for how the circadian system potentially shapes a temporal framework for daytime-dependent memory performance, and adds a novel facet to the versatility of the clock gene protein PER1. We provide evidence that the circadian clock gene Period1 (Per1) regulates CREB phosphorylation in the mouse hippocampus

The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

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Hurst, H C; Masson, N; Jones, N C; Lee, K A

1990-12-01

Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We demonstrated that CREB and ATF-47 are identical and that CREB and ATF-43 form protein-protein complexes. We also found that the cis requirements for stable DNA binding by ATF-43 and CREB are different. Using antibodies to ATF-43 we have identified a group of polypeptides (ATF-43) in the size range from 40 to 43 kDa. ATF-43 polypeptides are related by their reactivity with anti-ATF-43, DNA-binding specificity, complex formation with CREB, heat stability, and phosphorylation by protein kinase A. Certain cell types vary in their ATF-43 complement, suggesting that CREB activity is modulated in a cell-type-specific manner through interaction with ATF-43. ATF-43 polypeptides do not appear simply to correspond to the gene products of the ATF multigene family, suggesting that the size of the ATF family at the protein level is even larger than predicted from cDNA-cloning studies.

The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

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Zuloaga, R.; Fuentes, E.N.; Molina, A.; Valdés, J.A.

2013-01-01

Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP 3 /calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation

The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

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Zuloaga, R. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Fuentes, E.N.; Molina, A. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile); Valdés, J.A., E-mail: jvaldes@unab.cl [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile)

2013-10-18

Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

Impaired memory of eyeblink conditioning in CaMKIV KO mice.

Science.gov (United States)

Lee, Ka Hung; Chatila, Talal A; Ram, Rana A; Thompson, Richard F

2009-04-01

The calcium/calmodulin-dependent protein kinase type IV (CaMKIV) is highly expressed in cerebellar cortical granule cells and deep nuclear neurons in the cerebellum. It mediates the phosphorylation and activation of the cAMP-dependent response element binding protein (CREB). In several paradigms CREB-dependent transcription is required for cellular events underlying long-term memory processes. Also, CaMKIV deficiency results in impaired long-term depression (LTD) induction in cerebellar cortex. To investigate the function of CaMKIV in the cerebellum, Wild-type (WT) and CaMKIV KO mice were tested with delay eyeblink conditioning. KO and WT mice did not differ in acquisition, but the KO mice showed a significantly lower conditioned response (CR) percentage than the WT mice in the retention testing and retraining period. The CR peak latencies for the two groups did not differ in acquisition but were shorter for the KO mice in the testing period. No significant differences were found between KO and WT mice in spontaneous eyeblink activity, auditory brainstem response (ABR) amplitudes, and tail-flick latency. The results suggest an important role for CaMKIV in long-term memory in the cerebellum. (c) 2009 APA, all rights reserved.

Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide.

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Higuchi, Maiko; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

2008-01-01

Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenyl-methoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Ps-eom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a powerful antisense molecule to inhibit the expression of mRNA having a point mutation.

Cellular delivery and antisense effects of peptide nucleic acid conjugated to polyethyleneimine via disulfide linkers

DEFF Research Database (Denmark)

Berthold, Peter R; Shiraishi, Takehiko; Nielsen, Peter E

2010-01-01

Peptide nucleic acid (PNA) is potentially an attractive antisense and antigene agent for which more efficient cellular delivery systems are still warranted. The cationic polymer polyethylenimine (PEI) is commonly used for cellular transfection of DNA and RNA complexes, but is not readily applicable...... moiety) and further reacted this with a cysteine PNA. The level of modification was determined spectrophotometrically with high accuracy, and the PNA transfection efficiency of the conjugates was evaluated in an antisense luciferase splice-correction assay using HeLa pLuc705 cells. We find that PEI...... is an efficient vector for PNA delivery yielding significantly higher (up to 10-fold) antisense activity than an analogous PNA-octaarginine conjugate, even in the presence of chloroquine, which only slightly enhances the PEI-PNA activity. The PEI-PEG conjugates are preferred due to lower acute cellular toxicity...

Overlapping ETS and CRE Motifs (G/CCGGAAGTGACGTCA) Preferentially Bound by GABPα and CREB Proteins

Science.gov (United States)

Chatterjee, Raghunath; Zhao, Jianfei; He, Ximiao; Shlyakhtenko, Andrey; Mann, Ishminder; Waterfall, Joshua J.; Meltzer, Paul; Sathyanarayana, B. K.; FitzGerald, Peter C.; Vinson, Charles

2012-01-01

Previously, we identified 8-bps long DNA sequences (8-mers) that localize in human proximal promoters and grouped them into known transcription factor binding sites (TFBS). We now examine split 8-mers consisting of two 4-mers separated by 1-bp to 30-bps (X4-N1-30-X4) to identify pairs of TFBS that localize in proximal promoters at a precise distance. These include two overlapping TFBS: the ETS⇔ETS motif (C/GCCGGAAGCGGAA) and the ETS⇔CRE motif (C/GCGGAAGTGACGTCAC). The nucleotides in bold are part of both TFBS. Molecular modeling shows that the ETS⇔CRE motif can be bound simultaneously by both the ETS and the B-ZIP domains without protein-protein clashes. The electrophoretic mobility shift assay (EMSA) shows that the ETS protein GABPα and the B-ZIP protein CREB preferentially bind to the ETS⇔CRE motif only when the two TFBS overlap precisely. In contrast, the ETS domain of ETV5 and CREB interfere with each other for binding the ETS⇔CRE. The 11-mer (CGGAAGTGACG), the conserved part of the ETS⇔CRE motif, occurs 226 times in the human genome and 83% are in known regulatory regions. In vivo GABPα and CREB ChIP-seq peaks identified the ETS⇔CRE as the most enriched motif occurring in promoters of genes involved in mRNA processing, cellular catabolic processes, and stress response, suggesting that a specific class of genes is regulated by this composite motif. PMID:23050235

Treadmill exercise alleviates stress-induced impairment of social interaction through 5-hydroxytryptamine 1A receptor activation in rats.

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Kim, Tae-Woon; Lim, Baek-Vin; Kim, Kijeong; Seo, Jin-Hee; Kim, Chang-Ju

2015-08-01

Brain-derived neurotrophic factor (BDNF) and its receptors tyrosine kinase B (trkB), and cyclic adenosine monophosphate response element binding protein (CREB) have been suggested as the neurobiological risk factors causing depressive disorder. Serotonin (5-hydroxytryptamine, 5-HT) plays an important role in the pathogenesis of depression. We in-vestigated the effect of treadmill exercise on social interaction in relation with BDNF and 5-HT expressions following stress in rats. Stress was induced by applying inescapable 0.2 mA electric foot shock to the rats for 7 days. The rats in the exercise groups were forced to run on a motorized treadmill for 30 min once a day for 4 weeks. Social interaction test and western blot for BDNF, TrkB, pCREB, and 5-HT1A in the hippocampus were performed. The results indicate that the spend time with unfamiliar partner was decreased by stress, in contrast, treadmill exercise increased the spending time in the stress-induced rats. Expressions of BDNF, TrkB, and pCREB were decreased by stress, in contrast, treadmill exercise enhanced expressions of BDNF, TrkB, and pCREB in the stress-induced rats. In addition, 5-HT1A receptor expression was de-creased by stress, in contrast, treadmill exercise enhanced 5-HT1A expression in the stress-induced rats. In the present study, treadmill exercise alleviated stress-induced social interaction impairment through enhancing hippocampal plasticity and serotonergic function in the hippocampus. These effects of treadmill exercise are achieved through 5-HT1A receptor activation.

Cocaine alters Homer1 natural antisense transcript in the nucleus accumbens.

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Sartor, Gregory C; Powell, Samuel K; Velmeshev, Dmitry; Lin, David Y; Magistri, Marco; Wiedner, Hannah J; Malvezzi, Andrea M; Andrade, Nadja S; Faghihi, Mohammad A; Wahlestedt, Claes

2017-12-01

Natural antisense transcripts (NATs) are an abundant class of long noncoding RNAs that have recently been shown to be key regulators of chromatin dynamics and gene expression in nervous system development and neurological disorders. However, it is currently unclear if NAT-based mechanisms also play a role in drug-induced neuroadaptations. Aberrant regulation of gene expression is one critical factor underlying the long-lasting behavioral abnormalities that characterize substance use disorder, and it is possible that some drug-induced transcriptional responses are mediated, in part, by perturbations in NAT activity. To test this hypothesis, we used an automated algorithm that mines the NCBI AceView transcriptomics database to identify NAT overlapping genes linked to addiction. We found that 22% of the genes examined contain NATs and that expression of Homer1 natural antisense transcript (Homer1-AS) was altered in the nucleus accumbens (NAc) of mice 2h and 10days following repeated cocaine administration. In in vitro studies, depletion of Homer1-AS lead to an increase in the corresponding sense gene expression, indicating a potential regulatory mechanisms of Homer1 expression by its corresponding antisense transcript. Future in vivo studies are needed to definitely determine a role for Homer1-AS in cocaine-induced behavioral and molecular adaptations. Copyright © 2017 Elsevier Inc. All rights reserved.

Role of natural antisense transcripts pertaining to tumor suppressor genes in human carcinomas

International Nuclear Information System (INIS)

Pelicci, G.; Pierotti, M.

2009-01-01

Overlapping transcripts in opposite orientations can potentially form perfect sense-antisense duplex RNA. Recently, several studies have revealed the extent of natural antisense transcripts (NATs) and their role in important biological phenomena also in higher organisms. In order to test the hypothesis that the function of NATs in man might represent an essential element in the regulation of gene expression, especially at transcriptional level, in this study we planned to look for, systematically examine, and characterize NATs belonging in the human genome to the tumour suppressor class of genes, so to identify physiological (and potentially pathological) modulators in this gene class

Antisense RNA: a genetic approach to cell resistance against Parvovirus; RNA antisentido: una aproximacion de resistencia genetica a Parvovirus

Energy Technology Data Exchange (ETDEWEB)

Ramirez Martinez, J.C.

1992-12-31

The Minute Virus of Mice (MVMp), an autonomous Parvovirus that replicates cytolytically in the A9 mouse fibroblast cell line, was interfered by constitutive expression of an antisense RNA targeted against the major non-structural NS-1 protein. Permanently transfected A9 clones expressing NS-1 antisense, showed increased proliferative capacity upon virus infection, and likewise cultures infected at low multiplicity by MVMp reached confluence overcoming virus growth. Correspondingly, an inhibition in virus multiplication was demonstrated by a significant lower virus production and plaque forming ability in clones expressing antisense RNa. At the molecular level, several fold reduction in viral DNA, RNA and proteins was quantitated by respective analysis of Southern, RNase protection and bidimensional gels. Remarkably, the accumulation of all three viral messengers(R1,R2,R3) was decreased both in the cytoplasm and in the nucleus, suggesting that antisense-mediated inhibition is primarily exerted at the level of viral transcription or nuclear post-transcriptional events. Thus, this system illustrates the possibility to create an antisense-mediated protective stage to highly cytotoxic viruses in permissive cells, by down-modulation the expression of a transactivator of virus genes. (author)180 refs., 25 figs.

Antisense RNA: a genetic approach to cell resistance against Parvovirus. RNA antisentido: una aproximacion de resistencia genetica a Parvovirus

Energy Technology Data Exchange (ETDEWEB)

Ramirez Martinez, J.C.

1992-01-01

The Minute Virus of Mice (MVMp), an autonomous Parvovirus that replicates cytolytically in the A9 mouse fibroblast cell line, was interfered by constitutive expression of an antisense RNA targeted against the major non-structural NS-1 protein. Permanently transfected A9 clones expressing NS-1 antisense, showed increased proliferative capacity upon virus infection, and likewise cultures infected at low multiplicity by MVMp reached confluence overcoming virus growth. Correspondingly, an inhibition in virus multiplication was demonstrated by a significant lower virus production and plaque forming ability in clones expressing antisense RNa. At the molecular level, several fold reduction in viral DNA, RNA and proteins was quantitated by respective analysis of Southern, RNase protection and bidimensional gels. Remarkably, the accumulation of all three viral messengers(R1,R2,R3) was decreased both in the cytoplasm and in the nucleus, suggesting that antisense-mediated inhibition is primarily exerted at the level of viral transcription or nuclear post-transcriptional events. Thus, this system illustrates the possibility to create an antisense-mediated protective stage to highly cytotoxic viruses in permissive cells, by down-modulation the expression of a transactivator of virus genes. (author)180 refs., 25 figs.

Encapsulation of c-myc antisense oligodeoxynucleotides in lipid particles improves antitumoral efficacy in vivo in a human melanoma line.

Science.gov (United States)

Leonetti, C; Biroccio, A; Benassi, B; Stringaro, A; Stoppacciaro, A; Semple, S C; Zupi, G

2001-06-01

Phosphorothioate c-myc antisense oligodeoxynucleotides [S]ODNs (free INX-6295) were encapsulated in a new liposome formulation and the antitumor activity was compared to the unencapsulated antisense in a human melanoma xenograft. The systemic administration of INX-6295 encapsulated in stabilized antisense lipid particles (SALP INX-6295) improved plasma AUC (area under the plasma concentration-time curve) and initial half-life of free INX-6295, resulting in a significant enhancement in tumor accumulation and improvement in tumor distribution of antisense oligodeoxynucleotides. Animals treated with SALP INX-6295 exhibited a prolonged reduction of c-myc expression, reduced tumor growth and increased mice survival. When administered in combination with cisplatin (DDP), SALP INX-6295 produced a complete tumor regression in approximately 30% of treated mice, which persisted for at least 60 days following the first cycle of treatment. Finally, the median survival of mice treated with DDP/SALP INX-6295 increased by 105% compared to 84% for animals treated with the combination DDP/free INX-6295. These data indicate that the biological activity and the therapeutic efficacy of c-myc antisense therapy may be improved when these agents are administered in lipid-based delivery systems.

Curcumin modulates dopaminergic receptor, CREB and phospholipase c gene expression in the cerebral cortex and cerebellum of streptozotocin induced diabetic rats

Directory of Open Access Journals (Sweden)

George Naijil

2010-05-01

Full Text Available Abstract Curcumin, an active principle component in rhizome of Curcuma longa, has proved its merit for diabetes through its anti-oxidative and anti-inflammatory properties. This study aims at evaluating the effect of curcumin in modulating the altered dopaminergic receptors, CREB and phospholipase C in the cerebral cortex and cerebellum of STZ induced diabetic rats. Radioreceptor binding assays and gene expression was done in the cerebral cortex and cerebellum of male Wistar rats using specific ligands and probes. Total dopaminergic receptor binding parameter, Bmax showed an increase in cerebral cortex and decrease in the cerebellum of diabetic rats. Gene expression studies using real time PCR showed an increased expression of dopamine D1 and D2 receptor in the cerebral cortex of diabetic rats. In cerebellum dopamine D1 receptor was down regulated and D2 receptor showed an up regulation. Transcription factor CREB and phospholipase C showed a significant down regulation in cerebral cortex and cerebellum of diabetic rats. We report that curcumin supplementation reduces diabetes induced alteration of dopamine D1, D2 receptors, transcription factor CREB and phospholipase C to near control. Our results indicate that curcumin has a potential to regulate diabetes induced malfunctions of dopaminergic signalling, CREB and Phospholipase C expression in cerebral cortex and cerebellum and thereby improving the cognitive and emotional functions associated with these regions. Furthermore, in line with these studies an interaction between curcumin and dopaminergic receptors, CREB and phospholipase C is suggested, which attenuates the cortical and cerebellar dysfunction in diabetes. These results suggest that curcumin holds promise as an agent to prevent or treat CNS complications in diabetes.

Curcumin modulates dopaminergic receptor, CREB and phospholipase C gene expression in the cerebral cortex and cerebellum of streptozotocin induced diabetic rats.

Science.gov (United States)

Kumar, T Peeyush; Antony, Sherin; Gireesh, G; George, Naijil; Paulose, C S

2010-05-31

Curcumin, an active principle component in rhizome of Curcuma longa, has proved its merit for diabetes through its anti-oxidative and anti-inflammatory properties. This study aims at evaluating the effect of curcumin in modulating the altered dopaminergic receptors, CREB and phospholipase C in the cerebral cortex and cerebellum of STZ induced diabetic rats. Radioreceptor binding assays and gene expression was done in the cerebral cortex and cerebellum of male Wistar rats using specific ligands and probes. Total dopaminergic receptor binding parameter, B(max) showed an increase in cerebral cortex and decrease in the cerebellum of diabetic rats. Gene expression studies using real time PCR showed an increased expression of dopamine D1 and D2 receptor in the cerebral cortex of diabetic rats. In cerebellum dopamine D1 receptor was down regulated and D2 receptor showed an up regulation. Transcription factor CREB and phospholipase C showed a significant down regulation in cerebral cortex and cerebellum of diabetic rats. We report that curcumin supplementation reduces diabetes induced alteration of dopamine D1, D2 receptors, transcription factor CREB and phospholipase C to near control. Our results indicate that curcumin has a potential to regulate diabetes induced malfunctions of dopaminergic signalling, CREB and Phospholipase C expression in cerebral cortex and cerebellum and thereby improving the cognitive and emotional functions associated with these regions. Furthermore, in line with these studies an interaction between curcumin and dopaminergic receptors, CREB and phospholipase C is suggested, which attenuates the cortical and cerebellar dysfunction in diabetes. These results suggest that curcumin holds promise as an agent to prevent or treat CNS complications in diabetes.

Antisense silencing of the creA gene in Aspergillus nidulans

DEFF Research Database (Denmark)

Bautista, L. F.; Aleksenko, Alexei Y.; Hentzer, Morten

2000-01-01

Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was appr...

Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD)

DEFF Research Database (Denmark)

Brolin, Camilla; Shiraishi, Takehiko

2011-01-01

Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most...

Osthole Enhances Osteogenesis in Osteoblasts by Elevating Transcription Factor Osterix via cAMP/CREB Signaling In Vitro and In Vivo

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Zhong-Rong Zhang

2017-06-01

Full Text Available Anabolic anti-osteoporotic agents are desirable for treatment and prevention of osteoporosis and fragility fractures. Osthole is a coumarin derivative extracted from the medicinal herbs Cnidium monnieri (L. Cusson and Angelica pubescens Maxim.f. Osthole has been reported with osteogenic and anti-osteoporotic properties, whereas the underlying mechanism of its benefit still remains unclear. The objective of the present study was to investigate the osteopromotive action of osthole on mouse osteoblastic MC3T3-E1 cells and on mouse femoral fracture repair, and to explore the interaction between osthole-induced osteopromotive effect and cyclic adenosine monophosphate (cAMP elevating effect. Osthole treatment promoted osteogenesis in osteoblasts by enhancing alkaline phosphatase (ALP activity and mineralization. Oral gavage of osthole enhanced fracture repair and increased bone strength. Mechanistic study showed osthole triggered the cAMP/CREB pathway through the elevation of the intracellular cAMP level and activation of the phosphorylation of the cAMP response element-binding protein (CREB. Blockage of cAMP/CREB downstream signals with protein kinase A (PKA inhibitor KT5720 partially suppressed osthole-mediated osteogenesis by inhibiting the elevation of transcription factor, osterix. In conclusion, osthole shows osteopromotive effect on osteoblasts in vitro and in vivo. Osthole-mediated osteogenesis is related to activation of the cAMP/CREB signaling pathway and downstream osterix expression.

Polymerase-endonuclease amplification reaction (PEAR for large-scale enzymatic production of antisense oligonucleotides.

Directory of Open Access Journals (Sweden)

Xiaolong Wang

Full Text Available Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR, for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.

BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity

International Nuclear Information System (INIS)

Lu, Zheng; Ping, Liang; JianBo, Zhou; XiaoBing, Huang; Yu, Wen; Zheng, Wang; Jing, Li

2012-01-01

The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG 2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG 2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG 2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G 1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+) BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms

Inhibition of B cell proliferation by antisense DNA to both alpha and beta forms of Fc epsilon R II.

Science.gov (United States)

Bhatti, L; Behle, K; Stevens, R H

1992-10-01

Epstein-Barr Virus (EBV) infection activates B lymphocyte proliferation through partially understood mechanisms, resulting in phenotypic changes, including the appearance of new antigens. One such antigen is Fc epsilon R II/CD-23 which may be relevant for B cell proliferation. We have used anti-sense oligonucleotides to study the importance of the two forms of this molecule for proliferation in the EBV-transformed, Fc epsilon R II +ve lymphoblastoid B cell line, RPMI 8866. Anti-sense oligodeoxynucleotides were generated to the two forms of Fc epsilon R II; Fc epsilon R IIa (alpha) and IIb (beta) which differ only in their intracytoplasmic domains. Addition of increasing concentrations of anti-sense oligonucleotides, ranging from 1 to 30 microM, significantly decreased cellular proliferation as measured by the incorporation of [3H]thymidine (inhibition range 8-88%). Optimum inhibition of cellular proliferation was apparent at 15 microM concentration of both anti-sense Fc epsilon R IIa and IIb (Fc epsilon R IIa, mean +/- SE = 75 +/- 7% inhibition, p less than 0.001; Fc epsilon R IIb, mean +/- SE = 71 +/- 7% inhibition, p less than 0.001). Anti-sense oligonucleotides complementary to the common part of Fc epsilon R II resulted in a similar inhibition of proliferation. Sense oligonucleotides did not induce significant inhibition. Preincubation of sense and anti-sense oligonucleotides resulted in an abrogation of proliferation inhibition. Moreover, none of these oligonucleotides had any effect on a Fc epsilon R II -ve cell line. Incubation with both anti-sense IIa and IIb resulted in additive, but not synergistic inhibition of proliferation. Addition of soluble Fc epsilon R II did not reverse inhibition of proliferation, suggesting that membrane-bound or intracellular rather than soluble Fc epsilon R II was important for the induced proliferation. Analysis of cell surface expression for Fc epsilon II indicated that while there was a pronounced effect on cell number

Oxacillin sensitization of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius by antisense peptide nucleic acids in vitro.

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Goh, Shan; Loeffler, Anette; Lloyd, David H; Nair, Sean P; Good, Liam

2015-11-11

Antibiotic resistance genes can be targeted by antisense agents, which can reduce their expression and thus restore cellular susceptibility to existing antibiotics. Antisense inhibitors can be gene and pathogen specific, or designed to inhibit a group of bacteria having conserved sequences within resistance genes. Here, we aimed to develop antisense peptide nucleic acids (PNAs) that could be used to effectively restore susceptibility to β-lactams in methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP). Antisense PNAs specific for conserved regions of the mobilisable gene mecA, and the growth essential gene, ftsZ, were designed. Clinical MRSA and MRSP strains of high oxacillin resistance were treated with PNAs and assayed for reduction in colony forming units on oxacillin plates, reduction in target gene mRNA levels, and cell size. Anti-mecA PNA at 7.5 and 2.5 μM reduced mecA mRNA in MRSA and MRSP (p resistance in staphylococci. Further studies are warranted as clinical treatment alternatives are needed.

Effect of injection of antisense oligodeoxynucleotides of GAD isozymes into rat ventromedial hypothalamus on food intake and locomotor activity.

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Bannai, M; Ichikawa, M; Nishihara, M; Takahashi, M

1998-02-16

In the ventromedial hypothalamus (VMH), gamma-aminobutyric acid (GABA) plays a role in regulating feeding and running behaviors. The GABA synthetic enzyme, glutamic acid decarboxylase (GAD), consists of two isozymes, GAD65 and GAD67. In the present study, the phosphorothioated antisense oligodeoxynucleotides (ODNs) of each GAD isozyme were injected bilaterally into the VMH of male rats, and food intake, body weight and locomotor activity were monitored. ODNs were incorporated in the water-absorbent polymer (WAP, 0.2 nmol/microliter) so that ODNs were retained at the injection site. Each antisense ODN of GAD65 or GAD67 tended to reduce food intake on day 1 (day of injection=day 0) though not significantly. An injection combining both antisense ODNs significantly decreased food intake only on day 1, but body weight remained significantly lower than the control for 5 days. This suppression of body weight gain could be attributed to a significant increase in locomotor activity between days 3 and 5. Individual treatment with either ODNs did not change locomotor activity. The increase in daily locomotor activity in the group receiving the combined antisense ODNs occurred mainly during the light phase. Neither vehicle (WAP) nor control ODN affected food intake, body weight and locomotor activity. Histological studies indicated that antisense ODN distributed within 800 micron from the edge of the area where WAP was located 24 h after the injection gradually disappeared within days, but still remained within 300 micron m distance even 7 days after the injection. Antisense ODN was effectively incorporated by all the cell types examined, i.e., neurons, astrocytes and microglias. Further, HPLC analysis revealed that antisense ODNs of GAD isozymes, either alone or combined, decreased the content of GABA by 50% in VMH 24 h after the injection. These results indicate that suppression of GABA synthesis by either of the GAD isozymes is synergistically involved in suppressing food

Inhibition of the cAMP/PKA/CREB Pathway Contributes to the Analgesic Effects of Electroacupuncture in the Anterior Cingulate Cortex in a Rat Pain Memory Model.

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Shao, Xiao-Mei; Sun, Jing; Jiang, Yong-Liang; Liu, Bo-Yi; Shen, Zui; Fang, Fang; Du, Jun-Ying; Wu, Yuan-Yuan; Wang, Jia-Ling; Fang, Jian-Qiao

2016-01-01

Pain memory is considered as endopathic factor underlying stubborn chronic pain. Our previous study demonstrated that electroacupuncture (EA) can alleviate retrieval of pain memory. This study was designed to observe the different effects between EA and indomethacin (a kind of nonsteroid anti-inflammatory drugs, NSAIDs) in a rat pain memory model. To explore the critical role of protein kinase A (PKA) in pain memory, a PKA inhibitor was microinjected into anterior cingulate cortex (ACC) in model rats. We further investigated the roles of the cyclic adenosine monophosphate (cAMP), PKA, cAMP response element-binding protein (CREB), and cAMP/PKA/CREB pathway in pain memory to explore the potential molecular mechanism. The results showed that EA alleviates the retrieval of pain memory while indomethacin failed. Intra-ACC microinjection of a PKA inhibitor blocked the occurrence of pain memory. EA reduced the activation of cAMP, PKA, and CREB and the coexpression levels of cAMP/PKA and PKA/CREB in the ACC of pain memory model rats, but indomethacin failed. The present findings identified a critical role of PKA in ACC in retrieval of pain memory. We propose that the proper mechanism of EA on pain memory is possibly due to the partial inhibition of cAMP/PKA/CREB signaling pathway by EA.

CRTC2 Is a Coactivator of GR and Couples GR and CREB in the Regulation of Hepatic Gluconeogenesis.

Science.gov (United States)

Hill, Micah J; Suzuki, Shigeru; Segars, James H; Kino, Tomoshige

2016-01-01

Glucocorticoid hormones play essential roles in the regulation of gluconeogenesis in the liver, an adaptive response that is required for the maintenance of circulating glucose levels during fasting. Glucocorticoids do this by cooperating with glucagon, which is secreted from pancreatic islets to activate the cAMP-signaling pathway in hepatocytes. The cAMP-response element-binding protein (CREB)-regulated transcription coactivator 2 (CRTC2) is a coactivator known to be specific to CREB and plays a central role in the glucagon-mediated activation of gluconeogenesis in the early phase of fasting. We show here that CRTC2 also functions as a coactivator for the glucocorticoid receptor (GR). CRTC2 strongly enhances GR-induced transcriptional activity of glucocorticoid-responsive genes. CRTC2 physically interacts with the ligand-binding domain of the GR through a region spanning amino acids 561-693. Further, CRTC2 is required for the glucocorticoid-associated cooperative mRNA expression of the glucose-6-phosphatase, a rate-limiting enzyme for hepatic gluconeogenesis, by facilitating the attraction of GR and itself to its promoter region already occupied by CREB. CRTC2 is required for the maintenance of blood glucose levels during fasting in mice by enhancing the GR transcriptional activity on both the G6p and phosphoenolpyruvate carboxykinase (Pepck) genes. Finally, CRTC2 modulates the transcriptional activity of the progesterone receptor, indicating that it may influence the transcriptional activity of other steroid/nuclear receptors. Taken together, these results reveal that CRTC2 plays an essential role in the regulation of hepatic gluconeogenesis through coordinated regulation of the glucocorticoid/GR- and glucagon/CREB-signaling pathways on the key genes G6P and PEPCK.

Glutaredoxin 1 (GRX1) inhibits oxidative stress and apoptosis of chondrocytes by regulating CREB/HO-1 in osteoarthritis.

Science.gov (United States)

Sun, Jie; Wei, Xuelei; Lu, Yandong; Cui, Meng; Li, Fangguo; Lu, Jie; Liu, Yunjiao; Zhang, Xi

2017-10-01

GRX1 (glutaredoxin1), a sulfhydryl disulfide oxidoreductase, is involved in many cellular processes, including anti-oxidation, anti-apoptosis, and regulation of cell differentiation. However, the role of GRX1 in the oxidative stress and apoptosis of osteoarthritis chondrocytes remains unclear, prompting the current study. Protein and mRNA expressions were measured by Western blot and RT-qPCR. Oxidative stress was detected by the measurement of MDA and SOD contents. Cells apoptosis were detected by Annexin V-FITC/PI and caspase-3 activity assays. We found that the mRNA and protein expressions of GRX1 were significantly down-regulated in osteoarthritis tissues and cells. GRX1 overexpression increased the mRNA and protein expression of CREB and HO-1. Meanwhile, GRX1 overexpression inhibited oxidative stress and apoptosis in osteoarthritis chondrocytes. Furthermore, we found that GRX1 overexpression regulated HO-1 by increasing CREB, and that HO-1 regulated oxidative stress and apoptosis in osteoarthritis chondrocytes. Thus, GRX1 overexpression constrains oxidative stress and apoptosis in osteoarthritis chondrocytes by regulating CREB/HO-1, providing a novel insight into the molecular mechanism and potential treatment of osteoarthritis. Copyright © 2017. Published by Elsevier Ltd.

The neuroprotective effects of α-iso-cubebene on dopaminergic cell death: involvement of CREB/Nrf2 signaling.

Science.gov (United States)

Park, Sun Young; Son, Beung Gu; Park, Young Hoon; Kim, Cheol-Min; Park, Geuntae; Choi, Young-Whan

2014-09-01

As a part of ongoing studies to elucidate pharmacologically active components of Schisandra chinensis, we isolated and studied α-iso-cubebene. The neuroprotective mechanisms of α-iso-cubebene in human neuroblastoma SH-SY5Y cells were investigated. α-Iso-cubebene significantly inhibited cytotoxicity and apoptosis due to 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in dopaminergic SH-SY5Y cells. Pretreatment of cells with α-iso-cubebene reduced intracellular accumulation of ROS and calcium in response to 6-OHDA. The neuroprotective effects of α-iso-cubebene were found to result from protecting the mitochondrial membrane potential. Notably, α-iso-cubebene inhibited the release of apoptosis-inducing factor from the mitochondria into the cytosol and nucleus after 6-OHDA treatment. α-Iso-cubebene also induced the activation of PKA/PKB/CREB/Nrf2 and suppressed 6-OHDA-induced neurotoxicity. α-Iso-cubebene was found to induce phosphorylation of PKA and PKB and activate Nrf2 and CREB signaling pathways in a dose-dependent manner. Additionally, α-iso-cubebene stimulated the expression of the antioxidant response genes NQO1 and HO-1. Finally, α-iso-cubebene-mediated neuroprotective effects were found to be reversible after transfection with CREB and Nrf2 small interfering RNAs.

CREB binding protein is required for both short-term and long-term memory formation.

NARCIS (Netherlands)

Chen, G.; Zou, X.; Watanabe, H.; Deursen, J.M.A. van; Shen, J.

2010-01-01

CREB binding protein (CBP) is a transcriptional coactivator with histone acetyltransferase activity. Our prior study suggested that CBP might be a key target of presenilins in the regulation of memory formation and neuronal survival. To elucidate the role of CBP in the adult brain, we generated

Preparation and quality test of superparamagnetic iron oxide labeled antisense oligodeoxynucleotide probe: a preliminary study.

Science.gov (United States)

Wen, Ming; Li, Bibo; Ouyang, Yu; Luo, Yi; Li, Shaolin

2009-06-01

Molecular imaging of tumor antisense gene techniques have been applied to the study of magnetic resonance (MR) gene imaging associated with malignant tumors. In this study, we designed, synthesized, and tested a novel molecular probe, in which the antisense oligodeoxynucleotide (ASODN) was labeled with superparamagnetic iron oxide (SPIO), and its efficiency was examined by in vitro MR imaging after SK-Br-3 mammary carcinoma cell lines (oncocytes) transfection. The SPIO-labeled ASODN probe was prepared through SPIO conjugated to ASODN using a chemical cross linking method. Its morphology and size were detected by atomic force microscope, size distribution were detected by laser granulometer, the conjugating rate and biological activity were determined by high performance liquid chromatography, and the stability was determined by polyacrylamide gel electrophoresis. After that, the probes were transfected into the SK-Br-3 oncocytes, cellular iron uptake was analyzed qualitatively at light and electron microscopy and was quantified at atomic absorption spectrometry, and the signal change of the transfected cells was observed and measured using MR imaging. The morphology of the SPIO-labeled ASODN probe was mostly spherical with well-distributed scattering, and the diameters were between 25 and 40 nm (95%) by atomic force microscope and laser granulometer, the conjugating rate of the probe was 99%. Moreover, this probe kept its activity under physiological conditions and could conjugate with antisense oligodeoxynucleotide. In addition, light microscopy revealed an intracellular uptake of iron oxides in the cytosol and electron microscopic studies revealed a lysosomal deposition of iron oxides in the transfected SK-Br-3 oncocytes by antisense probes, some of them gathered stacks, and the iron content of the group of transfected SK-Br-3 oncocytes by antisense probe is significantly higher (18.37 +/- 0.42 pg) than other contrast groups, the MR imaging showed that

Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation

International Nuclear Information System (INIS)

Zhou, Wei-Jie; Wang, Sheng; Hu, Zhuang; Zhou, Zhen-Yu; Song, Cai-Juan

2015-01-01

Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP response element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer. - Highlights: • CREB and Caspase-3 signaling pathways are involved in the ASP induced breast cancer cells apoptosis. • ROCK1/Mlc signaling pathway plays a critical role in this ASP-mediated apoptosis. • Angelica sinensis polysaccharide (ASP) affected the PARP, Bax, Bcl-2, Bcl-xL and Apaf1 protein expression. • The activation of CREB and ROCK1 promotes caspase-3 activation and apoptosis induced

Angelica sinensis polysaccharides promotes apoptosis in human breast cancer cells via CREB-regulated caspase-3 activation

Energy Technology Data Exchange (ETDEWEB)

Zhou, Wei-Jie; Wang, Sheng [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Hu, Zhuang, E-mail: zhuanghu475000@sina.com [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Zhengzhou Center for Disease Control and Prevention, Zhengzhou 475000 (China); Zhou, Zhen-Yu; Song, Cai-Juan [Department of Breast and Thyroid Surgery, Huaihe Hospital, Henan University, Kaifeng 475000 (China); Zhengzhou Center for Disease Control and Prevention, Zhengzhou 475000 (China)

2015-11-20

Angelica sinensis polysaccharide (ASP) is purified from the fresh roots of Angelica sinensis (AS). This traditional Chinese medicine has been used for thousands of years for treating gynecological diseases and used in functional foods for the prevention and treatment of various diseases, such as inflammation and cancer. The antitumor activity of ASP is related to its biological activities, because it suppresses a variety of pro-proliferative or anti-apoptotic factors that are dramatically expressed in cancer cells of given types. In this study, we show that angelica sinensis polysaccharide induced apoptosis in breast cancer cells of T47D over-expressing the Cyclic AMP response element binding protein (CREB), inducing apoptosis-related signaling pathway activity. The result also found that ASP caused cell death was linked to caspase activity, accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. We found that ASP significantly affected the poly-ADP-ribose polymerase (PARP), Bcl-2 Associated X Protein (Bax), Bcl-2, Bcl-xL and apoptotic protease activating facter-1 (Apaf1) protein expression in a dose- and time-dependent manner. DAPI staining and Flow cytometry were used to analyze apoptosis. The nude mice xenograft model was used to evaluate the antitumor effect of ASP in vivo. ASP has profound antitumor effect on T47D cells, probably by inducing apoptosis through CREB signaling pathway. Thus, these results suggest that ASP would be a promising therapeutic agent for breast cancer. - Highlights: • CREB and Caspase-3 signaling pathways are involved in the ASP induced breast cancer cells apoptosis. • ROCK1/Mlc signaling pathway plays a critical role in this ASP-mediated apoptosis. • Angelica sinensis polysaccharide (ASP) affected the PARP, Bax, Bcl-2, Bcl-xL and Apaf1 protein expression. • The activation of CREB and ROCK1 promotes caspase-3 activation and apoptosis induced

cAMP-response Element-binding Protein (CREB) and NF-κB Transcription Factors Are Activated during Prolonged Hypoxia and Cooperatively Regulate the Induction of Matrix Metalloproteinase MMP1*

Science.gov (United States)

Nakayama, Koh

2013-01-01

Responses to low levels of oxygen (hypoxia) are essential to maintain homeostasis. During the hypoxic response, gene expression is altered by various transcription factors. The transcription factor, hypoxia-inducible factor (HIF), plays a central role in the hypoxic response. The α subunit of HIF, which is actively degraded during normoxia, becomes stabilized during hypoxia, which leads to HIF activation. A microarray analysis of HeLa cells showed that expression of matrix metalloproteinase 1 (MMP1) was markedly induced during prolonged hypoxia. CREB and NF-κB binding sites were identified in the MMP1 promoter region between 1945 and 1896 nucleotides upstream of the transcription start site. Assays with luciferase reporters demonstrated that HIF activity was induced during the early phase of hypoxia, whereas CREB and NF-κB were activated during the later (prolonged) phase. Depletion of CREB and/or NF-κB reduced MMP1 induction during prolonged hypoxia both at the mRNA and protein levels. A chromatin immunoprecipitation assay demonstrated binding of CREB and NF-κB to the MMP1 promoter. Finally, cell migration and invasion on a collagen matrix and pulmonary metastasis in nude mice were inhibited after depletion of CREB and NF-κB in MDA-MB-231 cells. Taken together, these results suggest that the cooperative action of CREB and NF-κB plays an important role to induce MMP1 expression during prolonged hypoxia and regulates cell migration and invasion in cancer cells. PMID:23775082

Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis

Directory of Open Access Journals (Sweden)

Lonardi Stefano

2008-01-01

Full Text Available Abstract Background In spite of large intergenic spaces in plant and animal genomes, 7% to 30% of genes in the genomes encode overlapping cis-natural antisense transcripts (cis-NATs. The widespread occurrence of cis-NATs suggests an evolutionary advantage for this type of genomic arrangement. Experimental evidence for the regulation of two cis-NAT gene pairs by natural antisense transcripts-generated small interfering RNAs (nat-siRNAs via the RNA interference (RNAi pathway has been reported in Arabidopsis. However, the extent of siRNA-mediated regulation of cis-NAT genes is still unclear in any genome. Results The hallmarks of RNAi regulation of NATs are 1 inverse regulation of two genes in a cis-NAT pair by environmental and developmental cues and 2 generation of siRNAs by cis-NAT genes. We examined Arabidopsis transcript profiling data from public microarray databases to identify cis-NAT pairs whose sense and antisense transcripts show opposite expression changes. A subset of the cis-NAT genes displayed negatively correlated expression profiles as well as inverse differential expression changes under at least one of the examined developmental stages or treatment conditions. By searching the Arabidopsis Small RNA Project (ASRP and Massively Parallel Signature Sequencing (MPSS small RNA databases as well as our stress-treated small RNA dataset, we found small RNAs that matched at least one gene in 646 pairs out of 1008 (64% protein-coding cis-NAT pairs, which suggests that siRNAs may regulate the expression of many cis-NAT genes. 209 putative siRNAs have the potential to target more than one gene and half of these small RNAs could target multiple members of a gene family. Furthermore, the majority of the putative siRNAs within the overlapping regions tend to target only one transcript of a given NAT pair, which is consistent with our previous finding on salt- and bacteria-induced nat-siRNAs. In addition, we found that genes encoding plastid- or

Notch1 regulates hippocampal plasticity through interaction with the Reelin pathway, glutamatergic transmission and CREB signaling

Directory of Open Access Journals (Sweden)

Emanuele eBrai

2015-11-01

Full Text Available Notch signaling plays a crucial role in adult brain function such as synaptic plasticity, memory and olfaction. Several reports suggest an involvement of this pathway in neurodegenerative dementia. Yet, to date, the mechanism underlying Notch activity in mature neurons remains unresolved. In this work, we investigate how Notch regulates synaptic potentiation and contributes to the establishment of memory in mice. We observe that Notch1 is a postsynaptic receptor with functional interactions with the Reelin receptor, ApoER2, and the ionotropic receptor, NMDAR. Targeted loss of Notch1 in the hippocampal CA fields affects Reelin signaling by influencing Dab1 expression and impairs the synaptic potentiation achieved through Reelin stimulation. Further analysis indicates that loss of Notch1 affects the expression and composition of the NMDAR but not AMPAR. Glutamatergic signaling is further compromised through downregulation of CamKII and its secondary and tertiary messengers resulting in reduced CREB signaling. Our results identify Notch1 as an important regulator of mechanisms involved in synaptic plasticity and memory formation. These findings emphasize the possible involvement of this signaling receptor in dementia.

Respirable antisense oligonucleotides: a new drug class for respiratory disease

Directory of Open Access Journals (Sweden)

Tanaka Makoto

2000-12-01

Full Text Available Abstract Respirable antisense oligonucleotides (RASONs, which attenuate specific disease-associated mRNAs, represent a new class of respiratory therapeutics with considerable potential. RASONs overcome previous obstacles that have impeded the development of antisense therapeutics targeting diseases in other organ systems. RASONs are delivered directly to the target tissue via inhalation; their uptake seems to be enhanced by cationic properties inherent in pulmonary surfactant, and, because of the markedly different target properties of mRNA and proteins, they can have very long durations of effect compared with traditional drugs targeting the protein of the same gene. RASONs contain chemical modifications that decrease their degradation by cellular nucleases. However, total insensitivity to nucleases is probably not an optimal design criterion for RASONs, because moderate nuclease sensitivity can prevent their systemic delivery, decreasing the potential for systemic toxicity. EPI-2010 is a 21-mer phosphorothioate RASON that attenuates bronchoconstriction, inflammation and surfactant depletion in preclinical models of human asthma, has a duration of effect of seven days, and seems to undergo minimal systemic delivery.

TLR4 induces CREB-mediated IL-6 production via upregulation of F-spondin to promote vascular smooth muscle cell migration

Energy Technology Data Exchange (ETDEWEB)

Lee, Guan-Lin [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Wu, Jing-Yiing [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Yeh, Chang-Ching [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Kuo, Cheng-Chin, E-mail: kuocc@nhri.org.tw [Institute of Cellular and System Medicine, National Health Research Institutes, Zhunan, Taiwan (China); Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan (China); Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan (China)

2016-05-13

Toll-like receptor 4 (TLR4) is important in promoting inflammation and vascular smooth muscle cell (VSMC) migration, both of which contribute to atherosclerosis development and progression. But the mechanism underlying the regulation of TLR4 in VSMC migration remains unclear. Stimulation of VSMCs with LPS increased the cellular level of F-spondin which is associated with the regulation of proinflammatory cytokine production. The LPS-induced F-spondin expression depended on TLR4-mediated PI3K/Akt pathway. Suppression of F-spondin level by siRNA inhibited not only F-spondin expression but also LPS-induced phosphorylation of cAMP response element binding protein (CREB) and IL-6 expression, VSMC migration and proliferation as well as MMP9 expression. Moreover, suppression of CREB level by siRNA inhibited TLR4-induced IL-6 production and VSMC migration. Inhibition of F-spondin siRNA on LPS-induced migration was restored by addition of exogenous recombinant mouse IL-6. We conclude that upon ligand binding, TLR4 activates PI3K/Akt signaling to induce F-spondin expression, subsequently control CREB-mediated IL-6 production to promote VSMC migration. These findings provide vital insights into the essential role of F-spondin in VSMC function and will be valuable for developing new therapeutic strategies against atherosclerosis. -- Highlights: •LPS-induced F-spondin expression of VSMCs is via a TLR4/PI3K/Akt signaling. •F-spondin is pivotal for LPS-induced CREB-mediated IL-6 production. •F-spondin is required for LPS-induced VSMC migration and proliferation.

TLR4 induces CREB-mediated IL-6 production via upregulation of F-spondin to promote vascular smooth muscle cell migration

International Nuclear Information System (INIS)

Lee, Guan-Lin; Wu, Jing-Yiing; Yeh, Chang-Ching; Kuo, Cheng-Chin

2016-01-01

Toll-like receptor 4 (TLR4) is important in promoting inflammation and vascular smooth muscle cell (VSMC) migration, both of which contribute to atherosclerosis development and progression. But the mechanism underlying the regulation of TLR4 in VSMC migration remains unclear. Stimulation of VSMCs with LPS increased the cellular level of F-spondin which is associated with the regulation of proinflammatory cytokine production. The LPS-induced F-spondin expression depended on TLR4-mediated PI3K/Akt pathway. Suppression of F-spondin level by siRNA inhibited not only F-spondin expression but also LPS-induced phosphorylation of cAMP response element binding protein (CREB) and IL-6 expression, VSMC migration and proliferation as well as MMP9 expression. Moreover, suppression of CREB level by siRNA inhibited TLR4-induced IL-6 production and VSMC migration. Inhibition of F-spondin siRNA on LPS-induced migration was restored by addition of exogenous recombinant mouse IL-6. We conclude that upon ligand binding, TLR4 activates PI3K/Akt signaling to induce F-spondin expression, subsequently control CREB-mediated IL-6 production to promote VSMC migration. These findings provide vital insights into the essential role of F-spondin in VSMC function and will be valuable for developing new therapeutic strategies against atherosclerosis. -- Highlights: •LPS-induced F-spondin expression of VSMCs is via a TLR4/PI3K/Akt signaling. •F-spondin is pivotal for LPS-induced CREB-mediated IL-6 production. •F-spondin is required for LPS-induced VSMC migration and proliferation.

Coordinated activation of the secretory pathway during notochord formation in the Xenopus embryo.

Science.gov (United States)

Tanegashima, Kosuke; Zhao, Hui; Rebbert, Martha L; Dawid, Igor B

2009-11-01

We compared the transcriptome in the developing notochord of Xenopus laevis embryos with that of other embryonic regions. A coordinated and intense activation of a large set of secretory pathway genes was observed in the notochord, but not in notochord precursors in the axial mesoderm at early gastrula stage. The genes encoding Xbp1 and Creb3l2 were also activated in the notochord. These two transcription factors are implicated in the activation of secretory pathway genes during the unfolded protein response, where cells react to the stress of a build-up of unfolded proteins in their endoplasmic reticulum. Xbp1 and Creb3l2 are differentially expressed but not differentially activated in the notochord. Reduction of expression of Xbp1 or Creb3l2 by injection of antisense morpholinos led to strong deficits in notochord but not somitic muscle development. In addition, the expression of some, but not all, genes encoding secretory proteins was inhibited by injection of xbp1 morpholinos. Furthermore, expression of activated forms of Xbp1 or Creb3l2 in animal explants could activate a similar subset of secretory pathway genes. We conclude that coordinated activation of a battery of secretory pathway genes mediated by Xbp1 and Creb/ATF factors is a characteristic and necessary feature of notochord formation.

Sodium Phenylbutyrate Enhances Astrocytic Neurotrophin Synthesis via Protein Kinase C (PKC)-mediated Activation of cAMP-response Element-binding Protein (CREB)

Science.gov (United States)

Corbett, Grant T.; Roy, Avik; Pahan, Kalipada

2013-01-01

Neurotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are believed to be genuine molecular mediators of neuronal growth and homeostatic synapse activity. However, levels of these neurotrophic factors decrease in different brain regions of patients with Alzheimer disease (AD). Induction of astrocytic neurotrophin synthesis is a poorly understood phenomenon but represents a plausible therapeutic target because neuronal neurotrophin production is aberrant in AD and other neurodegenerative diseases. Here, we delineate that sodium phenylbutyrate (NaPB), a Food and Drug Administration-approved oral medication for hyperammonemia, induces astrocytic BDNF and NT-3 expression via the protein kinase C (PKC)-cAMP-response element-binding protein (CREB) pathway. NaPB treatment increased the direct association between PKC and CREB followed by phosphorylation of CREB (Ser133) and induction of DNA binding and transcriptional activation of CREB. Up-regulation of markers for synaptic function and plasticity in cultured hippocampal neurons by NaPB-treated astroglial supernatants and its abrogation by anti-TrkB blocking antibody suggest that NaPB-induced astroglial neurotrophins are functionally active. Moreover, oral administration of NaPB increased the levels of BDNF and NT-3 in the CNS and improved spatial learning and memory in a mouse model of AD. Our results highlight a novel neurotrophic property of NaPB that may be used to augment neurotrophins in the CNS and improve synaptic function in disease states such as AD. PMID:23404502

Dual-Specificity Anti-HER-2/neu Antisense DNA Agents for Breast Cancer Therapy

National Research Council Canada - National Science Library

Stein, Stanley

2001-01-01

.... To achieve high avidity and specificity, we designed chimeric antisense molecules consisting of a short active DNA fused to a short "anchor" 2'-0-methyl RNA complementary to non-contiguous single...

Effects of curcumin (Curcuma longa) on learning and spatial memory as well as cell proliferation and neuroblast differentiation in adult and aged mice by upregulating brain-derived neurotrophic factor and CREB signaling.

Science.gov (United States)

Nam, Sung Min; Choi, Jung Hoon; Yoo, Dae Young; Kim, Woosuk; Jung, Hyo Young; Kim, Jong Whi; Yoo, Miyoung; Lee, Sanghee; Kim, Chul Jung; Yoon, Yeo Sung; Hwang, In Koo

2014-06-01

Aging is a progressive process, and it may lead to the initiation of neurological diseases. In this study, we investigated the effects of wild Indian Curcuma longa using a Morris water maze paradigm on learning and spatial memory in adult and D-galactose-induced aged mice. In addition, the effects on cell proliferation and neuroblast differentiation were assessed by immunohistochemistry for Ki67 and doublecortin (DCX) respectively. The aging model in mice was induced through the subcutaneous administration of D-galactose (100 mg/kg) for 10 weeks. C. longa (300 mg/kg) or its vehicle (physiological saline) was administered orally to adult and D-galactose-treated mice for the last three weeks before sacrifice. The administration of C. longa significantly shortened the escape latency in both adult and D-galactose-induced aged mice and significantly ameliorated D-galactose-induced reduction of cell proliferation and neuroblast differentiation in the subgranular zone of hippocampal dentate gyrus. In addition, the administration of C. longa significantly increased the levels of phosphorylated CREB and brain-derived neurotrophic factor in the subgranular zone of dentate gyrus. These results indicate that C. longa mitigates D-galactose-induced cognitive impairment, associated with decreased cell proliferation and neuroblast differentiation, by activating CREB signaling in the hippocampal dentate gyrus.

Comparison of three techniques for generation of tolerogenic dendritic cells: siRNA, oligonucleotide antisense, and antibody blocking.

Science.gov (United States)

Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Moazzeni, Mohammad; Soheili, Zahra Soheila; Samiee, Shahram

2010-12-01

In recent years, a new view of dendritic cells (DCs) as a main regulator of immunity to induce and maintain tolerance has been established. In vitro manipulation of their development and maturation is a topic of DC therapeutic application, which utilizes their inherent tolerogenicity. In this field, the therapeutic potential of antisense, siRNA, and blocking antibody are an interesting goal. In the present study, the efficiency of these three methods--siRNA, antisense, and blocking antibody--against CD40 molecule and its function in DCs and BCL1 cell line are compared. DCs were separated from mouse spleen and then cultured in vitro using Lipofectamine 2000 to deliver both silencers; the efficacy of transfection was estimated by flow cytometry. mRNA expression and protein synthesis were assessed by real time-PCR and flow cytometry, respectively. By Annexin V and propidium iodine staining, we could evaluate the viability of transfected cells. Knocking down the CD40 gene into separate groups of DCs by siRNA, antisense, and blocking antibody treated DCs can cause an increase in IL-4, decrease in IL-12, IFN-γ production, and allostimulation activity. Our results indicated that, in comparison to antisense and blocking antibody, siRNAs appear to be quantitatively more efficient in CD40 downregulation and their differences are significant.

Adenovirus DNA binding protein inhibits SrCap-activated CBP and CREB-mediated transcription

International Nuclear Information System (INIS)

Xu Xiequn; Tarakanova, Vera; Chrivia, John; Yaciuk, Peter

2003-01-01

The SNF2-related CBP activator protein (SrCap) is a potent activator of transcription mediated by CBP and CREB. We have previously demonstrated that the Adenovirus 2 DNA Binding Protein (DBP) binds to SrCap and inhibits the transcription mediated by the carboxyl-terminal region of SrCap (amino acids 1275-2971). We report here that DBP inhibits the ability of full-length SrCap (1-2971) to activate transcription mediated by Gal-CREB and Gal-CBP. In addition, DBP also inhibits the ability of SrCap to enhance Protein Kinase A (PKA) activated transcription of the enkaphalin promoter. DBP was found to dramatically inhibit transcription of a mammalian two-hybrid system that was dependent on the interaction of SrCap and CBP binding domains. We also found that DBP has no effect on transcription mediated by a transcriptional activator that is not related to SrCap, indicating that our reported transcriptional inhibition is specific for SrCap and not due to nonspecific effects of DBP's DNA binding activity on the CAT reporter plasmid. Taken together, these results suggest a model in which DBP inhibits cellular transcription mediated by the interaction between SrCap and CBP

Technetium-99m labeled antisense probes uptake in vascular smooth muscle cells

International Nuclear Information System (INIS)

Zhang, Y.X.; Qin, G.M.; An, R.; Cao, G.X.; Cao, W.; Gao, Z.R.

2002-01-01

In the arterial wall, smooth muscle cells (SMC) normally exist in a quiescent, differentiated state, representing the contractile phenotype. During the development of atherosclerosis SMC change towards the synthetic phenotype going along with proliferation, chemotactic response and increased monocyte binding. The Fas/Fas ligand/caspase death-signaling pathway, Bcl-2 protein family/mitochondria, the tumor suppressive gene p53, and the proto-oncogene c-myc may be activated in atherosclerotic lesions, and mediates vascular apoptosis during the development of atherosclerosis. The atherosclerotic plaques contained 3-4 fold more c-myc mRNA than those in the normal aortic arteries, while increased Bax and Bak coupled with lack/paucity of Bcl-2 and Bcl-xL are associated with SMC apoptosis in advanced lesions. Methods: 1 Oligonucleotide Conjugation: A solution of single stranded amine-derivatized DNA (100-1000μg) was prepared at a concentration of 2 mg/ml in 0.25M sodium bicarbonate, 1 M sodium chloride, 1mM EDTA, pH8.5. Cell uptake studies: 99m Tc- MAG 3 -DNA radioactivity incorporation into porcine coronary smooth muscle cells in the log and plateau phases, respectively, was determined after different times of incubation at 37. The influence of extracellular 99m Tc- MAG 3 -DNA concentration on SMC uptake was also analyzed. [Results] Essentially complete conjugation was achieved by reverse-phase Sep-Pak C18 chromatography analysis. The MAG 3 -DNA was labeled with 99m Tc at room temperature and neutral pH, with a mean labeling efficiency of 80.11%(s.d=2.96%,n=4). The labeled antisense DNA still remained the ability to hybridize with its complementary DNA. After labeling, the stability of the DNA in saline or serum was retained as determined by reverse-phase Sep-Pak C18 chromatography analysis, except a shift at 30 min in serum incubation that suggesting a short time serum protein binding. 99m Tc-MAG 3 -c-myc uptake plateaued at 60 min and was directly proportional to the

Calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides

DEFF Research Database (Denmark)

Shiraishi, Takehiko; Pankratova, Stanislava; Nielsen, Peter E

2005-01-01

Cell-penetrating peptides have been widely used to improve cellular delivery of a variety of proteins and antisense agents. However, recent studies indicate that such cationic peptides are predominantly entering cells via an endosomal pathway. We now show that the nuclear antisense effect in He......La cells of a variety of peptide nucleic acid (PNA) peptide conjugates is significantly enhanced by addition of 6 mM Ca(2+) (as well as by the lysosomotrophic agent chloroquine). In particular, the antisense activities of Tat(48-60) and heptaarginine-conjugated PNAs were increased 44-fold and 8.5-fold......, respectively. Evidence is presented that the mechanism involves endosomal release. The present results show that Ca(2+) can be used as an effective enhancer for in vitro cellular delivery of cationic peptide-conjugated PNA oligomers, and also emphasize the significance of the endosomal escape route...

Antisense expression of a gene encoding a calcium-binding protein ...

Indian Academy of Sciences (India)

PRAKASH

using the transgenic approach. The transformation of ... methods using EhCaBP or AtCaM3 gene-specific primers in ... acetone) was added, mixed and incubated for 15–18 h in the dark at .... as expected from the design of the AtCaM3 antisense construct .... Thus, there seems to be a positive qualitative correlation between ...

A Simple Three-Step Method for Design and Affinity Testing of New Antisense Peptides: An Example of Erythropoietin

OpenAIRE

Štambuk, Nikola; Manojlović, Zoran; Turčić, Petra; Martinić, Roko; Konjevoda, Paško; Weitner, Tin; Wardega, Piotr; Gabričević, Mario

2014-01-01

Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide–receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to ...

Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

International Nuclear Information System (INIS)

Tian, Hongmei; Ma, Leyuan; Zhao, Cong; Hao, Hui; Gong, Biao; Yu, Xiyan; Wang, Xiufeng

2010-01-01

To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.

Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

Energy Technology Data Exchange (ETDEWEB)

Tian, Hongmei; Ma, Leyuan; Zhao, Cong; Hao, Hui; Gong, Biao [College of Horticulture Science and Engineering, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai' an, Shandong 271018 (China); Yu, Xiyan, E-mail: yuxiyan@sdau.edu.cn [College of Horticulture Science and Engineering, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai' an, Shandong 271018 (China); Wang, Xiufeng, E-mail: xfwang@sdau.edu.cn [College of Horticulture Science and Engineering, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai' an, Shandong 271018 (China)

2010-03-12

To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.

Dopamine receptors modulate cytotoxicity of natural killer cells via cAMP-PKA-CREB signaling pathway.

Directory of Open Access Journals (Sweden)

Wei Zhao

Full Text Available Dopamine (DA, a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R with agonist SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R with agonist quinpirole attenuated NK cells. Simultaneously, SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of SKF38393 were blocked by SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA, prevented the SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC, counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The

Antisense oligonucleotide inhibition of apolipoprotein C-III reduces plasma triglycerides in rodents, nonhuman primates, and humans.

Science.gov (United States)

Graham, Mark J; Lee, Richard G; Bell, Thomas A; Fu, Wuxia; Mullick, Adam E; Alexander, Veronica J; Singleton, Walter; Viney, Nick; Geary, Richard; Su, John; Baker, Brenda F; Burkey, Jennifer; Crooke, Stanley T; Crooke, Rosanne M

2013-05-24

Elevated plasma triglyceride levels have been recognized as a risk factor for the development of coronary heart disease. Apolipoprotein C-III (apoC-III) represents both an independent risk factor and a key regulatory factor of plasma triglyceride concentrations. Furthermore, elevated apoC-III levels have been associated with metabolic syndrome and type 2 diabetes mellitus. To date, no selective apoC-III therapeutic agent has been evaluated in the clinic. To test the hypothesis that selective inhibition of apoC-III with antisense drugs in preclinical models and in healthy volunteers would reduce plasma apoC-III and triglyceride levels. Rodent- and human-specific second-generation antisense oligonucleotides were identified and evaluated in preclinical models, including rats, mice, human apoC-III transgenic mice, and nonhuman primates. We demonstrated the selective reduction of both apoC-III and triglyceride in all preclinical pharmacological evaluations. We also showed that inhibition of apoC-III was well tolerated and not associated with increased liver triglyceride deposition or hepatotoxicity. A double-blind, placebo-controlled, phase I clinical study was performed in healthy subjects. Administration of the human apoC-III antisense drug resulted in dose-dependent reductions in plasma apoC-III, concomitant lowering of triglyceride levels, and produced no clinically meaningful signals in the safety evaluations. Antisense inhibition of apoC-III in preclinical models and in a phase I clinical trial with healthy subjects produced potent, selective reductions in plasma apoC-III and triglyceride, 2 known risk factors for cardiovascular disease. This compelling pharmacological profile supports further clinical investigations in hypertriglyceridemic subjects.

Extensive polycistronism and antisense transcription in the mammalian Hox clusters.

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Gaëll Mainguy

Full Text Available The Hox clusters play a crucial role in body patterning during animal development. They encode both Hox transcription factor and micro-RNA genes that are activated in a precise temporal and spatial sequence that follows their chromosomal order. These remarkable collinear properties confer functional unit status for Hox clusters. We developed the TranscriptView platform to establish high resolution transcriptional profiling and report here that transcription in the Hox clusters is far more complex than previously described in both human and mouse. Unannotated transcripts can represent up to 60% of the total transcriptional output of a cluster. In particular, we identified 14 non-coding Transcriptional Units antisense to Hox genes, 10 of which (70% have a detectable mouse homolog. Most of these Transcriptional Units in both human and mouse present conserved sizeable sequences (>40 bp overlapping Hox transcripts, suggesting that these Hox antisense transcripts are functional. Hox clusters also display at least seven polycistronic clusters, i.e., different genes being co-transcribed on long isoforms (up to 30 kb. This work provides a reevaluated framework for understanding Hox gene function and dys-function. Such extensive transcriptions may provide a structural explanation for Hox clustering.

Development of a pluripotent stem cell derived neuronal model to identify chemically induced pathway perturbations in relation to neurotoxicity: Effects of CREB pathway inhibition

Energy Technology Data Exchange (ETDEWEB)

Pistollato, Francesca; Louisse, Jochem; Scelfo, Bibiana; Mennecozzi, Milena [Institute for Health and Consumer Protection (IHCP), JRC, Ispra (Italy); Accordi, Benedetta; Basso, Giuseppe [Oncohematology Laboratory, Department of Woman and Child Health, University of Padova, Padova (Italy); Gaspar, John Antonydas [Center of Physiology and Pathophysiology, Institute of Neurophysiology, University of Cologne, Cologne (Germany); Zagoura, Dimitra; Barilari, Manuela; Palosaari, Taina [Institute for Health and Consumer Protection (IHCP), JRC, Ispra (Italy); Sachinidis, Agapios [Center of Physiology and Pathophysiology, Institute of Neurophysiology, University of Cologne, Cologne (Germany); Bremer-Hoffmann, Susanne, E-mail: susanne.bremer@jrc.ec.europa.eu [Institute for Health and Consumer Protection (IHCP), JRC, Ispra (Italy)

2014-10-15

According to the advocated paradigm shift in toxicology, acquisition of knowledge on the mechanisms underlying the toxicity of chemicals, such as perturbations of biological pathways, is of primary interest. Pluripotent stem cells (PSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), offer a unique opportunity to derive physiologically relevant human cell types to measure molecular and cellular effects of such pathway modulations. Here we compared the neuronal differentiation propensity of hESCs and hiPSCs with the aim to develop novel hiPSC-based tools for measuring pathway perturbation in relation to molecular and cellular effects in vitro. Among other fundamental pathways, also, the cAMP responsive element binding protein (CREB) pathway was activated in our neuronal models and gave us the opportunity to study time-dependent effects elicited by chemical perturbations of the CREB pathway in relation to cellular effects. We show that the inhibition of the CREB pathway, using 2-naphthol-AS-E-phosphate (KG-501), induced an inhibition of neurite outgrowth and synaptogenesis, as well as a decrease of MAP2{sup +} neuronal cells. These data indicate that a CREB pathway inhibition can be related to molecular and cellular effects that may be relevant for neurotoxicity testing, and, thus, qualify the use of our hiPSC-derived neuronal model for studying chemical-induced neurotoxicity resulting from pathway perturbations. - Highlights: • HESCs derived neuronal cells serve as benchmark for iPSC based neuronal toxicity test development. • Comparisons between hESCs and hiPSCs demonstrated variability of the epigenetic state • CREB pathway modulation have been explored in relation to the neurotoxicant exposure KG-501 • hiPSC might be promising tools to translate theoretical AoPs into toxicological in vitro tests.

Increased production of biomass-degrading enzymes by double deletion of creA and creB genes involved in carbon catabolite repression in Aspergillus oryzae.

Science.gov (United States)

Ichinose, Sakurako; Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

2018-02-01

In a previous study, we reported that a double gene deletion mutant for CreA and CreB, which constitute the regulatory machinery involved in carbon catabolite repression, exhibited improved production of α-amylase compared with the wild-type strain and single creA or creB deletion mutants in Aspergillus oryzae. Because A. oryzae can also produce biomass-degrading enzymes, such as xylolytic and cellulolytic enzymes, we examined the production levels of those enzymes in deletion mutants in this study. Xylanase and β-glucosidase activities in the wild-type were hardly detected in submerged culture containing xylose as the carbon source, whereas those enzyme activities were significantly increased in the single creA deletion (ΔcreA) and double creA and creB deletion (ΔcreAΔcreB) mutants. In particular, the ΔcreAΔcreB mutant exhibited >100-fold higher xylanase and β-glucosidase activities than the wild-type. Moreover, in solid-state culture, the β-glucosidase activity of the double deletion mutant was >7-fold higher than in the wild-type. These results suggested that deletion of both creA and creB genes could also efficiently improve the production levels of biomass-degrading enzymes in A. oryzae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Potent Antibacterial Antisense Peptide-Peptide Nucleic Acid Conjugates Against Pseudomonas aeruginosa

DEFF Research Database (Denmark)

Ghosal, Anubrata; Nielsen, Peter E

2012-01-01

Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections in hospital settings, especially with immune compromised patients, and the increasing prevalence of multidrug resistant strains urges search for new drugs with novel mechanisms of action. In this study we introduce...... significantly reduced bacterial survival. These results open the possibility of development of antisense antibacterials for treatment of Pseudomonas infections....

Antisense-mediated suppression of C-hordein biosynthesis in the barley grain results in correlated changes in the transcriptome, protein profile, and amino acid composition

DEFF Research Database (Denmark)

Hansen, Mette; Lange, Marianne; Friis, Carsten

2007-01-01

Antisense- or RNAi-mediated suppression of the biosynthesis of nutritionally inferior storage proteins is a promising strategy for improving the amino acid profile of seeds. However, the potential pleiotropic effects of this on interconnected pathways and the agronomic quality traits need...... to be addressed. In the current study, a transcriptomic analysis of an antisense C-hordein line of barley was performed, using a grain-specific cDNA array. The C-hordein antisense line is characterized by marked changes in storage protein and amino acid profiles, while the seed weight is within the normal range...... and no external morphological irregularities were observed. The results of the transcriptome analysis showed excellent correlation with data on changes in the relative proportions of storage proteins and amino acid composition. The antisense line had a lower C-hordein level and down-regulated transcript encoding...

Ginger improves cognitive function via NGF-induced ERK/CREB activation in the hippocampus of the mouse.

Science.gov (United States)

Lim, Soonmin; Moon, Minho; Oh, Hyein; Kim, Hyo Geun; Kim, Sun Yeou; Oh, Myung Sook

2014-10-01

Ginger (the rhizome of Zingiber officinale Roscoe) has been used worldwide for many centuries in cooking and for treatment of several diseases. The main pharmacological properties of ginger include anti-inflammatory, antihyperglycemic, antiarthritic, antiemetic and neuroprotective actions. Recent studies demonstrated that ginger significantly enhances cognitive function in various cognitive disorders as well as in healthy brain. However, the biochemical mechanisms underlying the ginger-mediated enhancement of cognition have not yet been studied in normal or diseased brain. In the present study, we assessed the memory-enhancing effects of dried ginger extract (GE) in a model of scopolamine-induced memory deficits and in normal animals by performing a novel object recognition test. We found that GE administration significantly improved the ability of mice to recognize novel objects, indicating improvements in learning and memory. Furthermore, to elucidate the mechanisms of GE-mediated cognitive enhancement, we focused on nerve growth factor (NGF)-induced signaling pathways. NGF enzyme-linked immunosorbent assay analysis revealed that GE administration led to elevated NGF levels in both the mouse hippocampus and rat glioma C6 cells. GE administration also resulted in phosphorylation of extracellular-signal-regulated kinase (ERK) and cyclic AMP response element-binding protein (CREB), as revealed by Western blotting analysis. Neutralization of NGF with a specific NGF antibody inhibited GE-triggered activation of ERK and CREB in the hippocampus. Also, GE treatment significantly increased pre- and postsynaptic markers, synaptophysin and PSD-95, which are related to synapse formation in the brain. These data suggest that GE has a synaptogenic effect via NGF-induced ERK/CREB activation, resulting in memory enhancement. Copyright © 2014 Elsevier Inc. All rights reserved.

The effect of resveratrol on beta amyloid-induced memory impairment involves inhibition of phosphodiesterase-4 related signaling.

Science.gov (United States)

Wang, Gang; Chen, Ling; Pan, Xiaoyu; Chen, Jiechun; Wang, Liqun; Wang, Weijie; Cheng, Ruochuan; Wu, Fan; Feng, Xiaoqing; Yu, Yingcong; Zhang, Han-Ting; O'Donnell, James M; Xu, Ying

2016-04-05

Resveratrol, a natural polyphenol found in red wine, has wide spectrum of pharmacological properties including antioxidative and antiaging activities. Beta amyloid peptides (Aβ) are known to involve cognitive impairment, neuroinflammatory and apoptotic processes in Alzheimer's disease (AD). Activation of cAMP and/or cGMP activities can improve memory performance and decrease the neuroinflammation and apoptosis. However, it remains unknown whether the memory enhancing effect of resveratrol on AD associated cognitive disorders is related to the inhibition of phosphodiesterase 4 (PDE4) subtypes and subsequent increases in intracellular cAMP and/or cGMP activities. This study investigated the effect of resveratrol on Aβ1-42-induced cognitive impairment and the participation of PDE4 subtypes related cAMP or cGMP signaling. Mice microinfused with Aβ1-42 into bilateral CA1 subregions displayed learning and memory impairment, as evidenced by reduced memory acquisition and retrieval in the water maze and retention in the passive avoidance tasks; it was also significant that neuroinflammatory and pro-apoptotic factors were increased in Aβ1-42-treated mice. Aβ1-42-treated mice also increased in PDE4A, 4B and 4D expression, and decreased in PKA level. However, PKA inhibitor H89, but not PKG inhibitor KT5823, prevented resveratrol's effects on these parameters. Resveratrol also reversed Aβ1-42-induced decreases in phosphorylated cAMP response-element binding protein (pCREB), brain derived neurotrophic factor (BDNF) and anti-apoptotic factor BCl-2 expression, which were reversed by H89. These findings suggest that resveratrol reversing Aβ-induced learning and memory disorder may involve the regulation of neuronal inflammation and apoptosis via PDE4 subtypes related cAMP-CREB-BDNF signaling.

Nucleobase-modified antisense oligonucleotides containing 5-(phenyltriazol)-2′-deoxyuridine nucleotides induce exon-skipping

DEFF Research Database (Denmark)

Le, Bao T.; Hornum, Mick; Sharma, Pawan K.

2017-01-01

Chemically-modified antisense oligonucleotide-mediated exon-skipping has been validated as a therapeutic strategy for tackling several disease pathologies, particularly duchenne muscular dystrophy. To date, only sugar-modified and internucleotide linkage-modified oligonucleotide chemistries have...

CB1 receptor-mediated signaling underlies the hippocampal synaptic, learning, and memory deficits following treatment with JWH-081, a new component of spice/K2 preparations.

Science.gov (United States)

Basavarajappa, Balapal S; Subbanna, Shivakumar

2014-02-01

Recently, synthetic cannabinoids have been sprayed onto plant material, which is subsequently packaged and sold as "Spice" or "K2" to mimic the effects of marijuana. A recent report identified several synthetic additives in samples of "Spice/K2", including JWH-081, a synthetic ligand for the cannabinoid receptor 1 (CB1). The deleterious effects of JWH-081 on brain function are not known, particularly on CB1 signaling, synaptic plasticity, learning and memory. Here, we evaluated the effects of JWH-081 on pCaMKIV, pCREB, and pERK1/2 signaling events followed by long-term potentiation (LTP), hippocampal-dependent learning and memory tasks using CB1 receptor wild-type (WT) and knockout (KO) mice. Acute administration of JWH-081 impaired CaMKIV phosphorylation in a dose-dependent manner, whereas inhibition of CREB phosphorylation in CB1 receptor WT mice was observed only at higher dose of JWH-081 (1.25 mg/kg). JWH-081 at higher dose impaired CaMKIV and CREB phosphorylation in a time-dependent manner in CB1 receptor WT mice but not in KO mice and failed to alter ERK1/2 phosphorylation. In addition, SR treated or CB1 receptor KO mice have a lower pCaMKIV/CaMKIV ratio and higher pCREB/CREB ratio compared with vehicle or WT littermates. In hippocampal slices, JWH-081 impaired LTP in CB1 receptor WT but not in KO littermates. Furthermore, JWH-081 at higher dose impaired object recognition, spontaneous alternation and spatial memory on the Y-maze in CB1 receptor WT mice but not in KO mice. Collectively our findings suggest that deleterious effects of JWH-081 on hippocampal function involves CB1 receptor mediated impairments in CaMKIV and CREB phosphorylation, LTP, learning and memory in mice. © 2013 Wiley Periodicals, Inc.

Insulin/IGF-I regulation of necdin and brown adipocyte differentiation via CREB- and FoxO1-associated pathways

DEFF Research Database (Denmark)

Cypess, Aaron M; Zhang, Hongbin; Schulz, Tim J

2011-01-01

is regulated by the phosphoinositide 3 kinase-Akt pathway, increased necdin promoter activity. Based on reporter gene assays using truncations of the necdin promoter and chromatin immunoprecipitation studies, we demonstrated that CREB and FoxO1 are recruited to the necdin promoter, likely interacting......Brown adipose tissue plays an important role in obesity, insulin resistance, and diabetes. We have previously shown that the transition from brown preadipocytes to mature adipocytes is mediated in part by insulin receptor substrate (IRS)-1 and the cell cycle regulator protein necdin. In this study...... with specific consensus sequences in the proximal region. Based on these results, we propose that insulin/IGF-I act through IRS-1 phosphorylation to stimulate differentiation of brown preadipocytes via two complementary pathways: 1) the Ras-ERK1/2 pathway to activate CREB and 2) the phosphoinositide 3 kinase-Akt...

Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel Therapeutics

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Moizza Mansoor

2008-01-01

Full Text Available Antisense oligonucleotides (As-ODNs are single stranded, synthetically prepared strands of deoxynucleotide sequences, usually 18–21 nucleotides in length, complementary to the mRNA sequence of the target gene. As-ODNs are able to selectively bind cognate mRNA sequences by sequence-specific hybridization. This results in cleavage or disablement of the mRNA and, thus, inhibits the expression of the target gene. The specificity of the As approach is based on the probability that, in the human genome, any sequence longer than a minimal number of nucleotides (nt, 13 for RNA and 17 for DNA, normally occurs only once. The potential applications of As-ODNs are numerous because mRNA is ubiquitous and is more accessible to manipulation than DNA. With the publication of the human genome sequence, it has become theoretically possible to inhibit mRNA of almost any gene by As-ODNs, in order to get a better understanding of gene function, investigate its role in disease pathology and to study novel therapeutic targets for the diseases caused by dysregulated gene expression. The conceptual simplicity, the availability of gene sequence information from the human genome, the inexpensive availability of synthetic oligonucleotides and the possibility of rational drug design makes As-ODNs powerful tools for target identification, validation and therapeutic intervention. In this review we discuss the latest developments in antisense oligonucleotide design, delivery, pharmacokinetics and potential side effects, as well as its uses in target identification and validation, and finally focus on the current developments of antisense oligonucleotides in therapeutic intervention in various diseases.

Regulation of T cell activation by HIV-1 accessory proteins: Vpr acts via distinct mechanisms to cooperate with Nef in NFAT-directed gene expression and to promote transactivation by CREB

International Nuclear Information System (INIS)

Lahti, Anna L.; Manninen, Aki; Saksela, Kalle

2003-01-01

Nef and Vpr are lentiviral accessory proteins that have been implicated in regulation of cellular gene expression. We noticed that Vpr can potentiate Nef-induced activation of nuclear factor of activated T cells (NFAT)-dependent transcription. Unlike Nef, which stimulated calcium signaling to activate NFAT, Vpr functioned farther downstream. Similar to the positive effects of Vpr on most of the transcriptional test systems that we used, potentiation of NFAT-directed gene expression was relatively modest in magnitude (two- to threefold) and depended on the cell cycle-arresting capacity of Vpr. By contrast, we found that Vpr could cause more than fivefold upregulation of cyclic AMP response element (CRE)-directed transcription via a mechanism that did not require Vpr-induced G2/M arrest. This effect, however, was only evident under suboptimal conditions known to lead to serine phosphorylation of the CRE binding factor (CREB) but not to CREB-dependent gene expression. This suggested that Vpr may act by stabilizing interactions with CREB and its transcriptional cofactor CREB binding protein (CBP). Indeed, this effect could be blocked by cotransfection of the adenoviral CBP inhibitor E1A. These results provide additional evidence for cell cycle-independent regulation of gene expression by Vpr and implicate CREB as a potentially important target for Vpr action in HIV-infected host cells

Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

DEFF Research Database (Denmark)

Llovera Nadal, Laia; Berthold, Peter; Nielsen, Peter E

2012-01-01

have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA......Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating...... peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we...

Thiolated carboxymethyl dextran as a nanocarrier for colon delivery of hSET1 antisense: In vitro stability and efficiency study

Energy Technology Data Exchange (ETDEWEB)

Kiani, Melika, E-mail: Melika.kiani@gmail.com [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Mirzazadeh Tekie, Farnaz Sadat, E-mail: mirzazadehf@yahoo.com [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Dinarvand, Meshkat, E-mail: mdinarvand@hotmail.com [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Soleimani, Masoud, E-mail: soleim_m@modares.ac.ir [Stem Cell Technology Research Centre, P.O. Box 14155-3174, Tehran (Iran, Islamic Republic of); Department of Hematology, School of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-111, Tehran (Iran, Islamic Republic of); Dinarvand, Rassoul, E-mail: dinarvand@tums.ac.ir [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Atyabi, Fatemeh, E-mail: atyabifa@tums.ac.ir [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

2016-05-01

Gene therapy is an optimistic approach in cancer treatment. However, for efficient delivery of gene materials, designing an appropriate vector is necessary. Polyelectrolyte complexes (PECs) of chitosan and dextran could be considered a proper nanoparticulate carrier for sensitive biomaterials. In this study, PECs of chitosan and thiolated dextran were used as either an injectable or oral gene delivery system. hSET1 antisense was loaded into the PECs to suppress proliferation of colon cancer cell line. The prepared nanoparticles have ~ 115 nm diameter size and positive zeta potential with high mucoadhesion properties. They are able to protect antisense from degradation in serum and biorelevant fluids (FaSSIF and FaSSGF). Furthermore, prepared nanoparticles demonstrated superior cellular penetration and inhibitory effect on SW480 colon cancer cell proliferation. All nanoparticles significantly down regulated hSET1 in comparison with naked antisense. It can be concluded that thiolated PECs have potential use for injectable or oral delivery of nucleic acids such as antisense. - Highlights: • Formation of stable nanoparticle with dextran and chitosan derivatives for oral and intravenous gene delivery. • Satifactory cellular uptake of nanoparticles and approximately complete suppression of hSET1 expression in SW480 cell lines • Prolonged stability of nanoparticles against biorelevent media with desirable release rate.

Thiolated carboxymethyl dextran as a nanocarrier for colon delivery of hSET1 antisense: In vitro stability and efficiency study

International Nuclear Information System (INIS)

Kiani, Melika; Mirzazadeh Tekie, Farnaz Sadat; Dinarvand, Meshkat; Soleimani, Masoud; Dinarvand, Rassoul; Atyabi, Fatemeh

2016-01-01

Gene therapy is an optimistic approach in cancer treatment. However, for efficient delivery of gene materials, designing an appropriate vector is necessary. Polyelectrolyte complexes (PECs) of chitosan and dextran could be considered a proper nanoparticulate carrier for sensitive biomaterials. In this study, PECs of chitosan and thiolated dextran were used as either an injectable or oral gene delivery system. hSET1 antisense was loaded into the PECs to suppress proliferation of colon cancer cell line. The prepared nanoparticles have ~ 115 nm diameter size and positive zeta potential with high mucoadhesion properties. They are able to protect antisense from degradation in serum and biorelevant fluids (FaSSIF and FaSSGF). Furthermore, prepared nanoparticles demonstrated superior cellular penetration and inhibitory effect on SW480 colon cancer cell proliferation. All nanoparticles significantly down regulated hSET1 in comparison with naked antisense. It can be concluded that thiolated PECs have potential use for injectable or oral delivery of nucleic acids such as antisense. - Highlights: • Formation of stable nanoparticle with dextran and chitosan derivatives for oral and intravenous gene delivery. • Satifactory cellular uptake of nanoparticles and approximately complete suppression of hSET1 expression in SW480 cell lines • Prolonged stability of nanoparticles against biorelevent media with desirable release rate.

Essential role for cyclic-AMP responsive element binding protein 1 (CREB) in the survival of acute lymphoblastic leukemia

NARCIS (Netherlands)

van der Sligte, Naomi E.; Kampen, Kim R.; ter Elst, Arja; Scherpen, Frank J. G.; Meeuwsen-de Boer, Tiny G. J.; Guryev, Victor; van Leeuwen, Frank N.; Kornblau, Steven M.; de Bont, Eveline S. J. M.

2015-01-01

Acute lymphoblastic leukemia (ALL) relapse remains a leading cause of cancer related death in children, therefore, new therapeutic options are needed. Recently, we showed that a peptide derived from Cyclic-AMP Responsive Element Binding Protein (CREB) was highly phosphorylated in pediatric

A dystrophic Duchenne mouse model for testing human antisense oligonucleotides.

Directory of Open Access Journals (Sweden)

Marcel Veltrop

Full Text Available Duchenne muscular dystrophy (DMD is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.

Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria ananassa)

NARCIS (Netherlands)

Lunkenbein, S.; Coiner, H.; Vos, de C.H.; Schaart, J.G.; Boone, M.J.; Krens, F.A.; Schwab, W.; Salentijn, E.M.J.

2006-01-01

An octaploid (Fragaria × ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria × ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens.

Functional Analysis of Polyphenol Oxidases by Antisense/Sense Technology

Directory of Open Access Journals (Sweden)

Jutharat Attajarusit

2007-07-01

Full Text Available Polyphenol oxidases (PPOs catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum plants with modified PPO expression levels (suppressed PPO and overexpressing PPO. These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F., cotton bollworm (Helicoverpa armigera (Hübner and beet army worm (Spodoptera exigua (Hübner, whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants

Lanthanum chloride impairs spatial memory through ERK/MSK1 signaling pathway of hippocampus in rats.

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Liu, Huiying; Yang, Jinghua; Liu, Qiufang; Jin, Cuihong; Wu, Shengwen; Lu, Xiaobo; Zheng, Linlin; Xi, Qi; Cai, Yuan

2014-12-01

Rare earth elements (REEs) are used in many fields for their diverse physical and chemical properties. Surveys have shown that REEs can impair learning and memory in children and cause neurobehavioral defects in animals. However, the mechanism underlying these impairments has not yet been completely elucidated. Lanthanum (La) is often selected to study the effects of REEs. The aim of this study was to investigate the spatial memory impairments induced by lanthanum chloride (LaCl3) and the probable underlying mechanism. Wistar rats were exposed to LaCl3 in drinking water at 0 % (control, 0 mM), 0.25 % (18 mM), 0.50 % (36 mM), and 1.00 % (72 mM) from birth to 2 months after weaning. LaCl3 considerably impaired the spatial learning and memory of rats in the Morris water maze test, damaged the synaptic ultrastructure and downregulated the expression of p-MEK1/2, p-ERK1/2, p-MSK1, p-CREB, c-FOS and BDNF in the hippocampus. These results indicate that LaCl3 exposure impairs the spatial learning and memory of rats, which may be attributed to disruption of the synaptic ultrastructure and inhibition of the ERK/MSK1 signaling pathway in the hippocampus.

Roles of p300 and cyclic adenosine monophosphate response element binding protein in high glucose-induced hypoxia-inducible factor 1α inactivation under hypoxic conditions.

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Ding, Lingtao; Yang, Minlie; Zhao, Tianlan; Lv, Guozhong

2017-05-01

Given the high prevalence of diabetes and burn injuries worldwide, it is essential to dissect the underlying mechanism of delayed burn wound healing in diabetes patients, especially the high glucose-induced hypoxia-inducible factor 1 (HIF-1)-mediated transcription defects. Human umbilical vein endothelial cells were cultured with low or high concentrations of glucose. HIF-1α-induced vascular endothelial growth factor (VEGF) transcription was measured by luciferase assay. Immunofluorescence staining was carried out to visualize cyclic adenosine monophosphate response element binding protein (CREB) localization. Immunoprecipitation was carried out to characterize the association between HIF-1α/p300/CREB. To test whether p300, CREB or p300+CREB co-overexpression was sufficient to rescue the HIF-1-mediated transcription defect after high glucose exposure, p300, CREB or p300+CREB co-overexpression were engineered, and VEGF expression was quantified. Finally, in vitro angiogenesis assay was carried out to test whether the high glucose-induced angiogenesis defect is rescuable by p300 and CREB co-overexpression. Chronic high glucose treatment resulted in impaired HIF-1-induced VEGF transcription and CREB exclusion from the nucleus. P300 or CREB overexpression alone cannot rescue high glucose-induced HIF-1α transcription defects. In contrast, co-overexpression of p300 and CREB dramatically ameliorated high glucose-induced impairment of HIF-1-mediated VEGF transcription, as well as in vitro angiogenesis. Finally, we showed that co-overexpression of p300 and CREB rectifies the dissociation of HIF-1α-p300-CREB protein complex in chronic high glucose-treated cells. Both p300 and CREB are required for the function integrity of HIF-1α transcription machinery and subsequent angiogenesis, suggesting future studies to improve burn wound healing might be directed to optimization of the interaction between p300, CREB and HIF-1α. © 2016 The Authors. Journal of Diabetes

IL-1β Upregulates StAR and Progesterone Production Through the ERK1/2- and p38-Mediated CREB Signaling Pathways in Human Granulosa-Lutein Cells.

Science.gov (United States)

Dang, Xuan; Zhu, Qinling; He, Yaqiong; Wang, Yuan; Lu, Yao; Li, Xiaoxue; Qi, Jia; Wu, Hasiximuke; Sun, Yun

2017-10-01

The proinflammatory cytokine interleukin-1β (IL-1β) may be involved in several ovulation-associated events, such as protease synthesis, prostaglandin production, and steroidogenesis in granulosa cells. However, the exact effect of IL-1β on progesterone synthesis in granulosa cells and the underlying mechanism remain unclear. By using cultured granulosa-lutein cells collected from women undergoing in vitro fertilization or intracytoplasmic sperm injection, we found that IL-1β upregulated steroidogenic acute regulatory protein (StAR) expression and progesterone synthesis in granulosa-lutein cells, which was comparable with luteinizing hormone effect and could be abolished by an IL-1 receptor antagonist. Moreover, IL-1β activated the phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB), and knockdown of CREB attenuated the induction of StAR expression and progesterone synthesis by IL-1β in granulosa-lutein cells. Furthermore, IL-1β activated the extracellular signal-regulated kinase (ERK)1/2 and p38 pathways and inhibition of the ERK1/2 and p38 pathways attenuated the IL-1β-induced phosphorylation of CREB, StAR expression, and progesterone synthesis in granulosa-lutein cells. In conclusion, IL-1β could upregulate StAR expression and stimulate progesterone biosynthesis through increase in CREB phosphorylation via activating the ERK1/2 and p38 pathways in human granulosa-lutein cells. Copyright © 2017 Endocrine Society.

Resveratrol improves learning and memory in normally aged mice through microRNA-CREB pathway.

Science.gov (United States)

Zhao, Yong-Na; Li, Wei-Feng; Li, Fei; Zhang, Zhen; Dai, Yun-Dong; Xu, Ai-Li; Qi, Cui; Gao, Ji-Min; Gao, Jun

2013-06-14

Resveratrol (RSV) is a natural compound found in grapes and red wine. It has been well known for its beneficial effects as a dietary supplement in prevention of cardiovascular diseases and cancer. Recently, in vitro studies have reported the neuroprotective role of RSV in neurodegenerative process in Alzheimer's disease (AD). However, in vivo effects of RSV on the decline of brain function accompanying the aging process, especially those on cognitive loss, have not been not investigated. Here we report that, after intraventricular injection of RSV for one week in 8-9 month-old mice, the long-term memory formation and the LTP induction from hippocampus CA1 were improved. The RSV enhancement effects were blocked in SIRT1 mutant mice. Additional experiments suggest that RSV effects are likely to be mediated through reduced expressions of miR-134 and miR-124, which may in turn up-regulate CREB levels to subsequently promote BDNF synthesis. These findings demonstrate a role for RSV in cognition and a microRNA-CREB-BDNF mechanism by which RSV regulates these processes, demonstrating its value as a potential therapeutic target against CNS disorders in aging. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Schisandra chinensis produces the antidepressant-like effects in repeated corticosterone-induced mice via the BDNF/TrkB/CREB signaling pathway.

Science.gov (United States)

Yan, Tingxu; Xu, Mengjie; Wan, Shutong; Wang, Mengshi; Wu, Bo; Xiao, Feng; Bi, Kaishun; Jia, Ying

2016-09-30

The present study aimed to examine the antidepressant-like effects and the possible mechanisms of Schisandra chinensis on depressive-like behavior induced by repeated corticosterone injections in mice. Here we evaluated the effect of an ethanol extract of the dried fruit of S. chinensis (EESC) on BDNF/TrkB/CREB signaling in the hippocampus and the prefrontal cortex. Three weeks of corticosterone injections in mice resulted in depressive-like behavior, as indicated by the significant decrease in sucrose consumption and increase the immobility time in the forced swim test, but without any influence on the locomotor activity. Further, there was a significant increase in serum corticosterone level and a significant downregulation of BDNF/TrkB/CREB signaling pathway in the hippocampus and prefrontal cortex in CORT-treated mice. Treatment of mice with EESC (600mg/kg) significantly ameliorated all the behavioral and biochemical changes induced by corticosterone. Moreover, pharmacological inhibition of BDNF signaling by K252a abolished entirely the antidepressant-like effect triggered by chronic EESC treatment. These results suggest that EESC produces an antidepressant-like effect in CORT-induced depression in mice, which is possibly mediated, at least in part, by rectifying the stress-based hypothalamic-pituitary-adrenal (HPA) axis dysfunction paradigm and upregulation of BDNF/TrkB/CREB signaling pathway. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Sense and antisense transcripts of the developmentally regulated murine hsp70.2 gene are expressed in distinct and only partially overlapping areas in the adult brain

Science.gov (United States)

Murashov, A. K.; Wolgemuth, D. J.

1996-01-01

We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.

Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.

Science.gov (United States)

Alper, O; De Santis, M L; Stromberg, K; Hacker, N F; Cho-Chung, Y S; Salomon, D S

2000-11-15

Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness. Copyright 2000 Wiley-Liss, Inc.

Preliminary studies on gene therapy with TGF β1 antisense gene/liposome complexes and adenovirus transfer vector in RPF rats

International Nuclear Information System (INIS)

Liu Chunjie; Wang Dewen; Zhang Zhaoshan; Gao Yabing; Xiong Chengqi; Long Jianyin; Wang Huixin; Peng Ruiyun; Cui Xuemei

2001-01-01

Objective: To observed the efficiency of gene therapy with TGF β1 antisense gene/liposome complexes and adenovirus transfer vector in RPF rats. Methods: TGFβ1 sense and antisense gene expression vectors and adenovirus transfer vector were introduced into rat bronchus by way of intratracheal instillation. Results: At day 1.5 after TGFβ1 sense and antisense gene transfer, PCR amplification using neo gene-specific primer from lung tissue DNA was all positive. After day 5.5, 67% (2/3) of lung tissue DNA was positive. RNA dot blot hybridization indicated that TGFβ1 mRNA content of lung tissue transfected with pMAMneo-antiTGFβ1 gene decreased. Detection of lung hydroxyproline (Hyp) content after day 35 of gene transfer showed that even in lung of rats received pMAMneo-AntiTGFβ1 lipid complexes it raised remarkably (P 9 pfu/ml were instilled into bronchus at 0.5 ml per rat. After day 2 day 6, the lung tissues of all six rats (three per each group )expressed the transfected luciferase gene by luminometer. Conclusion: Cationic lipid-mediated TGFβ1 antisense gene therapy was a simple and easy method. It can slow down the course of pathogenesis of lung fibrosis. Replication-deficient recombinant adenovirus-mediated gene therapy of lung diseases is a good and efficient method

Period1 gates the circadian modulation of memory-relevant signaling in mouse hippocampus by regulating the nuclear shuttling of the CREB kinase pP90RSK

DEFF Research Database (Denmark)

Rawashdeh, Oliver; Jilg, Antje; Maronde, Erik

2016-01-01

, the presence of PER1 in hippocampal neurons is a prerequisite for the time-of-day-dependent phosphorylation of CREB, as it regulates the shuttling of pP90RSK into the nucleus. Representative immunofluorescence images show a temporal difference in phosphorylated cAMP response element-binding protein (p...... activation. Taken together, the PER1-dependent modulation of cytoplasmic-to-nuclear signaling in the murine hippocampus provides a molecular explanation for how the circadian system potentially shapes a temporal framework for daytime-dependent memory performance, and adds a novel facet to the versatility......CREB; green color) levels in all regions of the dorsal hippocampus between a wild-type C3H mouse (WT; left) and a Period1-knockout (Per1−/−; right) mouse. Images were taken 2 h after lights on, thus, when fluctuating levels of pCREB peak in WT mouse hippocampus. Insets show a representative hippocampal neuron...

Combination of vascular endothelial growth factor antisense oligonucleotide therapy and radiotherapy increases the curative effects against maxillofacial VX2 tumors in rabbits

Energy Technology Data Exchange (ETDEWEB)

Zheng Linfeng, E-mail: zhenglinfeng04@yahoo.com.cn [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Li Yujie, E-mail: yujieli01@yahoo.com.cn [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Wang Han, E-mail: bingowh@hotmail.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Zhao Jinglong, E-mail: jinglongz@yahoo.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Wang Xifu, E-mail: wangxiechen001@163.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Hu Yunsheng, E-mail: springmorninghu@163.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Zhang Guixiang, E-mail: guixiangzhang@sina.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China)

2011-05-15

Purpose: To study the effects of combination of vascular endothelial growth factor (VEGF) antisense oligonucleotide therapy and radiotherapy on maxillofacial VX2 tumors in rabbits. Methods: We used 24 New Zealand white rabbits as a model to induce maxillofacial VX2 tumor. The rabbits were randomly divided into the following 4 groups: radiotherapy group (group A), treated with 16 Gy of radiotherapy; VEGF antisense oligonucleotide treatment group (group B), treated with an injection of 150 {mu}g of VEGF antisense oligonucleotide into the local tumor; VEGF antisense oligonucleotide combined with radiotherapy group (group C), treated with an injection of 150 {mu}g of VEGF antisense oligonucleotide into the local tumor immediately after 16 Gy of radiotherapy; and control group (group D), treated with an injection of 300 {mu}l 5% aqueous glucose solution into the local tumor. On days 3 and 14 after treatment, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed to calculate maximal enhancement ratio (MER), slope of enhancement (SLE), and tumor volume change. Rabbits were killed on day 14 to obtain samples for pathological examination and immunohistochemical staining for VEGF. Results: In group C, tumor volume was significantly reduced on day 14 after treatment, and the difference was statistically different as compared to that before treatment, on day 3 after treatment and other groups (P < 0.01). Values of both MER and SLE after treatment were significantly lower than the values before treatment (P < 0.05). Pathological specimen revealed tumor cell edema, bleeding, necrosis, vascular wall thickening and occlusion, and decreased VEGF expression. The immunohistochemical score (IHS) of group C was significantly different from groups A and D respectively (P < 0.05). Conclusion: Injecting the tumor with VEGF antisense oligonucleotide immediately after radiotherapy can enhance the curative effect on rabbit maxillofacial VX2 tumor, and DCE-MRI can serve

Metabolite profiling of Arabidopsis thaliana (L.) plants transformed with an antisense chalcone synthase gene

DEFF Research Database (Denmark)

Le Gall, G.; Metzdorff, Stine Broeng; Pedersen, Jan W.

2005-01-01

A metabolite profiling study has been carried out on Arabidopsis thaliana (L.) Heynh. ecotype Wassilewskija and a series of transgenic lines of the ecotype transformed with a CHS (chalcone synthase) antisense construct. Compound identifications by LC/MS and H-1 NMR are discussed. The glucosinolate...

Sodium phenylbutyrate enhances astrocytic neurotrophin synthesis via protein kinase C (PKC)-mediated activation of cAMP-response element-binding protein (CREB): implications for Alzheimer disease therapy.

Science.gov (United States)

Corbett, Grant T; Roy, Avik; Pahan, Kalipada

2013-03-22

Neurotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are believed to be genuine molecular mediators of neuronal growth and homeostatic synapse activity. However, levels of these neurotrophic factors decrease in different brain regions of patients with Alzheimer disease (AD). Induction of astrocytic neurotrophin synthesis is a poorly understood phenomenon but represents a plausible therapeutic target because neuronal neurotrophin production is aberrant in AD and other neurodegenerative diseases. Here, we delineate that sodium phenylbutyrate (NaPB), a Food and Drug Administration-approved oral medication for hyperammonemia, induces astrocytic BDNF and NT-3 expression via the protein kinase C (PKC)-cAMP-response element-binding protein (CREB) pathway. NaPB treatment increased the direct association between PKC and CREB followed by phosphorylation of CREB (Ser(133)) and induction of DNA binding and transcriptional activation of CREB. Up-regulation of markers for synaptic function and plasticity in cultured hippocampal neurons by NaPB-treated astroglial supernatants and its abrogation by anti-TrkB blocking antibody suggest that NaPB-induced astroglial neurotrophins are functionally active. Moreover, oral administration of NaPB increased the levels of BDNF and NT-3 in the CNS and improved spatial learning and memory in a mouse model of AD. Our results highlight a novel neurotrophic property of NaPB that may be used to augment neurotrophins in the CNS and improve synaptic function in disease states such as AD.

Neuroprotective Effect of Arctigenin via Upregulation of P-CREB in Mouse Primary Neurons and Human SH-SY5Y Neuroblastoma Cells

Science.gov (United States)

Zhang, Nan; Wen, Qingping; Ren, Lu; Liang, Wenbo; Xia, Yang; Zhang, Xiaodan; Zhao, Dan; Sun, Dong; Hu, Yv; Hao, Haiguang; Yan, Yaping; Zhang, Guangxian; Yang, Jingxian; Kang, Tingguo

2013-01-01

Arctigenin (Arc) has been shown to act on scopolamine-induced memory deficit mice and to provide a neuroprotective effect on cultured cortical neurons from glutamate-induced neurodegeneration through mechanisms not completely defined. Here, we investigated the neuroprotective effect of Arc on H89-induced cell damage and its potential mechanisms in mouse cortical neurons and human SH-SY5Y neuroblastoma cells. We found that Arc prevented cell viability loss induced by H89 in human SH-SY5Y cells. Moreover, Arc reduced intracellular beta amyloid (Aβ) production induced by H89 in neurons and human SH-SY5Y cells, and Arc also inhibited the presenilin 1(PS1) protein level in neurons. In addition, neural apoptosis in both types of cells, inhibition of neurite outgrowth in human SH-SY5Y cells and reduction of synaptic marker synaptophysin (SYN) expression in neurons were also observed after H89 exposure. All these effects induced by H89 were markedly reversed by Arc treatment. Arc also significantly attenuated downregulation of the phosphorylation of CREB (p-CREB) induced by H89, which may contribute to the neuroprotective effects of Arc. These results demonstrated that Arc exerted the ability to protect neurons and SH-SY5Y cells against H89-induced cell injury via upregulation of p-CREB. PMID:24025424

Neuroprotective Effect of Arctigenin via Upregulation of P-CREB in Mouse Primary Neurons and Human SH-SY5Y Neuroblastoma Cells

Directory of Open Access Journals (Sweden)

Tingguo Kang

2013-09-01

Full Text Available Arctigenin (Arc has been shown to act on scopolamine-induced memory deficit mice and to provide a neuroprotective effect on cultured cortical neurons from glutamate-induced neurodegeneration through mechanisms not completely defined. Here, we investigated the neuroprotective effect of Arc on H89-induced cell damage and its potential mechanisms in mouse cortical neurons and human SH-SY5Y neuroblastoma cells. We found that Arc prevented cell viability loss induced by H89 in human SH-SY5Y cells. Moreover, Arc reduced intracellular beta amyloid (Aβ production induced by H89 in neurons and human SH-SY5Y cells, and Arc also inhibited the presenilin 1(PS1 protein level in neurons. In addition, neural apoptosis in both types of cells, inhibition of neurite outgrowth in human SH-SY5Y cells and reduction of synaptic marker synaptophysin (SYN expression in neurons were also observed after H89 exposure. All these effects induced by H89 were markedly reversed by Arc treatment. Arc also significantly attenuated downregulation of the phosphorylation of CREB (p-CREB induced by H89, which may contribute to the neuroprotective effects of Arc. These results demonstrated that Arc exerted the ability to protect neurons and SH-SY5Y cells against H89-induced cell injury via upregulation of p-CREB.

Lactoferricin B Inhibits the Phosphorylation of the Two-Component System Response Regulators BasR and CreB*

Science.gov (United States)

Ho, Yu-Hsuan; Sung, Tzu-Cheng; Chen, Chien-Sheng

2012-01-01

Natural antimicrobial peptides provide fundamental protection for multicellular organisms from microbes, such as Lactoferricin B (Lfcin B). Many studies have shown that Lfcin B penetrates the cell membrane and has intracellular activities. To elucidate the intracellular behavior of Lfcin B, we first used Escherichia coli K12 proteome chips to identify the intracellular targets of Lfcin B. The results showed that Lfcin B binds to two response regulators, BasR and CreB, of the two-component system. For further analysis, we conducted several in vitro and in vivo experiments and utilized bioinformatics methods. The electrophoretic mobility shift assays and kinase assays indicate that Lfcin B inhibits the phosphorylation of the response regulators (BasR and CreB) and their cognate sensor kinases (BasS and CreC). Antibacterial assays showed that Lfcin B reduced E. coli's tolerance to environmental stimuli, such as excessive ferric ions and minimal medium conditions. This is the first study to show that an antimicrobial peptide inhibits the growth of bacteria by influencing the phosphorylation of a two-component system directly. PMID:22138548

Lactoferricin B inhibits the phosphorylation of the two-component system response regulators BasR and CreB.

Science.gov (United States)

Ho, Yu-Hsuan; Sung, Tzu-Cheng; Chen, Chien-Sheng

2012-04-01

Natural antimicrobial peptides provide fundamental protection for multicellular organisms from microbes, such as Lactoferricin B (Lfcin B). Many studies have shown that Lfcin B penetrates the cell membrane and has intracellular activities. To elucidate the intracellular behavior of Lfcin B, we first used Escherichia coli K12 proteome chips to identify the intracellular targets of Lfcin B. The results showed that Lfcin B binds to two response regulators, BasR and CreB, of the two-component system. For further analysis, we conducted several in vitro and in vivo experiments and utilized bioinformatics methods. The electrophoretic mobility shift assays and kinase assays indicate that Lfcin B inhibits the phosphorylation of the response regulators (BasR and CreB) and their cognate sensor kinases (BasS and CreC). Antibacterial assays showed that Lfcin B reduced E. coli's tolerance to environmental stimuli, such as excessive ferric ions and minimal medium conditions. This is the first study to show that an antimicrobial peptide inhibits the growth of bacteria by influencing the phosphorylation of a two-component system directly.

Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.

Science.gov (United States)

Crosby, Jeff R; Zhao, Chenguang; Jiang, Chong; Bai, Dong; Katz, Melanie; Greenlee, Sarah; Kawabe, Hiroshi; McCaleb, Michael; Rotin, Daniela; Guo, Shuling; Monia, Brett P

2017-11-01

Epithelial sodium channel (ENaC, Scnn1) hyperactivity in the lung leads to airway surface dehydration and mucus accumulation in cystic fibrosis (CF) patients and in mice with CF-like lung disease. We identified several potent ENaC specific antisense oligonucleotides (ASOs) and tested them by inhalation in mouse models of CF-like lung disease. The inhaled ASOs distributed into lung airway epithelial cells and decreased ENaC expression by inducing RNase H1-dependent degradation of the targeted Scnn1a mRNA. Aerosol delivered ENaC ASO down-regulated mucus marker expression and ameliorated goblet cell metaplasia, inflammation, and airway hyper-responsiveness. Lack of systemic activity of ASOs delivered via the aerosol route ensures the safety of this approach. Our results demonstrate that antisense inhibition of ENaC in airway epithelial cells could be an effective and safe approach for the prevention and reversal of lung symptoms in CF and potentially other inflammatory diseases of the lung. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Conserved alternative and antisense transcripts at the programmed cell death 2 locus

Czech Academy of Sciences Publication Activity Database

Mihola, Ondřej; Forejt, Jiří; Trachtulec, Zdeněk

2007-01-01

Roč. 8, - (2007), s. 20 ISSN 1471-2164 R&D Projects: GA ČR(CZ) GA204/01/0997; GA ČR GA301/05/0738; GA AV ČR IAA5052406; GA MŠk(CZ) 1M0520 Institutional research plan: CEZ:AV0Z50520514 Keywords : Pdcd2 * antisense * alternative transcript * imprinting Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.180, year: 2007

Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium.

LENUS (Irish Health Repository)

Dooley, Ruth

2012-02-01

BACKGROUND: Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. RESULTS: Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. CONCLUSION: Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

LENUS (Irish Health Repository)

Dooley, Ruth

2011-08-22

Abstract Background Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. Results Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. Conclusion Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

Purinergic receptor antagonists inhibit odorant-mediated CREB phosphorylation in sustentacular cells of mouse olfactory epithelium

Directory of Open Access Journals (Sweden)

Hatt Hanns

2011-08-01

Full Text Available Abstract Background Extracellular nucleotides have long been known to play neuromodulatory roles and to be involved in intercellular signalling. In the olfactory system, ATP is released by olfactory neurons, and exogenous ATP can evoke an increase in intracellular calcium concentration in sustentacular cells, the nonneuronal supporting cells of the olfactory epithelium. Here we investigate the hypothesis that olfactory neurons communicate with sustentacular cells via extracellular ATP and purinergic receptor activation. Results Here we show that exposure of mice to a mixture of odorants induced a significant increase in the levels of the transcription factor CREB phosphorylated at Ser-133 in the nuclei of both olfactory sensory neurons and sustentacular cells. This activation was dependent on adenylyl cyclase III-mediated olfactory signaling and on activation of P2Y purinergic receptors on sustentacular cells. Purinergic receptor antagonists inhibited odorant-dependent CREB phosphorylation specifically in the nuclei of the sustentacular cells. Conclusion Our results point to a possible role for extracellular nucleotides in mediating intercellular communication between the neurons and sustentacular cells of the olfactory epithelium in response to odorant exposure. Maintenance of extracellular ionic gradients and metabolism of noxious chemicals by sustentacular cells may therefore be regulated in an odorant-dependent manner by olfactory sensory neurons.

G3139, a Bcl-2 antisense oligodeoxynucleotide, induces clinical responses in VAD refractory myeloma

NARCIS (Netherlands)

van de Donk, N. W. C. J.; de Weerdt, O.; Veth, G.; Eurelings, M.; van Stralen, E.; Frankel, S. R.; Hagenbeek, A.; Bloem, A. C.; Lokhorst, H. M.

2004-01-01

Expression of Bcl-2 in multiple myeloma is associated with resistance to chemotherapeutic drugs. Conversely, suppression of Bcl-2 enhanced the chemosensitivity of myeloma cells in vitro. G3139 is an antisense oligodeoxynucleotide targeted to the first six codons of the Bcl-2 mRNA open reading frame.

Targeting TGF-β Signaling by Antisense Oligonucleotide-mediated Knockdown of TGF-β Type I Receptor

Directory of Open Access Journals (Sweden)

Dwi U Kemaladewi

2014-01-01

Full Text Available Duchenne muscular dystrophy (DMD is caused by lack of functional dystrophin and results in progressive myofiber damage and degeneration. In addition, impaired muscle regeneration and fibrosis contribute to the progressive pathology of DMD. Importantly, transforming growth factor-β (TGF-β is implicated in DMD pathology and is known to stimulate fibrosis and inhibit muscle regeneration. In this study, we present a new strategy to target TGF-β signaling cascades by specifically inhibiting the expression of TGF-β type I receptor TGFBR1 (ALK5. Antisense oligonucleotides (AONs were designed to specifically induce exon skipping of mouse ALK5 transcripts. AON-induced exon skipping of ALK5 resulted in specific downregulation of full-length receptor transcripts in vitro in different cell types, repression of TGF-β activity, and enhanced C2C12 myoblast differentiation. To determine the effect of these AONs in dystrophic muscles, we performed intramuscular injections of ALK5 AONs in mdx mice, which resulted in a decrease in expression of fibrosis-related genes and upregulation of Myog expression compared to control AON-injected muscles. In summary, our study presents a novel method to target TGF-β signaling cascades with potential beneficial effects for DMD.

Physicochemical and biological properties of self-assembled antisense/poly(amidoamine dendrimer nanoparticles: the effect of dendrimer generation and charge ratio

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Alireza Nomani

2010-05-01

Full Text Available Alireza Nomani1,6, Ismaeil Haririan1,5, Ramin Rahimnia2,4, Shamileh Fouladdel2, Tarane Gazori1, Rassoul Dinarvand1, Yadollah Omidi3, Ebrahim Azizi2,41Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 2Molecular Research Lab, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 3Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran; 4Department of Medical Biotechnology, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran; 5Biomaterials Research Center (BRC Tehran, Iran; 6Department of Pharmaceutics, Faculty of Pharmacy, Zanjan University of Medical Sciences, Zanjan, IranAbstract: To gain a deeper understanding of the physicochemical phenomenon of self-assembled nanoparticles of different generations and ratios of poly (amidoamine dendrimer (PAMAM dendrimer and a short-stranded DNA (antisense oligonucleotide, multiple methods were used to characterize these nanoparticles including photon correlation spectroscopy (PCS; zeta potential measurement; and atomic force microscopy (AFM. PCS and AFM results revealed that, in contrast to larger molecules of DNA, smaller molecules produce more heterodisperse and large nanoparticles when they are condensed with a cationic dendrimer. AFM images also showed that such nanoparticles were spherical. The stability of the antisense content of the nanoparticles was investigated over different charge ratios using polyacrylamide gel electrophoresis. It was clear from such analyses that much more than charge neutrality point was required to obtain stable nanoparticles. For cell uptake, self-assembled nanoparticles were prepared with PAMAM G5 and 5’-FITC labeled antisense and the uptake experiment was carried out in T47D cell culture. This investigation also shows that the cytotoxicity of the nanoparticles was

Antisense to the glucocorticoid receptor in hippocampal dentate gyrus reduces immobility in forced swim test

NARCIS (Netherlands)

Korte, S.M.; de Kloet, E.R.; Buwalda, B; Bouman, S.D.; Bohus, B

1996-01-01

Immobility time of rats in the forced swim test was reduced after bilateral infusion of an 18-mer antisense phosphorothioate oligodeoxynucleotide targeted to the glucocorticoid receptor mRNA into the dentate gyrus of the hippocampus. Vehicle-, sense- and scrambled sequence-treated animals spent

CREB expression in the brains of two closely related parasitic wasp species that differ in long-term memory formation

NARCIS (Netherlands)

Van den Berg, M.; Verbaarschot, P.; Hontelez, S.; Vet, L.E.M.; Dicke, M.; Smid, H.M.

2010-01-01

The cAMP/PKA signalling pathway and transcription factor cAMP response element-binding protein (CREB) play key roles in long-term memory (LTM) formation. We used two closely related parasitic wasp species, Cotesia glomerata and Cotesia rubecula, which were previously shown to be different in LTM

Hippocampal Overexpression of Mutant CREB Blocks Long-Term, but Not Short-Term Memory for a Socially Transmitted Food Preference

Science.gov (United States)

Brightwell, Jennifer J.; Countryman, Renee A.; Neve, Rachael L.; Colombo, Paul J.; Smith, Clayton A.

2005-01-01

Phosphorylation of the transcription factor CREB on Ser133 is implicated in the establishment of long-term memory for hippocampus-dependent tasks, including spatial learning and contextual fear conditioning. We reported previously that training on a hippocampus-dependent social transmission of food preference (STFP) task increases CREB…

Deletion of PEA-15 in mice is associated with specific impairments of spatial learning abilities

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Hale Gregory

2009-11-01

Full Text Available Abstract Background PEA-15 is a phosphoprotein that binds and regulates ERK MAP kinase and RSK2 and is highly expressed throughout the brain. PEA-15 alters c-Fos and CREB-mediated transcription as a result of these interactions. To determine if PEA-15 contributes to the function of the nervous system we tested mice lacking PEA-15 in a series of experiments designed to measure learning, sensory/motor function, and stress reactivity. Results We report that PEA-15 null mice exhibited impaired learning in three distinct spatial tasks, while they exhibited normal fear conditioning, passive avoidance, egocentric navigation, and odor discrimination. PEA-15 null mice also had deficient forepaw strength and in limited instances, heightened stress reactivity and/or anxiety. However, these non-cognitive variables did not appear to account for the observed spatial learning impairments. The null mice maintained normal weight, pain sensitivity, and coordination when compared to wild type controls. Conclusion We found that PEA-15 null mice have spatial learning disabilities that are similar to those of mice where ERK or RSK2 function is impaired. We suggest PEA-15 may be an essential regulator of ERK-dependent spatial learning.

SCM-198 Ameliorates Cognitive Deficits, Promotes Neuronal Survival and Enhances CREB/BDNF/TrkB Signaling without Affecting Aβ Burden in AβPP/PS1 Mice.

Science.gov (United States)

Hong, Zhen-Yi; Yu, Shuang-Shuang; Wang, Zhi-Jun; Zhu, Yi-Zhun

2015-08-07

SCM-198 is an alkaloid found only in Herba leonuri and it has been reported to possess considerable neuroprotective effects in animal models of ischemic stroke, Parkinson's disease and Alzheimer's disease (AD). In this study, we demonstrated for the first time that 3-month oral SCM-198 treatment could significantly improve both recognition and spatial memory, inhibit microgliosis and promote neuronal survival in amyloid-β protein precursor and presenilin-1(AβPP/PS1) double-transgenic mice without affecting amyloid-β (Aβ) burden. In addition, decreases in cAMP-response element-binding protein (CREB) phosphorylation, brain-derived neurotrophic factor (BDNF) and tropomyosin-related kinase B (TrkB) phosphorylation were attenuated by SCM-198 both in vivo and in primary cortical neurons, which could be blocked by protein kinase A (PKA) inhibitors, suggesting the involvement of upstream PKA in enhancing the BDNF/TrkB/CREB signaling by SCM-198. Our results indicate that SCM-198, a drug that could promote neuronal survival and enhance BDNF/TrkB/CREB signaling, has beneficial effects on behavioral and biochemical alterations without affecting Aβ burden in AβPP/PS1 mice and might become a potential drug candidate for AD treatment in the future.

CREB1 is a strong genetic predictor of the variation in exercise heart rate response to regular exercise: the HERITAGE Family Study.

Science.gov (United States)

Rankinen, Tuomo; Argyropoulos, George; Rice, Treva; Rao, D C; Bouchard, Claude

2010-06-01

A genome-wide linkage scan identified a quantitative trait locus for exercise training-induced changes in submaximal exercise (50 W) heart rate (DeltaHR50) on chromosome 2q33.3-q34 in the HERITAGE Family Study (n=472). To fine-map the region, 1450 tag SNPs were genotyped between 205 and 215 Mb on chromosome 2. The strongest evidence of association with DeltaHR50 was observed with 2 single-nucleotide polymorphisms (SNPs) located in the 5' region of the cAMP-responsive element-binding protein 1 (CREB1) gene (rs2253206: P=1.6x10(-5) and rs2360969: P=4.3x10(-5)). The associations remained significant (P=0.01 and P=0.023, respectively) after accounting for multiple testing. Regression modeling of the 39 most significant SNPs in the single-SNP analysis identified 9 SNPs that collectively explained 20% of the DeltaHR50 variance. CREB1 SNP rs2253206 had the strongest effect (5.45% of variance), followed by SNPs in the FASTKD2 (3.1%), MAP2 (2.6%), SPAG16 (2.1%), ERBB4 (3 SNPs approximately 1.4% each), IKZF2 (1.4%), and PARD3B (1.0%) loci. In conditional linkage analysis, 6 SNPs from the final regression model (CREB1, FASTKD2, MAP2, ERBB4, IKZF2, and PARD3B) accounted for the original linkage signal: The log of the odds score dropped from 2.10 to 0.41 after adjusting for all 6 SNPs. Functional studies revealed that the common allele of rs2253206 exhibits significantly (P<0.05) lower promoter activity than the minor allele. Our data suggest that functional DNA sequence variation in the CREB1 locus is strongly associated with DeltaHR50 and explains a considerable proportion of the quantitative trait locus variance. However, at least 5 additional SNPs seem to be required to fully account for the original linkage signal.

Spirulina maxima Extract Prevents Neurotoxicity via Promoting Activation of BDNF/CREB Signaling Pathways in Neuronal Cells and Mice.

Science.gov (United States)

Koh, Eun-Jeong; Seo, Young-Jin; Choi, Jia; Lee, Hyeon Yong; Kang, Do-Hyung; Kim, Kui-Jin; Lee, Boo-Yong

2017-08-17

Spirulina maxima is a microalgae which contains flavonoids and other polyphenols. Although Spirulina maxima 70% ethanol extract (SM70EE) has diverse beneficial effects, its effects on neurotoxicity have not been fully understood. In this study, we investigated the neuroprotective effects of SM70EE against trimethyltin (TMT)-induced neurotoxicity in HT-22 cells. SM70EE inhibited the cleavage of poly-ADP ribose polymerase (PARP). Besides, ROS production was decreased by down-regulating oxidative stress-associated enzymes. SM70EE increased the factors of brain-derived neurotrophic factor (BDNF)/cyclic AMPresponsive elementbinding protein (CREB) signalling pathways. Additionally, acetylcholinesterase (AChE) was suppressed by SM70EE. Furthermore, we investigated whether SM70EE prevents cognitive deficits against scopolamine-induced neurotoxicity in mice by applying behavioral tests. SM70EE increased step-through latency time and decreased the escape latency time. Therefore, our data suggest that SM70EE may prevent TMT neurotoxicity through promoting activation of BDNF/CREB neuroprotective signaling pathways in neuronal cells. In vivo study, SM70EE would prevent cognitive deficits against scopolamine-induced neurotoxicity in mice.

Notch1 deficiency in postnatal neural progenitor cells in the dentate gyrus leads to emotional and cognitive impairment.

Science.gov (United States)

Feng, Shufang; Shi, Tianyao; Qiu, Jiangxia; Yang, Haihong; Wu, Yan; Zhou, Wenxia; Wang, Wei; Wu, Haitao

2017-10-01

It is well known that Notch1 signaling plays a crucial role in embryonic neural development and adult neurogenesis. The latest evidence shows that Notch1 also plays a critical role in synaptic plasticity in mature hippocampal neurons. So far, deeper insights into the function of Notch1 signaling during the different steps of adult neurogenesis are still lacking, and the mechanisms by which Notch1 dysfunction is associated with brain disorders are also poorly understood. In the current study, we found that Notch1 was highly expressed in the adult-born immature neurons in the hippocampal dentate gyrus. Using a genetic approach to selectively ablate Notch1 signaling in late immature precursors in the postnatal hippocampus by cross-breeding doublecortin (DCX) + neuron-specific proopiomelanocortin (POMC)-α Cre mice with floxed Notch1 mice, we demonstrated a previously unreported pivotal role of Notch1 signaling in survival and function of adult newborn neurons in the dentate gyrus. Moreover, behavioral and functional studies demonstrated that POMC-Notch1 -/- mutant mice showed anxiety and depressive-like behavior with impaired synaptic transmission properties in the dentate gyrus. Finally, our mechanistic study showed significantly compromised phosphorylation of cAMP response element-binding protein (CREB) in Notch1 mutants, suggesting that the dysfunction of Notch1 mutants is associated with the disrupted pCREB signaling in postnatally generated immature neurons in the dentate gyrus.-Feng, S., Shi, T., Qiu, J., Yang, H., Wu, Y., Zhou, W., Wang, W., Wu, H. Notch1 deficiency in postnatal neural progenitor cells in the dentate gyrus leads to emotional and cognitive impairment. © FASEB.

Nanoparticle for delivery of antisense γPNA oligomers targeting CCR5

OpenAIRE

Bahal, Raman; McNeer, Nicole Ali; Ly, Danith H.; Saltzman, W. Mark; Glazer, Peter M.

2013-01-01

The development of a new class of peptide nucleic acids (PNAs), i.e., gamma PNAs (γPNAs), creates the need for a general and effective method for its delivery into cells for regulating gene expression in mammalian cells. Here we report the antisense activity of a recently developed hydrophilic and biocompatible diethylene glycol (miniPEG)-based gamma peptide nucleic acid called MPγPNAs via its delivery by poly(lactide-co-glycolide) (PLGA)-based nanoparticle system. We show that MPγPNA oligome...

Diversity of antisense and other non-coding RNAs in Archaea revealed by comparative small RNA sequencing in four Pyrobaculum species

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David L Bernick

2012-07-01

Full Text Available A great diversity of small, non-coding RNA molecules with roles in gene regulation and RNA processing have been intensely studied in eukaryotic and bacterial model organisms, yet our knowledge of possible parallel roles for small RNAs in archaea is limited. We employed RNA-seq to identify novel small RNA across multiple species of the hyperthermophilic genus Pyrobaculum, known for unusual RNA gene characteristics. By comparing transcriptional data collected in parallel among four species, we were able to identify conserved RNA genes fitting into known and novel families. Among our findings, we highlight three novel cis-antisense small RNAs encoded opposite to key regulatory (ferric uptake regulator, metabolic (triose-phosphate isomerase, and core transcriptional apparatus genes (transcription factor B. We also found a large increase in the number of conserved C/D box small RNA genes over what had been previously recognized; many of these genes are encoded antisense to protein coding genes. The conserved opposition to orthologous genes across the Pyrobaculum genus suggests similarities to other cis-antisense regulatory systems. Furthermore, the genus-specific nature of these small RNAs indicates they are relatively recent, stable adaptations.

Caffeine suppresses exercise-enhanced long-term and location memory in middle-aged rats: Involvement of hippocampal Akt and CREB signaling.

Science.gov (United States)

Cechella, José L; Leite, Marlon R; da Rocha, Juliana T; Dobrachinski, Fernando; Gai, Bibiana M; Soares, Félix A A; Bresciani, Guilherme; Royes, Luiz F F; Zeni, Gilson

2014-11-05

The cognitive function decline is closely related with brain changes generated by age. The ability of caffeine and exercise to prevent memory impairment has been reported in animal models and humans. The purpose of the present study was to investigate whether swimming exercise and caffeine administration enhance memory in middle-aged Wistar rats. Male Wistar rats (18months) received caffeine at a dose of 30mg/kg, 5days per week by a period of 4weeks. Animals were subjected to swimming training with a workload (3% of body weight, 20min per day for 4weeks). After 4weeks, the object recognition test (ORT) and the object location test (OLT) were performed. The results of this study demonstrated that caffeine suppressed exercise-enhanced long-term (ORT) and spatial (OLT) memory in middle-aged and this effect may be related to a decrease in hippocampal p-CREB signaling. This study also provided evidence that the effects of this protocol on memory were not accompanied by alterations in the levels of activated Akt. The [(3)H] glutamate uptake was reduced in hippocampus of rats administered with caffeine and submitted to swimming protocol. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Modulation of opiate-related signaling molecules in morphine-dependent conditioned behavior: conditioned place preference to morphine induces CREB phosphorylation.

Science.gov (United States)

Morón, José A; Gullapalli, Srinivas; Taylor, Chirisse; Gupta, Achla; Gomes, Ivone; Devi, Lakshmi A

2010-03-01

Opiate addiction is a chronic, relapsing behavioral disorder where learned associations that develop between the abused opiate and the environment in which it is consumed are brought about through Pavlovian (classical) conditioning processes. However, the signaling mechanisms/pathways regulating the mechanisms that underlie the responses to opiate-associated cues or the development of sensitization as a consequence of repeated context-independent administration of opiates are unknown. In this study we examined the phosphorylation levels of various classic signaling molecules in brain regions implicated in addictive behaviors after acute and repeated morphine administration. An unbiased place conditioning protocol was used to examine changes in phosphorylation that are associated with (1) the expression of the rewarding effects of morphine and (2) the sensitization that develops to this effect. We also examined the effects of a delta-receptor antagonist on morphine-induced conditioned behavior and on the phosphorylation of classic signaling molecules in view of data showing that blockade of delta-opioid receptor (deltaOR) prevents the development of sensitization to the rewarding effects of morphine. We find that CREB phosphorylation is specifically induced upon the expression of a sensitized response to morphine-induced conditioned behavior in brain areas related to memory consolidation, such as the hippocampus and cortex. A similar effect is also observed, albeit to a lesser extent, in the case of the GluR1 subunit of AMPA glutamate receptor. These increases in the phosphorylation levels of CREB and pGluR1 are significantly blocked by pretreatment with a deltaOR antagonist. These results indicate a critical role for phospho-CREB, AMPA, and deltaOR activities in mediating the expression of a sensitized response to morphine-dependent conditioned behavior.

Rapid blockade of telomerase activity and tumor cell growth by the DPL lipofection of ribbon antisense to hTR.

Science.gov (United States)

Bajpai, Arun K; Park, Jeong-Hoh; Moon, Ik-Jae; Kang, Hyungu; Lee, Yun-Han; Doh, Kyung-Oh; Suh, Seong-Il; Chang, Byeong-Churl; Park, Jong-Gu

2005-09-29

Ribbon antisense (RiAS) to the hTR RNA, a component of the telomerase complex, was employed to inhibit telomerase activity and cancer cell growth. The antisense molecule, hTR-RiAS, combined with enhanced cellular uptake was shown to effectively inhibit telomerase activity and cause rapid cell death in various cancer cell lines. When cancer cells were treated with hTR-RiAS, the level of hTR RNA was reduced by more than 90% accompanied with reduction in telomerase activity. When checked for cancer cell viability, cancer cell lines treated with hTR-RiAS using DNA+Peptide+Lipid complex showed 70-80% growth inhibition in 3 days. The reduced cell viability was due to apoptosis as the percentage of cells exhibiting the sub-G0 arrest and DNA fragmentation increased after antisense treatment. Further, when subcutaneous tumors of a colon cancer cell line (SW480) were treated intratumorally with hTR-RiAS, tumor growth was markedly suppressed with almost total ablation of hTR RNA in the tumor tissue. Cells in the tumor tissue were also found to undergo apoptosis after hTR-RiAS treatment. These results suggest that hTR-RiAS is an effective anticancer reagent, with a potential for broad efficacy to diverse malignant tumors.

SCM-198 Ameliorates Cognitive Deficits, Promotes Neuronal Survival and Enhances CREB/BDNF/TrkB Signaling without Affecting Aβ Burden in AβPP/PS1 Mice

Directory of Open Access Journals (Sweden)

Zhen-Yi Hong

2015-08-01

Full Text Available SCM-198 is an alkaloid found only in Herba leonuri and it has been reported to possess considerable neuroprotective effects in animal models of ischemic stroke, Parkinson’s disease and Alzheimer’s disease (AD. In this study, we demonstrated for the first time that 3-month oral SCM-198 treatment could significantly improve both recognition and spatial memory, inhibit microgliosis and promote neuronal survival in amyloid-β protein precursor and presenilin-1(AβPP/PS1 double-transgenic mice without affecting amyloid-β (Aβ burden. In addition, decreases in cAMP-response element-binding protein (CREB phosphorylation, brain-derived neurotrophic factor (BDNF and tropomyosin-related kinase B (TrkB phosphorylation were attenuated by SCM-198 both in vivo and in primary cortical neurons, which could be blocked by protein kinase A (PKA inhibitors, suggesting the involvement of upstream PKA in enhancing the BDNF/TrkB/CREB signaling by SCM-198. Our results indicate that SCM-198, a drug that could promote neuronal survival and enhance BDNF/TrkB/CREB signaling, has beneficial effects on behavioral and biochemical alterations without affecting Aβ burden in AβPP/PS1 mice and might become a potential drug candidate for AD treatment in the future.

Influence of different chelators (HYNIC, MAG3 and DTPA) on tumor cell accumulation and mouse biodistribution of technetium-99m labeled to antisense DNA

International Nuclear Information System (INIS)

Zhang, Y.M.; Liu, N.; Zhu, Z.-H.; Rusckowski, M.; Hnatowich, D.J.

2000-01-01

We have shown recently that cell accumulation in culture of antisense DNA is strongly influenced by the presence of a 99m Tc-MAG 3 group for radiolabeling. We have now compared the in vitro and mouse in vivo behavior of 99m Tc when radiolabeled to one antisense phosphorothioate DNA by three different methods. The 18-mer antisense DNA against the RIα subunit of PKA was conjugated via a primary amine on the 5'-end with the NHS esters of HYNIC and MAG 3 and by the cyclic anhydride of DTPA. Surface plasmon resonance measurements revealed that the association rate constant for hybridization was unchanged for all three chelators as compared with that of the native DNA. Size exclusion HPLC showed rapid and quantitative protein binding for all three chelators upon incubation of labeled DNAs in 37 C serum and cell culture medium. However, in each case, radiolabeled and intact oligonucleotide was still detectable after 24 h. Cellular uptake was tested in an RIα mRNA-positive cancer cell line. The order of cellular accumulation of 99m Tc was DTPA>HYNIC(tricine)>MAG 3 , with the differences increasing with time between 4 and 24 h. The rate of 99m Tc egress from cells was found to be MAG 3 >HYNIC>DTPA, which may explain the order of cellular accumulation. The biodistribution in normal mice was heavily influenced by the labeling method and followed a pattern similar to that seen previously by us for peptides labeled with the same chelators. In conclusion, although these studies concerned only one antisense DNA in one cell line, the results suggest that the success of antisense imaging may depend, in part, on the method of radiolabeling. (orig.)

Study on biodistribution and imaging of radioiodinated antisense oligonucleotides in nude mice bearing human lymphoma

International Nuclear Information System (INIS)

Wang, R.F.; Shen, J.; Zhang, C.L.; Liu, M.; Guo, F.Q.

2005-01-01

The incidence of sporadic lymphoma has risen due to an increase in immunosuppressed patients, particularly those with human immunodeficiency virus (HIV) infection. Sometimes suspect lymphoma has an undetectable location and we can not get the pathological specimen. Management of lymphoma is also difficult because the persistence of a significant number of residual tumor cells after intensive treatment. These relative failures can be attributed to make us choose this study for opening a new diagnostic and therapeutic field of lymphoma from molecular level. Immunoglobulin (Ig) heavy chain framework region (FR) of V1 family have been verified to be a major determinant of malignant phenotype of V1 family B-cell lymphoma. Most of targets for tumor antisense therapy study are protooncogenes, such as c-myc, bc1-2, which are broad -spectrum tumor imaging agents. The aim of this study was to investigate the possibility of using radioiodine labeled FR antisense oligonucleotides (ASONs) as an imaging agent or antisense therapeutic radiopharmaceutical in lymphoma. A 18-mer partial phosphorothioate oligonucleotide sequence was synthesized and grafted in 5 ' with a tyramine group which was further labeled with 125 I or 131 I using the chloramine T method. Normal CD-1 mice were injected via a tail vein with 148 kBq of 125 I-FR-ASON (2∼3 μ g). Animals were sacrificed at 1, 2, 4 and 24 h and tissue samples were studied. Liposome-mediated 3.33 MBq of 131 I-FR-ASON (7 ∼ 9μ g) was injected intratumorally into tumor-bearing BALB/c mice (6 weeks after inoculation of 10 7 Namalwa cells) meanwhile liposome-mediated 131 I labeled sense oligonucleotides served as controls. Biodistribution was monitored by sequential scintigraphy and organ radioactivity measurement 24 h after injection. The percentage of the injected dose per gram (%ID/g) of tumor and tumor/ non-tumor tissue ratios (T/NT) were calculated for each group of mice and the difference between two groups was assessed. The 5

Association of MMP7 -181A→G Promoter Polymorphism with Gastric Cancer Risk: INFLUENCE OF NICOTINE IN DIFFERENTIAL ALLELE-SPECIFIC TRANSCRIPTION VIA INCREASED PHOSPHORYLATION OF cAMP-RESPONSE ELEMENT-BINDING PROTEIN (CREB).

Science.gov (United States)

Kesh, Kousik; Subramanian, Lakshmi; Ghosh, Nillu; Gupta, Vinayak; Gupta, Arnab; Bhattacharya, Samir; Mahapatra, Nitish R; Swarnakar, Snehasikta

2015-06-05

Elevated expression of matrix metalloproteinase7 (MMP7) has been demonstrated to play a pivotal role in cancer invasion. The -181A→G (rs11568818) polymorphism in the MMP7 promoter modulates gene expression and possibly affects cancer progression. Here, we evaluated the impact of -181A→G polymorphism on MMP7 promoter activity and its association with gastric cancer risk in eastern Indian case-control cohorts (n = 520). The GG genotype as compared with the AA genotype was predisposed (p = 0.02; odds ratio = 1.9, 95% confidence interval = 1.1-3.3) to gastric cancer risk. Stratification analysis showed that tobacco addiction enhanced gastric cancer risk in GG subjects when compared with AA subjects (p = 0.03, odds ratio = 2.46, and 95% confidence interval = 1.07-5.68). Meta-analysis revealed that tobacco enhanced the risk for cancer more markedly in AG and GG carriers. Activity and expression of MMP7 were significantly higher in GG than in AA carriers. In support, MMP7 promoter-reporter assays showed greater transcriptional activity toward A to G transition under basal/nicotine-induced/cAMP-response element-binding protein (CREB) overexpressed conditions in gastric adenocarcinoma cells. Moreover, nicotine (a major component of tobacco) treatment significantly up-regulated MMP7 expression due to enhanced CREB phosphorylation followed by its nuclear translocation in gastric adenocarcinoma cells. Furthermore, chromatin immunoprecipitation experiments revealed higher binding of phosphorylated CREB with the -181G than the -181A allele. Altogether, specific binding of phosphorylated CREB to the G allele-carrying promoter enhances MMP7 gene expression that is further augmented by nicotine due to increased CREB phosphorylation and thereby increases the risk for gastric cancer. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Maternal Exposure of Rats to Isoflurane during Late Pregnancy Impairs Spatial Learning and Memory in the Offspring by Up-Regulating the Expression of Histone Deacetylase 2.

Science.gov (United States)

Luo, Foquan; Hu, Yan; Zhao, Weilu; Zuo, Zhiyi; Yu, Qi; Liu, Zhiyi; Lin, Jiamei; Feng, Yunlin; Li, Binda; Wu, Liuqin; Xu, Lin

2016-01-01

Increasing evidence indicates that most general anesthetics can harm developing neurons and induce cognitive dysfunction in a dose- and time-dependent manner. Histone deacetylase 2 (HDAC2) has been implicated in synaptic plasticity and learning and memory. Our previous results showed that maternal exposure to general anesthetics during late pregnancy impaired the offspring's learning and memory, but the role of HDAC2 in it is not known yet. In the present study, pregnant rats were exposed to 1.5% isoflurane in 100% oxygen for 2, 4 or 8 hours or to 100% oxygen only for 8 hours on gestation day 18 (E18). The offspring born to each rat were randomly subdivided into 2 subgroups. Thirty days after birth, the Morris water maze (MWM) was used to assess learning and memory in the offspring. Two hours before each MWM trial, an HDAC inhibitor (SAHA) was given to the offspring in one subgroup, whereas a control solvent was given to those in the other subgroup. The results showed that maternal exposure to isoflurane impaired learning and memory of the offspring, impaired the structure of the hippocampus, increased HDAC2 mRNA and downregulated cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) mRNA, N-methyl-D-aspartate receptor 2 subunit B (NR2B) mRNA and NR2B protein in the hippocampus. These changes were proportional to the duration of the maternal exposure to isoflurane and were reversed by SAHA. These results suggest that exposure to isoflurane during late pregnancy can damage the learning and memory of the offspring rats via the HDAC2-CREB -NR2B pathway. This effect can be reversed by HDAC2 inhibition.

Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo

DEFF Research Database (Denmark)

Nudelman, Aaron Samuel; DiRocco, Derek P; Lambert, Talley J

2010-01-01

Activity-dependent changes in gene-expression are believed to underlie the molecular representation of memory. In this study, we report that in vivo activation of neurons rapidly induces the CREB-regulated microRNA miR-132. To determine if production of miR-132 is regulated by neuronal activity its......, olfactory bulb, and striatum by contextual fear conditioning, odor-exposure, and cocaine-injection, respectively, also increased pri-miR-132. Induction kinetics of pri-miR-132 were monitored and found to parallel those of immediate early genes, peaking at 45 min and returning to basal levels within 2 h...

Tongqiao Huoxue Decoction ameliorates learning and memory defects in rats with vascular dementia by up-regulating the Ca(2+)-CaMKII-CREB pathway.

Science.gov (United States)

Ge, Chao-Liang; Wang, Xin-Ming; Huang, Zhao-Gang; Xia, Quan; Wang, Ning; Xu, Du-Juan

2015-11-01

The present study was aimed at determining the effects of Tongqiao Huoxue Decoction (TQHXD) on the Ca(2+)-CaMKII-CREB pathway and the memory and learning capacities of rats with vascular dementia (VD). The rat VD model was established by using an improved bilateral carotid artery ligation method. The Morris water maze experiment was used to evaluate the ethology of the VD rats following treatments with TQHXD at 3.01, 6.02, and 12.04 g·kg(-1) per day for 31 days. At the end of experiment, the hippocampus were harvested and analyzed. Western blotting and RT-PCR were used to measure the expression levels of calmodulin-binding protein kinase II(CaMKII), protein kinase A(PKA), cAMP-response element binding protein(CREB), and three N-methyl-D-aspartic acid receptor subunits (NR1, NR2A, and NR2B). Our results revealed that TQHXD could alleviate the loss of learning abilities and increase the memory capacity (P < 0.05 and P < 0.01 vs the model group, respectively). The treatment with 6.02 and 12.04 g·kg(-1) of TQHXD significantly up-regulated the Ca(2+)-CaMKII-CREB pathway in the hippocampus. In conclusion, TQHXD showed therapeutic effects on a bilateral carotid artery ligation-induced vascular dementia model, through the up-regulation of calcium signalling pathways. Copyright © 2015 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles

Energy Technology Data Exchange (ETDEWEB)

Murphy, Jane; Hall, William W. [Centre for Research in Infectious Diseases, School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4 (Ireland); Ratner, Lee [Department of Medicine, Division of Molecular Oncology, Washington University School of Medicine, Saint Louis, Missouri, United States of America (United States); Sheehy, Noreen [Centre for Research in Infectious Diseases, School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4 (Ireland)

2016-07-15

The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we found that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells. - Highlights: • This study demonstrates for the first time interactions between NF90/110 and the HTLV antisense proteins HBZ and APH-2. • We show that NF90/110 significantly enhance LTR activation by the HTLV Tax protein, an effect that is abolished by HBZ but enhanced by APH-2. • The study shows that even though the HTLV antisense proteins activate survivin expression they antagonize the ability of NF90/110 to do so. • Overall we found that NF90/110 positively regulate HTLV infection and as such might represent a therapeutic target in infected cells.

Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles

International Nuclear Information System (INIS)

Murphy, Jane; Hall, William W.; Ratner, Lee; Sheehy, Noreen

2016-01-01

The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we found that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells. - Highlights: • This study demonstrates for the first time interactions between NF90/110 and the HTLV antisense proteins HBZ and APH-2. • We show that NF90/110 significantly enhance LTR activation by the HTLV Tax protein, an effect that is abolished by HBZ but enhanced by APH-2. • The study shows that even though the HTLV antisense proteins activate survivin expression they antagonize the ability of NF90/110 to do so. • Overall we found that NF90/110 positively regulate HTLV infection and as such might represent a therapeutic target in infected cells.

A Jacob/Nsmf Gene Knockout Results in Hippocampal Dysplasia and Impaired BDNF Signaling in Dendritogenesis.

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Christina Spilker

2016-03-01

Full Text Available Jacob, the protein encoded by the Nsmf gene, is involved in synapto-nuclear signaling and docks an N-Methyl-D-Aspartate receptor (NMDAR-derived signalosome to nuclear target sites like the transcription factor cAMP-response-element-binding protein (CREB. Several reports indicate that mutations in NSMF are related to Kallmann syndrome (KS, a neurodevelopmental disorder characterized by idiopathic hypogonadotropic hypogonadism (IHH associated with anosmia or hyposmia. It has also been reported that a protein knockdown results in migration deficits of Gonadotropin-releasing hormone (GnRH positive neurons from the olfactory bulb to the hypothalamus during early neuronal development. Here we show that mice that are constitutively deficient for the Nsmf gene do not present phenotypic characteristics related to KS. Instead, these mice exhibit hippocampal dysplasia with a reduced number of synapses and simplification of dendrites, reduced hippocampal long-term potentiation (LTP at CA1 synapses and deficits in hippocampus-dependent learning. Brain-derived neurotrophic factor (BDNF activation of CREB-activated gene expression plays a documented role in hippocampal CA1 synapse and dendrite formation. We found that BDNF induces the nuclear translocation of Jacob in an NMDAR-dependent manner in early development, which results in increased phosphorylation of CREB and enhanced CREB-dependent Bdnf gene transcription. Nsmf knockout (ko mice show reduced hippocampal Bdnf mRNA and protein levels as well as reduced pCREB levels during dendritogenesis. Moreover, BDNF application can rescue the morphological deficits in hippocampal pyramidal neurons devoid of Jacob. Taken together, the data suggest that the absence of Jacob in early development interrupts a positive feedback loop between BDNF signaling, subsequent nuclear import of Jacob, activation of CREB and enhanced Bdnf gene transcription, ultimately leading to hippocampal dysplasia.

The Dynamics of Compound, Transcript, and Protein Effects After Treatment With 2OMePS Antisense Oligonucleotides in mdx Mice

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Ingrid E C Verhaart

2014-01-01

Full Text Available Antisense-mediated exon skipping is currently in clinical development for Duchenne muscular dystrophy (DMD to amend the consequences of the underlying genetic defect and restore dystrophin expression. Due to turnover of compound, transcript, and protein, chronic treatment with effector molecules (antisense oligonucleotides will be required. To investigate the dynamics and persistence of antisense 2′-O-methyl phosphorothioate oligonucleotides, exon skipping, and dystrophin expression after dosing was concluded, mdx mice were treated subcutaneously for 8 weeks with 100 mg/kg oligonucleotides twice weekly. Thereafter, mice were sacrificed at different time points after the final injection (36 hours–24 weeks. Oligonucleotide half-life was longer in heart (~65 days compared with that in skeletal muscle, liver, and kidney (~35 days. Exon skipping half-lives varied between 33 and 53 days, whereas dystrophin protein showed a long half-life (>100 days. Oligonucleotide and exon-skipping levels peaked in the first week and declined thereafter. By contrast, dystrophin expression peaked after 3–8 weeks and then slowly declined, remaining detectable after 24 weeks. Concordance between levels of oligonucleotides, exon skipping, and proteins was observed, except in heart, wherein high oligonucleotide levels but low exon skipping and dystrophin expression were seen. Overall, these results enhance our understanding of the pharmacokinetics and pharmacodynamics of 2′-O-methyl phosphorothioate oligos used for the treatment of DMD.

The Effect of Hippocampal Cognitive Impairment and XIAP on Glucose and Lipids Metabolism in Rats

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Chunbo Xia

2016-02-01

Full Text Available Background/Aims: To investigate the effect of cognitive impairment and X-linked inhibitor of apoptosis protein (XIAP on glucolipid metabolism. Materials and Methods: β-amyloid (Aβ 1-42 was injected into the hippocampus of rats to establish a cognitive impairment model. Trans-activator of transcription (TAT-XIAP fusion protein (the TAT-XIAP group, PBS (the model group, or XIAP antisense oligonucleotides (the ASODN group was injected into the lateral ventricles of the rats to increase and decrease the activity of XIAP in the hippocampus. To determine the level of blood glucose and lipids, adenosine monophosphate-activated protein kinase (AMPK expression of liver and hipppocamual neuronal apoptosis. Results: The levels of FPG, TG, TC and LDL were significantly higher in the TAT-XIAP group, the model group and the ASODN group than in the blank group (P Conclusion: Cognitive impairment and hippocampal neuron apoptosis can cause glucose and lipids metabolic abnormalities, possibly by regulating gastrointestinal motility and AMPK expression in the liver. The changes in the function of XIAP, which is an anti-apoptotic protein in the hippocampus, may affect the metabolism of glucose and lipids.

The roles of BDNF, pCREB and Wnt3a in the latent period preceding activation of progenitor cell mitosis in the adult dentate gyrus by fluoxetine.

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Scarlett B Pinnock

2010-10-01

Full Text Available The formation of new neurons continues into adult life in the dentate gyrus of the rat hippocampus, as in many other species. Neurogenesis itself turns out to be highly labile, and is regulated by a number of factors. One of these is the serotoninergic system: treatment with drugs (such as the SSRI fluoxetine markedly stimulates mitosis in the progenitor cells of the dentate gyrus. But this process has one remarkable feature: it takes at least 14 days of continuous treatment to be effective. This is despite the fact that the pharmacological action of fluoxetine occurs within an hour or so of first administration. This paper explores the role of BDNF in this process, using the effect of a Trk antagonist (K252a on the labelling of progenitor cells with the mitosis marker Ki67 and the associated expression of pCREB and Wnt3a. These experiments show that (i Fluoxetine increased Ki67 counts, as well as pCREB and Wnt3a expression in the dentate gyrus. The action of fluoxetine on the progenitor cells and on pCREB (but not Wnt3a depends upon Trk receptor activation, since it was prevented by icv infusion of K252a. (ii These receptors are required for both the first 7 days of fluoxetine action, during which no apparent change in progenitor mitosis occurs, as well as the second 7 days. Increased pCREB was always associated with progenitor cell mitosis, but Wnt3a expression may be necessary but not sufficient for increased progenitor cell proliferation. These results shed new light on the action of fluoxetine on neurogenesis in the adult dentate gyrus, and have both clinical and experimental interest.

NMDA receptor adjusted co-administration of ecstasy and cannabinoid receptor-1 agonist in the amygdala via stimulation of BDNF/Trk-B/CREB pathway in adult male rats.

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Ashabi, Ghorbangol; Sadat-Shirazi, Mitra-Sadat; Khalifeh, Solmaz; Elhampour, Laleh; Zarrindast, Mohammad-Reza

2017-04-01

Consumption of cannabinoid receptor-1 (CB-1) agonist such as cannabis is widely taken in 3,4- methylenedioxymethamphetamine (MDMA) or ecstasy users; it has been hypothesized that co-consumption of CB-1 agonist might protect neurons against MDMA toxicity. N-methyl-d-aspartate (NMDA) receptors regulate neuronal plasticity and firing rate in the brain through Tyrosine-kinase B (Trk-B) activation. The molecular and electrophysiological association among NMDA and MDMA/Arachidonylcyclopropylamide (ACPA, a selective CB-1 receptor agonist) co-consumption was not well-known. Here, neuronal spontaneous activity, Brain-derived neurotrophic factor (BDNF), Trk-B and cAMP response element binding protein (CREB) phosphorylation levels were recognized in ACPA and MDMA co-injected rats. Besides, we proved the role of NMDA receptor on MDMA and ACPA combination on neuronal spontaneous activity and Trk-B/BDNF pathway in the central amygdala (CeA). Male rats were anesthetized with intra-peritoneal injections of urethane; MDMA, D-2-amino-5-phosphonopentanoate (D-AP5, NMDA receptor antagonist) were injected into CeA. ACPA was administrated by intra-cerebroventricular injection. Thirty minutes following injections, neuronal firing rate was recorded from CeA. Two hours after drug injection, amygdala was collected from brain for molecular evaluations. Single administration of MDMA and/or ACPA reduced firing rates compared with sham group in the CeA dose-dependently. Injection of D-AP5, ACPA and MDMA reduced firing rate compared with sham group (P<0.001). Interestingly, injection of ACPA+MDMA enhanced BDNF, Trk-B and CREB phosphorylation compared with MDMA groups. D-AP5, ACPA and MDMA co-injection decreased BDNF, Trk-B and CREB phosphorylation levels compared with ACPA+MDMA in the amygdala (P<0.01). Probably, NMDA receptors are involved in the protective role of acute MDMA+ACPA co-injection via BDNF/Trk-B/CREB pathways. Copyright © 2017 Elsevier Inc. All rights reserved.

Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy

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van Ommen Gert-Jan B

2007-07-01

Full Text Available Abstract Background Antisense-mediated exon skipping is currently one of the most promising therapeutic approaches for Duchenne muscular dystrophy (DMD. Using antisense oligonucleotides (AONs targeting specific exons the DMD reading frame is restored and partially functional dystrophins are produced. Following proof of concept in cultured muscle cells from patients with various deletions and point mutations, we now focus on single and multiple exon duplications. These mutations are in principle ideal targets for this approach since the specific skipping of duplicated exons would generate original, full-length transcripts. Methods Cultured muscle cells from DMD patients carrying duplications were transfected with AONs targeting the duplicated exons, and the dystrophin RNA and protein were analyzed. Results For two brothers with an exon 44 duplication, skipping was, even at suboptimal transfection conditions, so efficient that both exons 44 were skipped, thus generating, once more, an out-of-frame transcript. In such cases, one may resort to multi-exon skipping to restore the reading frame, as is shown here by inducing skipping of exon 43 and both exons 44. By contrast, in cells from a patient with an exon 45 duplication we were able to induce single exon 45 skipping, which allowed restoration of wild type dystrophin. The correction of a larger duplication (involving exons 52 to 62, by combinations of AONs targeting the outer exons, appeared problematic due to inefficient skipping and mistargeting of original instead of duplicated exons. Conclusion The correction of DMD duplications by exon skipping depends on the specific exons targeted. Its options vary from the ideal one, restoring for the first time the true, wild type dystrophin, to requiring more 'classical' skipping strategies, while the correction of multi-exon deletions may need the design of tailored approaches.

In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts

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Ling, King-Hwa; Brautigan, Peter J.; Moore, Sarah; Fraser, Rachel; Leong, Melody Pui-Yee; Leong, Jia-Wen; Zainal Abidin, Shahidee; Lee, Han-Chung; Cheah, Pike-See; Raison, Joy M.; Babic, Milena; Lee, Young Kyung; Daish, Tasman; Mattiske, Deidre M.; Mann, Jeffrey R.; Adelson, David L.; Thomas, Paul Q.; Hahn, Christopher N.; Scott, Hamish S.

2016-01-01

SRY (Sex Determining Region Y)-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1], [2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1. PMID:26958646

In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts

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King-Hwa Ling

2016-06-01

Full Text Available SRY (Sex Determining Region Y-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1,2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH, Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR, gain-of-function and in situ hybridization (ISH experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1.

Retroviral gene transfer of an antisense construct against membrane type 1 matrix metalloproteinase reduces the invasiveness of rheumatoid arthritis synovial fibroblasts.

Science.gov (United States)

Rutkauskaite, Edita; Volkmer, Dagmar; Shigeyama, Yukio; Schedel, Jörg; Pap, Geza; Müller-Ladner, Ulf; Meinecke, Ingmar; Alexander, Dorothea; Gay, Renate E; Drynda, Susanne; Neumann, Wolfram; Michel, Beat A; Aicher, Wilhelm K; Gay, Steffen; Pap, Thomas

2005-07-01

Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed prominently in rheumatoid arthritis synovial fibroblasts (RASFs), but the specific contribution of MT1-MMP to fibroblast-mediated destruction of articular cartilage is incompletely understood. This study used gene transfer of an antisense expression construct to assess the effects of MT1-MMP inhibition on the invasiveness of RASFs. Retroviral gene transfer of a pLXIN vector-based antisense RNA expression construct (MT1-MMPalphaS) to MT1-MMP was used to stably transduce RASFs. Levels of MT1-MMP RNA and protein were determined by quantitative polymerase chain reaction, Western blotting, and immunocytochemistry in MT1-MMPalphaS-transduced RASFs as well as in control cells, with monitoring for 60 days. The effects of MT1-MMPalphaS on the invasiveness of RASFs were analyzed in the SCID mouse co-implantation model of RA. MT1-MMPalphaS-transduced RASFs produced high levels of antisense RNA that exceeded endogenous levels of MT1-MMP messenger RNA by 15-fold and resulted in a down-regulation of MT1-MMP at the protein level. Inhibition of MT1-MMP production was maintained for 60 days and significantly reduced the invasiveness of RASFs in the SCID mouse model. Whereas prominent invasion into cartilage by non-transduced and mock-transduced RASFs was observed (mean invasion scores 3.0 and 3.1, respectively), MT1-MMPalphaS-transduced cells showed only moderate invasiveness (mean invasion score 1.8; P < 0.05). The data demonstrate that an antisense RNA expression construct against MT1-MMP can be generated and expressed in RASFs for at least 60 days. Inhibition of MT1-MMP significantly reduces the cartilage degradation by RASFs.

Influence of the dopaminergic system, CREB, and transcription factor-κB on cocaine neurotoxicity

International Nuclear Information System (INIS)

Planeta, C.S.; Lepsch, L.B.; Alves, R.; Scavone, C.

2013-01-01

Cocaine is a widely used drug and its abuse is associated with physical, psychiatric and social problems. Abnormalities in newborns have been demonstrated to be due to the toxic effects of cocaine during fetal development. The mechanism by which cocaine causes neurological damage is complex and involves interactions of the drug with several neurotransmitter systems, such as the increase of extracellular levels of dopamine and free radicals, and modulation of transcription factors. The aim of this review was to evaluate the importance of the dopaminergic system and the participation of inflammatory signaling in cocaine neurotoxicity. Our study showed that cocaine activates the transcription factors NF-κB and CREB, which regulate genes involved in cellular death. GBR 12909 (an inhibitor of dopamine reuptake), lidocaine (a local anesthetic), and dopamine did not activate NF-κB in the same way as cocaine. However, the attenuation of NF-κB activity after the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, suggests that the activation of NF-κB by cocaine is, at least partially, due to activation of D1 receptors. NF-κB seems to have a protective role in these cells because its inhibition increased cellular death caused by cocaine. The increase in BDNF (brain-derived neurotrophic factor) mRNA can also be related to the protective role of both CREB and NF-κB transcription factors. An understanding of the mechanisms by which cocaine induces cell death in the brain will contribute to the development of new therapies for drug abusers, which can help to slow down the progress of degenerative processes

Influence of the dopaminergic system, CREB, and transcription factor-κB on cocaine neurotoxicity

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Planeta, C.S. [Laboratório de Neuropsicofarmacologia, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, Araraquara, SP (Brazil); Lepsch, L.B.; Alves, R.; Scavone, C. [Departamento de Farmacologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil)

2013-10-15

Cocaine is a widely used drug and its abuse is associated with physical, psychiatric and social problems. Abnormalities in newborns have been demonstrated to be due to the toxic effects of cocaine during fetal development. The mechanism by which cocaine causes neurological damage is complex and involves interactions of the drug with several neurotransmitter systems, such as the increase of extracellular levels of dopamine and free radicals, and modulation of transcription factors. The aim of this review was to evaluate the importance of the dopaminergic system and the participation of inflammatory signaling in cocaine neurotoxicity. Our study showed that cocaine activates the transcription factors NF-κB and CREB, which regulate genes involved in cellular death. GBR 12909 (an inhibitor of dopamine reuptake), lidocaine (a local anesthetic), and dopamine did not activate NF-κB in the same way as cocaine. However, the attenuation of NF-κB activity after the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, suggests that the activation of NF-κB by cocaine is, at least partially, due to activation of D1 receptors. NF-κB seems to have a protective role in these cells because its inhibition increased cellular death caused by cocaine. The increase in BDNF (brain-derived neurotrophic factor) mRNA can also be related to the protective role of both CREB and NF-κB transcription factors. An understanding of the mechanisms by which cocaine induces cell death in the brain will contribute to the development of new therapies for drug abusers, which can help to slow down the progress of degenerative processes.

Expression of antisense small RNAs in response to stress in Pseudomonas aeruginosa

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Gómez Lozano, María; Marvig, Rasmus Lykke; Tulstrup, Monica Vera-Lise

2014-01-01

Background: RNA sequencing technologies reveal that bacteria express RNA molecules other than mRNA, rRNA or tRNA. During the last years genome-wide bacterial transcriptomes have been shown to comprise intergenic RNA, antisense RNA, and untranslated regions, all capable of performing diverse...... that the extent of overlap between the studies is very limited. Conclusions: RNA-seq experiments are revealing hundreds of novel transcripts in all bacterial genomes investigated. The comparison between independent studies that used RNA-seq to detect novel asRNAs in P. aeruginosa shows that the overlap between...

Quantitative Antisense Screening and Optimization for Exon 51 Skipping in Duchenne Muscular Dystrophy.

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Echigoya, Yusuke; Lim, Kenji Rowel Q; Trieu, Nhu; Bao, Bo; Miskew Nichols, Bailey; Vila, Maria Candida; Novak, James S; Hara, Yuko; Lee, Joshua; Touznik, Aleksander; Mamchaoui, Kamel; Aoki, Yoshitsugu; Takeda, Shin'ichi; Nagaraju, Kanneboyina; Mouly, Vincent; Maruyama, Rika; Duddy, William; Yokota, Toshifumi

2017-11-01

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder, is caused by mutations in the dystrophin (DMD) gene. Exon skipping is a therapeutic approach that uses antisense oligonucleotides (AOs) to modulate splicing and restore the reading frame, leading to truncated, yet functional protein expression. In 2016, the US Food and Drug Administration (FDA) conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based AO drug, eteplirsen, developed for DMD exon 51 skipping. Eteplirsen remains controversial with insufficient evidence of its therapeutic effect in patients. We recently developed an in silico tool to design antisense morpholino sequences for exon skipping. Here, we designed morpholino AOs targeting DMD exon 51 using the in silico tool and quantitatively evaluated the effects in immortalized DMD muscle cells in vitro. To our surprise, most of the newly designed morpholinos induced exon 51 skipping more efficiently compared with the eteplirsen sequence. The efficacy of exon 51 skipping and rescue of dystrophin protein expression were increased by up to more than 12-fold and 7-fold, respectively, compared with the eteplirsen sequence. Significant in vivo efficacy of the most effective morpholino, determined in vitro, was confirmed in mice carrying the human DMD gene. These findings underscore the importance of AO sequence optimization for exon skipping. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Search for antisense copies of beta-globin mRNA in anemic mouse spleen

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Taylor John M

2001-03-01

Full Text Available Abstract Background Previous studies by Volloch and coworkers have reported that during the expression of high levels of β-globin mRNA in the spleen of anemic mice, they could also detect small but significant levels of an antisense (AS globin RNA species, which they postulated might have somehow arisen by RNA-directed RNA synthesis. For two reasons we undertook to confirm and possibly extend these studies. First, previous studies in our lab have focussed on what is an unequivocal example of host RNA-directed RNA polymerase activity on the RNA genome of human hepatitis delta virus. Second, if AS globin species do exist they could in turn form double-stranded RNA species which might induce post-transcriptional gene silencing, a phenomenon somehow provoked in eukaryotic cells by AS RNA sequences. Results We reexamined critical aspects of the previous globin studies. We used intraperitoneal injections of phenylhydrazine to induce anemia in mice, as demonstrated by the appearance and ultimate disappearance of splenomegaly. While a 30-fold increase in globin mRNA was detected in the spleen, the relative amount of putative AS RNA could be no more than 0.004%. Conclusions Contrary to earlier reports, induction of a major increase in globin transcripts in the mouse spleen was not associated with a detectable level of antisense RNA to globin mRNA.

Effect of antisense c-raf-1 on tumorigenicity and radiation sensitivity of a human squamous carcinoma

International Nuclear Information System (INIS)

Kasid, U.; Pfeifer, A.; Brennan, T.; Beckett, M.; Weichselbaum, R.R.; Dritschilo, A.; Mark, G.E.

1989-01-01

Antisense RNA-mediated inhibition of gene expression was used to investigate the biological function of the c-raf-1 gene in a radiation-resistant human squamous carcinoma cell line, SQ-20B. S1 nuclease protection assays revealed that transfection of full-length raf complementary DNA in the antisense orientation (AS) leads to a specific reduction (greater than tenfold) of steady-state levels of the endogenous c-raf-1 sense (S) transcript in SQ-20B cells. In nude mice, the malignant potential of SQ-20B cells transfected with raf (S) was significantly increased relative to that of SQ-20B cells transfected with raf (AS). SQ-20B cells containing transfected raf (S) maintained a radiation-resistant phenotype as compared to those cells harboring the AS version, which appeared to have enhanced radiation sensitivity. These data indicate that the reduced expression of endogenous c-raf-1 is sufficient to modulate the tumorigenicity and the radiation-resistant phenotype of SQ-20B cells, thus implicating c-raf-1 in a pathway important to the genesis of this type of cancer

HSP90 and pCREB alterations are linked to mancozeb-dependent behavioral and neurodegenerative effects in a marine teleost

International Nuclear Information System (INIS)

Zizza, Merylin; Di Lorenzo, Mariana; Laforgia, Vincenza; Furia, Emilia; Sindona, Giovanni; Canonaco, Marcello; Facciolo, Rosa Maria

2017-01-01

The pesticide mancozeb (mz) is recognized as a potent inducer of oxidative stress due to its ability to catalyze the production of reactive oxygen species plus inhibiting mitochondrial respiration thus becoming an environmental risk for neurodegenerative diseases. Despite numerous toxicological studies on mz have been directed to mammals, attention on marine fish is still lacking. Thus, it was our intention to evaluate neurobehavioral activities of ornate wrasses (Thalassoma pavo) exposed to 0.2 mg/l of mz after a preliminary screening test (0.07–0.3 mg/l). Treated fish exhibited an evident (p < 0.001) latency to reach T-maze arms (> 1000%) while exploratory attitudes (total arm entries) diminished (− 50%; p < 0.05) versus controls during spontaneous exploration tests. Moreover, they showed evident enhancements (+ 111%) of immobility in the cylinder test. Contextually, strong (− 88%; p < 0.01) reductions of permanence in light zone of the Light/Dark apparatus along with diminished crossings (− 65%) were also detected. Conversely, wrasses displayed evident enhancements (160%) of risk assessment consisting of fast entries in the dark side of this apparatus. From a molecular point of view, a notable activation (p < 0.005) of the brain transcription factor pCREB occurred during mz-exposure. Similarly, in situ hybridization supplied increased HSP90 mRNAs in most brain areas such as the lateral part of the dorsal telencephalon (Dl; + 68%) and valvula of the cerebellum (VCe; + 35%) that also revealed evident argyrophilic signals. Overall, these first indications suggest a possible protective role of the early biomarkers pCREB and HSP90 against fish toxicity. - Highlights: • Fish exposed to mancozeb exhibited an evident latency to reach T-maze arms. • Mancozeb caused immobility and reduction of explorative attitudes. • Fish exposed to mancozeb showed anxiogenic performances in the Light/Dark apparatus. • The brain of fish exposed to mancozeb supplied pCREB

HSP90 and pCREB alterations are linked to mancozeb-dependent behavioral and neurodegenerative effects in a marine teleost

Energy Technology Data Exchange (ETDEWEB)

Zizza, Merylin [Comparative Neuroanatomy Laboratory, Biology, Ecology and Earth Science Department (DiBEST), University of Calabria, Arcavacata of Rende, 87036, CS (Italy); Di Lorenzo, Mariana; Laforgia, Vincenza [Department of Biology, Section of Evolutionary and Comparative Biology, University of Naples Federico II, 80134 Naples (Italy); Furia, Emilia; Sindona, Giovanni [Department of Chemistry and Chemical Technologies (DCTC), University of Calabria, Arcavacata of Rende, 87036, CS (Italy); Canonaco, Marcello [Comparative Neuroanatomy Laboratory, Biology, Ecology and Earth Science Department (DiBEST), University of Calabria, Arcavacata of Rende, 87036, CS (Italy); Facciolo, Rosa Maria, E-mail: rm.facciolo@unical.it [Comparative Neuroanatomy Laboratory, Biology, Ecology and Earth Science Department (DiBEST), University of Calabria, Arcavacata of Rende, 87036, CS (Italy)

2017-05-15

The pesticide mancozeb (mz) is recognized as a potent inducer of oxidative stress due to its ability to catalyze the production of reactive oxygen species plus inhibiting mitochondrial respiration thus becoming an environmental risk for neurodegenerative diseases. Despite numerous toxicological studies on mz have been directed to mammals, attention on marine fish is still lacking. Thus, it was our intention to evaluate neurobehavioral activities of ornate wrasses (Thalassoma pavo) exposed to 0.2 mg/l of mz after a preliminary screening test (0.07–0.3 mg/l). Treated fish exhibited an evident (p < 0.001) latency to reach T-maze arms (> 1000%) while exploratory attitudes (total arm entries) diminished (− 50%; p < 0.05) versus controls during spontaneous exploration tests. Moreover, they showed evident enhancements (+ 111%) of immobility in the cylinder test. Contextually, strong (− 88%; p < 0.01) reductions of permanence in light zone of the Light/Dark apparatus along with diminished crossings (− 65%) were also detected. Conversely, wrasses displayed evident enhancements (160%) of risk assessment consisting of fast entries in the dark side of this apparatus. From a molecular point of view, a notable activation (p < 0.005) of the brain transcription factor pCREB occurred during mz-exposure. Similarly, in situ hybridization supplied increased HSP90 mRNAs in most brain areas such as the lateral part of the dorsal telencephalon (Dl; + 68%) and valvula of the cerebellum (VCe; + 35%) that also revealed evident argyrophilic signals. Overall, these first indications suggest a possible protective role of the early biomarkers pCREB and HSP90 against fish toxicity. - Highlights: • Fish exposed to mancozeb exhibited an evident latency to reach T-maze arms. • Mancozeb caused immobility and reduction of explorative attitudes. • Fish exposed to mancozeb showed anxiogenic performances in the Light/Dark apparatus. • The brain of fish exposed to mancozeb supplied pCREB

Hepatic Insulin Resistance Following Chronic Activation of the CREB Coactivator CRTC2

DEFF Research Database (Denmark)

Hogan, Meghan F; Ravnskjaer, Kim; Matsumura, Shigenobu

2015-01-01

and dephosphorylation of the cAMP regulated CREB coactivators CRTC2 and CRTC3. In parallel, decreases in circulating insulin also increase gluconeogenic gene expression via the de-phosphorylation and activation of the forkhead transcription factor FOXO1. Hepatic gluconeogenesis is increased in insulin resistance where...... increased gluconeogenic gene expression under fasting as well as feeding conditions. Circulating glucose concentrations were constitutively elevated in CRTC2S171,275A expressing mice, leading to compensatory increases in circulating insulin concentrations that enhance FOXO1 phosphorylation. Despite...... accompanying decreases in FOXO1 activity, hepatic gluconeogenic gene expression remained elevated in CRTC2S171,275A mice demonstrating that chronic increases in CRTC2 activity in the liver are indeed sufficient to promote hepatic insulin resistance and to disrupt glucose homeostasis....

The endogenous retroviral insertion in the human complement C4 gene modulates the expression of homologous genes by antisense inhibition.

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Schneider, P M; Witzel-Schlömp, K; Rittner, C; Zhang, L

2001-02-01

Intron 9 contains the complete endogenous retrovirus HERV-K(C4) as a 6.4-kb insertion in 60% of human C4 genes. The retroviral insertion is in reverse orientation to the C4 coding sequence. Therefore, expression of C4 could lead to the transcription of an antisense RNA, which might protect against exogenous retroviral infections. To test this hypothesis, open reading frames from the HERV sequence were subcloned in sense orientiation into a vector allowing expression of a beta-galactosidase fusion protein. Mouse L cells which had been stably transfected with either the human C4A or C4B gene both carrying the HERV insertion (LC4 cells), and L(Tk-) cells without the C4 gene were transiently transfected either with a retroviral construct or with the wild-type vector. Expression was monitored using an enzymatic assay. We demonstrated that (1) HERV-K(C4) antisense mRNA transcripts are present in cells constitutively expressing C4, (2) expression of retroviral-like constructs is significantly downregulated in cells expressing C4, and (3) this downregulation is further modulated in a dose-dependent fashion following interferon-gamma stimulation of C4 expression. These results support the hypothesis of a genomic antisense strategy mediated by the HERV-K(C4) insertion as a possible defense mechanism against exogenous retroviral infections.

Antidepressant-like activity of red wine phenolic extracts in repeated corticosterone-induced depression mice via BDNF/TrkB/CREB signaling pathway

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Jia Ying

2016-01-01

Full Text Available The aim of this study was to investigate the antidepressant-like effect of red wine phenolic extracts in mouse model exposed to exogenous corticosterone. The results showed that 3-week corticosterone injections caused depression-like behavior in mice, as indicated by the significant decrease in sucrose consumption and increase immobility time in the forced swim test. Red wine phenolic extracts treatment significantly reduced serum corticosterone levels. Moreover, it was found that red wine phenolic extract increased the brain-derived neurotrophic factor protein (BNDF and Tropomyosin-related kinase B (TrkB phosphorylation and cAMP-responsive element binding protein (CREB phosphorylation levels in the hippocampus and prefrontal cortex. However, K252a, an inhibitor of TrkB, completely abolished those antidepressant-like effects. These results suggested that the red wine phenolic extracts produce an antidepressant-like effect in corticosterone-treated mice, at least in part, which is possibly mediated by modulating hypothalamic-pituitary-adrenal (HPA axis, BDNF, TrkB and CREB phosphorylation levels in the brain region of mice.

Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors.

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Lobos-González, Lorena; Silva, Verónica; Araya, Mariela; Restovic, Franko; Echenique, Javiera; Oliveira-Cruz, Luciana; Fitzpatrick, Christopher; Briones, Macarena; Villegas, Jaime; Villota, Claudio; Vidaurre, Soledad; Borgna, Vincenzo; Socias, Miguel; Valenzuela, Sebastián; Lopez, Constanza; Socias, Teresa; Varas, Manuel; Díaz, Jorge; Burzio, Luis O; Burzio, Verónica A

2016-09-06

We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown in syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy.

Improved cellular uptake of antisense Peptide nucleic acids by conjugation to a cell-penetrating Peptide and a lipid domain

DEFF Research Database (Denmark)

Shiraishi, Takehiko; Nielsen, Peter E

2011-01-01

based on a splicing correction of a mutated luciferase gene in HeLa pLuc705 cells by targeting antisense oligonucleotides to a cryptic splice site. Further improvement in the delivery of CatLip-PNA conjugates is achieved by using auxiliary agents/treatments (e.g., chloroquine, calcium ions......Unaided cellular uptake of RNA interference agents such as antisense oligonucleotides and siRNA is extremely poor, and in vivo bioavailability is also limited. Thus, effective delivery strategies for such potential drugs are in high demand. Recently, a novel approach using a class of short cationic....... We have found, however, that this low -bioavailability can be significantly improved by chemical conjugation to a lipid domain ("Lip," such as a fatty acid), thereby creating "CatLip"-conjugates. The cellular uptake of these conjugates is conveniently evaluated using a sensitive cellular assay system...

PLGA-PEG-PLGA microspheres as a delivery vehicle for antisense oligonucleotides to CTGF: Implications on post-surgical peritoneal adhesion prevention

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Azeke, John Imuetinyan-Jesu, Jr.

Abdominal adhesions are the aberrant result of peritoneal wound healing commonly associated with surgery and inflammation. A subject of a large number of studies since the first half of the last century, peritoneal adhesion prevention has, for the most part, evaded the scientific community and continues to cost Americans an estimated $2-4 billion annually. It is known that transforming growth factor-beta (TGF-beta) plays a key role in the wound healing cascade; however, suppression of this multifunctional growth factor's activity may have more harmful consequences than can be tolerated. As a result, much attention has fallen on connective tissue growth factor (CTGF), a downstream mediator of TGF-beta's fibrotic action. It has been demonstrated in several in vitro models, that the suppression of CTGF hinders fibroblast proliferation, a necessary condition for fibrosis. Furthermore, antisense oligonucleotides (antisense oligos, AO) to CTGF have been shown to knock down CTGF mRNA levels by specifically hindering the translation of CTGF protein. Antisense technologies have met with a great deal of excitement as a viable means of preventing diseases such as adhesions by hindering protein translation at the mRNA level. However, the great challenge associated with the use of these drugs lies in the short circulation time when administered "naked". Viral delivery systems, although excellent platforms in metabolic studies, are not ideal for diagnostic use because of the inherent danger associated with viral vectors. Microparticles made of biodegradable polymers have therefore presented themselves as a viable means of delivering these drugs to target cells over extended periods. Herein, we present two in vivo studies confirming the up-regulation of TGF-beta protein and CTGF mRNA following injury to the uterine tissues of female rats. We were able to selectively knockdown post-operative CTGF protein levels following surgery, however, our observations led us to conclude that

Antisense Phosphorodiamidate Morpholino Oligomers as Novel Antiviral Compounds

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Yuchen Nan

2018-04-01

Full Text Available Phosphorodiamidate morpholino oligomers (PMO are short single-stranded DNA analogs that are built upon a backbone of morpholine rings connected by phosphorodiamidate linkages. As uncharged nucleic acid analogs, PMO bind to complementary sequences of target mRNA by Watson–Crick base pairing to block protein translation through steric blockade. PMO interference of viral protein translation operates independently of RNase H. Meanwhile, PMO are resistant to a variety of enzymes present in biologic fluids, a characteristic that makes them highly suitable for in vivo applications. Notably, PMO-based therapy for Duchenne muscular dystrophy (DMD has been approved by the United States Food and Drug Administration which is now a hallmark for PMO-based antisense therapy. In this review, the development history of PMO, delivery methods for improving cellular uptake of neutrally charged PMO molecules, past studies of PMO antagonism against RNA and DNA viruses, PMO target selection, and remaining questions of PMO antiviral strategies are discussed in detail and new insights are provided.

Connexin31.1 deficiency in the mouse impairs object memory and modulates open-field exploration, acetylcholine esterase levels in the striatum, and cAMP response element-binding protein levels in the striatum and piriform cortex.

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Dere, E; Zheng-Fischhöfer, Q; Viggiano, D; Gironi Carnevale, U A; Ruocco, L A; Zlomuzica, A; Schnichels, M; Willecke, K; Huston, J P; Sadile, A G

2008-05-02

Neuronal gap junctions in the brain, providing intercellular electrotonic signal transfer, have been implicated in physiological and behavioral correlates of learning and memory. In connexin31.1 (Cx31.1) knockout (KO) mice the coding region of the Cx31.1 gene was replaced by a LacZ reporter gene. We investigated the impact of Cx31.1 deficiency on open-field exploration, the behavioral response to an odor, non-selective attention, learning and memory performance, and the levels of memory-related proteins in the hippocampus, striatum and the piriform cortex. In terms of behavior, the deletion of the Cx31.1 coding DNA in the mouse led to increased exploratory behaviors in a novel environment, and impaired one-trial object recognition at all delays tested. Despite strong Cx31.1 expression in the peripheral and central olfactory system, Cx31.1 KO mice exhibited normal behavioral responses to an odor. We found increased levels of acetylcholine esterase (AChE) and cAMP response element-binding protein (CREB) in the striatum of Cx31.1 KO mice. In the piriform cortex the Cx31.1 KO mice had an increased heterogeneity of CREB expression among neurons. In conclusion, gap-junctions featuring the Cx31.1 protein might be involved in open-field exploration as well as object memory and modulate levels of AChE and CREB in the striatum and piriform cortex.

Oral administration of sitagliptin activates CREB and is neuroprotective in murine model of brain trauma

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Brian Dellavalle

2016-12-01

Full Text Available Introduction: Traumatic brain injury is a major cause of mortality and morbidity. We have previously shown that the injectable glucagon-like peptide-1 (GLP-1 analogue, liraglutide, significantly improved the outcome in mice after severe traumatic brain injury (TBI. In this study we are interested in the effects of oral treatment of a different class of GLP-1 based therapy, dipeptidyl peptidase IV (DPP-IV inhibition on mice after TBI. DPP-IV inhibitors reduce the degradation of endogenous GLP-1 and extend circulation of this protective peptide in the bloodstream. This class has yet to be investigated as a potential therapy for TBI. Methods: Mice were administrated once-daily 50 mg/kg of sitagliptin in a Nutella® ball or Nutella® alone throughout the study, beginning two days before severe trauma was induced with a stereotactic cryo-lesion. At two days post trauma, lesion size was determined. Brains were isolated for immunoblotting for assessment of selected biomarkers for pathology and protection.Results: Sitagliptin treatment reduced lesion size at day 2 post-injury by ~28% (p0.05. Conversely, apoptotic tone (alpha-spectrin fragmentation, Bcl-2 levels and the neuroinflammatory markers IL-6, and Iba-1 were not affected by treatment.Conclusions: This study shows, for the first time, that DPP-IV inhibition ameliorates both anatomical and biochemical consequences of TBI and activates CREB in the brain. Moreover, this work supports previous studies suggesting that the effect of GLP-1 analogues in models of brain damage relates to GLP-1 receptor stimulation in a dose-dependent manner.Keywords: GLP-1, Traumatic Brain Injury, TBI, sitagliptin, liraglutide, CREB, Oxidative Stress, GIP, DPP-IV, DPP-4

Chemical modifications of antisense morpholino oligomers enhance their efficacy against Ebola virus infection.

Science.gov (United States)

Swenson, Dana L; Warfield, Kelly L; Warren, Travis K; Lovejoy, Candace; Hassinger, Jed N; Ruthel, Gordon; Blouch, Robert E; Moulton, Hong M; Weller, Dwight D; Iversen, Patrick L; Bavari, Sina

2009-05-01

Phosphorodiamidate morpholino oligomers (PMOs) are uncharged nucleic acid-like molecules designed to inactivate the expression of specific genes via the antisense-based steric hindrance of mRNA translation. PMOs have been successful at knocking out viral gene expression and replication in the case of acute viral infections in animal models and have been well tolerated in human clinical trials. We propose that antisense PMOs represent a promising class of therapeutic agents that may be useful for combating filoviral infections. We have previously shown that mice treated with a PMO whose sequence is complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebola virus challenge. In the present study, we report on the abilities of two additional VP24-specific PMOs to reduce the cell-free translation of a VP24 reporter, to inhibit the in vitro replication of Ebola virus, and to protect mice against lethal challenge when the PMOs are delivered prior to infection. Additionally, structure-activity relationship evaluations were conducted to assess the enhancement of antiviral efficacy associated with PMO chemical modifications that included conjugation with peptides of various lengths and compositions, positioning of conjugated peptides to either the 5' or the 3' terminus, and the conferring of charge modifications by the addition of piperazine moieties. Conjugation with arginine-rich peptides greatly enhanced the antiviral efficacy of VP24-specific PMOs in infected cells and mice during lethal Ebola virus challenge.

Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells

DEFF Research Database (Denmark)

Shiraishi, Takehiko; Hamzavi, Ramin; Nielsen, Peter E

2008-01-01

oligomer. This modification of the PNA does not interfere with the nucleic acid target binding affinity based on thermal stability of the PNA/RNA duplexes. When delivered to cultured HeLa pLuc705 cells by Lipofectamine, the PNAs showed dose-dependent nuclear antisense activity in the nanomolar range...

Antisense RNA Controls LRP1 Sense Transcript Expression through Interaction with a Chromatin-Associated Protein, HMGB2

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Yasunari Yamanaka

2015-05-01

Full Text Available Long non-coding RNAs (lncRNAs, including natural antisense transcripts (NATs, are expressed more extensively than previously anticipated and have widespread roles in regulating gene expression. Nevertheless, the molecular mechanisms of action of the majority of NATs remain largely unknown. Here, we identify a NAT of low-density lipoprotein receptor-related protein 1 (Lrp1, referred to as Lrp1-AS, that negatively regulates Lrp1 expression. We show that Lrp1-AS directly binds to high-mobility group box 2 (Hmgb2 and inhibits the activity of Hmgb2 to enhance Srebp1a-dependent transcription of Lrp1. Short oligonucleotides targeting Lrp1-AS inhibit the interaction of antisense transcript and Hmgb2 protein and increase Lrp1 expression by enhancing Hmgb2 activity. Quantitative RT-PCR analysis of brain tissue samples from Alzheimer’s disease patients and aged-matched controls revealed upregulation of LRP1-AS and downregulation of LRP1. Our data suggest a regulatory mechanism whereby a NAT interacts with a ubiquitous chromatin-associated protein to modulate its activity in a locus-specific fashion.

Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures.

Science.gov (United States)

Semple, S C; Klimuk, S K; Harasym, T O; Dos Santos, N; Ansell, S M; Wong, K F; Maurer, N; Stark, H; Cullis, P R; Hope, M J; Scherrer, P

2001-02-09

Typical methods used for encapsulating antisense oligodeoxynucleotides (ODN) and plasmid DNA in lipid vesicles result in very low encapsulation efficiencies or employ cationic lipids that exhibit unfavorable pharmacokinetic and toxicity characteristics when administered intravenously. In this study, we describe and characterize a novel formulation process that utilizes an ionizable aminolipid (1,2-dioleoyl-3-dimethylammonium propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities (0.15--0.25 g ODN/g lipid) of polyanionic ODN in lipid vesicles. This process requires the presence of up to 40% ethanol (v/v) and initial formulation at acidic pH values where the DODAP is positively charged. In addition, the presence of a poly(ethylene glycol)-lipid was required during the formulation process to prevent aggregation. The 'stabilized antisense-lipid particles' (SALP) formed are stable on adjustment of the external pH to neutral pH values and the formulation process allows encapsulation efficiencies of up to 70%. ODN encapsulation was confirmed by nuclease protection assays and (31)P NMR measurements. Cryo-electron microscopy indicated that the final particles consisted of a mixed population of unilamellar and small multilamellar vesicles (80--140 nm diameter), the relative proportion of which was dependent on the initial ODN to lipid ratio. Finally, SALP exhibited significantly enhanced circulation lifetimes in mice relative to free antisense ODN, cationic lipid/ODN complexes and SALP prepared with quaternary aminolipids. Given the small particle sizes and improved encapsulation efficiency, ODN to lipid ratios, and circulation times of this formulation compared to others, we believe SALP represent a viable candidate for systemic applications involving nucleic acid therapeutics.

Antisense imaging of epidermal growth factor-induced p21{sup WAF-1/CIP-1} gene expression in MDA-MB-468 human breast cancer xenografts

Energy Technology Data Exchange (ETDEWEB)

Wang, Judy; Chen, Paul; Mrkobrada, Marko [Leslie Dan Faculty of Pharmacy, University of Toronto, 19 Russell Street, M5S 2S2, Toronto, Ontario (Canada); Hu, Meiduo [Leslie Dan Faculty of Pharmacy, University of Toronto, 19 Russell Street, M5S 2S2, Toronto, Ontario (Canada); Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario (Canada); Vallis, Katherine A. [Department of Radiation Oncology, Princess Margaret Hospital, University Health Network, 610 University Avenue, Toronto, Ontario (Canada); Department of Radiation Oncology, University of Toronto, Toronto, Ontario (Canada); Department of Medical Biophysics, University of Toronto, Toronto, Ontario (Canada); Reilly, Raymond M. [Department of Medical Imaging, University of Toronto, Toronto, Ontario (Canada)

2003-09-01

Molecular imaging of the expression of key genes which determine the response to DNA damage following cancer treatment may predict the effectiveness of a particular treatment strategy. A prominent early response gene for DNA damage is the gene encoding p21{sup WAF-1/CIP-1}, a cyclin-dependent kinase inhibitor that regulates progression through the cell cycle. In this study, we explored the feasibility of imaging p21{sup WAF-1/CIP-1} gene expression at the mRNA level using an 18-mer phosphorothioated antisense oligodeoxynucleotide (ODN) labeled with {sup 111}In. The known induction of the p21{sup WAF-1/CIP-1} gene in MDA-MB-468 human breast cancer cells following exposure to epidermal growth factor (EGF) was used as an experimental tool. Treatment of MDA-MB-468 cells in vitro with EGF (20 nM) increased the ratio of p21{sup WAF-1/CIP-1} mRNA/{beta}-actin mRNA threefold within 2 h as measured by the reverse transcription polymerase chain reaction (RT-PCR). A concentration-dependent inhibition of EGF-induced p21{sup WAF-1/CIP-1} protein expression was achieved in MDA-MB-468 cells by treatment with antisense ODNs with up to a tenfold decrease observed at 1 {mu}M. There was a fourfold lower inhibition of p21{sup WAF-1/CIP-1} protein expression by control sense or random sequence ODNs. Intratumoral injections of EGF (15 {mu}g/day x 3 days) were employed to induce p21{sup WAF-1/CIP-1} gene expression in MDA-MB-468 xenografts implanted subcutaneously into athymic mice. RT-PCR of explanted tumors showed a threefold increased level of p21{sup WAF-1/CIP-1} mRNA compared with normal saline-treated tumors. Successful imaging of EGF-induced p21{sup WAF-1/CIP-1} gene expression in MDA-MB-468 xenografts was achieved at 48 h post injection of {sup 111}In-labeled antisense ODNs (3.7 MBq; 2 {mu}g). Tumors displaying basal levels of p21{sup WAF-1/CIP-1} gene expression in the absence of EGF treatment could not be visualized. Biodistribution studies showed a significantly higher tumor

Nanomolar Cellular Antisense Activity of Peptide Nucleic Acid (PNA) Cholic Acid ("Umbrella") and Cholesterol Conjugates Delivered by Cationic Lipids

DEFF Research Database (Denmark)

Shiraishi, Takehiko; Nielsen, Peter E

2012-01-01

of cholesterol and cholic acid ("umbrella") derivatives of splice correction antisense PNA oligomers. While the conjugates alone were practically inactive up to 1 µM, their activity was dramatically improved when delivered by a cationic lipid transfection agent (LipofectAMINE2000). In particular, PNAs...

Feasibility of SPECT-CT imaging to study the pharmacokinetics of antisense oligonucleotides in a mouse model of Duchenne muscular dystrophy

NARCIS (Netherlands)

Steeg, E. van de; Läppchen, T.; Aguilera, B.; Jansen, H.T.; Muilwijk, D.; Vermue, R.; Hoorn, J.W. van der; Donato, K.; Rossin, R.; Visser, P.C. de; Vlaming, M.L.H.

2017-01-01

Antisense oligonucleotides (AONs) are promising candidates for treatment of Duchenne muscular dystrophy (DMD), a severe and progressive disease resulting in premature death. However, more knowledge on the pharmacokinetics of new AON drug candidates is desired for effective application in the clinic.

SINEUPs are modular antisense long-non coding RNAs that increase synthesis of target proteins in cells

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Silvia eZucchelli

2015-05-01

Full Text Available Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson’s disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1, is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD while the embedded inverted SINEB2 element is the Effector Domain (ED. By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. SINEUPs function in an array of cell lines and can be efficiently directed towards N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson’s disease-associated DJ-1 and proved to be active in different neuronal cell lines.In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

Os DNA sintéticos anti-sentido Antisense Synthtetic DNA

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Alfredo Cravador

1998-07-01

Full Text Available One old dream of the chemist in the field of the drug research is to create molecules capable of reaching their target with the precision of a missile. To accomplish it these molecules must have the propriety of distinguishing qualitative differences between healthy and diseased cells. A therapy based on this principle, able of eradicating specifically defective cells, or cells affected by a pathogen has an enormous advantage with the regard to the classical approach in which the cytotoxic drugs merely exploit quantitative biochemical and kinetic differences between abnormal and normal cells. We present in this article a review on the chemical synthesis of analogues of desoxyribonucleotides and on results obtained on the specific and irreversible inhibition of undesired genetic expression using the antisense principle.

Neural Plasticity Associated with Hippocampal PKA-CREB and NMDA Signaling Is Involved in the Antidepressant Effect of Repeated Low Dose of Yueju Pill on Chronic Mouse Model of Learned Helplessness.

Science.gov (United States)

Zou, Zhilu; Chen, Yin; Shen, Qinqin; Guo, Xiaoyan; Zhang, Yuxuan; Chen, Gang

2017-01-01

Yueju pill is a traditional Chinese medicine formulated to treat syndromes of mood disorders. Here, we investigated the therapeutic effect of repeated low dose of Yueju in the animal model mimicking clinical long-term depression condition and the role of neural plasticity associated with PKA- (protein kinase A-) CREB (cAMP response element binding protein) and NMDA (N-methyl-D-aspartate) signaling. We showed that a single low dose of Yueju demonstrated antidepressant effects in tests of tail suspension, forced swim, and novelty-suppressed feeding. A chronic learned helplessness (LH) protocol resulted in a long-term depressive-like condition. Repeated administration of Yueju following chronic LH remarkably alleviated all of depressive-like symptoms measured, whereas conventional antidepressant fluoxetine only showed a minor improvement. In the hippocampus, Yueju and fluoxetine both normalized brain-derived neurotrophic factor (BDNF) and PKA level. Only Yueju, not fluoxetine, rescued the deficits in CREB signaling. The chronic LH upregulated the expression of NMDA receptor subunits NR1, NR2A, and NR2B, which were all attenuated by Yueju. Furthermore, intracerebraventricular administration of NMDA blunted the antidepressant effect of Yueju. These findings supported the antidepressant efficacy of repeated routine low dose of Yueju in a long-term depression model and the critical role of CREB and NMDA signaling.

In mpkCCD cells, long-term regulation of aquaporin-2 by vasopressin occurs independent of protein kinase A and CREB but may involve Epac

DEFF Research Database (Denmark)

Kortenoeven, Marleen; Trimpert, Christiane; van den Brand, Michiel

2012-01-01

kinase A (PKA) inhibitor H89. Moreover, phosphorylation of the cAMP-responsive element binding protein (CREB) and CRE-dependent transcription was observed after short-term dDAVP stimulation. With 4 days of dDAVP, AQP2 transcription remained elevated, but this was not blocked by H89, and CRE...

Strand-specific RNA-seq reveals widespread occurrence of novel cis-natural antisense transcripts in rice

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Lu Tingting

2012-12-01

Full Text Available Abstract Background Cis-natural antisense transcripts (cis-NATs are RNAs transcribed from the antisense strand of a gene locus, and are complementary to the RNA transcribed from the sense strand. Common techniques including microarray approach and analysis of transcriptome databases are the major ways to globally identify cis-NATs in various eukaryotic organisms. Genome-wide in silico analysis has identified a large number of cis-NATs that may generate endogenous short interfering RNAs (nat-siRNAs, which participate in important biogenesis mechanisms for transcriptional and post-transcriptional regulation in rice. However, the transcriptomes are yet to be deeply sequenced to comprehensively investigate cis-NATs. Results We applied high-throughput strand-specific complementary DNA sequencing technology (ssRNA-seq to deeply sequence mRNA for assessing sense and antisense transcripts that were derived under salt, drought and cold stresses, and normal conditions, in the model plant rice (Oryza sativa. Combined with RAP-DB genome annotation (the Rice Annotation Project Database build-5 data set, 76,013 transcripts corresponding to 45,844 unique gene loci were assembled, in which 4873 gene loci were newly identified. Of 3819 putative rice cis-NATs, 2292 were detected as expressed and giving rise to small RNAs from their overlapping regions through integrated analysis of ssRNA-seq data and small RNA data. Among them, 503 cis-NATs seemed to be associated with specific conditions. The deep sequence data from isolated epidermal cells of rice seedlings further showed that 54.0% of cis-NATs were expressed simultaneously in a population of homogenous cells. Nearly 9.7% of rice transcripts were involved in one-to-one or many-to-many cis-NATs formation. Furthermore, only 17.4-34.7% of 223 many-to-many cis-NAT groups were all expressed and generated nat-siRNAs, indicating that only some cis-NAT groups may be involved in complex regulatory networks. Conclusions

Electroporation increases antitumoral efficacy of the bcl-2 antisense G3139 and chemotherapy in a human melanoma xenograft

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Baldi Alfonso

2011-07-01

Full Text Available Abstract Background Nucleic acids designed to modulate the expression of target proteins remain a promising therapeutic strategy in several diseases, including cancer. However, clinical success is limited by the lack of efficient intracellular delivery. In this study we evaluated whether electroporation could increase the delivery of antisense oligodeoxynucleotides against bcl-2 (G3139 as well as the efficacy of combination chemotherapy in human melanoma xenografts. Methods Melanoma-bearing nude mice were treated i.v. with G3139 and/or cisplatin (DDP followed by the application of trains of electric pulses to tumors. Western blot, immunohistochemistry and real-time PCR were performed to analyze protein and mRNA expression. The effect of electroporation on muscles was determined by histology, while tumor apoptosis and the proliferation index were analyzed by immunohistochemistry. Antisense oligodeoxynucleotides tumor accumulation was measured by FACS and confocal microscopy. Results The G3139/Electroporation combined therapy produced a significant inhibition of tumor growth (TWI, more than 50% accompanied by a marked tumor re-growth delay (TRD, about 20 days. The efficacy of this treatment was due to the higher G3139 uptake in tumor cells which led to a marked down-regulation of bcl-2 protein expression. Moreover, the G3139/EP combination treatment resulted in an enhanced apoptotic index and a decreased proliferation rate of tumors. Finally, an increased tumor response was observed after treatment with the triple combination G3139/DDP/EP, showing a TWI of about 75% and TRD of 30 days. Conclusions These results demonstrate that electroporation is an effective strategy to improve the delivery of antisense oligodeoxynucleotides within tumor cells in vivo and it may be instrumental in optimizing the response of melanoma to chemotherapy. The high response rate observed in this study suggest to apply this strategy for the treatment of melanoma patients.

Intravenous administration of stabilized antisense lipid particles (SALP) leads to activation and expansion of liver natural killer cells.

Science.gov (United States)

Bramson, J L; Bodner, C A; Johnson, J; Semple, S; Hope, M J

2000-06-01

Stabilized antisense lipid particles (SALP) have been developed for the systemic delivery of oligonucleotides. The impact of intravenous SALP administration was measured with respect to activation of natural killer (NK) and NK1.1+ T (NKT) cells in the livers of immunocompetent mice. Treatment with a SALP containing a highly mitogenic oligonucleotide (INX-6295) generated an increase in NK cytolytic activity and cell number within the liver but did not appear to affect the number of hepatic NKT cells or their cytolytic activity. The same results were observed after intravenous administration of the mitogenic oligonucleotide alone. Interestingly, treatment with a SALP containing a weakly mitogenic oligonucleotide (INX-6300) also activated the liver NK cells, whereas the oligonucleotide alone was unable to elicit these effects. The NK stimulatory activity of a SALP containing INX-6300 required both lipid and oligonucleotide components. These results demonstrate that in addition to modifying the pharmacokinetics and biodistribution of intravenously administered oligonucleotides, SALP possess immunostimulatory activity independent of oligonucleotide mitogenicity, which can serve as an adjuvant to antisense therapies for cancer.

Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe

Energy Technology Data Exchange (ETDEWEB)

Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H. [Kobe Univ. School of Medicine and Science (Japan)

1994-09-01

Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

Activity-dependent expression of miR-132 regulates immediate-early gene induction during olfactory learning in the greater short-nosed fruit bat, Cynopterus sphinx.

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Mukilan, Murugan; Ragu Varman, Durairaj; Sudhakar, Sivasubramaniam; Rajan, Koilmani Emmanuvel

2015-04-01

The activity-dependent expression of immediate-early genes (IEGs) and microRNA (miR)-132 has been implicated in synaptic plasticity and the formation of long-term memory (LTM). In the present study, we show that olfactory training induces the expression of IEGs (EGR-1, C-fos, C-jun) and miR-132 at similar time scale in olfactory bulb (OB) of Cynopterus sphinx. We examined the role of miR-132 in the OB using antisense oligodeoxynucleotide (AS-ODN) and demonstrated that a local infusion of AS-ODN in the OB 2h prior to training impaired olfactory memory formation in C. sphinx. However, the infusion of AS-ODN post-training did not cause a deficit in memory formation. Furthermore, the inhibition of miR-132 reduced the olfactory training-induced expression of IEGs and post synaptic density protein-95 (PSD-95) in the OB. Additionally, we show that miR-132 regulates the activation of calcium/calmodulin-dependent protein kinase-II (CaMKII) and cAMP response element binding protein (CREB), possibly through miR-148a. These data suggest that olfactory training induces the expression of miR-132 and IEGs, which in turn activates post-synaptic proteins that regulate olfactory memory formation. Copyright © 2015 Elsevier Inc. All rights reserved.

An essential GT motif in the lamin A promoter mediates activation by CREB-binding protein

International Nuclear Information System (INIS)

Janaki Ramaiah, M.; Parnaik, Veena K.

2006-01-01

Lamin A is an important component of nuclear architecture in mammalian cells. Mutations in the human lamin A gene lead to highly degenerative disorders that affect specific tissues. In studies directed towards understanding the mode of regulation of the lamin A promoter, we have identified an essential GT motif at -55 position by reporter gene assays and mutational analysis. Binding of this sequence to Sp transcription factors has been observed in electrophoretic mobility shift assays and by chromatin immunoprecipitation studies. Further functional analysis by co-expression of recombinant proteins and ChIP assays has shown an important regulatory role for CREB-binding protein in promoter activation, which is mediated by the GT motif

Regulation of Peripheral Myelination through Transcriptional Buffering of Egr2 by an Antisense Long Non-coding RNA

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Margot Martinez-Moreno

2017-08-01

Full Text Available Precise regulation of Egr2 transcription is fundamentally important to the control of peripheral myelination. Here, we describe a long non-coding RNA antisense to the promoter of Egr2 (Egr2-AS-RNA. During peripheral nerve injury, the expression of Egr2-AS-RNA is increased and correlates with decreased Egr2 transcript and protein levels. Ectopic expression of Egr2-AS-RNA in dorsal root ganglion (DRG cultures inhibits the expression of Egr2 mRNA and induces demyelination. In vivo inhibition of Egr2-AS-RNA using oligonucleotide GapMers released from a biodegradable hydrogel following sciatic nerve injury reverts the EGR2-mediated gene expression profile and significantly delays demyelination. Egr2-AS-RNA gradually recruits H3K27ME3, AGO1, AGO2, and EZH2 on the Egr2 promoter following sciatic nerve injury. Furthermore, expression of Egr2-AS-RNA is regulated through ERK1/2 signaling to YY1, while loss of Ser184 of YY1 regulates binding to Egr2-AS-RNA. In conclusion, we describe functional exploration of an antisense long non-coding RNA in peripheral nervous system (PNS biology.

Rescue of Outer Hair Cells with Antisense Oligonucleotides in Usher Mice Is Dependent on Age of Treatment.

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Ponnath, Abhilash; Depreux, Frederic F; Jodelka, Francine M; Rigo, Frank; Farris, Hamilton E; Hastings, Michelle L; Lentz, Jennifer J

2018-02-01

The absence of functional outer hair cells is a component of several forms of hereditary hearing impairment, including Usher syndrome, the most common cause of concurrent hearing and vision loss. Antisense oligonucleotide (ASO) treatment of mice with the human Usher mutation, Ush1c c.216G>A, corrects gene expression and significantly improves hearing, as measured by auditory-evoked brainstem responses (ABRs), as well as inner and outer hair cell (IHC and OHC) bundle morphology. However, it is not clear whether the improvement in hearing achieved by ASO treatment involves the functional rescue of outer hair cells. Here, we show that Ush1c c.216AA mice lack OHC function as evidenced by the absence of distortion product otoacoustic emissions (DPOAEs) in response to low-, mid-, and high-frequency tone pairs. This OHC deficit is rescued by treatment with an ASO that corrects expression of Ush1c c.216G>A. Interestingly, although rescue of inner hairs cells, as measured by ABR, is achieved by ASO treatment as late as 7 days after birth, rescue of outer hair cells, measured by DPOAE, requires treatment before post-natal day 5. These results suggest that ASO-mediated rescue of both IHC and OHC function is age dependent and that the treatment window is different for the different cell types. The timing of treatment for congenital hearing disorders is of critical importance for the development of drugs such ASO-29 for hearing rescue.

Attenuation of pCREB and Egr1 expression in the insular and anterior cingulate cortices associated with enhancement of CFA-evoked mechanical hypersensitivity after repeated forced swim stress.

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Imbe, Hiroki; Kimura, Akihisa

2017-09-01

The perception and response to pain are severely impacted by exposure to stressors. In some animal models, stress increases pain sensitivity, which is termed stress-induced hyperalgesia (SIH). The insular cortex (IC) and anterior cingulate cortex (ACC), which are typically activated by noxious stimuli, affect pain perception through the descending pain modulatory system. In the present study, we examined the expression of phospho-cAMP response element-binding protein (pCREB) and early growth response 1 (Egr1) in the IC and ACC at 3h (the acute phase of peripheral tissue inflammation) after complete Freund's adjuvant (CFA) injection in naïve rats and rats preconditioned with forced swim stress (FS) to clarify the effect of FS, a stressor, on cortical cell activities in the rats showing SIH induced by FS. The CFA injection into the hindpaw induced mechanical hypersensitivity and increased the expression of the pCREB and Egr1 in the IC and ACC at 3h after the injection. FS (day 1, 10min; days 2-3, 20min) prior to the CFA injection enhanced the CFA-induced mechanical hypersensitivity and attenuated the increase in the expression of pCREB and Egr1 in the IC and ACC. These findings suggested that FS modulates the CFA injection-induced neuroplasticity in the IC and ACC to enhance the mechanical hypersensitivity. These findings are thought to signify stressor-induced dysfunction of the descending pain modulatory system. Copyright © 2017 Elsevier Inc. All rights reserved.

Biodegradable polymer nanocarriers for therapeutic antisense microRNA delivery in living animals

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Paulmurugan, Ramasamy; Sekar, Narayana M.; Sekar, Thillai V.

2012-03-01

MicroRNAs are endogenous regulators of gene expression, deregulated in several cellular diseases including cancer. Altering the cellular microenvironment by modulating the microRNAs functions can regulate different genes involved in major cellular processes, and this approach is now being investigated as a promising new generation of molecularly targeted anti-cancer therapies. AntagomiRs (Antisense-miRNAs) are a novel class of chemically modified stable oligonucleotides used for blocking the functions of endogenous microRNAs, which are overexpressed. A key challenge in achieving effective microRNAbased therapeutics lies in the development of an efficient delivery system capable of specifically delivering antisense oligonucleotides and target cancer cells in living animals. We are now developing an effective delivery system designed to selectively deliver antagomiR- 21 and antagomiR-10b to triple negative breast cancer cells, and to revert tumor cell metastasis and invasiveness. The FDA-approved biodegradable PLGA-nanoparticles were selected as a carrier for antagomiRs delivery. Chemically modified antagomiRs (antagomiR-21 and antagomiR-10b) were co-encapsulated in PEGylated-PLGA-nanoparticles by using the double-emulsification (W/O/W) solvent evaporation method, and the resulting average particle size of 150-200nm was used for different in vitro and in vivo experiments. The antagomiR encapsulated PLGA-nanoparticles were evaluated for their in vitro antagomiRs delivery, intracellular release profile, and antagomiRs functional effects, by measuring the endogenous cellular targets, and the cell growth and metastasis. The xenografts of tumor cells in living mice were used for evaluating the anti-metastatic and anti-invasive properties of cells. The results showed that the use of PLGA for antagomiR delivery is not only efficient in crossing cell membrane, but can also maintain functional intracellular antagomiRs level for a extended period of time and achieve

The cellular uptake of antisense oligonucleotid of E6 mRNA into cervical cancer cells by DOPE-modified hydroxyapatite nanoparticles

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Negin Saffarzadeh

2014-10-01

Full Text Available Objective(s: Although several chemical and physical methods for gene delivery have been introduced, their cytotoxicity, non-specific immune responses and the lack of biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles (NPs and 1,2-dioleoyl-sn-glycero-3-phosphoethanol​amine (DOPE-modified hydroxyapatite NPs was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical cancer cell line were evaluated. Materials and Methods: Calcium nitrate and diammonium phosphate were used for the synthesis of the hydroxyapatite nanoparticle. Thus, they were coated with polyethylene glycol (PEG, DOPE and antisense oligonucleotide of E6 mRNA using a cross-linker. Then, hydroxyapatite NPs and DOPE-modified hydroxyapatite NPs were incubated 48 hours with cervical cancer cells and their uptakes were evaluated by fluorescent microscopy. Results: The hydroxyapatite NPs had different shapes and some agglomeration with average size of 100 nm. The results showed DOPE-modified hydroxyapatite NPs had higher uptake than hydroxyapatite NPs (P

Γ-Ionizing radiation activated EGFR-p38/ERK-STAT3/CREB-1-EMT pathway for promotion of the migration/invasion of lung cancer cell and its inhibition by podophyllotoxin acetate

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Cho, Jeong Hyun; Um, Hong Duck; Park, Jong Kuk [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

2016-05-15

In this study, we sought to identify the intracellular machinery responsible for IR induced cancer invasion/migration. We report that IR activates the EGFR - p38/ERK - CREB-1/STAT3 pathway, which triggers EMT and increases invasion/migration of lung cancer. Moreover, we show that podophyllotoxin acetate (PA) inhibits IR-induced invasion/migration at least partly by blocking EGFR - p38/ERK - STAT3/ CREB-1signaling and thereby suppressing EMT. Our results revealed that IR increased the invasion/migration of A549 cells, and this effect was decreased by 10 nM PA treatment. PA also inhibited the expressions/activities of matrix metalloprotase (MMP) -2, MMP-9, and vimentin, suggesting that PA could block the IR-induced epithelial-mesenchymal transition (EMT). The IR induced increases in invasion/migration were associated with the activation of EGFR-AKT, and PA inhibited this effect. P38 and p44/42 ERK were also involved in IR induced invasion/migration, and combined treatments with PA plus inhibitors of each MAPK synergistically blocked this invasion/migration. In terms of transcription factors (TFs), IR-induced increases in cyclic AMP response element-binding protein-1 (CREB-1) and signal transducer and activator of transcription 3 (STAT3) increased invasion/migration and EMT. PA also inhibited these transcription factors and then blocked IR-induced invasion/migration.

Γ-Ionizing radiation activated EGFR-p38/ERK-STAT3/CREB-1-EMT pathway for promotion of the migration/invasion of lung cancer cell and its inhibition by podophyllotoxin acetate

International Nuclear Information System (INIS)

Cho, Jeong Hyun; Um, Hong Duck; Park, Jong Kuk

2016-01-01

In this study, we sought to identify the intracellular machinery responsible for IR induced cancer invasion/migration. We report that IR activates the EGFR - p38/ERK - CREB-1/STAT3 pathway, which triggers EMT and increases invasion/migration of lung cancer. Moreover, we show that podophyllotoxin acetate (PA) inhibits IR-induced invasion/migration at least partly by blocking EGFR - p38/ERK - STAT3/ CREB-1signaling and thereby suppressing EMT. Our results revealed that IR increased the invasion/migration of A549 cells, and this effect was decreased by 10 nM PA treatment. PA also inhibited the expressions/activities of matrix metalloprotase (MMP) -2, MMP-9, and vimentin, suggesting that PA could block the IR-induced epithelial-mesenchymal transition (EMT). The IR induced increases in invasion/migration were associated with the activation of EGFR-AKT, and PA inhibited this effect. P38 and p44/42 ERK were also involved in IR induced invasion/migration, and combined treatments with PA plus inhibitors of each MAPK synergistically blocked this invasion/migration. In terms of transcription factors (TFs), IR-induced increases in cyclic AMP response element-binding protein-1 (CREB-1) and signal transducer and activator of transcription 3 (STAT3) increased invasion/migration and EMT. PA also inhibited these transcription factors and then blocked IR-induced invasion/migration

Neural Plasticity Associated with Hippocampal PKA-CREB and NMDA Signaling Is Involved in the Antidepressant Effect of Repeated Low Dose of Yueju Pill on Chronic Mouse Model of Learned Helplessness

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Zhilu Zou

2017-01-01

Full Text Available Yueju pill is a traditional Chinese medicine formulated to treat syndromes of mood disorders. Here, we investigated the therapeutic effect of repeated low dose of Yueju in the animal model mimicking clinical long-term depression condition and the role of neural plasticity associated with PKA- (protein kinase A- CREB (cAMP response element binding protein and NMDA (N-methyl-D-aspartate signaling. We showed that a single low dose of Yueju demonstrated antidepressant effects in tests of tail suspension, forced swim, and novelty-suppressed feeding. A chronic learned helplessness (LH protocol resulted in a long-term depressive-like condition. Repeated administration of Yueju following chronic LH remarkably alleviated all of depressive-like symptoms measured, whereas conventional antidepressant fluoxetine only showed a minor improvement. In the hippocampus, Yueju and fluoxetine both normalized brain-derived neurotrophic factor (BDNF and PKA level. Only Yueju, not fluoxetine, rescued the deficits in CREB signaling. The chronic LH upregulated the expression of NMDA receptor subunits NR1, NR2A, and NR2B, which were all attenuated by Yueju. Furthermore, intracerebraventricular administration of NMDA blunted the antidepressant effect of Yueju. These findings supported the antidepressant efficacy of repeated routine low dose of Yueju in a long-term depression model and the critical role of CREB and NMDA signaling.

Peripheral reduction of FGFR4 with antisense oligonucleotides increases metabolic rate and lowers adiposity in diet-induced obese mice.

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Xing Xian Yu

Full Text Available Obesity is a primary risk factor for multiple metabolic disorders. Many drugs for the treatment of obesity, which mainly act through CNS as appetite suppressants, have failed during development or been removed from the market due to unacceptable adverse effects. Thus, there are very few efficacious drugs available and remains a great unmet medical need for anti-obesity drugs that increase energy expenditure by acting on peripheral tissues without severe side effects. Here, we report a novel approach involving antisense inhibition of fibroblast growth factor receptor 4 (FGFR4 in peripheral tissues. Treatment of diet-induce obese (DIO mice with FGFR4 antisense oligonucleotides (ASO specifically reduced liver FGFR4 expression that not only resulted in decrease in body weight (BW and adiposity in free-feeding conditions, but also lowered BW and adiposity under caloric restriction. In addition, combination treatment with FGFR4 ASO and rimonabant showed additive reduction in BW and adiposity. FGFR4 ASO treatment increased basal metabolic rate during free-feeding conditions and, more importantly, prevented adaptive decreases of metabolic rate induced by caloric restriction. The treatment increased fatty acid oxidation while decreased lipogenesis in both liver and fat. Mechanistic studies indicated that anti-obesity effect of FGFR4 ASO was mediated at least in part through an induction of plasma FGF15 level resulted from reduction of hepatic FGFR4 expression. The anti-obesity effect was accompanied by improvement in plasma glycemia, whole body insulin sensitivity, plasma lipid levels and liver steatosis. Therefore, FGFR4 could be a potential novel target and antisense reduction of hepatic FGFR4 expression could be an efficacious therapy as an adjunct to diet restriction or to an appetite suppressant for the treatment of obesity and related metabolic disorders.

Peripheral reduction of FGFR4 with antisense oligonucleotides increases metabolic rate and lowers adiposity in diet-induced obese mice.

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Yu, Xing Xian; Watts, Lynnetta M; Manchem, Vara Prasad; Chakravarty, Kaushik; Monia, Brett P; McCaleb, Michael L; Bhanot, Sanjay

2013-01-01

Obesity is a primary risk factor for multiple metabolic disorders. Many drugs for the treatment of obesity, which mainly act through CNS as appetite suppressants, have failed during development or been removed from the market due to unacceptable adverse effects. Thus, there are very few efficacious drugs available and remains a great unmet medical need for anti-obesity drugs that increase energy expenditure by acting on peripheral tissues without severe side effects. Here, we report a novel approach involving antisense inhibition of fibroblast growth factor receptor 4 (FGFR4) in peripheral tissues. Treatment of diet-induce obese (DIO) mice with FGFR4 antisense oligonucleotides (ASO) specifically reduced liver FGFR4 expression that not only resulted in decrease in body weight (BW) and adiposity in free-feeding conditions, but also lowered BW and adiposity under caloric restriction. In addition, combination treatment with FGFR4 ASO and rimonabant showed additive reduction in BW and adiposity. FGFR4 ASO treatment increased basal metabolic rate during free-feeding conditions and, more importantly, prevented adaptive decreases of metabolic rate induced by caloric restriction. The treatment increased fatty acid oxidation while decreased lipogenesis in both liver and fat. Mechanistic studies indicated that anti-obesity effect of FGFR4 ASO was mediated at least in part through an induction of plasma FGF15 level resulted from reduction of hepatic FGFR4 expression. The anti-obesity effect was accompanied by improvement in plasma glycemia, whole body insulin sensitivity, plasma lipid levels and liver steatosis. Therefore, FGFR4 could be a potential novel target and antisense reduction of hepatic FGFR4 expression could be an efficacious therapy as an adjunct to diet restriction or to an appetite suppressant for the treatment of obesity and related metabolic disorders.

The human PINK1 locus is regulated in vivo by a non-coding natural antisense RNA during modulation of mitochondrial function

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Wahlestedt Claes

2007-03-01

Full Text Available Abstract Background Mutations in the PTEN induced putative kinase 1 (PINK1 are implicated in early-onset Parkinson's disease. PINK1 is expressed abundantly in mitochondria rich tissues, such as skeletal muscle, where it plays a critical role determining mitochondrial structural integrity in Drosophila. Results Herein we characterize a novel splice variant of PINK1 (svPINK1 that is homologous to the C-terminus regulatory domain of the protein kinase. Naturally occurring non-coding antisense provides sophisticated mechanisms for diversifying genomes and we describe a human specific non-coding antisense expressed at the PINK1 locus (naPINK1. We further demonstrate that PINK1 varies in vivo when human skeletal muscle mitochondrial content is enhanced, supporting the idea that PINK1 has a physiological role in mitochondrion. The observation of concordant regulation of svPINK1 and naPINK1 during in vivo mitochondrial biogenesis was confirmed using RNAi, where selective targeting of naPINK1 results in loss of the PINK1 splice variant in neuronal cell lines. Conclusion Our data presents the first direct observation that a mammalian non-coding antisense molecule can positively influence the abundance of a cis-transcribed mRNA under physiological abundance conditions. While our analysis implies a possible human specific and dsRNA-mediated mechanism for stabilizing the expression of svPINK1, it also points to a broader genomic strategy for regulating a human disease locus and increases the complexity through which alterations in the regulation of the PINK1 locus could occur.

2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside confers neuroprotection in cell and animal models of ischemic stroke through calpain1/PKA/CREB-mediated induction of neuronal glucose transporter 3

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Yu, Shu; Cheng, Qiong; Li, Lu; Liu, Mei; Yang, Yumin; Ding, Fei, E-mail: dingfei@ntu.edu.cn

2014-06-15

Salidroside is proven to be a neuroprotective agent of natural origin, and its analog, 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside (named SalA-4 g), has been synthesized in our lab. In this study, we showed that SalA-4 g promoted neuronal survival and inhibited neuronal apoptosis in primary hippocampal neurons exposed to oxygen and glucose deprivation (OGD) and in rats subjected to ischemia by transient middle cerebral artery occlusion (MCAO), respectively, and that SalA-4 g was more neuroprotective than salidroside. We further found that SalA-4 g elevated glucose uptake in OGD-injured primary hippocampal neurons and increased the expression and recruitment of glucose transporter 3 (GLUT3) in ischemic brain. Signaling analysis revealed that SalA-4 g triggered the phosphorylation of CREB, and increased the expression of PKA RII in primary hippocampal neurons exposed to OGD injury, while inhibition of PKA/CREB by H-89 alleviated the elevation in glucose uptake and GLUT3 expression, and blocked the protective effects of SalA-4 g. Moreover, SalA-4 g was noted to inhibit intracellular Ca{sup 2+} influx and calpain1 activation in OGD-injured primary hippocampal neurons. Our results suggest that SalA-4 g neuroprotection might be mediated by increased glucose uptake and elevated GLUT3 expression through calpain1/PKA/CREB pathway. - Highlights: • A salidroside (Sal) analog (SalA-4 g) is prepared to be more neuroprotective than Sal. • SalA-4 g protected hippocampal neurons from oxygen and glucose deprivation insult. • SalA-4 g reduced ischemic injury after transient middle cerebral artery occlusion in rats. • Neuroprotection of SalA-4 g was mediated by GLUT3 level via calpain/PKA/CREB pathway.

2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside confers neuroprotection in cell and animal models of ischemic stroke through calpain1/PKA/CREB-mediated induction of neuronal glucose transporter 3

International Nuclear Information System (INIS)

Yu, Shu; Cheng, Qiong; Li, Lu; Liu, Mei; Yang, Yumin; Ding, Fei

2014-01-01

Salidroside is proven to be a neuroprotective agent of natural origin, and its analog, 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside (named SalA-4 g), has been synthesized in our lab. In this study, we showed that SalA-4 g promoted neuronal survival and inhibited neuronal apoptosis in primary hippocampal neurons exposed to oxygen and glucose deprivation (OGD) and in rats subjected to ischemia by transient middle cerebral artery occlusion (MCAO), respectively, and that SalA-4 g was more neuroprotective than salidroside. We further found that SalA-4 g elevated glucose uptake in OGD-injured primary hippocampal neurons and increased the expression and recruitment of glucose transporter 3 (GLUT3) in ischemic brain. Signaling analysis revealed that SalA-4 g triggered the phosphorylation of CREB, and increased the expression of PKA RII in primary hippocampal neurons exposed to OGD injury, while inhibition of PKA/CREB by H-89 alleviated the elevation in glucose uptake and GLUT3 expression, and blocked the protective effects of SalA-4 g. Moreover, SalA-4 g was noted to inhibit intracellular Ca 2+ influx and calpain1 activation in OGD-injured primary hippocampal neurons. Our results suggest that SalA-4 g neuroprotection might be mediated by increased glucose uptake and elevated GLUT3 expression through calpain1/PKA/CREB pathway. - Highlights: • A salidroside (Sal) analog (SalA-4 g) is prepared to be more neuroprotective than Sal. • SalA-4 g protected hippocampal neurons from oxygen and glucose deprivation insult. • SalA-4 g reduced ischemic injury after transient middle cerebral artery occlusion in rats. • Neuroprotection of SalA-4 g was mediated by GLUT3 level via calpain/PKA/CREB pathway

Depression, anxiety-like behavior and memory impairment are associated with increased oxidative stress and inflammation in a rat model of social stress.

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Patki, Gaurav; Solanki, Naimesh; Atrooz, Fatin; Allam, Farida; Salim, Samina

2013-11-20

In the present study, we have examined the behavioral and biochemical effect of induction of psychological stress using a modified version of the resident-intruder model for social stress (social defeat). At the end of the social defeat protocol, body weights, food and water intake were recorded, depression and anxiety-like behaviors as well as memory function was examined. Biochemical analysis including oxidative stress measurement, inflammatory markers and other molecular parameters, critical to behavioral effects were examined. We observed a significant decrease in the body weight in the socially defeated rats as compared to the controls. Furthermore, social defeat increased anxiety-like behavior and caused memory impairment in rats (PSocially defeated rats made significantly more errors in long term memory tests (Psocially defeated rats, when compared to control rats. We suggest that social defeat stress alters ERK1/2, IL-6, GLO1, GSR1, CAMKIV, CREB, and BDNF levels in specific brain areas, leading to oxidative stress-induced anxiety-depression-like behaviors and as well as memory impairment in rats. © 2013 Published by Elsevier B.V.

Quantitative Analysis of Survivin Protein Expression and Its Therapeutic Depletion by an Antisense Oligonucleotide in Human Lung Tumors

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Anna L Olsen

2012-01-01

Full Text Available RNA-directed antisense and interference therapeutics are a promising treatment option for cancer. The demonstration of depletion of target proteins within human tumors in vivo using validated methodology will be a key to the application of this technology. Here, we present a flow cytometric-based approach to quantitatively determine protein levels in solid tumor material derived by fiber optic brushing (FOB of non-small cell lung cancer (NSCLC patients. Focusing upon the survivin protein, and its depletion by an antisense oligonucleotide (ASO (LY2181308, we show that we can robustly identify a subpopulation of survivin positive tumor cells in FOB samples, and, moreover, detect survivin depletion in tumor samples from a patient treated with LY2181308. Survivin depletion appears to be a result of treatment with this ASO, because a tumor treated with conventional cytotoxic chemotherapy did not exhibit a decreased percentage of survivin positive cells. Our approach is likely to be broadly applicable to, and useful for, the quantification of protein levels in tumor samples obtained as part of clinical trials and studies, facilitating the proof-of-principle testing of novel targeted therapies.

Estrogen receptor α L429 and A430 regulate 17β-estradiol-induced cell proliferation via CREB1.

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Pesiri, Valeria; Totta, Pierangela; Segatto, Marco; Bianchi, Fabrizio; Pallottini, Valentina; Marino, Maria; Acconcia, Filippo

2015-12-01

17β-Estradiol (E2)-dependent cell proliferation requires both estrogen receptor α (ERα)-based integrated control of gene transcription and kinase pathways activation. Such coordination of intracellular E2:ERα-dependent signaling mechanisms is finely tuned by receptor association with specific partner proteins. Recently, we identified the leucine (L) 429 and alanine (A) 430 within the ERα ligand binding domain as important residues for receptor non-covalent interaction to ubiquitinated species [i.e., ERα ubiquitin-binding surface (ERα UBS)] and for E2-induced ERα activation. To date, if these two ERα amino acids are involved in the control of E2-dependent pathways required for cell proliferation is unknown. Here, by using stably expressing ERα mutated in L429 and A430 (i.e., L429A,A430G-LAAG) cell lines, we show that L429 and A430 are critical for E2-induced cell proliferation, PI3K/AKT pathway activation, and ERα-mediated transcriptional changes. Moreover, we demonstrate that these two receptor structural determinants direct the E2-induced PI3K/AKT/CREB1 pathway activation and CREB1-mediated transcriptional activity that in turn control the hormone-induced cell proliferation. As a whole, our data demonstrate for the first time that the ERα UBS contributes to the modulation of E2-induced ERα-mediated cell proliferation and provide a novel connection between the receptor structure and the functional molecular mechanisms by which E2:ERα complex can regulate cell processes. Copyright © 2015 Elsevier Inc. All rights reserved.

Piper sarmentosum Roxb. produces antidepressant-like effects in rodents, associated with activation of the CREB-BDNF-ERK signaling pathway and reversal of HPA axis hyperactivity.

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Li, Qing; Qu, Fa-Lin; Gao, Yue; Jiang, Yi-Ping; Rahman, Khalid; Lee, Kuo-Hsiung; Han, Ting; Qin, Lu-Ping

2017-03-06

There are many plants of genus Piper which have been reported to induce antidepressant-like effects, Piper sarmentosum (PS) is one of them. PS is a Chinese herbal medicine and a traditional edible vegetable. In the present study, the antidepressant-like effects of PS extracts and the ethyl acetate fraction of PS extracts (PSY) were assessed using the open field test (OFT), forced swimming test (FST), and tail suspension test (TST) in mice. Furthermore, we applied a 4 consecutive weeks of chronic unpredictable mild stress (CUMS) as a model of depression in rats, followed by a sucrose preference test. Then we examined the possible mechanisms of this action. The activity of the hypothalamic-pituitary-adrenal (HPA) axis was evaluated by detecting the serum corticosterone (CORT) concentrations, and the protein expression levels of brain-derived neurotrophic factor (BDNF), the phosphorylated form CREB and ERK1/2 were detected by qRT-PCR or Western blot. The results showed that PS extracts (100, 200mg/kg) and PSY (12.5, 25, 50mg/kg) treatment produced antidepressant-like effects in mice similar to fluoxetine (20mg/kg), indicated by the reduced immobility time in the FST and TST, while both had no influence on the locomotor activity in the OFT. PSY treatment significantly increased sucrose preference and reduced serum CORT levels in CUMS rats. Moreover, PSY up-regulated BDNF protein levels, and increased CREB and ERK phosphorylation levels in the hippocampus on CUMS rats. These findings suggest that the antidepressant-like effects of PS extracts and PSY are mediated, at least in part, by modulating HPA axis, BDNF, CREB and ERK phosphorylation and expression in the hippocampus. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Upregulated miR-132 in Lgr5+ gastric cancer stem cell-like cells contributes to cisplatin-resistance via SIRT1/CREB/ABCG2 signaling pathway.

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Zhang, Lanfang; Guo, Xiaohe; Zhang, Dezhong; Fan, Yingying; Qin, Lei; Dong, Shuping; Zhang, Lanfang

2017-09-01

Cisplatin resistance has long been a major problem that restricts its use. A novel paradigm in tumor biology suggests that gastric tumor chemo-resistance is driven by gastric cancer stem cell-like (GCSCs). Growing evidence has indicated that microRNAs (miRNAs) contributes to chemo-resistance in gastric cancer (GC). Here, Lgr5 + cells derived from gastric cancer cell lines displayed stem cell-like features. Flow cytometry demonstrated the presence of a variable fraction of Lgr5 in 19 out of 20 GC specimens. By comparing the miRNA expression profiles of Lgr5 + GCSCs and Lrg5 - cells, we established the upregulation of miR-132 in Lgr5 + GCSCs. The enhanced miR-132 expression correlated chemo-resistance in GC patients. Kaplan-Meier survival curve showed that patients with low miR-132 expression survived obviously longer. Functional assays results indicated that miR-132 promoted cisplatin resistance in Lgr5 + GCSCs in vitro and in vivo. Further dual-luciferase reporter gene assays revealed that SIRT1 was the direct target of miR-132. The expression of miR-132 was inversely correlated with SIRT1 in gastric cancer specimens. Furthermore, through PCR array we discovered ABCG2 was one of the downstream targets of SIRT1. Overexpression of SIRT1 down-regulated ABCG2 expression by promoting the de-acetylation of the transcription factor CREB. CREB was further activated ABCG2 via binding to the promoter of ABCG2 to induce transcription. Thus, we concluded that miR-132 regulated SIRT1/CREB/ABCG2 signaling pathway contributing to the cisplatin resistance and might serve as a novel therapeutic target against gastric cancer. © 2017 Wiley Periodicals, Inc.

Widespread anti-sense transcription in apple is correlated with siRNA production and indicates a large potential for transcriptional and/or post-transcriptional control.

Science.gov (United States)

Celton, Jean-Marc; Gaillard, Sylvain; Bruneau, Maryline; Pelletier, Sandra; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Navarro, Lionel; Laurens, François; Renou, Jean-Pierre

2014-07-01

Characterizing the transcriptome of eukaryotic organisms is essential for studying gene regulation and its impact on phenotype. The realization that anti-sense (AS) and noncoding RNA transcription is pervasive in many genomes has emphasized our limited understanding of gene transcription and post-transcriptional regulation. Numerous mechanisms including convergent transcription, anti-correlated expression of sense and AS transcripts, and RNAi remain ill-defined. Here, we have combined microarray analysis and high-throughput sequencing of small RNAs (sRNAs) to unravel the complexity of transcriptional and potential post-transcriptional regulation in eight organs of apple (Malus × domestica). The percentage of AS transcript expression is higher than that identified in annual plants such as rice and Arabidopsis thaliana. Furthermore, we show that a majority of AS transcripts are transcribed beyond 3'UTR regions, and may cover a significant portion of the predicted sense transcripts. Finally we demonstrate at a genome-wide scale that anti-sense transcript expression is correlated with the presence of both short (21-23 nt) and long (> 30 nt) siRNAs, and that the sRNA coverage depth varies with the level of AS transcript expression. Our study provides a new insight on the functional role of anti-sense transcripts at the genome-wide level, and a new basis for the understanding of sRNA biogenesis in plants. © 2014 INRA. New Phytologist © 2014 New Phytologist Trust.

Dexamethasone-induced and estradiol-induced CREB activation and annexin 1 expression in CCRF-CEM lymphoblastic cells: evidence for the involvement of cAMP and p38 MAPK

Directory of Open Access Journals (Sweden)

M. Castro-caldas

2003-01-01

Full Text Available Aims: Annexin 1 (ANXA1, a member of the annexin family of calcium-binding and phospholipid-binding proteins, is a key mediator of the anti-inflammatory actions of steroid hormones. We have previously demonstrated that, in the human lymphoblastic CCRF-CEM cell line, both the synthetic glucocorticoid hormone, dexamethasone (Dex, and the estrogen hormone, 17β-estradiol (E2β, induce the synthesis of ANXA1, by a mechanism independent of the activation of their nuclear receptors. Recently, it was reported that the gene coding for ANXA1 contains a cAMP-responsive element (CRE. In this work, we investigated whether Dex and E2β were able to induce the activation of CRE binding proteins (CREB in the CCRF-CEM cells. Moreover, we studied the intracellular signalling pathways involved in CREB activation and ANXA1 synthesis in response to Dex and E2β; namely, the role of cAMP and the p38 mitogen-activated protein kinase (MAPK.

Long-term Exon Skipping Studies With 2′-O-Methyl Phosphorothioate Antisense Oligonucleotides in Dystrophic Mouse Models

Directory of Open Access Journals (Sweden)

Christa L Tanganyika-de Winter

2012-01-01

Full Text Available Antisense-mediated exon skipping for Duchenne muscular dystrophy (DMD is currently tested in phase 3 clinical trials. The aim of this approach is to modulate splicing by skipping a specific exon to reframe disrupted dystrophin transcripts, allowing the synthesis of a partly functional dystrophin protein. Studies in animal models allow detailed analysis of the pharmacokinetic and pharmacodynamic profile of antisense oligonucleotides (AONs. Here, we tested the safety and efficacy of subcutaneously administered 2′-O-methyl phosphorothioate AON at 200 mg/kg/week for up to 6 months in mouse models with varying levels of disease severity: mdx mice (mild phenotype and mdx mice with one utrophin allele (mdx/utrn+/−; more severe phenotype. Long-term treatment was well tolerated and exon skipping and dystrophin restoration confirmed for all animals. Notably, in the more severely affected mdx/utrn+/− mice the therapeutic effect was larger: creatine kinase (CK levels were more decreased and rotarod running time was more increased. This suggests that the mdx/utrn+/− model may be a more suitable model to test potential therapies than the regular mdx mouse. Our results also indicate that long-term subcutaneous treatment in dystrophic mouse models with these AONs is safe and beneficial.

Divergently overlapping cis-encoded antisense RNA regulating toxin-antitoxin systems from E. coli: hok/sok, ldr/rdl, symE/symR.

Science.gov (United States)

Kawano, Mitsuoki

2012-12-01

Toxin-antitoxin (TA) systems are categorized into three classes based on the type of antitoxin. In type I TA systems, the antitoxin is a small antisense RNA that inhibits translation of small toxic proteins by binding to the corresponding mRNAs. Those type I TA systems were originally identified as plasmid stabilization modules rendering a post-segregational killing (PSK) effect on the host cells. The type I TA loci also exist on the Escherichia coli chromosome but their biological functions are less clear. Genetic organization and regulatory elements of hok/sok and ldr/rdl families are very similar and the toxins are predicted to contain a transmembrane domain, but otherwise share no detectable sequence similarity. This review will give an overview of the type I TA modules of E. coli K-12, especially hok/sok, ldr/rdl and SOS-inducible symE/symR systems, which are regulated by divergently overlapping cis-encoded antisense RNAs.

Electroacupuncture Ameliorates Learning and Memory via Activation of the CREB Signaling Pathway in the Hippocampus to Attenuate Apoptosis after Cerebral Hypoperfusion

OpenAIRE

Han, Xiaohua; Zhao, Xiuxiu; Lu, Min; Liu, Fang; Guo, Feng; Zhang, Jinghui; Huang, Xiaolin

2013-01-01

Studies have shown that electroacupuncture (EA) ameliorates learning and memory after ischemic injury. However, there have been few studies elucidating the mechanisms of EA on learning and memory in cerebral hypoperfusion. In this study, we explored the cAMP response element-binding protein (CREB) signaling pathway-mediated antiapoptotic action involved in EA-induced improvement of learning and memory. EA at GV20 and GV14 acupoints was applied in cerebral hypoperfusion rats. A Morris water ma...

Hydrogel-Assisted Antisense LNA Gapmer Delivery for In Situ Gene Silencing in Spinal Cord Injury

DEFF Research Database (Denmark)

Moreno, Pedro M.D.; Ferreira, Ana R.; Salvador, Daniela

2018-01-01

)-modified AON gapmers in combination with a fibrin hydrogel bridging material to induce gene silencing in situ at a SCI lesion site. LNA gapmers were effectively developed against two promising gene targets aiming at enhancing axonal regeneration—RhoA and GSK3β. The fibrin-matrix-assisted AON delivery system......After spinal cord injury (SCI), nerve regeneration is severely hampered due to the establishment of a highly inhibitory microenvironment at the injury site, through the contribution of multiple factors. The potential of antisense oligonucleotides (AONs) to modify gene expression at different levels...

Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels.

Science.gov (United States)

Gschwentner, M; Nagl, U O; Wöll, E; Schmarda, A; Ritter, M; Paulmichl, M

1995-08-01

Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.

PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

Science.gov (United States)

Zinker, Bradley A.; Rondinone, Cristina M.; Trevillyan, James M.; Gum, Rebecca J.; Clampit, Jill E.; Waring, Jeffrey F.; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E.; Reilly, Regina M.; Koterski, Sandra; Opgenorth, Terry J.; Ulrich, Roger G.; Crosby, Seth; Butler, Madeline; Murray, Susan F.; McKay, Robert A.; Bhanot, Sanjay; Monia, Brett P.; Jirousek, Michael R.

2002-01-01

The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes. PMID:12169659

Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

KAUST Repository

Ková cs, Krisztiá n A.; Steinmann, Myriam; Halfon, Olivier; Magistretti, Pierre J.; Cardinaux, Jean René

2015-01-01

CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

Complex regulation of CREB-binding protein by homeodomain-interacting protein kinase 2

KAUST Repository

Kovács, Krisztián A.

2015-11-01

CREB-binding protein (CBP) and p300 are transcriptional coactivators involved in numerous biological processes that affect cell growth, transformation, differentiation, and development. In this study, we provide evidence of the involvement of homeodomain-interacting protein kinase 2 (HIPK2) in the regulation of CBP activity. We show that HIPK2 interacts with and phosphorylates several regions of CBP. We demonstrate that serines 2361, 2363, 2371, 2376, and 2381 are responsible for the HIPK2-induced mobility shift of CBP C-terminal activation domain. Moreover, we show that HIPK2 strongly potentiates the transcriptional activity of CBP. However, our data suggest that HIPK2 activates CBP mainly by counteracting the repressive action of cell cycle regulatory domain 1 (CRD1), located between amino acids 977 and 1076, independently of CBP phosphorylation. Our findings thus highlight a complex regulation of CBP activity by HIPK2, which might be relevant for the control of specific sets of target genes involved in cellular proliferation, differentiation and apoptosis. © 2015 Elsevier Inc.

An antisense oligodeoxynucleotide targeted against the type IIβ regulatory subunit mRNA of protein kinase inhibits cAMP-induced differentiation in HL-60 leukemia cells without affecting phorbol ester effects

International Nuclear Information System (INIS)

Tortora, G.; Clair, T.; Cho-Chung, Y.S.

1990-01-01

The type II β regulatory subunit of cAMP-dependent protein kinase (RII β ) has been hypothesized to play an important role in the growth inhibition and differentiation induced by site-selective cAMP analogs in human cancer cells, but direct proof of this function has been lacking. To address this tissue, HL-60 human promyelocytic leukemia cells were exposed to RII β antisense synthetic oligodeoxynucleotide, and the effects on cAMP-induced growth regulation were examined. Exposure of these cells to RII β antisense oligodeoxynucleotide resulted in a decrease in cAMP analog-induced growth inhibition and differentiation without apparent effect on differentiation induced by phorbol esters. This loss in cAMP growth regulatory function correlated with a decrease in basal and induced levels of RII β protein. Exposure to RII β sense, RI α and RII α antisense, or irrelevant oligodeoxynucleotides had no such effect. These results show that the RII β regulatory subunit of protein kinase plays a critical role in the cAMP-induced growth regulation of HL-60 leukemia cells

Antisense oligonucleotide therapy rescues disruptions in organization of exploratory movements associated with Usher syndrome type 1C in mice.

Science.gov (United States)

Donaldson, Tia N; Jennings, Kelsey T; Cherep, Lucia A; McNeela, Adam M; Depreux, Frederic F; Jodelka, Francine M; Hastings, Michelle L; Wallace, Douglas G

2018-02-15

Usher syndrome, Type 1C (USH1C) is an autosomal recessive inherited disorder in which a mutation in the gene encoding harmonin is associated with multi-sensory deficits (i.e., auditory, vestibular, and visual). USH1C (Usher) mice, engineered with a human USH1C mutation, exhibit these multi-sensory deficits by circling behavior and lack of response to sound. Administration of an antisense oligonucleotide (ASO) therapeutic that corrects expression of the mutated USH1C gene, has been shown to increase harmonin levels, reduce circling behavior, and improve vestibular and auditory function. The current study evaluates the organization of exploratory movements to assess spatial organization in Usher mice and determine the efficacy of ASO therapy in attenuating any such deficits. Usher and heterozygous mice received the therapeutic ASO, ASO-29, or a control, non-specific ASO treatment at postnatal day five. Organization of exploratory movements was assessed under dark and light conditions at two and six-months of age. Disruptions in exploratory movement organization observed in control-treated Usher mice were consistent with impaired use of self-movement and environmental cues. In general, ASO-29 treatment rescued organization of exploratory movements at two and six-month testing points. These observations are consistent with ASO-29 rescuing processing of multiple sources of information and demonstrate the potential of ASO therapies to ameliorate topographical disorientation associated with other genetic disorders. Copyright © 2017 Elsevier B.V. All rights reserved.

Regulatory motifs for CREB-binding protein and Nfe2l2 transcription factors in the upstream enhancer of the mitochondrial uncoupling protein 1 gene.

Science.gov (United States)

Rim, Jong S; Kozak, Leslie P

2002-09-13

Thermogenesis against cold exposure in mammals occurs in brown adipose tissue (BAT) through mitochondrial uncoupling protein (UCP1). Expression of the Ucp1 gene is unique in brown adipocytes and is regulated tightly. The 5'-flanking region of the mouse Ucp1 gene contains cis-acting elements including PPRE, TRE, and four half-site cAMP-responsive elements (CRE) with BAT-specific enhancer elements. In the course of analyzing how these half-site CREs are involved in Ucp1 expression, we found that a DNA regulatory element for NF-E2 overlaps CRE2. Electrophoretic mobility shift assay and competition assays with the CRE2 element indicates that nuclear proteins from BAT, inguinal fat, and retroperitoneal fat tissue interact with the CRE2 motif (CGTCA) in a specific manner. A supershift assay using an antibody against the CRE-binding protein (CREB) shows specific affinity to the complex from CRE2 and nuclear extract of BAT. Additionally, Western blot analysis for phospho-CREB/ATF1 shows an increase in phosphorylation of CREB/ATF1 in HIB-1B cells after norepinephrine treatment. Transient transfection assay using luciferase reporter constructs also indicates that the two half-site CREs are involved in transcriptional regulation of Ucp1 in response to norepinephrine and cAMP. We also show that a second DNA regulatory element for NF-E2 is located upstream of the CRE2 region. This element, which is found in a similar location in the 5'-flanking region of the human and rodent Ucp1 genes, shows specific binding to rat and human NF-E2 by electrophoretic mobility shift assay with nuclear extracts from brown fat. Co-transfections with an Nfe2l2 expression vector and a luciferase reporter construct of the Ucp1 enhancer region provide additional evidence that Nfe2l2 is involved in the regulation of Ucp1 by cAMP-mediated signaling.

REM sleep enhancement and behavioral cataplexy following orexin (hypocretin)-II receptor antisense perfusion in the pontine reticular formation.

Science.gov (United States)

Thakkar, M M; Ramesh, V; Cape, E G; Winston, S; Strecker, R E; McCarley, R W

1999-01-01

Orexin (hypocretin)-containing neurons of the hypothalamus project to brainstem sites that are involved in the neural control of REM sleep, including the locus coeruleus, the dorsal raphe nucleus, the cholinergic zone of the mesopontine tegmentum, and the pontine reticular formation (PRF). Orexin knockout mice exhibit narcolepsy/cataplexy, and a mutant and defective gene for the orexin type II receptor is present in dogs with an inherited form of narcolepsy/cataplexy. However, the physiological systems mediating these effects have not been described. We reasoned that, since the effector neurons for the majority of REM sleep signs, including muscle atonia, were located in the PRF, this region was likely implicated in the production of these orexin-related abnormalities. To test this possibility, we used microdialysis perfusion of orexin type II receptor antisense in the PRF of rats. Ten to 24 hours after antisense perfusion, REM sleep increased two- to three-fold during both the light period (quiescent phase) and the dark period (active phase), and infrared video showed episodes of behavioral cataplexy. Moreover, preliminary data indicated no REM-related effects following perfusion with nonsense DNA, or when perfusion sites were outside the PRF. More work is needed to provide precise localization of the most effective site of orexin-induced inhibition of REM sleep phenomena.

Rescue of peripheral vestibular function in Usher syndrome mice using a splice-switching antisense oligonucleotide.

Science.gov (United States)

Vijayakumar, Sarath; Depreux, Frederic F; Jodelka, Francine M; Lentz, Jennifer J; Rigo, Frank; Jones, Timothy A; Hastings, Michelle L

2017-09-15

Usher syndrome type 1C (USH1C/harmonin) is associated with profound retinal, auditory and vestibular dysfunction. We have previously reported on an antisense oligonucleotide (ASO-29) that dramatically improves auditory function and balance behavior in mice homozygous for the harmonin mutation Ush1c c.216G > A following a single systemic administration. The findings were suggestive of improved vestibular function; however, no direct vestibular assessment was made. Here, we measured vestibular sensory evoked potentials (VsEPs) to directly assess vestibular function in Usher mice. We report that VsEPs are absent or abnormal in Usher mice, indicating profound loss of vestibular function. Strikingly, Usher mice receiving ASO-29 treatment have normal or elevated vestibular response thresholds when treated during a critical period between postnatal day 1 and 5, respectively. In contrast, treatment of mice with ASO-29 treatment at P15 was minimally effective at rescuing vestibular function. Interestingly, ASO-29 treatment at P1, P5 or P15 resulted in sufficient vestibular recovery to support normal balance behaviors, suggesting a therapeutic benefit to balance with ASO-29 treatment at P15 despite the profound vestibular functional deficits that persist with treatment at this later time. These findings provide the first direct evidence of an effective treatment of peripheral vestibular function in a mouse model of USH1C and reveal the potential for using antisense technology to treat vestibular dysfunction. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Specific RNP capture with antisense LNA/DNA mixmers.

Science.gov (United States)

Rogell, Birgit; Fischer, Bernd; Rettel, Mandy; Krijgsveld, Jeroen; Castello, Alfredo; Hentze, Matthias W

2017-08-01

RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins. © 2017 Rogell et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

The antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 prevents premature expression of the flv4-2 operon upon shift in inorganic carbon supply.

Science.gov (United States)

Eisenhut, Marion; Georg, Jens; Klähn, Stephan; Sakurai, Isamu; Mustila, Henna; Zhang, Pengpeng; Hess, Wolfgang R; Aro, Eva-Mari

2012-09-28

The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (C(i)), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the Q(B) site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by C(i) limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in C(i) conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon.

The Antisense RNA As1_flv4 in the Cyanobacterium Synechocystis sp. PCC 6803 Prevents Premature Expression of the flv4-2 Operon upon Shift in Inorganic Carbon Supply*

Science.gov (United States)

Eisenhut, Marion; Georg, Jens; Klähn, Stephan; Sakurai, Isamu; Mustila, Henna; Zhang, Pengpeng; Hess, Wolfgang R.; Aro, Eva-Mari

2012-01-01

The functional relevance of natural cis-antisense transcripts is mostly unknown. Here we have characterized the association of three antisense RNAs and one intergenically encoded noncoding RNA with an operon that plays a crucial role in photoprotection of photosystem II under low carbon conditions in the cyanobacterium Synechocystis sp. PCC 6803. Cyanobacteria show strong gene expression dynamics in response to a shift of cells from high carbon to low levels of inorganic carbon (Ci), but the regulatory mechanisms are poorly understood. Among the most up-regulated genes in Synechocystis are flv4, sll0218, and flv2, which are organized in the flv4-2 operon. The flavodiiron proteins encoded by this operon open up an alternative electron transfer route, likely starting from the QB site in photosystem II, under photooxidative stress conditions. Our expression analysis of cells shifted from high carbon to low carbon demonstrated an inversely correlated transcript accumulation of the flv4-2 operon mRNA and one antisense RNA to flv4, designated as As1_flv4. Overexpression of As1_flv4 led to a decrease in flv4-2 mRNA. The promoter activity of as1_flv4 was transiently stimulated by Ci limitation and negatively regulated by the AbrB-like transcription regulator Sll0822, whereas the flv4-2 operon was positively regulated by the transcription factor NdhR. The results indicate that the tightly regulated antisense RNA As1_flv4 establishes a transient threshold for flv4-2 expression in the early phase after a change in Ci conditions. Thus, it prevents unfavorable synthesis of the proteins from the flv4-2 operon. PMID:22854963

Simultaneous Expression from Both the Sense and Antisense Strand of the Erythropoietin Receptor Gene Mitigates Acute Lung Injury

Science.gov (United States)

2017-09-01

concept efficacy that increasing EpoR or RopE expression by cDNA delivery to lung cells in vitro enhances cytoprotection against hyperoxia-induced injury...oxidative damage, cell culture, rodent model, inhalation cDNA delivery, sense and antisense erythropoietin receptor transcripts 16. SECURITY...prevention of acute lung injury. 1-6 50% Subtask 1: Prepare plasmid cDNA of EpoR and RopE in nanoparticle formulation. 1 Completed 06.2017 Subtask 2

Inhibiting the growth of methicillin-resistant Staphylococcus aureus in vitro with antisense peptide nucleic acid conjugates targeting the ftsZ gene

Directory of Open Access Journals (Sweden)

Shumei Liang

2015-01-01

Conclusion: Our results demonstrate that the potent effects of PNAs on bacterial growth and cell viability were mediated by the down-regulation or even knock-out of ftsZ gene expression. This highlights the utility of ftsZ as a promising target for the development of new antisense antibacterial agents to treat MRSA infections.

Decreased Hippocampal Neuroplasticity and Behavioral Impairment in an Animal Model of Inhalant Abuse

Directory of Open Access Journals (Sweden)

Hanaa Malloul

2018-02-01

Full Text Available Thinners are highly toxic chemicals widely employed as organic solvents in industrial and domestic use. They have psychoactive properties when inhaled, and their chronic abuse as inhalants is associated with severe long-term health effects, including brain damage and cognitive-behavioral alterations. Yet, the sites and mechanisms of action of these compounds on the brain are far from being fully understood. Here, we investigated the consequences of paint thinner inhalation in adult male mice. Depression-like behaviors and an anxiolytic effect were found following repeated exposure in chronic treatments lasting 12 weeks. Both subchronic (6 weeks and chronic treatments impaired learning and memory functions, while no changes were observed after acute treatment. To investigate possible molecular/structural alterations underlying such behavioral changes, we focused on the hippocampus. Notably, prolonged, but not acute thinner inhalation strongly affected adult neurogenesis in the dentate gyrus (DG, reducing progenitor cell proliferation after chronic treatments and impairing the survival of newborn neurons following both chronic and subchronic treatments. Furthermore, a down-regulation in the expression of BDNF and NMDA receptor subunits as well as a reduction in CREB expression/phosphorylation were found in the hippocampi of chronically treated mice. Our findings demonstrate for the first time significant structural and molecular changes in the adult hippocampus after prolonged paint thinner inhalation, indicating reduced hippocampal neuroplasticity and strongly supporting its implication in the behavioral dysfunctions associated to inhalant abuse.

Growth inhibition of human pancreatic cancer cells by lipofection mediated IGF-1R antisense oligodeoxynucletides in combination with ionizing radiation

International Nuclear Information System (INIS)

Pan Yaozhen; Sun Chengyi; Wang Yuzhi

2004-01-01

Objective: To study the growth inhibition of human pancreatic cancer cells (PC-3) by lipofection-mediated and ionizing radiation improving transfection of IGF-1R antisense oligodeoxynucletides (ASON) in vitro. Methods: Colonigenicity of PC-3 cells in vitro after 60 Co γ-radiation was observed for ascertaining their radiosensitivity and optimal radiation dose was selected according to the radiation sensitivity. PC-3 cells were transfected by two ways: 1) by lipofection-mediated IGF-1R ASON combined with ionizing radiation. 2) by lipo-ASON alone without ionizing radiation. Cell growth was assessed by MTT method. The expression of IGF-1R at mRNA level was examined by RT-PCR. Flow cytometry was used to demonstrate apoptotic changes in lipo-ASON-treated cells. Results: The inhibitory efficiency of lipo-ASON combined with ionizing radiation was higher than that without ionizing radiation (P < 0.05). The apoptotic efficiency and the decreased level of IGF-1R at mRNA were significantly improved (P < 0.05). Conclusion: Lipofection-mediated and ionizing radiation-promoted transfection of IGF-1R antisense oligodeoxynucletides (ASON) significantly decreases IGF-1R at mRNA level and induces apoptosis of human pancreatic cancer cells in vitro

Depletion of key protein components of the RISC pathway impairs pre-ribosomal RNA processing.

Science.gov (United States)

Liang, Xue-Hai; Crooke, Stanley T

2011-06-01

Little is known about whether components of the RNA-induced silencing complex (RISC) mediate the biogenesis of RNAs other than miRNA. Here, we show that depletion of key proteins of the RISC pathway by antisense oligonucleotides significantly impairs pre-rRNA processing in human cells. In cells depleted of Drosha or Dicer, different precursors to 5.8S rRNA strongly accumulated, without affecting normal endonucleolytic cleavages. Moderate yet distinct processing defects were also observed in Ago2-depleted cells. Physical links between pre-rRNA and these proteins were identified by co-immunoprecipitation analyses. Interestingly, simultaneous depletion of Dicer and Drosha led to a different processing defect, causing slower production of 28S rRNA and its precursor. Both Dicer and Ago2 were detected in the nuclear fraction, and reduction of Dicer altered the structure of the nucleolus, where pre-rRNA processing occurs. Together, these results suggest that Drosha and Dicer are implicated in rRNA biogenesis.

CREB mediates ICAM-3: inducing radio-resistance, cell growth and migration/invasion of the human nonsmall cell lung cancer cell

Energy Technology Data Exchange (ETDEWEB)

Park, Jong Kuk; So, Kwang Sup; Bae, In Hwa; Um, Hong Duck [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

2009-05-15

The ICAM family proteins comprises cell surface molecules that are homologous to NCAM and are members of the single passed type 1 immunoglobulin superfamily (IgSF) that are anchored at the cellular membrane. The ICAM family consists of five subfamilies (ICAM-1 to ICAM-5) of heavily glycosylated cell surface receptors with common functional or structural homology. The extracellular domains of ICAM protein have roles in immune response and inflammation through various cell-cell interactions. The cytoplasmic tail residues of ICAM-3 participate in intracellular signaling such as calcium mobilization and tyrosine phosphorylation. Interestingly, the ICAM proteins appear to have a dual role in cancer. ICAM molecules may target and block tumor progression by stimulation of an immune response such as leukocyte activation. Conversely, other investigations have shown that ICAM molecules are involved in cancer malignancy because their increased expressions are associated with a poor diagnosis, lower survival rates and invasion in several cancers including melanoma, breast cancer and leukemia. We have also reported that an increase of ICAM-3 expression in several cancer cells and specimens of cervical cancer patient induce enhanced radio-resistance by the activation of focal adhesion kinase (FAK) and promote cancer cell proliferation by the activation of Akt and p44/42 MAPK. Therefore, these previous reports imply that ICAM-3 has various undefined roles in cancer. In this study, we investigated whether ICAM-3 increase cell migration and invasion through CREB activation and CREB has a role of increase of radioresistance and cell growth.

Alcohol exposure decreases CREB binding protein expression and histone acetylation in the developing cerebellum.

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Weixiang Guo

Full Text Available Fetal alcohol exposure affects 1 in 100 children making it the leading cause of mental retardation in the US. It has long been known that alcohol affects cerebellum development and function. However, the underlying molecular mechanism is unclear.We demonstrate that CREB binding protein (CBP is widely expressed in granule and Purkinje neurons of the developing cerebellar cortex of naïve rats. We also show that exposure to ethanol during the 3(rd trimester-equivalent of human pregnancy reduces CBP levels. CBP is a histone acetyltransferase, a component of the epigenetic mechanism controlling neuronal gene expression. We further demonstrate that the acetylation of both histone H3 and H4 is reduced in the cerebellum of ethanol-treated rats.These findings indicate that ethanol exposure decreases the expression and function of CBP in the developing cerebellum. This effect of ethanol may be responsible for the motor coordination deficits that characterize fetal alcohol spectrum disorders.

Translational inhibition of CTX M extended spectrum β-lactamase in clinical strains of Escherichia coli by synthetic antisense oligonucleotides partially restores sensitivity to cefotaxime.

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John Benedict Readman

2016-03-01

Full Text Available Synthetic antisense oligomers are DNA mimics that can specifically inhibit gene expression at the translational level by ribosomal steric hindrance. They bind to their mRNA targets by Watson Crick base pairing and are resistant to degradation by both nucleases and proteases. A 25 mer phosphorodiamidate morpholino oligomer (PMO and a 13 mer polyamide (peptide nucleic acid (PNA were designed to target mRNA (positions -4 to +21, and –17 to –5 respectively close to the translational initiation site of the extended spectrum β lactamase resistance genes of CTX M group 1. These antisense oligonucleotides were found to inhibit β lactamase activity by up to 96% in a cell free translation transcription coupled system using an expression vector carrying a blaCTX-M-15 gene cloned from a clinical isolate. Despite evidence for up regulation of CTX-M gene expression, they were both found to significantly restore sensitivity to cefotaxime in E. coli AS19, an atypical cell wall permeable mutant, in a dose dependant manner (0 - 40 nM. The PMO and PNA were covalently bound to the cell penetrating peptide (KFF3K and both significantly (P<0.05 increased sensitivity to cefotaxime in a dose dependent manner (0 - 40 nM in field isolates harbouring CTX-M group 1 β-lactamases. Antisense oligonucleotides targeted to the translational initiation site and Shine Dalgarno region of blaCTX-M-15 inhibited gene expression, and when conjugated to a cell penetrating delivery vehicle, partially restored antibiotic sensitivity to both field and clinical isolates.

Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle.

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Pandorf, Clay E; Jiang, Weihua; Qin, Anqi X; Bodell, Paul W; Baldwin, Kenneth M; Haddad, Fadia

2012-04-01

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development.

Cognitive Ameliorating Effect of Acanthopanax koreanum Against Scopolamine-Induced Memory Impairment in Mice.

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Lee, Sunhee; Park, Ho Jae; Jeon, Se Jin; Kim, Eunji; Lee, Hyung Eun; Kim, Haneul; Kwon, Yubeen; Zhang, Jiabao; Jung, In Ho; Ryu, Jong Hoon

2017-03-01

Acanthopanax koreanum Nakai (Araliaceae) is one of the most widely cultivated medicinal plants in Jeju Island, Korea, and the roots and stem bark of A. koreanum have been traditionally used as a tonic agent for general weakness. However, the use of A. koreanum for general weakness observed in the elderly, including those with declined cognitive function, has not been intensively investigated. This study was performed to investigate the effect of the ethanol extract of A. koreanum (EEAK) on cholinergic blockade-induced memory impairment in mice. To evaluate the ameliorating effects of EEAK against scopolamine-induced memory impairment, mice were orally administered EEAK (25, 50, 100, or 200 mg/kg), and several behavioral tasks, including a passive avoidance task, the Y-maze, and a novel object recognition task, were employed. Besides, western blot analysis was conducted to examine whether EEAK affected memory-associated signaling molecules, such as protein kinase B (Akt), Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), and cAMP response element-binding protein (CREB). The administration of EEAK (100 or 200 mg/kg, p.o.) significantly ameliorated the scopolamine-induced cognitive impairment in the passive avoidance task, the Y-maze, and the novel object recognition task. The phosphorylation levels of both Akt and CaMKII were significantly increased by approximately two-fold compared with the control group because of the administration of EEAK (100 or 200 mg/kg) (p cognitive dysfunction induced by the cholinergic blockade, in part, via several memory-associated signaling molecules and may hold therapeutic potential against cognitive dysfunction, such as that presented in neurodegenerative diseases, for example, Alzheimer's disease. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Physicochemical and biological properties of self-assembled antisense/poly(amidoamine) dendrimer nanoparticles: the effect of dendrimer generation and charge ratio

OpenAIRE

Nomani, Alireza; Haririan, Ismaeil; Rahimnia, Ramin; Fouladdel, Shamileh; Gazori, Tarane; Dinarvand, Rassoul; Omidi, Yadollah; Azizi, Ebrahim

2010-01-01

To gain a deeper understanding of the physicochemical phenomenon of self-assembled nanoparticles of different generations and ratios of poly (amidoamine) dendrimer (PAMAM) dendrimer and a short-stranded DNA (antisense oligonucleotide), multiple methods were used to characterize these nanoparticles including photon correlation spectroscopy (PCS); zeta potential measurement; and atomic force microscopy (AFM). PCS and AFM results revealed that, in contrast to larger molecules of DNA, smaller mol...

Fluorinated Nucleotide Modifications Modulate Allele Selectivity of SNP-Targeting Antisense Oligonucleotides

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Michael E. Østergaard

2017-06-01

Full Text Available Antisense oligonucleotides (ASOs have the potential to discriminate between subtle RNA mismatches such as SNPs. Certain mismatches, however, allow ASOs to bind at physiological conditions and result in RNA cleavage mediated by RNase H. We showed that replacing DNA nucleotides in the gap region of an ASO with other chemical modification can improve allele selectivity. Herein, we systematically substitute every position in the gap region of an ASO targeting huntingtin gene (HTT with fluorinated nucleotides. Potency is determined in cell culture against mutant HTT (mtHTT and wild-type HTT (wtHTT mRNA and RNase H cleavage intensities, and patterns are investigated. This study profiled five different fluorinated nucleotides and showed them to have predictable, site-specific effects on RNase H cleavage, and the cleavage patterns were rationalized from a published X-ray structure of human RNase H1. The results herein can be used as a guide for future projects where ASO discrimination of SNPs is important.

Increased cell survival by inhibition of BRCA1 using an antisense approach in an estrogen responsive ovarian carcinoma cell line

International Nuclear Information System (INIS)

Annab, Lois A; Hawkins, Rebecca E; Solomon, Greg; Barrett, J Carl; Afshari, Cynthia A

2000-01-01

We tested the hypothesis that BRCA1 may play a role in the regulation of ovarian tumor cell death as well as the inhibition of ovarian cell proliferation. Introduction of BRCA1 antisense retroviral constructs into BG-1 estrogen-dependent ovarian adenocarcinoma cells resulted in reduced BRCA1 expression. BRCA1 antisense pooled populations and derived subclones were able to proliferate in monolayer culture without estrogen, whereas control cells began to die after 10 days of estrogen deprivation. In addition, both populations and subclones of BRCA1 antisense infected cells demonstrated a growth advantage in monolayer culture in the presence of estrogen and were able to proliferate in monolayer culture without estrogen, while control cells did not. Furthermore, clonal studies demonstrated that reduced levels of BRCA1 protein correlated with growth in soft agar and greater tumor formation in nude mice in the absence of estrogen. These data suggest that reduction of BRCA1 protein in BG-1 ovarian adenocarcinoma cells may have an effect on cell survival during estrogen deprivation both in vitro and in vivo. Germline mutations in the breast and ovarian cancer susceptibility gene BRCA1, which is located on chromosome 17q21, are associated with a predisposition to the development of cancer in these organs [1,2]. No mutations in the BRCA1 gene have been detected in sporadic breast cancer cases, but mutations have been detected in sporadic cases of ovarian cancer [3,4]. Although there is debate regarding the level of cancer risk associated with mutations in BRCA1 and the significance of the lack of mutations in sporadic tumors, it is possible that alterations in the function of BRCA1 may occur by mechanisms other than mutation, leading to an underestimation of risk when it is calculated solely on the basis of mutational analysis. Such alterations cannot be identified until the function and regulation of BRCA1 are better understood. The BRCA1 gene encodes a 220-kDa nuclear

Data in support of transcriptional regulation and function of Fas-antisense long noncoding RNA during human erythropoiesis

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Olga Villamizar

2016-06-01

Full Text Available This paper describes data related to a research article titled, “Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death” [1]. Long noncoding RNAs (lncRNAs are increasingly appreciated for their capacity to regulate many steps of gene expression. While recent studies suggest that many lncRNAs are functional, the scope of their actions throughout human biology is largely undefined including human red blood cell development (erythropoiesis. Here we include expression data for 82 lncRNAs during early, intermediate and late stages of human erythropoiesis using a commercial qPCR Array. From these data, we identified lncRNA Fas-antisense 1 (Fas-AS1 or Saf described in the research article. Also included are 5′ untranslated sequences (UTR for lncRNA Saf with transcription factor target sequences identified. Quantitative RT-PCR data demonstrate relative levels of critical erythroid transcription factors, GATA-1 and KLF1, in K562 human erythroleukemia cells and maturing erythroblasts derived from human CD34+ cells. End point and quantitative RT-PCR data for cDNA prepared using random hexamers versus oligo(dT18 revealed that lncRNA Saf is not effectively polyadenylated. Finally, we include flow cytometry histograms demonstrating Fas levels on maturing erythroblasts derived from human CD34+ cells transduced using mock conditions or with lentivirus particles encoding for Saf.

High dose tetrabromobisphenol A impairs hippocampal neurogenesis and memory retention.

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Kim, Ah Hyun; Chun, Hye Jeong; Lee, Seulah; Kim, Hyung Sik; Lee, Jaewon

2017-08-01

Tetrabromobisphenol A (TBBPA) is a brominated flame retardant that is commonly used in commercial and household products, such as, computers, televisions, mobile phones, and electronic boards. TBBPA can accumulate in human body fluids, and it has been reported that TBBPA possesses endocrine disruptive activity. However, the neurotoxic effect of TBBPA on hippocampal neurogenesis has not yet been investigated. Accordingly, the present study was undertaken to evaluate the effect of TBBPA on adult hippocampal neurogenesis and cognitive function. Male C57BL/6 mice were orally administrated vehicle or TBBPA (20 mg/kg, 100 mg/kg, or 500 mg/kg daily) for two weeks. TBBPA was observed to significantly and dose-dependently reduce the survival of newly generated cells in the hippocampus but not to affect the proliferation of newly generated cells. Numbers of hippocampal BrdU and NeuN positive cells were dose-dependently reduced by TBBPA, indicating impaired neurogenesis in the hippocampus. Interestingly, glial activation without neuronal death was observed in hippocampi exposed to TBBPA. Furthermore, memory retention was found to be adversely affected by TBBPA exposure by a mechanism involving suppression of the BDNF-CREB signaling pathway. The study suggests high dose TBBPA disrupts hippocampal neurogenesis and induces associated memory deficits. Copyright © 2017 Elsevier Ltd. All rights reserved.

Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

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Marsollier, Anne-Charlotte; Ciszewski, Lukasz; Mariot, Virginie; Popplewell, Linda; Voit, Thomas; Dickson, George; Dumonceaux, Julie

2016-04-15

Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Nucleus incertus inactivation impairs spatial learning and memory in rats.

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Nategh, Mohsen; Nikseresht, Sara; Khodagholi, Fariba; Motamedi, Fereshteh

2015-02-01

Nucleus incertus (NI) is a pontine nucleus which releases mainly GABA and relaxin-3 in rats. Its suggested functions include response to stress, arousal, and modulation of hippocampal theta rhythm. Since the role of NI in learning and memory has not been well characterized, therefore the involvement of this nucleus in spatial learning and memory and the aftermath hippocampal levels of c-fos and pCREB were evaluated. NI was targeted by implanting cannula in male rats. For reference memory, NI was inactivated by lidocaine (0.4 μl, 4%) at three stages of acquisition, consolidation and retrieval in Morris water maze paradigm. For working memory, NI was inactivated in acquisition and retrieval phases. Injection of lidocaine prior to the first training session of reference memory significantly increased the distance moved, suggesting that inactivation of NI delays acquisition in this spatial task. Inactivation also interfered with the retrieval phase of spatial reference memory, as the time in target quadrant for lidocaine group was less, and the escape latency was higher compared to the control group. However, no difference was observed in the consolidation phase. In the working memory task, with inter-trial intervals of 75 min, the escape latency was higher when NI was inactivated in the retrieval phase. In addition, c-fos and pCREB/CREB levels decreased in NI-inhibited rats. This study suggests that nucleus incertus might participate in acquisition of spatial reference, and retrieval of both spatial reference and working memory. Further studies should investigate possible roles of NI in the hippocampal plasticity. Copyright © 2014 Elsevier Inc. All rights reserved.

Antitumor effects of radioiodinated antisense oligonucleotide mediated by VIP receptor

International Nuclear Information System (INIS)

Ou Xiaohong; Tan Tianzhi; Li Yunchun; Kuang Anren

2004-01-01

Purpose: we had constructed a targeting delivery system based on intestinal peptide (VIP) for antisense oligonucleotide (ASON) transfer into VIP receptor-positive cells in previous study. The aims of present studies are to observe the antitumor effect of VIP-131I-ASON in HT29 human colon adenocarcinoma xenografts. Methods: A 15-met phosphorothioate ASON, which was complementary to the translation start region of the C-myc oncogene mRNA, was labeled with 131I and the labelled compound was linked to the VIP bound covalently 'to a polylysine chain so as to deliver oligonucleotide into tumor cells. Distribution experiments for evaluating the radiolabeled antisense complexe uptake in tumor tissue were performed in BALB/c nude mice bearing with HT29 tumor xenografts. Nude mice beating HT29 tumor xenografts were adminstered VIP-131I-ASON (3.7,7.4 MBq) or 131I-ASON (3.7 MBq), 131I labeled control sense and nosense DNA (3.7 MBq), or saline. Antitumor effects were assessed using endpoints of tumor growth delay. C-myc-encoded protein expression of tumor was measured by immunocytohistochemical staining. Results: Distribution experiment performed with athymic mice bearing human colon tumor xenografts revealed maximal accumulation of conjugated ASON in the tumor tissue 2 h after administration and significantly higher than that in nude mice injected unconjngated ASON [(5.89±1.03)%ID/g and(1.56±0.31)%ID/g, respectively; t=7.7954 P<0.001]. The radioratio of tumor to muscle was peaked 4h after administration. VIP-131I-ASON exhibited strong antitumor effects against HT29 xenografts, decreasing their growth rate 7-fold compare with that in saline-treated mice(tumor growth delay, 25.4±0.89 day). The antitumor effects of unconjugated 131I-ASON were much less profound than VIP-131I-ASON (tumor growth delay, 3.2±1.3 and 25.4±0.89 day, respectively; q=51.4126 P<0.01). Sense, nosense control ON with VIP carder caused no therapeutic effect. There was no progressive weight loss or

Preventive role of social interaction for cocaine conditioned place preference: correlation with FosB/DeltaFosB and pCREB expression in rat mesocorticolimbic areas.

Science.gov (United States)

El Rawas, Rana; Klement, Sabine; Salti, Ahmad; Fritz, Michael; Dechant, Georg; Saria, Alois; Zernig, Gerald

2012-01-01

The worsening of drug abuse by drug-associated social interaction is a well-studied phenomenon. In contrast, the molecular mechanisms of the beneficial effect of social interaction, if offered as a mutually exclusive choice to drugs of abuse, are under-investigated. In a rat place preference conditioning (CPP) paradigm, four 15 min episodes of social interaction with a gender- and weight-matched male early-adult conspecific inhibited cocaine-induced reinstatement of cocaine CPP, a model of relapse. These protective effects of social interaction were paralleled by a reduced activation, as assessed by Zif268 expression, in brain areas known to play pivotal roles in drug-seeking behavior. Here we show that social interaction during extinction of cocaine CPP also reduced cocaine-CPP-stimulated FosB expression in the nucleus accumbens shell and core. In addition, social interaction during cocaine CPP extinction increased pCREB (cAMP response element binding protein) expression in the nucleus accumbens shell and the cingulate cortex area 1 (Cg1). Our results show that FosB and pCREB may be implicated in the protective effect of social interaction against cocaine-induced reinstatement of CPP. Thus, social interaction, if offered in a context that is clearly distinct from the previously drug-associated one, may profoundly inhibit relapse to cocaine addiction.

The aPKC-CBP Pathway Regulates Adult Hippocampal Neurogenesis in an Age-Dependent Manner

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Ayden Gouveia

2016-10-01

Full Text Available While epigenetic modifications have emerged as attractive substrates to integrate environmental changes into the determination of cell identity and function, specific signals that directly activate these epigenetic modifications remain unknown. Here, we examine the role of atypical protein kinase C (aPKC-mediated Ser436 phosphorylation of CBP, a histone acetyltransferase, in adult hippocampal neurogenesis and memory. Using a knockin mouse strain (CbpS436A in which the aPKC-CBP pathway is deficient, we observe impaired hippocampal neuronal differentiation, maturation, and memory and diminished binding of CBP to CREB in 6-month-old CbpS436A mice, but not at 3 months of age. Importantly, elevation of CREB activity rescues these deficits, and CREB activity is reduced whereas aPKC activity is increased in the murine hippocampus as they age from 3 to 6 months regardless of genotype. Thus, the aPKC-CBP pathway is a homeostatic compensatory mechanism that modulates hippocampal neurogenesis and memory in an age-dependent manner in response to reduced CREB activity.

Limonin, a Component of Dictamni Radicis Cortex, Inhibits Eugenol-Induced Calcium and cAMP Levels and PKA/CREB Signaling Pathway in Non-Neuronal 3T3-L1 Cells

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Yeo Cho Yoon

2015-12-01

Full Text Available Limonin, one of the major components in dictamni radicis cortex (DRC, has been shown to play various biological roles in cancer, inflammation, and obesity in many different cell types and tissues. Recently, the odorant-induced signal transduction pathway (OST has gained attention not only because of its function in the perception of smell but also because of its numerous physiological functions in non-neuronal cells. However, little is known about the effects of limonin and DRC on the OST pathway in non-neuronal cells. We investigated odorant-stimulated increases in Ca2+ and cAMP, major second messengers in the OST pathway, in non-neuronal 3T3-L1 cells pretreated with limonin and ethanol extracts of DRC. Limonin and the extracts significantly decreased eugenol-induced Ca2+ and cAMP levels and upregulated phosphorylation of CREB and PKA. Our results demonstrated that limonin and DRC extract inhibit the OST pathway in non-neuronal cells by modulating Ca2+ and cAMP levels and phosphorylation of CREB.

Low-Intensity Pulsed Ultrasound Enhances Nerve Growth Factor-Induced Neurite Outgrowth through Mechanotransduction-Mediated ERK1/2-CREB-Trx-1 Signaling.

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Zhao, Lu; Feng, Yi; Hu, Hong; Shi, Aiwei; Zhang, Lei; Wan, Mingxi

2016-12-01

Enhancing the action of nerve growth factor (NGF) is a potential therapeutic approach to neural regeneration. To facilitate neural regeneration, we investigated whether combining low-intensity pulsed ultrasound (LIPUS) and NGF could promote neurite outgrowth, an essential process in neural regeneration. In the present study, PC12 cells were subjected to a combination of LIPUS (1 MHz, 30 or 50 mW/cm 2 , 20% duty cycle and 100-Hz pulse repetition frequency, 10 min every other day) and NGF (50 ng/mL) treatment, and then neurite outgrowth was compared. Our findings indicated that the combined treatment with LIPUS (50 mW/cm 2 ) and NGF (50 ng/mL) promotes neurite outgrowth that is comparable to that achieved by NGF (100 ng/mL) treatment alone. LIPUS significantly increased NGF-induced neurite length, but not neurite branching. These effects were attributed to the enhancing effects of LIPUS on NGF-induced phosphorylation of ERK1/2 and CREB and the expression of thioredoxin (Trx-1). Furthermore, blockage of stretch-activated ion channels with Gd 3+ suppressed the stimulating effects of LIPUS on NGF-induced neurite outgrowth and the downstream signaling activation. Taken together, our findings suggest that LIPUS enhances NGF-induced neurite outgrowth through mechanotransduction-mediated signaling of the ERK1/2-CREB-Trx-1 pathway. The combination of LIPUS and NGF could potentially be used for the treatment of nerve injury and neurodegenerative diseases. Copyright © 2016 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Plant 7SL RNA and tRNA(Tyr) genes with inserted antisense sequences are efficiently expressed in an in vitro transcription system from Nicotiana tabacum cells

Czech Academy of Sciences Publication Activity Database

Yukawa, Y.; Matoušek, Jaroslav; Grimm, M.; Vrba, Lukáš; Steger, G.; Sugiura, M.; Beier, H.

2002-01-01

Roč. 50, - (2002), s. 713-723 ISSN 0167-4412 R&D Projects: GA ČR GA521/99/1591; GA MŠk ME 463 Keywords : antisense RNA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.529, year: 2002

Suppression of type I interferon production by porcine epidemic diarrhea virus and degradation of CREB-binding protein by nsp1

Energy Technology Data Exchange (ETDEWEB)

Zhang, Qingzhan; Shi, Kaichuang; Yoo, Dongwan, E-mail: dyoo@illinois.edu

2016-02-15

Type I interferons (IFN-α/β) are the major components of the innate immune response of hosts, and in turn many viruses have evolved to modulate the host response during infection. We found that the IFN-β production was significantly suppressed during PEDV infection in cells. To identify viral IFN antagonists and to study their suppressive function, viral coding sequences for the entire structural and nonstructural proteins were cloned and expressed. Of 16 PEDV nonstructural proteins (nsps), nsp1, nsp3, nsp7, nsp14, nsp15 and nsp16 were found to inhibit the IFN-β and IRF3 promoter activities. The sole accessory protein ORF3, structure protein envelope (E), membrane (M), and nucleocapsid (N) protein were also shown to inhibit such activities. PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP) by degrading CBP. A further study showed that the CBP degradation by nsp1 was proteasome-dependent. Our data demonstrate that PEDV modulates the host innate immune responses by degrading CBP and suppressing ISGs expression. - Highlights: • PEDV modulates the host innate immune system by suppressing the type I interferon production and ISGs expression. • Ten viral proteins were identified as IFN antagonists, and nsp1 was the most potent viral IFN antagonist. • PEDV nsp1 did not interfere the IRF3 phosphorylation and nuclear translocation but interrupted the enhanceosome assembly of IRF3 and CREB-binding protein (CBP). • PEDV nsp1 caused the CBP degradation in the nucleus, which may be the key mechanism for PEDV-mediated IFN downregulation.

An Approach to Detect and Study DNA Double-Strand Break Repair by Transcript RNA Using a Spliced-Antisense RNA Template.

Science.gov (United States)

Keskin, Havva; Storici, Francesca

2018-01-01

A double-strand break (DSB) is one of the most dangerous DNA lesion, and its repair is crucial for genome stability. Homologous recombination is considered the safest way to repair a DNA DSB and requires an identical or nearly identical DNA template, such as a sister chromatid or a homologous chromosome for accurate repair. Can transcript RNA serve as donor template for DSB repair? Here, we describe an approach that we developed to detect and study DNA repair by transcript RNA. Key features of the method are: (i) use of antisense (noncoding) RNA as template for DSB repair by RNA, (ii) use of intron splicing to distinguish the sequence of the RNA template from that of the DNA that generates the RNA template, and (iii) use of a trans and cis system to study how RNA repairs a DSB in homologous but distant DNA or in its own DNA, respectively. This chapter provides details on how to use a spliced-antisense RNA template to detect and study DSB repair by RNA in trans or cis in yeast cells. Our approach for detection of DSB repair by RNA in cells can be applied to cell types other than yeast, such as bacteria, mammalian cells, or other eukaryotic cells. © 2018 Elsevier Inc. All rights reserved.

The cellular uptake of antisense oligonucleotid of E6 mRNA into cervical cancer cells by DOPE-modified hydroxyapatite nanoparticles

OpenAIRE

Negin Saffarzadeh; Seyed Mehdi Kalantar; Ali Jebali; Seyed Hossein Hekmatimoghaddam; Mohammad Hassan Sheikhha; Ehsan Farashahi

2014-01-01

Objective(s): Although several chemical and physical methods for gene delivery have been introduced, their cytotoxicity, non-specific immune responses and the lack of biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles (NPs) and 1,2-dioleoyl-sn-glycero-3-phosphoethanol​amine (DOPE)-modified hydroxyapatite NPs was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical cancer cell line were evaluated. Materials and Methods: Calcium...

Recent advances in antisense oligonucleotide therapy in genetic neuromuscular diseases

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Ashok Verma

2018-01-01

Full Text Available Genetic neuromuscular diseases are caused by defective expression of nuclear or mitochondrial genes. Mutant genes may reduce expression of wild-type proteins, and strategies to activate expression of the wild-type proteins might provide therapeutic benefits. Also, a toxic mutant protein may cause cell death, and strategies that reduce mutant gene expression may provide therapeutic benefit. Synthetic antisense oligonucleotide (ASO can recognize cellular RNA and control gene expression. In recent years, advances in ASO chemistry, creation of designer ASO molecules to enhance their safety and target delivery, and scientific controlled clinical trials to ascertain their therapeutic safety and efficacy have led to an era of plausible application of ASO technology to treat currently incurable neuromuscular diseases. Over the past 1 year, for the first time, the United States Food and Drug Administration has approved two ASO therapies in genetic neuromuscular diseases. This overview summarizes the recent advances in ASO technology, evolution and use of synthetic ASOs as a therapeutic platform, and the mechanism of ASO action by exon-skipping in Duchenne muscular dystrophy and exon-inclusion in spinal muscular atrophy, with comments on their advantages and limitations.

Antisense-MDM2 Sensitizes LNCaP Prostate Cancer Cells to Androgen Deprivation, Radiation, and the Combination In Vivo

International Nuclear Information System (INIS)

Stoyanova, Radka; Hachem, Paul; Hensley, Harvey; Khor, L.-Y.; Mu Zhaomei; Hammond, M. Elizabeth H.; Agrawal, Sudhir; Pollack, Alan

2007-01-01

Purpose: To test the effects of antisense (AS)-MDM2 alone and with androgen deprivation (AD), radiotherapy (RT), and AD + RT on wild-type LNCaP cells in an orthotopic in vivo model. Methods: Androgen-sensitive LNCaP cells were grown in the prostates of nude mice. Magnetic resonance imaging-based tumor volume and serum prostate-specific antigen (PSA) measurements were used to assess effects on tumor response. Tumor response was measured by biochemical and tumor volume failure definitions and doubling time estimates from fitted PSA and tumor volume growth curves. Expression of MDM2, p53, p21, and Ki-67 was quantified using immunohistochemical staining and image analysis of formalin-fixed tissue, analogous to methods used clinically. Results: Antisense-MDM2 significantly inhibited the growth of LNCaP tumors over the mismatch controls. The most significant increase in tumor growth delay and tumor doubling time was from AS-MDM2 + AD + RT, although the effect of AS-MDM2 + AD was substantial. Expression of MDM2 was significantly reduced by AS-MDM2 in the setting of RT. Conclusions: This is the first in vivo investigation of the effects of AS-MDM2 in an orthotopic model and the first to demonstrate incremental sensitization when added to AD and AD + RT. The results with AD underscore the potential to affect micrometastatic disease, which is probably responsible for treatment failure in 30-40% of men with high-risk disease

The stimulatory adenosine receptor ADORA2B regulates serotonin (5-HT synthesis and release in oxygen-depleted EC cells in inflammatory bowel disease.

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Rikard Dammen

Full Text Available We recently demonstrated that hypoxia, a key feature of IBD, increases enterochromaffin (EC cell 5-HT secretion, which is also physiologically regulated by the ADORA2B mechanoreceptor. Since hypoxia is associated with increased extracellular adenosine, we wanted to examine whether this nucleotide amplifies HIF-1α-mediated 5-HT secretion.The effects of hypoxia were studied on IBD mucosa, isolated IBD-EC cells, isolated normal EC cells and the EC cell tumor derived cell line KRJ-1. Hypoxia (0.5% O2 was compared to NECA (adenosine agonist, MRS1754 (ADORA2B receptor antagonist and SCH442146 (ADORA2A antagonist on HIF signaling and 5-HT secretion. Antisense approaches were used to mechanistically evaluate EC cells in vitro. PCR and western blot were used to analyze transcript and protein levels of HIF-1α signaling and neuroendocrine cell function. An animal model of colitis was evaluated to confirm hypoxia:adenosine signaling in vivo.HIF-1α is upregulated in IBD mucosa and IBD-EC cells, the majority (~90% of which express an activated phenotype in situ. Hypoxia stimulated 5-HT release maximally at 30 mins, an effect amplified by NECA and selectively inhibited by MRS1754, through phosphorylation of TPH-1 and activation of VMAT-1. Transient transfection with Renilla luciferase under hypoxia transcriptional response element (HRE control identified that ADORA2B activated HIF-1α signaling under hypoxic conditions. Additional signaling pathways associated with hypoxia:adenosine included MAP kinase and CREB. Antisense approaches mechanistically confirmed that ADORA2B signaling was linked to these pathways and 5-HT release under hypoxic conditions. Hypoxia:adenosine activation which could be reversed by 5'-ASA treatment was confirmed in a TNBS-model.Hypoxia induced 5-HT synthesis and secretion is amplified by ADORA2B signaling via MAPK/CREB and TPH-1 activation. Targeting ADORA2s may decrease EC cell 5-HT production and secretion in IBD.

Sp1 and CREB regulate basal transcription of the human SNF2L gene

International Nuclear Information System (INIS)

Xia Yu; Jiang Baichun; Zou Yongxin; Gao Guimin; Shang Linshan; Chen Bingxi; Liu Qiji; Gong Yaoqin

2008-01-01

Imitation Switch (ISWI) is a member of the SWI2/SNF2 superfamily of ATP-dependent chromatin remodelers, which are involved in multiple nuclear functions, including transcriptional regulation, replication, and chromatin assembly. Mammalian genomes encode two ISWI orthologs, SNF2H and SNF2L. In order to clarify the molecular mechanisms governing the expression of human SNF2L gene, we functionally examined the transcriptional regulation of human SNF2L promoter. Reporter gene assays demonstrated that the minimal SNF2L promoter was located between positions -152 to -86 relative to the transcription start site. In this region we have identified a cAMP-response element (CRE) located at -99 to -92 and a Sp1-binding site at -145 to -135 that play a critical role in regulating basal activity of human SNF2L gene, which were proven by deletion and mutation of specific binding sites, EMSA, and down-regulating Sp1 and CREB via RNAi. This study provides the first insight into the mechanisms that control basal expression of human SNF2L gene

The cellular transcription factor CREB corresponds to activating transcription factor 47 (ATF-47) and forms complexes with a group of polypeptides related to ATF-43.

OpenAIRE

Hurst, H C; Masson, N; Jones, N C; Lee, K A

1990-01-01

Promoter elements containing the sequence motif CGTCA are important for a variety of inducible responses at the transcriptional level. Multiple cellular factors specifically bind to these elements and are encoded by a multigene family. Among these factors, polypeptides termed activating transcription factor 43 (ATF-43) and ATF-47 have been purified from HeLa cells and a factor referred to as cyclic AMP response element-binding protein (CREB) has been isolated from PC12 cells and rat brain. We...

PlantNATsDB: a comprehensive database of plant natural antisense transcripts.

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Chen, Dijun; Yuan, Chunhui; Zhang, Jian; Zhang, Zhao; Bai, Lin; Meng, Yijun; Chen, Ling-Ling; Chen, Ming

2012-01-01

Natural antisense transcripts (NATs), as one type of regulatory RNAs, occur prevalently in plant genomes and play significant roles in physiological and pathological processes. Although their important biological functions have been reported widely, a comprehensive database is lacking up to now. Consequently, we constructed a plant NAT database (PlantNATsDB) involving approximately 2 million NAT pairs in 69 plant species. GO annotation and high-throughput small RNA sequencing data currently available were integrated to investigate the biological function of NATs. PlantNATsDB provides various user-friendly web interfaces to facilitate the presentation of NATs and an integrated, graphical network browser to display the complex networks formed by different NATs. Moreover, a 'Gene Set Analysis' module based on GO annotation was designed to dig out the statistical significantly overrepresented GO categories from the specific NAT network. PlantNATsDB is currently the most comprehensive resource of NATs in the plant kingdom, which can serve as a reference database to investigate the regulatory function of NATs. The PlantNATsDB is freely available at http://bis.zju.edu.cn/pnatdb/.

The successes and future prospects of the linear antisense RNA amplification methodology.

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Li, Jifen; Eberwine, James

2018-05-01

It has been over a quarter of a century since the introduction of the linear RNA amplification methodology known as antisense RNA (aRNA) amplification. Whereas most molecular biology techniques are rapidly replaced owing to the fast-moving nature of development in the field, the aRNA procedure has become a base that can be built upon through varied uses of the technology. The technique was originally developed to assess RNA populations from small amounts of starting material, including single cells, but over time its use has evolved to include the detection of various cellular entities such as proteins, RNA-binding-protein-associated cargoes, and genomic DNA. In this Perspective we detail the linear aRNA amplification procedure and its use in assessing various components of a cell's chemical phenotype. This procedure is particularly useful in efforts to multiplex the simultaneous detection of various cellular processes. These efforts are necessary to identify the quantitative chemical phenotype of cells that underlies cellular function.

Protective effects of a green tea polyphenol, epigallocatechin-3-gallate, against sevoflurane-induced neuronal apoptosis involve regulation of CREB/BDNF/TrkB and PI3K/Akt/mTOR signalling pathways in neonatal mice.

Science.gov (United States)

Ding, Mei-Li; Ma, Hui; Man, Yi-Gang; Lv, Hong-Yan

2017-12-01

Epigallocatechin-3-gallate (EGCG), a polyphenol in green tea, is an effective antioxidant and possesses neuroprotective effects. Brain-derived neurotrophic factor (BDNF) and cyclic AMP response element-binding protein (CREB) are crucial for neurogenesis and synaptic plasticity. In this study, we aimed to assess the protective effects of EGCG against sevoflurane-induced neurotoxicity in neonatal mice. Distinct groups of C57BL/6 mice were given EGCG (25, 50, or 75 mg/kg body weight) from postnatal day 3 (P3) to P21 and were subjected to sevoflurane (3%; 6 h) exposure on P7. EGCG significantly inhibited sevoflurane-induced neuroapoptosis as determined by Fluoro-Jade B staining and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Increased levels of cleaved caspase-3, downregulated Bad and Bax, and significantly enhanced Bcl-2, Bcl-xL, xIAP, c-IAP-1, and survivin expression were observed. EGCG induced activation of the PI3K/Akt pathway as evidenced by increased Akt, phospho-Akt, GSK-3β, phospho-GSK-3β, and mTORc1 levels. Sevoflurane-mediated downregulation of cAMP/CREB and BDNF/TrkB signalling was inhibited by EGCG. Reverse transcription PCR analysis revealed enhanced BDNF and TrkB mRNA levels upon EGCG administration. Improved performance of mice in Morris water maze tests suggested enhanced learning and memory. The study indicates that EGCG was able to effectively inhibit sevoflurane-induced neurodegeneration and improve learning and memory retention of mice via activation of CREB/BDNF/TrkB-PI3K/Akt signalling.

High-fat diet induces hepatic insulin resistance and impairment of synaptic plasticity.

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Zhigang Liu

Full Text Available High-fat diet (HFD-induced obesity is associated with insulin resistance, which may affect brain synaptic plasticity through impairment of insulin-sensitive processes underlying neuronal survival, learning, and memory. The experimental model consisted of 3 month-old C57BL/6J mice fed either a normal chow diet (control group or a HFD (60% of calorie from fat; HFD group for 12 weeks. This model was characterized as a function of time in terms of body weight, fasting blood glucose and insulin levels, HOMA-IR values, and plasma triglycerides. IRS-1/Akt pathway was assessed in primary hepatocytes and brain homogenates. The effect of HFD in brain was assessed by electrophysiology, input/output responses and long-term potentiation. HFD-fed mice exhibited a significant increase in body weight, higher fasting glucose- and insulin levels in plasma, lower glucose tolerance, and higher HOMA-IR values. In liver, HFD elicited (a a significant decrease of insulin receptor substrate (IRS-1 phosphorylation on Tyr608 and increase of Ser307 phosphorylation, indicative of IRS-1 inactivation; (b these changes were accompanied by inflammatory responses in terms of increases in the expression of NFκB and iNOS and activation of the MAP kinases p38 and JNK; (c primary hepatocytes from mice fed a HFD showed decreased cellular oxygen consumption rates (indicative of mitochondrial functional impairment; this can be ascribed partly to a decreased expression of PGC1α and mitochondrial biogenesis. In brain, HFD feeding elicited (a an inactivation of the IRS-1 and, consequentially, (b a decreased expression and plasma membrane localization of the insulin-sensitive neuronal glucose transporters GLUT3/GLUT4; (c a suppression of the ERK/CREB pathway, and (d a substantial decrease in long-term potentiation in the CA1 region of hippocampus (indicative of impaired synaptic plasticity. It may be surmised that 12 weeks fed with HFD induce a systemic insulin resistance that impacts

Effects of CD49d-targeted antisense-oligonucleotide on α4 integrin expression and function of acute lymphoblastic leukemia cells: Results of in vitro and in vivo studies.

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Yann Duchartre

Full Text Available We recently demonstrated the effectiveness of blocking CD49d with anti-functional antibodies or small molecule inhibitors as a rational targeted approach to the treatment of acute leukemia in combination with chemotherapy. Antisense oligonucleotide promises to be no less specific than antibodies and inhibitors, but more interesting for pharmacokinetics and pharmacodynamics. We addressed this using the published CD49d antisense drug ATL1102. In vitro, we incubated/nucleofected the ALL cell line Kasumi-2 with ATL1102. In vivo, immunodeficient hosts were engrafted with primary ALL cells and treated with ATL1102. Changes in expression of CD49d mRNA and CD49d protein, and of cooperating gene products, including ß1 integrin and CXCR4, as well as survival in the mouse experiments were quantified. We observed dose-dependent down-regulation of CD49d mRNA and protein levels and its partner integrin ß1 cell surface protein level and, up-regulation of CXCR4 surface expression. The suppression was more pronounced after nucleofection than after incubation, where down-regulation was significant only at the higher doses. In vivo effects of ATL1102 were not sufficient to translate into "clinical" benefit in the leukemia model. In summary, antisense oligonucleotides are successful tools for specifically modulating gene expression but sufficient delivery to down-regulate CD49d in vivo may be difficult to achieve.

Antisense myb inhibition of purified erythroid progenitors in development and differentiation is linked to cycling activity and expression of DNA polymerase alpha

International Nuclear Information System (INIS)

Valtieri, M.; Venturelli, D.; Care, A.; Fossati, C.; Pelosi, E.; Labbaye, C.; Mattia, G.; Gewirtz, A.M.; Calabretta, B.; Peschle, C.

1991-01-01

These studies aimed to determine the expression and functional role of c-myb in erythroid progenitors with different cycling activities. In the first series of experiments the erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU-E) populations from adult peripheral blood (PB), bone marrow (BM), and embryonic-fetal liver (FL) were treated with either c-myb antisense oligomers or 3H-thymidine (3H-TdR). A direct correlation was always observed between the inhibitory effect of anti-myb oligomers and the level of cycling activity. Thus, the inhibitory effect of antisense c-myb on the number of BFU-E colonies was 28.3% +/- 15.8% in PB, 53.4% +/- 9.3% in BM, and 68.2% +/- 24.5% in FL. Both adult and embryonic CFU-E were markedly inhibited. Using purified PB progenitors, we observed a similar pattern, although with slightly lower inhibitory effects. In the 3H-TdR suicide assay the killing index of BFU-E was 8.9% +/- 4.2% in PB, 29.4% +/- 6.5% in BM, and 40.1% +/- 9.6% in FL. The values for adult and embryonic CFU-E were 55.7% +/- 7.9% and 60.98% +/- 6.6%, respectively. We then investigated the kinetics of c-myb mRNA level during the erythroid differentiation of purified adult PB and FL BFU-E, as evaluated in liquid-phase culture by reverse transcription-polymerase chain reaction. Adult erythroid precursors showed a gradual increase of c-myb mRNA from day 4 through day 8 of culture and a sharp decrease at later times, whereas the expression of c-myb mRNA and protein in differentiation embryonic precursors peaked 2 days earlier. In both cases, c-myb mRNA level peaked at the CFU-E stage of differentiation. Finally, highly purified adult PB BFU-E were stimulated into cycling by a 3-day treatment with interleukin-3 in liquid phase: both the sensitivity to c-myb antisense oligomers and the 3H-TdR suicide index showed a gradual, strictly parallel increase

Preventive role of social interaction for cocaine conditioned place preference: correlation with FosB/DeltaFosB and pCREB expression in rat mesocorticolimbic areas

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El Rawas, Rana; Klement, Sabine; Salti, Ahmad; Fritz, Michael; Dechant, Georg; Saria, Alois; Zernig, Gerald

2012-01-01

The worsening of drug abuse by drug-associated social interaction is a well-studied phenomenon. In contrast, the molecular mechanisms of the beneficial effect of social interaction, if offered as a mutually exclusive choice to drugs of abuse, are under-investigated. In a rat place preference conditioning (CPP) paradigm, four 15 min episodes of social interaction with a gender- and weight-matched male early-adult conspecific inhibited cocaine-induced reinstatement of cocaine CPP, a model of relapse. These protective effects of social interaction were paralleled by a reduced activation, as assessed by Zif268 expression, in brain areas known to play pivotal roles in drug-seeking behavior. Here we show that social interaction during extinction of cocaine CPP also reduced cocaine-CPP-stimulated FosB expression in the nucleus accumbens shell and core. In addition, social interaction during cocaine CPP extinction increased pCREB (cAMP response element binding protein) expression in the nucleus accumbens shell and the cingulate cortex area 1 (Cg1). Our results show that FosB and pCREB may be implicated in the protective effect of social interaction against cocaine-induced reinstatement of CPP. Thus, social interaction, if offered in a context that is clearly distinct from the previously drug-associated one, may profoundly inhibit relapse to cocaine addiction. PMID:22403532

Preventive role of social interaction for cocaine conditioned place preference: correlation with FosB/DeltaFosB and pCREB expression in rat mesocorticolimbic areas

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Rana eEl Rawas

2012-03-01

Full Text Available The worsening of drug abuse by drug-associated social interaction is a well-studied phenomenon. In contrast, the molecular mechanisms of the beneficial effect of social interaction, if offered as a mutually exclusive choice to drugs of abuse, are under-investigated. In a rat place preference conditioning (CPP paradigm, four 15 min episodes of social interaction with a gender- and weight matched male early-adult conspecific inhibited cocaine-induced reinstatement of cocaine CPP, a model of relapse. These protective effects of social interaction were paralleled by a reduced activation, as assessed by Zif268 expression in brain areas known to play pivotal roles in drug-seeking behavior. Here we show that social interaction during extinction of cocaine CPP also reduced cocaine-CPP-stimulated FosB expression in the nucleus accumbens shell and core. In addition, social interaction during cocaine CPP extinction increased pCREB (cAMP response element binding protein expression in the nucleus accumbens shell and the cingulate cortex area 1 (Cg1. Our results show that FosB and pCREB may be implicated in the protective effect of social interaction against cocaine-induced reinstatement of CPP. Thus, social interaction, if offered in a context that is clearly distinct from the previously drug-associated one, may profoundly inhibit relapse to cocaine addiction.

Involvement of TORC2, a CREB co-activator, in the in vivo-specific transcriptional control of HTLV-1

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Furuta Rika A

2009-08-01

Full Text Available Abstract Background Human T-cell leukemia virus type 1 (HTLV-1 causes adult T -cell leukemia (ATL but the expression of HTLV-1 is strongly suppressed in the peripheral blood of infected people. However, such suppression, which may explain the long latency in the development of ATL, is readily reversible, and viral expression resumes quickly with ex vivo culture of infected T -cells. To investigate the mechanism of in vivo -specific transcriptional suppression, we established a mouse model in which mice were intraperitoneally administered syngeneic EL4 T -lymphoma cells transduced with a recombinant retrovirus expressing a GFP-Tax fusion protein, Gax, under the control of the HTLV-1 enhancer (EL4-Gax. Results Gax gene transcription was silenced in vivo but quickly up-regulated in ex vivo culture. Analysis of integrated Gax reporter gene demonstrated that neither CpG methylation of the promoter DNA nor histone modification was associated with the reversible suppression. ChIP-analysis of LTR under suppression revealed reduced promoter binding of TFIIB and Pol-II, but no change in the binding of CREB or CBP/p300 to the viral enhancer sequence. However, the expression of TORC2, a co-activator of CREB, decreased substantially in the EL4-Gax cells in vivo, and this returned to normal levels in ex vivo culture. The reduced expression of TORC2 was associated with translocation from the nucleus to the cytoplasm. A knock-down experiment with siRNA confirmed that TORC2 was the major functional protein of the three TORC-family proteins (TORC1, 2, 3 in EL4-Gax cells. Conclusion These results suggest that the TORC2 may play an important role in the in vivo -specific transcriptional control of HTLV-1. This study provides a new model for the reversible mechanism that suppresses HTLV-1 expression in vivo without the DNA methylation or hypoacetylated histones that is observed in the primary cells of most HTLV-1 -infected carriers and a substantial number of ATL

Topiramate via NMDA, AMPA/kainate, GABAA and Alpha2 receptors and by modulation of CREB/BDNF and Akt/GSK3 signaling pathway exerts neuroprotective effects against methylphenidate-induced neurotoxicity in rats.

Science.gov (United States)

Motaghinejad, Majid; Motevalian, Manijeh; Fatima, Sulail; Beiranvand, Tabassom; Mozaffari, Shiva

2017-11-01

Chronic abuse of methylphenidate (MPH) often causes neuronal cell death. Topiramate (TPM) carries neuroprotective effects, but its exact mechanism of action remains unclear. In the present study, the role of various doses of TPM and its possible mechanisms, receptors and signaling pathways involved against MPH-induced hippocampal neurodegeneration were evaluated in vivo. Thus, domoic acid (DOM) was used as AMPA/kainate receptor agonist, bicuculline (BIC) as GABA A receptor antagonist, ketamine (KET) as NMDA receptor antagonist, yohimbine (YOH) as α 2 adrenergic receptor antagonist and haloperidol (HAL) was used as dopamine D 2 receptor antagonist. Open field test (OFT) was used to investigate the disturbances in motor activity. Hippocampal neurodegenerative parameters were evaluated. Protein expressions of CREB/BDNF and Akt/GSK3 signaling pathways were also evaluated. Cresyl violet staining was performed to show and confirm the changes in the shape of the cells. TPM (70 and 100 mg/kg) reduced MPH-induced rise in lipid peroxidation, oxidized form of glutathione (GSSG), IL-1β and TNF-α levels, Bax expression and motor activity disturbances. In addition, TPM treatment increased Bcl-2 expression, the level of reduced form of glutathione (GSH) and the levels and activities of superoxide dismutase, glutathione peroxidase and glutathione reductase enzymes. TPM also inhibited MPH-induced hippocampal degeneration. Pretreatment of animals with DOM, BIC, KET and YOH inhibited TPM-induced neuroprotection and increased oxidative stress, neuroinflammation, neuroapoptosis and neurodegeneration while reducing CREB, BDNF and Akt protein expressions. Also pretreatment with DOM, BIC, KET and YOH inhibited TPM-induced decreases in GSK3. It can be concluded that the mentioned receptors by modulation of CREB/BDNF and Akt/GSK3 pathways, are involved in neuroprotection of TPM against MPH-induced neurodegeneration.

Endogenous Parathyroid Hormone Promotes Fracture Healing by Increasing Expression of BMPR2 through cAMP/PKA/CREB Pathway in Mice

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Wei Zhou

2017-06-01

Full Text Available Background/Aims: Endogenous parathyroid hormone (PTH plays an important role in fracture healing. This study investigated whether endogenous PTH regulates fracture healing by bone morphogenetic protein (BMP and/or the transforming growth factor-β (TGF-β signaling pathway. Methods: Eight-week-old wild-type (WT and PTH-knockout (PTH KO male mice were selected, and models of open right-femoral fracture were constructed. Fracture healing and callus characteristics of mice in the two groups were compared by X-ray, micro-computed tomography, histological, and immunohistochemical examinations. Bone marrow mesenchymal stem cells (BMMSCs of 8-week-old WT and PTHKO male mice were obtained and induced into osteoblasts and chondrocytes. Results: We found that expression levels of Runt-related transcription factor (RUNX2, bone morphogenetic protein-receptor-type Ⅱ (BMPR2, phosphorylated Smad 1/5/8, and phosphorylated cyclic adenosine monophosphate-responsive element binding protein (CREB in the callus of PTHKO mice were significantly decreased, whereas no significant difference in expression of SOX9, TGF-βR2,or pSMAD2/3 was observed between PTHKO and WT mice. Additionally, the activity of osteoblast alkaline phosphatase was low at 7 days post-induction, and was upregulated by addition of PTH or dibutyryl cyclic adenosine monophosphate (dbcAMP to the cell culture. Furthermore, H89 (protein kinase A inhibitoreliminated the simulating effects of PTH and dbcAMP, and a low concentration of cyclic adenosine monophosphate (cAMP was observed in PTHKO mouse BMMSCs. Conclusion: These results suggested that endogenous PTH enhanced BMPR2 expression by a cAMP/PKA/CREB pathway in osteoblasts, and increased RUNX2 expression through transduction of the BMP/pSMAD1/5/8 signaling pathway.

Developmental exposure to paraquat and maneb can impair cognition, learning and memory in Sprague-Dawley rats.

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Li, Bai; He, Xi; Sun, Yan; Li, Baixiang

2016-10-20

Paraquat and maneb are identified environmental pollutants. Combined exposure to paraquat and maneb is a latent risk factor for many diseases, particularly those of the central nervous system, including Parkinson's disease and Alzheimer's disease. Hippocampus is the key structure in memory formation and babies are more sensitive to environmental stimuli than adults, so we investigated the neurotoxicity of paraquat and maneb on the hippocampi of rat pups. Female and male Sprague-Dawley rats were mated (female : male = 2 : 1) every night for a week. The gravid rats were randomly divided into three groups (one control and two experimental groups). A mixed solution of paraquat-maneb was administered twice a week by lavage at a dose of 10 or 15 mg kg(-1) bodyweight (containing 30 or 45 mg kg(-1) bodyweight maneb, respectively) from day 6 after pregnancy till ablactation. Maternal weight gain and offspring bodyweights were not affected by the drugs. However, behavioral tests showed that reaction latency and mistake frequency increased after treatment. Intuitively, we found significant changes in the hippocampal neurons in the morphological observation. Taking into account the interaction of the related genes in the cAMP-PKA-CREB pathway, we used a variety of methods to detect the gene and protein levels. Reduced expression of cAMP and related genes and proteins in the hippocampus and serum was also observed. These results indicate that PQ-MB stimulates cAMP to reduce the production of PKA, thus reducing the phosphorylation of CREB and inhibiting the activation of other elements (BDNF, C-JUN, and C-FOS). These changes lead to hippocampal damage and impaired abilities (learning, cognition, and memory). Our results demonstrate that PQ-MB induces hippocampal toxicity in the early life of rats, and they thus provide a theoretical foundation for further investigation of the bathypelagic mechanism involved and measures that can be taken to avoid PQ-MB neurotoxicity.

Dose-Dependent Lowering of Mutant Huntingtin Using Antisense Oligonucleotides in Huntington Disease Patients.

Science.gov (United States)

van Roon-Mom, Willeke M C; Roos, Raymund A C; de Bot, Susanne T

2018-04-01

On December 11 of 2017, Ionis Pharmaceuticals published a press release announcing dose-dependent reductions of mutant huntingtin protein in their HTTRx Phase 1/2a study in Huntington disease (HD) patients. The results from this Ionis trial have gained much attention from the patient community and the oligonucleotide therapeutics field, since it is the first trial targeting the cause of HD, namely the mutant huntingtin protein, using antisense oligonucleotides (ASOs). The press release also states that the primary endpoints of the study (safety and tolerability) were met, but does not contain data. This news follows the approval of another therapeutic ASO nusinersen (trade name Spinraza) for a neurological disease, spinal muscular atrophy, by the U.S. Food and Drug Administration and European Medicines Agency, in 2016 and 2017, respectively. Combined, this offers hope for the development of the HTTRx therapy for HD patients.

Down-regulation of Survivin by Antisense Oligonucleotides Increases Apoptosis, Inhibits Cytokinesis and Anchorage-Independent Growth

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Jun Chen

2000-05-01

Full Text Available Survivin, a member of the inhibitor of apoptosis protein (IAP family, is detected in most common human cancers but not in adjacent normal cells. Previous studies suggest that survivin associates with the mitotic spindle and directly inhibits caspase activity. To further investigate the function of survivin, we used a survivin antisense (AS oligonucleotide to downregulate survivin expression in normal and cancer cells. We found that inhibition of survivin expression increased apoptosis and polyploidy while decreasing colony formation in soft agar. Immunohistochemistry showed that cells without survivin can initiate the cleavage furrow and contractile ring, but cannot complete cytokinesis, thus resulting in multinucleated cells. These findings indicate that survivin plays important roles in a late stage of cytokinesis, as well as in apoptosis.

HTLV Deregulation of the NF-κB Pathway: An Update on Tax and Antisense Proteins Role

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Fochi, Stefania; Mutascio, Simona; Bertazzoni, Umberto; Zipeto, Donato; Romanelli, Maria G.

2018-01-01

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), an aggressive CD4+/CD25+ T-cell malignancy and of a severe neurodegenerative disease, HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The chronic activation or deregulation of the canonical and non-canonical nuclear factor kappa B (NF-κB) pathways play a crucial role in tumorigenesis. The HTLV-1 Tax-1 oncoprotein is a potent activator of the NF-κB transcription factors and the NF-κB response is required for promoting the development of HTLV-1 transformed cell lines. The homologous retrovirus HTLV-2, which also expresses a Tax-2 transforming protein, is not associated with ATL. In this review, we provide an updated synopsis of the role of Tax-1 in the deregulation of the NF-κB pathway, highlighting the differences with the homologous Tax-2. Special emphasis is directed toward the understanding of the molecular mechanisms involved in NF-κB activation resulting from Tax interaction with host factors affecting several cellular processes, such as cell cycle, apoptosis, senescence, cell proliferation, autophagy, and post-translational modifications. We also discuss the current knowledge on the role of the antisense viral protein HBZ in down-regulating the NF-κB activation induced by Tax, and its implication in cellular senescence. In addition, we review the recent studies on the mechanism of HBZ-mediated inhibition of NF-κB activity as compared to that exerted by the HTLV-2 antisense protein, APH-2. Finally, we discuss recent advances aimed at understanding the role exerted in the development of ATL by the perturbation of NF-κB pathway by viral regulatory proteins. PMID:29515558

HTLV Deregulation of the NF-κB Pathway: An Update on Tax and Antisense Proteins Role.

Science.gov (United States)

Fochi, Stefania; Mutascio, Simona; Bertazzoni, Umberto; Zipeto, Donato; Romanelli, Maria G

2018-01-01

Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), an aggressive CD4 + /CD25 + T-cell malignancy and of a severe neurodegenerative disease, HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The chronic activation or deregulation of the canonical and non-canonical nuclear factor kappa B (NF-κB) pathways play a crucial role in tumorigenesis. The HTLV-1 Tax-1 oncoprotein is a potent activator of the NF-κB transcription factors and the NF-κB response is required for promoting the development of HTLV-1 transformed cell lines. The homologous retrovirus HTLV-2, which also expresses a Tax-2 transforming protein, is not associated with ATL. In this review, we provide an updated synopsis of the role of Tax-1 in the deregulation of the NF-κB pathway, highlighting the differences with the homologous Tax-2. Special emphasis is directed toward the understanding of the molecular mechanisms involved in NF-κB activation resulting from Tax interaction with host factors affecting several cellular processes, such as cell cycle, apoptosis, senescence, cell proliferation, autophagy, and post-translational modifications. We also discuss the current knowledge on the role of the antisense viral protein HBZ in down-regulating the NF-κB activation induced by Tax, and its implication in cellular senescence. In addition, we review the recent studies on the mechanism of HBZ-mediated inhibition of NF-κB activity as compared to that exerted by the HTLV-2 antisense protein, APH-2. Finally, we discuss recent advances aimed at understanding the role exerted in the development of ATL by the perturbation of NF-κB pathway by viral regulatory proteins.

NATpipe: an integrative pipeline for systematical discovery of natural antisense transcripts (NATs) and phase-distributed nat-siRNAs from de novo assembled transcriptomes

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Yu, Dongliang; Meng, Yijun; Zuo, Ziwei; Xue, Jie; Wang, Huizhong

2016-01-01

Nat-siRNAs (small interfering RNAs originated from natural antisense transcripts) are a class of functional small RNA (sRNA) species discovered in both plants and animals. These siRNAs are highly enriched within the annealed regions of the NAT (natural antisense transcript) pairs. To date, great research efforts have been taken for systematical identification of the NATs in various organisms. However, developing a freely available and easy-to-use program for NAT prediction is strongly demanded by researchers. Here, we proposed an integrative pipeline named NATpipe for systematical discovery of NATs from de novo assembled transcriptomes. By utilizing sRNA sequencing data, the pipeline also allowed users to search for phase-distributed nat-siRNAs within the perfectly annealed regions of the NAT pairs. Additionally, more reliable nat-siRNA loci could be identified based on degradome sequencing data. A case study on the non-model plant Dendrobium officinale was performed to illustrate the utility of NATpipe. Finally, we hope that NATpipe would be a useful tool for NAT prediction, nat-siRNA discovery, and related functional studies. NATpipe is available at www.bioinfolab.cn/NATpipe/NATpipe.zip. PMID:26858106

Effective photo-enhancement of cellular activity of fluorophore-octaarginine antisense PNA conjugates correlates with singlet oxygen formation, endosomal escape and chromophore lipophilicity

DEFF Research Database (Denmark)

Yarani, Reza; Shiraishi, Takehiko; Nielsen, Peter E.

2018-01-01

Photochemical internalization (PCI) is a cellular drug delivery method based on the generation of light-induced reactive oxygen species (ROS) causing damage to the endosomal membrane and thereby resulting in drug release to the cytoplasm. In our study a series of antisense fluorophore octaarginin...... indicate that efficient photodynamic endosomal escape is strongly dependent on the quantum yield for photochemical singlet oxygen formation, photostability as well as the lipophilicity of the chromophore....

A small molecule for a big transformation: Topical application of a 20-nucleotide-long antisense fragment of the DIAP-2 gene inhibits the development of Drosophila melanogaster female imagos

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Nyadar Palmah M.

2018-01-01

Full Text Available Several genes have been identified to play important roles associated with sex selection in Drosophila melanogaster. An essential part is attributed to the sex-lethal gene that depends on the expression of the X:A (number of chromosomes to autosomes ratio signal controlling both sex selection and dosage compensation processes in D. melanogaster. Interestingly, for sex selection in D. melanogaster there are no documented data addressing the role of the inhibitor of apoptosis (IAP genes and their signaling influence on this biological process. In this study, we found that topical application of a 20-nucleotide-long antisense DNA fragment (oligoDIAP-2 from the death-associated inhibitor of apoptosis (DIAP-2 gene interferes with D. melanogaster development and significantly decreases the number of female imagos and their biomass. We show that the applied antisense oligoDIAP-2 fragment downregulates the target DIAP-2 gene whose normal concentration is necessary for the development of female D. melanogaster. These data correspond to the results on downregulation of the target host IAP-Z gene of Lymantria dispar L. female imagos after topical treatment with an 18-nucleotide-long antisense DNA fragment from the L. dispar multicapsid nuclear polyhedrosis virus IAP-3 gene at the larval stage. The observed novel phenomenon linking the downregulation of insect IAP genes and the low rate of female imago development could have practical application, especially in insect pest control and molecular pathology.

Structure and expression of two nuclear receptor genes in marsupials: insights into the evolution of the antisense overlap between the α-thyroid hormone receptor and Rev-erbα

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Brown M Scott

2010-12-01

Full Text Available Abstract Background Alternative processing of α-thyroid hormone receptor (TRα, NR1A1 mRNAs gives rise to two functionally antagonistic nuclear receptors: TRα1, the α-type receptor, and TRα2, a non-hormone binding variant that is found only in mammals. TRα2 shares an unusual antisense coding overlap with mRNA for Rev-erbα (NR1D1, another nuclear receptor protein. In this study we examine the structure and expression of these genes in the gray short-tailed opossum, Monodelphis domestica, in comparison with that of eutherian mammals and three other marsupial species, Didelphis virginiana, Potorous tridactylus and Macropus eugenii, in order to understand the evolution and regulatory role of this antisense overlap. Results The sequence, expression and genomic organization of mRNAs encoding TRα1 and Rev-erbα are very similar in the opossum and eutherian mammals. However, the sequence corresponding to the TRα2 coding region appears truncated by almost 100 amino acids. While expression of TRα1 and Rev-erbα was readily detected in all tissues of M. domestica ages 0 days to 18 weeks, TRα2 mRNA was not detected in any tissue or stage examined. These results contrast with the widespread and abundant expression of TRα2 in rodents and other eutherian mammals. To examine requirements for alternative splicing of TRα mRNAs, a series of chimeric minigenes was constructed. Results show that the opossum TRα2-specific 5' splice site sequence is fully competent for splicing but the sequence homologous to the TRα2 3' splice site is not, even though the marsupial sequences are remarkably similar to core splice site elements in rat. Conclusions Our results strongly suggest that the variant nuclear receptor isoform, TRα2, is not expressed in marsupials and that the antisense overlap between TRα and Rev-erbα thus is unique to eutherian mammals. Further investigation of the TRα and Rev-erbα genes in marsupial and eutherian species promises to yield

Structure and expression of two nuclear receptor genes in marsupials: insights into the evolution of the antisense overlap between the α-thyroid hormone receptor and Rev-erbα

Science.gov (United States)

2010-01-01

Background Alternative processing of α-thyroid hormone receptor (TRα, NR1A1) mRNAs gives rise to two functionally antagonistic nuclear receptors: TRα1, the α-type receptor, and TRα2, a non-hormone binding variant that is found only in mammals. TRα2 shares an unusual antisense coding overlap with mRNA for Rev-erbα (NR1D1), another nuclear receptor protein. In this study we examine the structure and expression of these genes in the gray short-tailed opossum, Monodelphis domestica, in comparison with that of eutherian mammals and three other marsupial species, Didelphis virginiana, Potorous tridactylus and Macropus eugenii, in order to understand the evolution and regulatory role of this antisense overlap. Results The sequence, expression and genomic organization of mRNAs encoding TRα1 and Rev-erbα are very similar in the opossum and eutherian mammals. However, the sequence corresponding to the TRα2 coding region appears truncated by almost 100 amino acids. While expression of TRα1 and Rev-erbα was readily detected in all tissues of M. domestica ages 0 days to 18 weeks, TRα2 mRNA was not detected in any tissue or stage examined. These results contrast with the widespread and abundant expression of TRα2 in rodents and other eutherian mammals. To examine requirements for alternative splicing of TRα mRNAs, a series of chimeric minigenes was constructed. Results show that the opossum TRα2-specific 5' splice site sequence is fully competent for splicing but the sequence homologous to the TRα2 3' splice site is not, even though the marsupial sequences are remarkably similar to core splice site elements in rat. Conclusions Our results strongly suggest that the variant nuclear receptor isoform, TRα2, is not expressed in marsupials and that the antisense overlap between TRα and Rev-erbα thus is unique to eutherian mammals. Further investigation of the TRα and Rev-erbα genes in marsupial and eutherian species promises to yield additional insight into the

Antisense pre-treatment increases gene therapy efficacy in dystrophic muscles.

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Peccate, Cécile; Mollard, Amédée; Le Hir, Maëva; Julien, Laura; McClorey, Graham; Jarmin, Susan; Le Heron, Anita; Dickson, George; Benkhelifa-Ziyyat, Sofia; Piétri-Rouxel, France; Wood, Matthew J; Voit, Thomas; Lorain, Stéphanie

2016-08-15

In preclinical models for Duchenne muscular dystrophy, dystrophin restoration during adeno-associated virus (AAV)-U7-mediated exon-skipping therapy was shown to decrease drastically after six months in treated muscles. This decline in efficacy is strongly correlated with the loss of the therapeutic AAV genomes, probably due to alterations of the dystrophic myofiber membranes. To improve the membrane integrity of the dystrophic myofibers at the time of AAV-U7 injection, mdx muscles were pre-treated with a single dose of the peptide-phosphorodiamidate morpholino (PPMO) antisense oligonucleotides that induced temporary dystrophin expression at the sarcolemma. The PPMO pre-treatment allowed efficient maintenance of AAV genomes in mdx muscles and enhanced the AAV-U7 therapy effect with a ten-fold increase of the protein level after 6 months. PPMO pre-treatment was also beneficial to AAV-mediated gene therapy with transfer of micro-dystrophin cDNA into muscles. Therefore, avoiding vector genome loss after AAV injection by PPMO pre-treatment would allow efficient long-term restoration of dystrophin and the use of lower and thus safer vector doses for Duchenne patients. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Changes of Tc-99m sestamibi uptake in P-glycoprotein expressing leukaemia cells treated in vivo with antisense oligodeoxynucleotide complementary to mdr1 mRNA

International Nuclear Information System (INIS)

Kinuya, S.; Yokoyama, K; Fukuoka, M.; Michigishi, T.; Tonami, N.; Shiba, K.; Mori, H.; Watanabe, N.; Shuke, N.

2006-01-01

We examined the feasibility of Tc-99m sestamibi to monitor changes of mRNA expression of MDRl/P-glycoprotein (Pgp) following antisense oligodeoxynucleotide (AS-ODN) treatment in vivo. Three days after the intraperitoneal inoculation of murine leukaemia P388/R cells expressing MDR1/P-gp in CDFI mice, 15-mer phosphorothioate ASODN to the initiation codon of mouse mdr1 mRNA was administered intraperitoneally at 10 mg/kg daily for 3 or 4 days. Cells collected from ascites were suspended in medium for Tc-99m sestamibi uptake studies. To know the duration of antisense effects, cells were harvested 2 days later after the 3-day treatment. AS-ODN treatment increased Tc-99m sestamibi uptake. Effects of 3-day treatment and 4-day treatment were the same. Treatment effects were not detected when uptake was observed 2 days after 3-day treatment. Based on the results it was concluded that in vivo treatment with AS-ODN specific to the coding portion of mdr1 mRNA increased Tc-99m sestamibi uptake in leukaemia cells possessing MDR function. (author)

Andrographolide - A promising therapeutic agent, negatively regulates glial cell derived neurodegeneration of prefrontal cortex, hippocampus and working memory impairment.

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Das, Sudeshna; Mishra, K P; Ganju, Lilly; Singh, S B

2017-12-15

Over activation of glial cell derived innate immune factors induces neuro-inflammation that results in neurodegenerative disease, like working memory impairment. In this study, we have investigated the role of andrographolide, a major constituent of Andrographis paniculata plant, in reduction of reactive glial cell derived working memory impairment. Real time PCR, Western bloting, flow cytometric and immunofluorescence studies demonstrated that andrographolide inhibited lipopolysaccharide (LPS)-induced overexpression of HMGB1, TLR4, NFκB, COX-2, iNOS, and release of inflammatory mediators in primary mix glial culture, adult mice prefrontal cortex and hippocampus region. Active microglial and reactive astrocytic makers were also downregulated after andrographolide treatment. Andrographolide suppressed overexpression of microglial MIP-1α, P2X7 receptor and its downstream signaling mediators including-inflammasome NLRP3, caspase1 and mature IL-1β. Furthermore, in vivo maze studies suggested that andrographolide treatment reversed LPS-induced behavioural and working memory disturbances including regulation of expression of protein markers like PKC, p-CREB, amyloid beta, APP, p-tau, synapsin and PSD-95. Andrographolide, by lowering expression of pro apoptotic genes and enhancing the expression of anti-apoptotic gene showed its anti-apoptotic nature that in turn reduces neurodegeneration. Morphology studies using Nissl and FJB staining also showed the neuroprotective effect of andrographolide in the prefrontal cortex region. The above studies indicated that andrographolide prevented neuroinflammation-associated neurodegeneration and improved synaptic plasticity markers in cortical as well as hippocampal region which suggests that andrographolide could be a novel pharmacological countermeasure for the treatment of neuroinflammation and neurological disorders related to memory impairment. Copyright © 2017 Elsevier B.V. All rights reserved.

Anti-sense expression of a metallopeptidase gene enhances nuclear entry of HBV-DNA

International Nuclear Information System (INIS)

Yeh, C.-T.; Lai, H.-Y.; Chu, S.-P.; Tseng, I-Chu

2004-01-01

Although several putative hepatitis B virus (HBV) receptors have been identified, none of them is capable of initiating HBV replication in a non-permissive human cell line. Using an Epstein-Barr virus-based extrachromosomal replication system, we have screened through a human liver cDNA library and successfully identified a clone capable of facilitating nuclear transport of HBV-DNA during the early phase of HBV infection. This clone contained a cDNA encoding a metallopeptidase-like protein in anti-sense orientation. Pretreatment of naive HepG2 cells with 1,10-phenanthroline, an inhibitor for liver metallopeptidases, led to nuclear entry of HBV-DNA after HBV infection. However, cccDNA was still undetectable in the nuclei, indicating other cellular factors required to complete the replication cycle were still missing. Our present data suggest that in the initial stage of HBV infection, liver metallopeptidase constitutes a barrier for effective nuclear entry of HBV genomic DNA. Attenuation of metallopeptidase activity may facilitate HBV infection

A long natural-antisense RNA is accumulated in the conidia of Aspergillus oryzae.

Science.gov (United States)

Tsujii, Masaru; Okuda, Satoshi; Ishi, Kazutomo; Madokoro, Kana; Takeuchi, Michio; Yamagata, Youhei

2016-01-01

Analysis of expressed sequence tag libraries from various culture conditions revealed the existence of conidia-specific transcripts assembled to putative conidiation-specific reductase gene (csrA) in Aspergillus oryzae. However, the all transcripts were transcribed with opposite direction to the gene csrA. The sequence analysis of the transcript revealed that the RNA overlapped mRNA of csrA with 3'-end, and did not code protein longer than 60 amino acid residues. We designated the transcript Conidia Specific Long Natural-antisense RNA (CSLNR). The real-time PCR analysis demonstrated that the CSLNR is conidia-specific transcript, which cannot be transcribed in the absence of brlA, and the amount of CSLNR was much more than that of the transcript from csrA in conidia. Furthermore, the csrA deletion, also lacking coding region of CSLNR in A. oryzae reduced the number of conidia. Overexpression of CsrA demonstrated the inhibition of growth and conidiation, while CSLNR did not affect conidiation.

Molecular imaging of atherosclerotic plaques with technetium-99m-labelled antisense oligonucleotides

International Nuclear Information System (INIS)

Qin Guangming; Zhang Yongxue; Cao Wei; An Rui; Gao Zairong; Xu Wendai; Zhang Kaijun; Li Guiling; Li Shuren

2005-01-01

The purpose of this study was to visualise experimental atherosclerotic lesions using radiolabelled antisense oligonucleotides (ASONs). Atherosclerosis was induced in New Zealand White rabbits fed 1% cholesterol for approximately 60 days. In vivo and ex vivo imaging was performed in atherosclerotic rabbits and normal control rabbits after i.v. injection of 92.5±18.5 MBq 99m Tc-labelled ASON or 99m Tc-labelled sense oligonucleotides. Immediately after the in vivo imaging, the animals were sacrificed and ex vivo imaging of the aortic specimens was performed. Biodistribution of radiolabelled c-mycASON was evaluated in vivo in atherosclerotic rabbits. Planar imaging revealed accumulation of 99m Tc-labelled c-mycASON in atherosclerotic lesions along the artery wall. Ex vivo imaging further demonstrated that the area of activity accumulation matched the area of atherosclerotic lesions. In contrast, no atherosclerotic lesions were found in the vessel wall and no positive imaging results were obtained in animals of the control group. This molecular imaging approach has potential for non-invasive imaging of atherosclerotic plaques at an early stage. (orig.)

Beneficial Effects of Gagam-Palmultang on Scopolamine-Induced Memory Deficits in Mice

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Yu Ri Kim

2018-01-01

Full Text Available From text mining of Dongeuibogam, the 7 herbs in Palmultang can be considered effective candidates for memory enhancement. We sought to determine whether Gagam-Palmultang, comprising these 7 herbs, ameliorates scopolamine-induced memory impairment in mice, by focusing on the central cholinergic system and memory-related signaling molecules. Behavioral tests were performed after inducing memory impairment by scopolamine administration. The cholinergic system activity and memory-related molecules were examined in the hippocampus by enzyme-linked immunosorbent, western blot, and immunofluorescence assays. Gagam-Palmultang ameliorated scopolamine-induced memory impairment in the Morris water maze test, producing a significant improvement in the mean time required to find the hidden platform. Treatment with Gagam-Palmultang reduced acetylcholinesterase activity and expression in the hippocampus induced by scopolamine. The diminished phosphorylated phosphatidylinositide 3-kinase (PI3K, extracellular signal-regulated kinase (ERK, cAMP response element-binding protein (CREB, and mature brain-derived neurotrophic factor (mBDNF expressions caused by scopolamine administration were attenuated by treatment with Gagam-Palmultang. This treatment also promoted neuronal cell proliferation in the hippocampus. Gagam-Palmultang has beneficial effects against scopolamine-induced memory impairments, which are exerted via modulation of the cholinergic system as well as the PI3K and ERK/CREB/BDNF signaling pathway. Therefore, this multiherb formula may be a useful therapeutic agent for diseases associated with memory impairments.

Delivery of a survivin promoter-driven antisense survivin-expressing plasmid DNA as a cancer therapeutic: a proof-of-concept study

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Lin KY

2016-05-01

Full Text Available Kun-Yuan Lin,1 Siao Muk Cheng,2 Shing-Ling Tsai,2 Ju-Ya Tsai,1 Chun-Hui Lin,1 Chun Hei Antonio Cheung1,2 1Department of Pharmacology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC; 2Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, Tainan, Taiwan, ROC Abstract: Survivin is a member of the inhibitor-of-apoptosis proteins family. It is overexpressed in many different cancer types but not in the differentiated normal tissue. In addition, overexpression of survivin promotes cancer cell survival and induces chemotherapeutic drug resistance, making it an attractive target for new anticancer interventions. Despite survivin being a promising molecular target for anticancer treatment, it is widely accepted that survivin is only a “semi-druggable” target. Therefore, it is important to develop a new strategy to target survivin for anticancer treatment. In this study, we constructed a novel survivin promoter-driven full-length antisense survivin (pSur/AS-Sur expression plasmid DNA. Promoter activity assay revealed that the activity of the survivin promoter of pSur/AS-Sur correlated with the endogenous expression of survivin at the transcriptional level in the transfected A549, MDA-MB-231, and PANC-1 cancer cells. Western blot analysis showed that liposomal delivery of pSur/AS-Sur successfully downregulated the expression of survivin in A549, MBA-MB-231, and PANC-1 cells in vitro. In addition, delivery of pSur/AS-Sur induced autophagy, caspase-dependent apoptosis, and caspase-independent apoptosis as indicated by the increased LC3B-II conversion, autophagosome formation, caspase-9/-3 and poly(ADP-ribose polymerase-1 cleavage, and apoptosis-inducing factor nuclear translocation in A549, MBA-MB-231, and PANC-1 cells. Importantly, liposomal delivery of pSur/AS-Sur was also capable of decreasing the proliferation of the survivin/MDR1 coexpressing multidrug-resistant KB-TAX50 cancer cells and

Hippocampal Focal Knockout of CBP Affects Specific Histone Modifications, Long-Term Potentiation, and Long-Term Memory

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Barrett, Ruth M; Malvaez, Melissa; Kramar, Eniko; Matheos, Dina P; Arrizon, Abraham; Cabrera, Sara M; Lynch, Gary; Greene, Robert W; Wood, Marcelo A

2011-01-01

To identify the role of the histone acetyltransferase (HAT) CREB-binding protein (CBP) in neurons of the CA1 region of the hippocampus during memory formation, we examine the effects of a focal homozygous knockout of CBP on histone modifications, gene expression, synaptic plasticity, and long-term memory. We show that CBP is critical for the in vivo acetylation of lysines on histones H2B, H3, and H4. CBP's homolog p300 was unable to compensate for the loss of CBP. Neurons lacking CBP maintained phosphorylation of the transcription factor CREB, yet failed to activate CREB:CBP-mediated gene expression. Loss of CBP in dorsal CA1 of the hippocampus resulted in selective impairments to long-term potentiation and long-term memory for contextual fear and object recognition. Together, these results suggest a necessary role for specific chromatin modifications, selectively mediated by CBP in the consolidation of memories. PMID:21508930

Poly(ester amine Composed of Polyethylenimine and Pluronic Enhance Delivery of Antisense Oligonucleotides In Vitro and in Dystrophic mdx Mice

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Mingxing Wang

2016-01-01

Full Text Available A series of poly(esteramines (PEAs constructed from low molecular weight polyethyleneimine (LPEI and Pluronic were evaluated for the delivery of antisense oligonuclotides (AOs, 2′-O-methyl phosphorothioate RNA (2′-OMePS and phosphorodiamidate morpholino oligomer (PMO in cell culture and dystrophic mdx mice. Improved exon-skipping efficiency of both 2′-OMePS and PMO was observed in the C2C12E50 cell line with all PEA polymers compared with PEI 25k or LF-2k. The degree of efficiency was found in the order of PEA 01, PEA 04 > PEA 05 > others. The in vivo study in mdx mice demonstrated enhanced exon-skipping of 2′-OMePS with the order of PEA 06 > PEA 04, PEA 07 > PEA 03 > PEA 01 > others, and much higher than PEI 25k formulated 2′-OMePS. Exon-skipping efficiency of PMO in formulation with the PEAs were significantly enhanced in the order of PEA 02 > PEA 10 > PEA 01, PEA 03 > PEA 05, PEA 07, PEA 08 > others, with PEA 02 reaching fourfold of Endo-porter formulated PMO. PEAs improve PMO delivery more effectively than 2′-OMePS delivery in vivo, and the systemic delivery evaluation further highlight the efficiency of PEA for PMO delivery in all skeletal muscle. The results suggest that the flexibility of PEA polymers could be explored for delivery of different AO chemistries, especially for antisense therapy.

The antisense expression of AhPEPC1 increases seed oil production in peanuts (Arachis hypogaea L.)

Energy Technology Data Exchange (ETDEWEB)

Pan, L.; Zhang, J.; Chi, X.; Chen, N.; Chen, M.; Wang, M.; Wang, T.; Yang, Z.; Zhang, Z.; Wan, Y.; Yu, S.; Liu, F.

2016-07-01

Although phosphoenolpyruvate carboxylases (PEPCs) are reported to be involved in fatty acid accumulation, nitrogen assimilation, and salt and drought stresses, knowledge regarding PEPC gene functions is still limited, particularly in peanuts (Arachis hypogaea L.). In this study, the antisense expression of the peanut PEPC isoform 1 (AhPEPC1) gene increased the lipid content by 5.7%–10.3%. This indicated that AhPEPC1 might be related to plant lipid accumulation. The transgenic plants underwent more root elongation than the wild-type under salinity stress. Additionally, the specific down regulation of the AhPEPC1 gene improved the salt tolerance in peanuts. This is the first report on the role of PEPC in lipid accumulation and salt tolerance in peanuts.

Formulation and drug-content assay of microencapsulated antisense oligonucleotide to NF-κB using ATR-FTIR

International Nuclear Information System (INIS)

Siwale, Rodney; Meadows, Fred; Mody, Vicky V; Shah, Samit

2013-01-01

Antisense oligonucleotide to NF-κB sequence: 5′-GGA AAC ACA TCC TCC ATG-3′, was microencapsulated in an albumin matrix by the method of spray drying TM . Spectral analysis was performed on varying drug loading formulations of both drugs by mid-IR attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). An out of plane O–H bending vibration at 948 cm −1 , unique to both the native and microencapsulated drugs was identified. The calculated peak areas corresponded to the drug loadings in the microsphere formulations. A standard curve could then be used to determine the drug content of an unknown microsphere formulation. Accuracy and precision were determined to be comparable to other analytical techniques such as HPLC. (paper)

Latency of Epstein-Barr virus is stabilized by antisense-mediated control of the viral immediate-early gene BZLF-1.

Science.gov (United States)

Prang, N; Wolf, H; Schwarzmann, F

1999-12-01

The ability of the Epstein-Barr virus (EBV) to avoid lytic replication and to establish a latent infection in B-lymphocytes is fundamental for its lifelong persistence and the pathogenesis of various EBV-associated diseases. The viral immediate-early gene BZLF-1 plays a key role for the induction of lytic replication and its activity is strictly regulated on different levels of gene expression. Recently, it was demonstrated that BZLF-1 is also controlled by a posttranscriptional mechanism. Transient synthesis of a mutated competitor RNA saturated this mechanism and caused both expression of the BZLF-1 protein and the induction of lytic viral replication. Using short overlapping fragments of the competitor, it is shown that this control acts on the unspliced primary transcript. RT-PCR demonstrated unspliced BZLF-1 RNA in latently infected B-lymphocytes in the absence of BZLF-1 protein. Due to the complementarity of the gene BZLF-1 and the latency-associated gene EBNA-1 on the opposite strand of the genome, we propose an antisense-mediated mechanism. RNase protection assays demonstrated transcripts in antisense orientation to the BZLF-1 transcript during latency, which comprise a comparable constellation to other herpesviruses. A combined RNAse protection/RT-PCR assay detected the double-stranded hybrid RNA, consisting of the unspliced BZLF-1 transcript and a noncoding intron of the EBNA-1 gene. Binding of BZLF-1 transcripts is suggested to be an important backup control mechanism in addition to transcriptional regulation, stabilizing latency and preventing inappropriate lytic viral replication in vivo. Copyright 1999 Wiley-Liss, Inc.

[Subchronic toxicity test of genetically modified rice with double antisense starch-branching enzyme gene].

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Li, Min; Piao, Jianhua; Yang, Xiaoguang

2010-07-01

To observe the sub-chronic toxic effects of the genetically modified rice with double antisense SBE gene. Based on gender and weight, weanling Wistar rats were randomly sorted into five groups: non-genetically modified rice group (group A), genetically modified rice group (group B), half genetically modified rice group (group C), quarter genetically modified rice group (group D) and AIN-93G normal diet group (group E). Indicators were the followings: body weight, food consumption, blood routine, blood biochemical test, organ weight, bone density and pathological examination of organs. At the middle of the experiment, the percentage of monocyte of female group B was less than that of group E (P 0.05), and no notable abnormity in the pathological examination of main organs (P > 0.05). There were no enough evidence to confirm the sub-chronic toxicity of genetically modified rice on rats.

Attenuation of alpha2A-adrenergic receptor expression in neonatal rat brain by RNA interference or antisense oligonucleotide reduced anxiety in adulthood.

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Shishkina, G T; Kalinina, T S; Dygalo, N N

2004-01-01

Brain alpha2-adrenergic receptors (alpha2-ARs) have been implicated in the regulation of anxiety, which is associated with stress. Environmental treatments during neonatal development could modulate the level of brain alpha2-AR expression and alter anxiety in adults, suggesting possible involvement of these receptors in early-life programming of anxiety state. The present study was undertaken to determine whether the reduction of the expression of A subtype of these receptors most abundant in the neonatal brain affects anxiety-related behavior in adulthood. We attenuated the expression of alpha2A-ARs during neonatal life by two different sequence specific approaches, antisense technology and RNA interference. Treatment of rats with the antisense oligodeoxynucleotide or short interfering RNA (siRNA) against alpha2A-ARs on the days 2-4 of their life, produced a marked acute decrease in the levels of both alpha2A-AR mRNA and [3H]RX821002 binding sites in the brainstem into which drugs were injected. The decrease of alpha2A-AR expression in the neonatal brainstem influenced the development of this receptor system in the brain regions as evidenced by the increased number of [3H]RX821002 binding sites in the hypothalamus of adult animals with both neonatal alpha2A-AR knockdown treatments; also in the frontal cortex of antisense-treated, and in the hippocampus of siRNA-treated adult rats. These adult animals also demonstrated a decreased anxiety in the elevated plus-maze as evidenced by an increased number of the open arm entries, greater proportion of time spent in the open arms, and more than a two-fold increase in the number of exploratory head dips. The results provide the first evidence that the reduction in the brain expression of a gene encoding for alpha2A-AR during neonatal life led to the long-term neurochemical and behavioral alterations. The data suggests that alterations in the expression of the receptor-specific gene during critical periods of brain

Endogenous Msx1 antisense transcript: In vivo and in vitro evidences, structure, and potential involvement in skeleton development in mammals

OpenAIRE

Blin-Wakkach, C.; Lezot, F.; Ghoul-Mazgar, S.; Hotton, D.; Monteiro, S.; Teillaud, C.; Pibouin, L.; Orestes-Cardoso, S.; Papagerakis, P.; Macdougall, M.; Robert, B.; Berdal, A.

2001-01-01

Msx1 is a key factor for the development of tooth and craniofacial skeleton and has been proposed to play a pivotal role in terminal cell differentiation. In this paper, we demonstrated the presence of an endogenous Msx1 antisense RNA (Msx1-AS RNA) in mice, rats, and humans. In situ analysis revealed that this RNA is expressed only in differentiated dental and bone cells with an inverse correlation with Msx1 protein. These in vivo data and overexpression of Msx1 sense and AS RNA in an odontob...

Repeated forced swim stress enhances CFA-evoked thermal hyperalgesia and affects the expressions of pCREB and c-Fos in the insular cortex.

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Imbe, H; Kimura, A; Donishi, T; Kaneoke, Y

2014-02-14

Stress affects brain activity and promotes long-term changes in multiple neural systems. Exposure to stressors causes substantial effects on the perception and response to pain. In several animal models, chronic stress produces lasting hyperalgesia. The insular (IC) and anterior cingulate cortices (ACC) are the regions exhibiting most reliable pain-related activity. And the IC and ACC play an important role in pain modulation via the descending pain modulatory system. In the present study we examined the expression of phospho-cAMP response element-binding protein (pCREB) and c-Fos in the IC and ACC after forced swim stress (FS) and complete Freund's adjuvant (CFA) injection to clarify changes in the cerebral cortices that affect the activity of the descending pain modulatory system in the rats with stress-induced hyperalgesia. FS (day 1, 10min; days 2-3, 20min) induced an increase in the expression of pCREB and c-Fos in the anterior IC (AIC). CFA injection into the hindpaw after the FS shows significantly enhanced thermal hyperalgesia and induced a decrease in the expression of c-Fos in the AIC and the posterior IC (PIC). Quantitative image analysis showed that the numbers of c-Fos-immunoreactive neurons in the left AIC and PIC were significantly lower in the FS+CFA group (L AIC, 95.9±6.8; L PIC, 181.9±23.1) than those in the naive group (L AIC, 151.1±19.3, pCFA-induced thermal hyperalgesia through dysfunction of the descending pain modulatory system. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Endogenous BDNF is required for long-term memory formation in the rat parietal cortex.

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Alonso, Mariana; Bekinschtein, Pedro; Cammarota, Martín; Vianna, Monica R M; Izquierdo, Iván; Medina, Jorge H

2005-01-01

Information storage in the brain is a temporally graded process involving different memory phases as well as different structures in the mammalian brain. Cortical plasticity seems to be essential to store stable long-term memories, although little information is available at the moment regarding molecular and cellular events supporting memory consolidation in the neocortex. Brain-derived neurotrophic factor (BDNF) modulates both short-term synaptic function and activity-dependent synaptic plasticity in hippocampal and cortical neurons. We have recently demonstrated that endogenous BDNF in the hippocampus is involved in memory formation. Here we examined the role of BDNF in the parietal cortex (PCx) in short-term (STM) and long-term memory (LTM) formation of a one-trial fear-motivated learning task in rats. Bilateral infusions of function-blocking anti-BDNF antibody into the PCx impaired both STM and LTM retention scores and decreased the phosphorylation state of cAMP response element-binding protein (CREB). In contrast, intracortical administration of recombinant human BDNF facilitated LTM and increased CREB activation. Moreover, inhibitory avoidance training is associated with a rapid and transient increase in phospho-CREB/total CREB ratio in the PCx. Thus, our results indicate that endogenous BDNF is required for both STM and LTM formation of inhibitory avoidance learning, possibly involving CREB activation-dependent mechanisms. The present data support the idea that early sensory areas constitute important components of the networks subserving memory formation and that information processing in neocortex plays an important role in memory formation.

Linderane Suppresses Hepatic Gluconeogenesis by Inhibiting the cAMP/PKA/CREB Pathway Through Indirect Activation of PDE 3 via ERK/STAT3

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Wei Xie

2018-05-01

Full Text Available The role of phosphodiesterase 3 (PDE3, a cyclic AMP (cAMP-degrading enzyme, in modulating gluconeogenesis remains unknown. Here, linderane, a natural compound, was found to inhibit gluconeogenesis by activating hepatic PDE3 in rat primary hepatocytes. The underlying molecular mechanism and its effects on whole-body glucose and lipid metabolism were investigated. The effect of linderane on gluconeogenesis, cAMP content, phosphorylation of cAMP-response element-binding protein (CREB and PDE activity were examined in cultured primary hepatocytes and C57BL/6J mice. The precise mechanism by which linderane activates PDE3 and inhibits the cAMP pathway was explored using pharmacological inhibitors. The amelioration of metabolic disorders was observed in ob/ob mice. Linderane inhibited gluconeogenesis, reduced phosphoenolpyruvate carboxykinase (Pck1 and glucose-6-phosphatase (G6pc gene expression, and decreased intracellular cAMP concentration and CREB phosphorylation in rat primary hepatocytes under both basal and forskolin-stimulated conditions. In rat primary hepatocytes, it also increased total PDE and PDE3 activity but not PDE4 activity. The suppressive effect of linderane on the cAMP pathway and gluconeogenesis was abolished by the non-specific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX and the specific PDE3 inhibitor cilostazol. Linderane indirectly activated PDE3 through extracellular regulated protein kinase 1/2 (ERK1/2 and signal transducer and activator of transcription 3 (STAT3 activation. Linderane improved glucose and lipid metabolism after chronic oral administration in ob/ob mice. Our findings revealed linderane as an indirect PDE3 activator that suppresses gluconeogenesis through cAMP pathway inhibition and has beneficial effects on metabolic syndromes in ob/ob mice. This investigation highlighted the potential for PDE3 activation in the treatment of type 2 diabetes.

Synaptotagmin 3 deficiency in T cells impairs recycling of the chemokine receptor CXCR4 and thereby inhibits CXCL12 chemokine-induced migration.

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Masztalerz, Agnieszka; Zeelenberg, Ingrid S; Wijnands, Yvonne M; de Bruijn, Rosalie; Drager, Angelika M; Janssen, Hans; Roos, Ed

2007-01-15

Synaptotagmins regulate vesicle trafficking and fusion of vesicles with membranes - processes that have been implicated in cell migration. We therefore hypothesized that synaptotagmins play a role in T-cell migration. Amongst synaptotagmins 1-11, we found synaptotagmin 3 (SYT3) to be the only one that is expressed in T cells. CXCR4-triggered migration was inhibited by antisense synaptotagmin 3 mRNA and by the isolated C2B domain, known to impair oligomerization of all synaptotagmins, but not by a C2B mutant that binds Ca(2+) but does not block oligomerization. The C2B domain also blocked CXCR4-triggered actin polymerization and invasion. However, CXCR4-dependent adhesion in flow was not affected. Surprisingly, we found that little or no SYT3 is present near the plasma membrane but that it is mainly localized in multivesicular bodies, which also contained much of the CXCR4. Impaired SYT3 function blocked CXCR4 recycling and thus led to reduced surface levels of CXCR4. Migration was restored by overexpression of CXCR4. We conclude that STT3 is essential for CXCR4 recycling in T cells and thereby for the maintenance of high CXCR4 surface levels required for migration.

L-3-n-Butylphthalide Regulates Proliferation, Migration, and Differentiation of Neural Stem Cell In Vitro and Promotes Neurogenesis in APP/PS1 Mouse Model by Regulating BDNF/TrkB/CREB/Akt Pathway.

Science.gov (United States)

Lei, Hui; Zhang, Yu; Huang, Longjian; Xu, Shaofeng; Li, Jiang; Yang, Lichao; Wang, Ling; Xing, Changhong; Wang, Xiaoliang; Peng, Ying

2018-05-04

Alzheimer's disease (AD) is characterized by extracellular accumulation of β-amyloid peptides (Aβ) and intracellular neurofibrillary tangles, along with cognitive decline and neurodegeneration. The cognitive deficit is considered to be due to the dysfunction of hippocampal neurogenesis. Although L-3-n-butylphthalide (L-NBP) has been shown beneficial effects in multiple AD animal models, the underlying molecular mechanisms are still elusive. In this study, we investigated the effects of L-NBP on neurogenesis both in vitro and in vivo. L-NBP promoted proliferation and migration of neural stem cells and induced neuronal differentiation in vitro. In APP/PS1 mice, L-NBP induced neurogenesis in the dentate gyrus and improved cognitive functions. In addition, L-NBP significantly increased the expressions of BDNF and NGF, tyrosine phosphorylation of its cognate receptor, and phosphorylation of Akt as well as CREB at Ser133 in the hippocampus of APP/PS1 mice. These results indicated that L-NBP might stimulate the proliferation, migration, and differentiation of hippocampal neural stem cells and reversed cognitive deficits in APP/PS1 mice. BDNF/TrkB/CREB/Akt signaling pathway might be involved.

Inactivation of JAK2/STAT3 Signaling Axis and Downregulation of M1 mAChR Cause Cognitive Impairment in klotho Mutant Mice, a Genetic Model of Aging

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Park, Seok-Joo; Shin, Eun-Joo; Min, Sun Seek; An, Jihua; Li, Zhengyi; Hee Chung, Yoon; Hoon Jeong, Ji; Bach, Jae-Hyung; Nah, Seung-Yeol; Kim, Won-Ki; Jang, Choon-Gon; Kim, Yong-Sun; Nabeshima, Yo-ichi; Nabeshima, Toshitaka; Kim, Hyoung-Chun

2013-01-01

We previously reported cognitive dysfunction in klotho mutant mice. In the present study, we further examined novel mechanisms involved in cognitive impairment in these mice. Significantly decreased janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3) phosphorylation were observed in the hippocampus of klotho mutant mice. A selective decrease in protein expression and binding density of the M1 muscarinic cholinergic receptor (M1 mAChR) was observed in these mice. Cholinergic parameters (ie, acetylcholine (ACh), choline acetyltransferase (ChAT), and acetylcholinesterase (AChE)) and NMDAR-dependent long-term potentiation (LTP) were significantly impaired in klotho mutant mice. McN-A-343 (McN), an M1 mAChR agonist, significantly attenuated these impairments. AG490 (AG), a JAK2 inhibitor, counteracted the attenuating effects of McN, although AG did not significantly alter the McN-induced effect on AChE. Furthermore, AG significantly inhibited the attenuating effects of McN on decreased NMDAR-dependent LTP, protein kinase C βII, p-ERK, p-CREB, BDNF, and p-JAK2/p-STAT3-expression in klotho mutant mice. In addition, k252a, a BDNF receptor tyrosine kinase B (TrkB) inhibitor, significantly counteracted McN effects on decreased ChAT, ACh, and M1 mAChR and p-JAK2/p-STAT3 expression. McN-induced effects on cognitive impairment in klotho mutant mice were consistently counteracted by either AG or k252a. Our results suggest that inactivation of the JAK2/STAT3 signaling axis and M1 mAChR downregulation play a critical role in cognitive impairment observed in klotho mutant mice. PMID:23389690

Prelimbic cortex extracellular signal-regulated kinase 1/2 activation is required for memory retrieval of long-term inhibitory avoidance.

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Luo, Fei; Zheng, Jian; Sun, Xuan; Deng, Wei-Ke; Li, Bao Ming; Liu, Fang

2017-04-15

Neural mechanism underlying memory retrieval has been extensively studied in the hippocampus and amygdala. However, little is known about the role of medial prefrontal cortex in long-term memory retrieval. We evaluate this issue in one-trial step-through inhibitory avoidance (IA) paradigm. Our results showed that, 1) inactivation of mPFC by local infusion of GABA A -receptor agonist muscimol caused severe deficits in retrieval of 1-day and 7-day but had no effects on 2-h inhibitory avoidance memory; 2) the protein level of phosphorylated-ERK1/2 in mPFC were significantly increased following retrieval of 1-day and 7-day IA memory, so did the numbers of phosphorylated-ERK (pERK) and phosphorylated-CREB (pCREB) labeled neurons; 3) intra-mPFC infusion of ERK kinase inhibitor PD98095 significantly reduced phosphorylated ERK1/2 levels and phosphorylated-ERK1/2 and phosphorylated-CREB labeled cells, and severely impaired retrieval of 7-day IA memory when the drugs were administrated 30min prior to test. The present study provides evidence that retrieval of long-lasting memory for inhibitory avoidance requires mPFC and involves the ERK-CREB signaling cascade. Copyright © 2017 Elsevier B.V. All rights reserved.

Vliv hipokampální aplikace Nr1/Nr2 antisense oligodeoxynukleotidů na expresi proteinů postsynaptické denzity a na prepulzní inhibici

Czech Academy of Sciences Publication Activity Database

Vrajová, M.; Klaschka, Jan; Tejkalová, H.; Bubeníková-Valešová, V.

2011-01-01

Roč. 15, Suppl. 2 (2011), s. 11-14 ISSN 1211-7579 R&D Projects: GA MŠk(CZ) 1M0517 Institutional research plan: CEZ:AV0Z10300504 Keywords : NMDA receptor * PSD proteins * antisense oligodeoxynucleotides for NMDA-NR1/NR2 subunits * prepulse inhibition Subject RIV: FL - Psychiatry, Sexuology http://www.tigis.cz/images/stories/psychiatrie/2011/s2/03_vrajova_cns_2-11.pdf

The dense core vesicle protein IA-2, but not IA-2β, is required for active avoidance learning.

Science.gov (United States)

Carmona, G N; Nishimura, T; Schindler, C W; Panlilio, L V; Notkins, A L

2014-06-06

The islet-antigens IA-2 and IA-2β are major autoantigens in type-1 diabetes and transmembrane proteins in dense core vesicles (DCV). Recently we showed that deletion of both IA-2 and IA-2β alters the secretion of hormones and neurotransmitters and impairs behavior and learning. The present study was designed to evaluate the contribution to learning of each of these genes by using single knockout (SKO) and double knockout (DKO) mice in an active avoidance test. After 5 days of training, wild-type (WT) mice showed 60-70% active avoidance responses, whereas the DKO mice showed only 10-15% active avoidance responses. The degree of active avoidance responses in the IA-2 SKO mice was similar to that of the DKO mice, but in contrast, the IA-2β SKO mice behaved like WT mice showing 60-70% active avoidance responses. Molecular studies revealed a marked decrease in the phosphorylation of the cAMP response element-binding protein (CREB) and Ca(2+)/calmodulin-dependent protein kinase II (CAMKII) in the striatum and hippocampus of the IA-2 SKO and DKO mice, but not in the IA-2β SKO mice. To evaluate the role of CREB and CAMKII in the SKO and DKO mice, GBR-12909, which selectively blocks the dopamine uptake transporter and increases CREB and CAMKII phosphorylation, was administered. GBR-12909 restored the phosphorylation of CREB and CAMKII and increased active avoidance learning in the DKO and IA-2 SKO to near the normal levels found in the WT and IA-2β SKO mice. We conclude that in the absence of the DCV protein IA-2, active avoidance learning is impaired. Published by Elsevier Ltd.

Development of Multiexon Skipping Antisense Oligonucleotide Therapy for Duchenne Muscular Dystrophy

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Yokota, Toshifumi; Wood, Matthew J. A.

2013-01-01

Duchenne muscular dystrophy (DMD) is an incurable, X-linked progressive muscle degenerative disorder that results from the absence of dystrophin protein and leads to premature death in affected individuals due to respiratory and/or cardiac failure typically by age of 30. Very recently the exciting prospect of an effective oligonucleotide therapy has emerged which restores dystrophin protein expression to affected tissues in DMD patients with highly promising data from a series of clinical trials. This therapeutic approach is highly mutation specific and thus is personalised. Therefore DMD has emerged as a model genetic disorder for understanding and overcoming of the challenges of developing personalised genetic medicines. One of the greatest weaknesses of the current oligonucleotide approach is that it is a mutation-specific therapy. To address this limitation, we have recently demonstrated that exons 45–55 skipping therapy has the potential to treat clusters of mutations that cause DMD, which could significantly reduce the number of compounds that would need to be developed in order to successfully treat all DMD patients. Here we discuss and review the latest preclinical work in this area as well as a variety of accompanying issues, including efficacy and potential toxicity of antisense oligonucleotides, prior to human clinical trials. PMID:23984357

Effects of Antisense Oligonucleotides against C-Reactive Protein on the Development of Atherosclerosis in WHHL Rabbits

Directory of Open Access Journals (Sweden)

Qi Yu

2014-01-01

Full Text Available Increased plasma levels of C-reactive protein (CRP are closely associated with cardiovascular diseases, but whether CRP is directly involved in the pathogenesis of atherosclerosis is still under debate. Many controversial and contradictory results using transgenic mice and rabbits have been published but it is also unclear whether CRP lowering can be used for the treatment of atherosclerosis. In the current study, we examined the effects of the rabbit CRP antisense oligonucleotides (ASO on the development of atherosclerosis in WHHL rabbits. CRP ASO treatment led to a significant reduction of plasma CRP levels; however, both aortic and coronary atherosclerotic lesions were not significantly changed compared to those of control WHHL rabbits. These results suggest that inhibition of plasma CRP does not affect the development of atherosclerosis in WHHL rabbits.

CREB and FoxO1: two transcription factors for the regulation of hepatic gluconeogenesis

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Oh, Kyoung-Jin; Han, Hye-Sook; Kim, Min-Jung; Koo, Seung-Hoi

2013-01-01

Liver plays a major role in maintaining glucose homeostasis in mammals. Under fasting conditions, hepatic glucose production is critical as a source of fuel to maintain the basic functions in other tissues, including skeletal muscle, red blood cells, and the brain. Fasting hormones glucagon and cortisol play major roles during the process, in part by activating the transcription of key enzyme genes in the gluconeogenesis such as phosphoenol pyruvate carboxykinase (PEPCK) and glucose 6 phosphatase catalytic subunit (G6Pase). Conversely, gluconeogenic transcription is repressed by pancreatic insulin under feeding conditions, which effectively inhibits transcriptional activator complexes by either promoting post-translational modifications or activating transcriptional inhibitors in the liver, resulting in the reduction of hepatic glucose output. The transcriptional regulatory machineries have been highlighted as targets for type 2 diabetes drugs to control glycemia, so understanding of the complex regulatory mechanisms for transcription circuits for hepatic gluconeogenesis is critical in the potential development of therapeutic tools for the treatment of this disease. In this review, the current understanding regarding the roles of two key transcriptional activators, CREB and FoxO1, in the regulation of hepatic gluconeogenic program is discussed. [BMB Reports 2013; 46(12): 567-574] PMID:24238363

Histone acetylation and CREB binding protein are required for neuronal resistance against ischemic injury.

Directory of Open Access Journals (Sweden)

Ferah Yildirim

Full Text Available Epigenetic transcriptional regulation by histone acetylation depends on the balance between histone acetyltransferase (HAT and deacetylase activities (HDAC. Inhibition of HDAC activity provides neuroprotection, indicating that the outcome of cerebral ischemia depends crucially on the acetylation status of histones. In the present study, we characterized the changes in histone acetylation levels in ischemia models of focal cerebral ischemia and identified cAMP-response element binding protein (CREB-binding protein (CBP as a crucial factor in the susceptibility of neurons to ischemic stress. Both neuron-specific RNA interference and neurons derived from CBP heterozygous knockout mice showed increased damage after oxygen-glucose deprivation (OGD in vitro. Furthermore, we demonstrated that ischemic preconditioning by a short (5 min subthreshold occlusion of the middle cerebral artery (MCA, followed 24 h afterwards by a 30 min occlusion of the MCA, increased histone acetylation levels in vivo. Ischemic preconditioning enhanced CBP recruitment and histone acetylation at the promoter of the neuroprotective gene gelsolin leading to increased gelsolin expression in neurons. Inhibition of CBP's HAT activity attenuated neuronal ischemic preconditioning. Taken together, our findings suggest that the levels of CBP and histone acetylation determine stroke outcome and are crucially associated with the induction of an ischemia-resistant state in neurons.

MicroRNA 26a (miR-26a/KLF4 and CREB-C/EBPβ regulate innate immune signaling, the polarization of macrophages and the trafficking of Mycobacterium tuberculosis to lysosomes during infection.

Directory of Open Access Journals (Sweden)

Sanjaya Kumar Sahu

2017-05-01

Full Text Available For efficient clearance of Mycobacterium tuberculosis (Mtb, macrophages tilt towards M1 polarization leading to the activation of transcription factors associated with the production of antibacterial effector molecules such as nitric oxide (NO and proinflammatory cytokines such as interleukin 1 β (IL-1β and tumor necrosis factor α (TNF-α. At the same time, resolution of inflammation is associated with M2 polarization with increased production of arginase and cytokines such as IL-10. The transcriptional and post-transcriptional mechanisms that govern the balance between M1 and M2 polarization, and bacteria-containing processes such as autophagy and trafficking of Mtb to lysosomes, are incompletely understood. Here we report for the first time, that the transcription factor KLF4 is targeted by microRNA-26a (miR-26a. During Mtb infection, downregulation of miR-26a (observed both ex vivo and in vivo facilitates upregulation of KLF4 which in turn favors increased arginase and decreased iNOS activity. We further demonstrate that KLF4 prevents trafficking of Mtb to lysosomes. The CREB-C/EBPβ signaling axis also favors M2 polarization. Downregulation of miR-26a and upregulation of C/ebpbeta were observed both in infected macrophages as well as in infected mice. Knockdown of C/ebpbeta repressed the expression of selected M2 markers such as Il10 and Irf4 in infected macrophages. The importance of these pathways is substantiated by observations that expression of miR-26a mimic or knockdown of Klf4 or Creb or C/ebpbeta, attenuated the survival of Mtb in macrophages. Taken together, our results attribute crucial roles for the miR-26a/KLF4 and CREB-C/EBPβsignaling pathways in regulating the survival of Mtb in macrophages. These studies expand our understanding of how Mtb hijacks host signaling pathways to survive in macrophages, and open up new exploratory avenues for host-targeted interventions.

MicroRNA 26a (miR-26a)/KLF4 and CREB-C/EBPβ regulate innate immune signaling, the polarization of macrophages and the trafficking of Mycobacterium tuberculosis to lysosomes during infection.

Science.gov (United States)

Sahu, Sanjaya Kumar; Kumar, Manish; Chakraborty, Sohini; Banerjee, Srijon Kaushik; Kumar, Ranjeet; Gupta, Pushpa; Jana, Kuladip; Gupta, Umesh D; Ghosh, Zhumur; Kundu, Manikuntala; Basu, Joyoti

2017-05-01

For efficient clearance of Mycobacterium tuberculosis (Mtb), macrophages tilt towards M1 polarization leading to the activation of transcription factors associated with the production of antibacterial effector molecules such as nitric oxide (NO) and proinflammatory cytokines such as interleukin 1 β (IL-1β) and tumor necrosis factor α (TNF-α). At the same time, resolution of inflammation is associated with M2 polarization with increased production of arginase and cytokines such as IL-10. The transcriptional and post-transcriptional mechanisms that govern the balance between M1 and M2 polarization, and bacteria-containing processes such as autophagy and trafficking of Mtb to lysosomes, are incompletely understood. Here we report for the first time, that the transcription factor KLF4 is targeted by microRNA-26a (miR-26a). During Mtb infection, downregulation of miR-26a (observed both ex vivo and in vivo) facilitates upregulation of KLF4 which in turn favors increased arginase and decreased iNOS activity. We further demonstrate that KLF4 prevents trafficking of Mtb to lysosomes. The CREB-C/EBPβ signaling axis also favors M2 polarization. Downregulation of miR-26a and upregulation of C/ebpbeta were observed both in infected macrophages as well as in infected mice. Knockdown of C/ebpbeta repressed the expression of selected M2 markers such as Il10 and Irf4 in infected macrophages. The importance of these pathways is substantiated by observations that expression of miR-26a mimic or knockdown of Klf4 or Creb or C/ebpbeta, attenuated the survival of Mtb in macrophages. Taken together, our results attribute crucial roles for the miR-26a/KLF4 and CREB-C/EBPβsignaling pathways in regulating the survival of Mtb in macrophages. These studies expand our understanding of how Mtb hijacks host signaling pathways to survive in macrophages, and open up new exploratory avenues for host-targeted interventions.

Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation

DEFF Research Database (Denmark)

Taskova, Maria; Madsen, Charlotte S.; Jensen, Knud J.

2017-01-01

Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, m...... and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media.......Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide......, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides...

Mechanical loading stimulates chondrogenesis via the PKA/CREB-Sox9 and PP2A pathways in chicken micromass cultures.

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Juhász, Tamás; Matta, Csaba; Somogyi, Csilla; Katona, Éva; Takács, Roland; Soha, Rudolf Ferenc; Szabó, István A; Cserháti, Csaba; Sződy, Róbert; Karácsonyi, Zoltán; Bakó, Eva; Gergely, Pál; Zákány, Róza

2014-03-01

Biomechanical stimuli play important roles in the formation of articular cartilage during early foetal life, and optimal mechanical load is a crucial regulatory factor of adult chondrocyte metabolism and function. In this study, we undertook to analyse mechanotransduction pathways during in vitro chondrogenesis. Chondroprogenitor cells isolated from limb buds of 4-day-old chicken embryos were cultivated as high density cell cultures for 6 days. Mechanical stimulation was carried out by a self-designed bioreactor that exerted uniaxial intermittent cyclic load transmitted by the culture medium as hydrostatic pressure and fluid shear to differentiating cells. The loading scheme (0.05 Hz, 600 Pa; for 30 min) was applied on culturing days 2 and 3, when final commitment and differentiation of chondroprogenitor cells occurred in this model. The applied mechanical load significantly augmented cartilage matrix production and elevated mRNA expression of several cartilage matrix constituents, including collagen type II and aggrecan core protein, as well as matrix-producing hyaluronan synthases through enhanced expression, phosphorylation and nuclear signals of the main chondrogenic transcription factor Sox9. Along with increased cAMP levels, a significantly enhanced protein kinase A (PKA) activity was also detected and CREB, the archetypal downstream transcription factor of PKA signalling, exhibited elevated phosphorylation levels and stronger nuclear signals in response to mechanical stimuli. All the above effects were diminished by the PKA-inhibitor H89. Inhibition of the PKA-independent cAMP-mediators Epac1 and Epac2 with HJC0197 resulted in enhanced cartilage formation, which was additive to that of the mechanical stimulation, implying that the chondrogenesis-promoting effect of mechanical load was independent of Epac. At the same time, PP2A activity was reduced following mechanical load and treatments with the PP2A-inhibitor okadaic acid were able to mimic the effects of

Defective synaptic transmission and structure in the dentate gyrus and selective fear memory impairment in the Rsk2 mutant mouse model of Coffin-Lowry syndrome.

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Morice, Elise; Farley, Séverine; Poirier, Roseline; Dallerac, Glenn; Chagneau, Carine; Pannetier, Solange; Hanauer, André; Davis, Sabrina; Vaillend, Cyrille; Laroche, Serge

2013-10-01

The Coffin-Lowry syndrome (CLS) is a syndromic form of intellectual disability caused by loss-of-function of the RSK2 serine/threonine kinase encoded by the rsk2 gene. Rsk2 knockout mice, a murine model of CLS, exhibit spatial learning and memory impairments, yet the underlying neural mechanisms are unknown. In the current study, we examined the performance of Rsk2 knockout mice in cued, trace and contextual fear memory paradigms and identified selective deficits in the consolidation and reconsolidation of hippocampal-dependent fear memories as task difficulty and hippocampal demand increase. Electrophysiological, biochemical and electron microscopy analyses were carried out in the dentate gyrus of the hippocampus to explore potential alterations in neuronal functions and structure. In vivo and in vitro electrophysiology revealed impaired synaptic transmission, decreased network excitability and reduced AMPA and NMDA conductance in Rsk2 knockout mice. In the absence of RSK2, standard measures of short-term and long-term potentiation (LTP) were normal, however LTP-induced CREB phosphorylation and expression of the transcription factors EGR1/ZIF268 were reduced and that of the scaffolding protein SHANK3 was blocked, indicating impaired activity-dependent gene regulation. At the structural level, the density of perforated and non-perforated synapses and of multiple spine boutons was not altered, however, a clear enlargement of spine neck width and post-synaptic densities indicates altered synapse ultrastructure. These findings show that RSK2 loss-of-function is associated in the dentate gyrus with multi-level alterations that encompass modifications of glutamate receptor channel properties, synaptic transmission, plasticity-associated gene expression and spine morphology, providing novel insights into the mechanisms contributing to cognitive impairments in CLS. Copyright © 2013 Elsevier Inc. All rights reserved.

Effect of bcl-2 antisense oligodexynucleotides on chemotherapy efficacy of Vp-16 on human small cell lung cancer cell line NCI-H69

International Nuclear Information System (INIS)

He Wenqian; Liu Zhonghua

2007-01-01

Objective: To study the effect of bcl-2 antisense oligodexynucleotides on chemotherapy efficacy of Vp-16 on human small cell lung cancer cell line NCI-H69. Methods: Cultured NCI-H69 cells were derided into 4 groups: bcl-2 antisense oligodexynucleotides (ASODN) added, sense oligodexynucleotides (SODN) added, nonsense oligodexynucleotides (NSODN) added and control (no nucleotides added), the oligodexynucleotides were transfected into the cultured cells with oligofectamine. The cellular expression of Bcl-2 protein 72h later was examined with Western-Blot. The four different groups of cultured tumor cells were treated with etopside(Vp-16) at different concentrations (0, 0.25, 0.5, 1.0, 2.0 and 4.0 μg/ml) for 48hr then the cell survival fraction was assessed with MTY test. Results: The apoptotic rate of cells in the ASODN group was significantly higher than that of the control group, also, the survival fraction of cells in ASODN group was significantly lower than that of the control group. The Bcl-2 protein expression in ASODN group was significantly lower than that in the control group, but no inhibition was observed in SODN and NSODN groups. Conclusion: The bcl-2 ASODN could enhance the sensitivity to chemotherapy with Vp-16 in small cell lung cancer cell line NCI-H69 by effectively blocking bcl-2 gene expression. (authors)

New function of the myostatin/activin type I receptor (ALK4) as a mediator of muscle atrophy and muscle regeneration.

NARCIS (Netherlands)

Pasteuning-Vuhman, S.; Boertje-van der Meulen, J.; van Putten, M.; Overzier, M.; ten Dijke, P; Kiełbasa, S.M.; Arindrarto, W.; Wolterbeek, R.; Lezhnina, K.V.; Ozerov, I.V.; Aliper, A.M.; Hoogaars, W.; Aartsma-Rus, A; Loomans, C.J.

Skeletal muscle fibrosis and impaired muscle regeneration are major contributors to muscle wasting in Duchenne muscular dystrophy (DMD). Muscle growth is negatively regulated by myostatin (MSTN) and activins. Blockage of these pathways may improve muscle quality and function in DMD. Antisense

Identification of a novel antisense long non-coding RNA PLA2G16-AS that regulates the expression of PLA2G16 in pigs.

Science.gov (United States)

Liu, Pengliang; Jin, Long; Zhao, Lirui; Long, Keren; Song, Yang; Tang, Qianzi; Ma, Jideng; Wang, Xun; Tang, Guoqing; Jiang, Yanzhi; Zhu, Li; Li, Xuewei; Li, Mingzhou

2018-05-31

Natural antisense transcripts (NATs) are widely present in mammalian genomes and act as pivotal regulator molecules to control gene expression. However, studies on the NATs of pigs are relatively rare. Here, we identified a novel antisense transcript, designated PLA2G16-AS, transcribed from the phospholipase A2 group XVI locus (PLA2G16) in the porcine genome, which is a well-known regulatory molecule of fat deposition. PLA2G16-AS and PLA2G16 were dominantly expressed in porcine adipose tissue, and were differentially expressed between Tibetan pigs and Rongchang pigs. In addition, PLA2G16-AS has a weak sequence conservation among different vertebrates. PLA2G16-AS was also shown to form an RNA-RNA duplex with PLA2G16, and to regulate PLA2G16 expression at the mRNA level. Moreover, the overexpression of PLA2G16-AS increased the stability of PLA2G16 mRNA in porcine cells. We envision that our findings of a NAT for a regulatory gene associated with lipolysis might further our understanding of the molecular regulation of fat deposition. Copyright © 2017. Published by Elsevier B.V.

Rufinamide, an antiepileptic drug, improves cognition and increases neurogenesis in the aged gerbil hippocampal dentate gyrus via increasing expressions of IGF-1, IGF-1R and p-CREB.

Science.gov (United States)

Chen, Bai Hui; Ahn, Ji Hyeon; Park, Joon Ha; Song, Minah; Kim, Hyunjung; Lee, Tae-Kyeong; Lee, Jae Chul; Kim, Young-Myeong; Hwang, In Koo; Kim, Dae Won; Lee, Choong-Hyun; Yan, Bing Chun; Kang, Il Jun; Won, Moo-Ho

2018-04-25

Rufinamide is a novel antiepileptic drug and commonly used in the treatment of Lennox-Gastaut syndrome. In the present study, we investigated effects of rufinamide on cognitive function using passive avoidance test and neurogenesis in the hippocampal dentate gyrus using Ki-67 (a marker for cell proliferation), doublecortin (DCX, a marker for neuroblast) and BrdU/NeuN (markers for newly generated mature neurons) immunohistochemistry in aged gerbils. Aged gerbils (24-month old) were treated with 1 mg/kg and 3 mg/kg rufinamide for 4 weeks. Treatment with 3 mg/kg rufinamide, not 1 mg/kg rufinamide, significantly improved cognitive function and increased neurogenesis, showing that proliferating cells (Ki-67-immunoreactive cells), differentiating neuroblasts (DCX-immunoreactive neuroblasts) and mature neurons (BrdU/NeuN-immunoreactive cells) in the aged dentate gyrus compared with those in the control group. When we examined its mechanisms, rufinamide significantly increased immunoreactivities of insulin-like growth factor-1 (IGF-1), its receptor (IGF-1R), and phosphorylated cAMP response element binding protein (p-CREB). However, rufinamide did not show any increase in immunoreactivities of brain-derived neurotrophic factor and its receptor. Therefore, our results indicate that rufinamide can improve cognitive function and increase neurogenesis in the hippocampus of the aged gerbil via increasing expressions of IGF-1, IGF-1R and p-CREB. Copyright © 2018 Elsevier B.V. All rights reserved.

Antioxidative and Anti-Melanogenic Activities of Bamboo Stems (Phyllostachys nigra variety henosis) via PKA/CREB-Mediated MITF Downregulation in B16F10 Melanoma Cells.

Science.gov (United States)

Choi, Moon-Hee; Jo, Han-Gyo; Yang, Ji Hye; Ki, Sung Hwan; Shin, Hyun-Jae

2018-01-30

Phyllostachys nigra var. henosis, a domestic bamboo species, has been attracting much attention; its bioactive compounds (especially in the leaf) show antioxidant, anti-inflammatory, and anti-obesity activities. Little information is available on the antioxidative and anti-melanogenetic activities of the bioactive compounds in bamboo stems. The anti-melanogenic and antioxidative activities of the EtOAc fraction (PN3) of a P. nigra stem extract were investigated in a cell-free system and in B16F10 melanoma cells. PN3 consisted of a mixture of flavonoids, such as catechin, chlorogenic acid, caffeic acid, and p -coumaric acid. The antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS)), and hydroxyl radical scavenging) was evaluated, as well as the inhibition of reactive oxygen species (ROS) produced by the Fenton reaction. PN3 showed in vitro tyrosinase inhibition activity with the half maximal inbihitory concentration (IC 50 ) values of 240 μg/mL, and in vivo cytotoxic concentration ranges > 100 μg/mL. The protein expression levels and mRNA transcription levels of TYR , TRP-1 , and MITF were decreased in a dose-dependent manner by the treatment with PN3. PN3 interfered with the phosphorylation of intracellular protein kinase A (PKA)/cAMP response element-binding protein (CREB), demonstrating potent anti-melanogenic effects. PN3 could inhibit PKA/CREB and the subsequent degradation of microphthalmia-associated transcription factor (MITF), resulting in the suppression of melanogenic enzymes and melanin production, probably because of the presence of flavonoid compounds. These properties make it a candidate as an additive to whitening cosmetics.

Mevastatin ameliorates sphingosine 1‐phosphate‐induced COX‐2/PGE2‐dependent cell migration via FoxO1 and CREB phosphorylation and translocation

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Hsu, Chih‐Kai; Lin, Chih‐Chung; Hsiao, Li‐Der

2015-01-01

Background and Purpose Sphingosine 1‐phosphate (S1P), an important inflammatory mediator, has been shown to regulate COX‐2 production and promote various cellular responses such as cell migration. Mevastatin, an inhibitor of 3‐hydroxy‐3‐methylglutaryl‐CoA reductase (HMG‐CoA), effectively inhibits inflammatory responses. However, the mechanisms underlying S1P‐evoked COX‐2‐dependent cell migration, which is modulated by mevastatin in human tracheal smooth muscle cells (HTSMCs) remain unclear. Experimental Approach The expression of COX‐2 was determined by Western blotting, real time‐PCR and promoter analyses. The signalling molecules were investigated by pretreatment with respective pharmacological inhibitors or transfection with siRNAs. The interaction between COX‐2 promoter and transcription factors was determined by chromatin immunoprecipitation assay. Finally, the effect of mevastatin on HTSMC migration and leukocyte counts in BAL fluid and COX‐2 expression induced by S1P was determined by a cell migration assay, cell counting and Western blot. Key Results S1P stimulated mTOR activation through the Nox2/ROS and PI3K/Akt pathways, which can further stimulate FoxO1 phosphorylation and translocation to the cytosol. We also found that S1P induced CREB activation and translocation via an mTOR‐independent signalling pathway. Finally, we showed that pretreatment with mevastatin markedly reduced S1P‐induced cell migration and COX‐2/PGE2 production via a PPARγ‐dependent signalling pathway. Conclusions and Implications Mevastatin attenuates the S1P‐induced increased expression of COX‐2 and cell migration via the regulation of FoxO1 and CREB phosphorylation and translocation by PPARγ in HTSMCs. Mevastatin could be beneficial for prevention of airway inflammation in the future. PMID:26359950

Effects of different scent on learning and memory ability and hippocampal CREB protein in mice%气味对小鼠学习记忆能力及海马cAMP反应元件结合蛋白的影响

Institute of Scientific and Technical Information of China (English)

王小艳; 董军; 陆大祥

2004-01-01

目的:探讨不同气味(苹果、香水、樟脑)对小鼠学习记忆能力及海马cAMP反应元件结合蛋白(CREB)和磷酸化的CREB(pCREB)的影响.方法:让小鼠在不同气味的环境下生活14d,在第7d开始方形水迷宫训练,3d后进行测试,连续测试5d.测试完后断髓处死动物,取出脑组织,用免疫组织化学染色观察海马pCREB和CREB表达情况,并进行图像分析.结果:樟脑组和香水组水迷宫的潜伏期较对照组延长,错误次数增多(P＜0.05).免疫组化染色显示鼠海马CREB的磷酸化水平大大降低(P＜0.05),但对CREB的表达无明显影响.苹果组与对照组比各指标均无显著差异(P＞0.05).结论:樟脑气味和香水气味对小鼠记忆能力有负面作用,且这种作用可能是通过降低CREB磷酸化水平而实现的.苹果气味对小鼠记忆能力无明显影响.

Regulation of hippocampus-dependent memory by the zinc finger protein Zbtb20 in mature CA1 neurons.

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Ren, Anjing; Zhang, Huan; Xie, Zhifang; Ma, Xianhua; Ji, Wenli; He, David Z Z; Yuan, Wenjun; Ding, Yu-Qiang; Zhang, Xiao-Hui; Zhang, Weiping J

2012-10-01

The mammalian hippocampus harbours neural circuitry that is crucial for associative learning and memory. The mechanisms that underlie the development and regulation of this complex circuitry are not fully understood. Our previous study established an essential role for the zinc finger protein Zbtb20 in the specification of CA1 field identity in the developing hippocampus. Here, we show that conditionally deleting Zbtb20 specifically in mature CA1 pyramidal neurons impaired hippocampus-dependent memory formation, without affecting hippocampal architecture or the survival, identity and basal excitatory synaptic activity of CA1 pyramidal neurons. We demonstrate that mature CA1-specific Zbtb20 knockout mice exhibited reductions in long-term potentiation (LTP) and NMDA receptor (NMDAR)-mediated excitatory post-synaptic currents. Furthermore, we show that activity-induced phosphorylation of ERK and CREB is impaired in the hippocampal CA1 of Zbtb20 mutant mice. Collectively, these results indicate that Zbtb20 in mature CA1 plays an important role in LTP and memory by regulating NMDAR activity, and activation of ERK and CREB.

Serum brain-derived neurotrophic factor and glucocorticoid receptor levels in lymphocytes as markers of antidepressant response in major depressive patients: a pilot study.

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Rojas, Paulina Soledad; Fritsch, Rosemarie; Rojas, Romina Andrea; Jara, Pablo; Fiedler, Jenny Lucy

2011-09-30

Depressive patients often have altered cortisol secretion, an effect that likely derives from impaired activity of the glucocorticoid receptor (GR), the main regulator of the hypothalamus-pituitary-adrenal (HPA) axis. Glucocorticoids reduce the levels of brain-derived neurotrophic factor (BDNF), a downstream target of antidepressants. Antidepressants promote the transcriptional activity of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB), a regulator of BDNF expression. To identify potential biomarkers for the onset of antidepressant action in depressive patients, GR and phospho-CREB (pCREB) levels in lymphocytes and serum BDNF levels were repeatedly measured during the course of antidepressant treatment. Thirty-four depressed outpatients (10 male and 24 female) were treated with venlafaxine (75mg/day), and individuals exhibiting a 50% reduction in their baseline 17-Item Hamilton Depression Rating Scale score by the 6th week of treatment were considered responders. Responders showed an early improvement in parallel with a rise in BDNF levels during the first two weeks of treatment. Non-responders showed increased GR levels by the third week and reduced serum BDNF by the sixth week of treatment. In contrast, venlafaxine did not affect levels of pCREB. We conclude that levels of BDNF in serum and GR levels in lymphocytes may represent biomarkers that could be used to predict responses to venlafaxine treatment. Copyright © 2011 Elsevier Ltd. All rights reserved.

Rare genomic variants link bipolar disorder to CREB regulated intracellular signaling pathways

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Berit eKerner

2013-11-01

Full Text Available Bipolar disorder is a common, complex, and severe psychiatric disorder with cyclical disturbances of mood and a high suicide rate. Here, we describe a family with four siblings, three affected females and one unaffected male. The disease course was characterized by early-onset bipolar disorder and co-morbid anxiety spectrum disorders that followed the onset of bipolar disorder. Genetic risk factors were suggested by the early onset of the disease, the severe disease course, including multiple suicide attempts, and lack of adverse prenatal or early life events. In particular, drug and alcohol abuse did not contribute to the disease onset. Exome sequencing identified very rare, heterozygous, and likely protein-damaging variants in eight brain-expressed genes: IQUB, JMJD1C, GADD45A, GOLGB1, PLSCR5, VRK2, MESDC2, and FGGY. The variants were shared among all three affected family members but absent in the unaffected sibling and in more than 200 controls. The genes encode proteins with significant regulatory roles in the ERK/MAPK and CREB-regulated intracellular signaling pathways. These pathways are central to neuronal and synaptic plasticity, cognition, affect regulation and response to chronic stress. In addition, proteins in these pathways are the target of commonly used mood stabilizing drugs, such as tricyclic antidepressants, lithium and valproic acid. The combination of multiple rare, damaging mutations in these central pathways could lead to reduced resilience and increased vulnerability to stressful life events. Our results support a new model for psychiatric disorders, in which multiple rare, damaging mutations in genes functionally related to a common signaling pathway contribute to the manifestation of bipolar disorder.

Profiled support vector machines for antisense oligonucleotide efficacy prediction

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Martín-Guerrero José D

2004-09-01

Full Text Available Abstract Background This paper presents the use of Support Vector Machines (SVMs for prediction and analysis of antisense oligonucleotide (AO efficacy. The collected database comprises 315 AO molecules including 68 features each, inducing a problem well-suited to SVMs. The task of feature selection is crucial given the presence of noisy or redundant features, and the well-known problem of the curse of dimensionality. We propose a two-stage strategy to develop an optimal model: (1 feature selection using correlation analysis, mutual information, and SVM-based recursive feature elimination (SVM-RFE, and (2 AO prediction using standard and profiled SVM formulations. A profiled SVM gives different weights to different parts of the training data to focus the training on the most important regions. Results In the first stage, the SVM-RFE technique was most efficient and robust in the presence of low number of samples and high input space dimension. This method yielded an optimal subset of 14 representative features, which were all related to energy and sequence motifs. The second stage evaluated the performance of the predictors (overall correlation coefficient between observed and predicted efficacy, r; mean error, ME; and root-mean-square-error, RMSE using 8-fold and minus-one-RNA cross-validation methods. The profiled SVM produced the best results (r = 0.44, ME = 0.022, and RMSE= 0.278 and predicted high (>75% inhibition of gene expression and low efficacy (http://aosvm.cgb.ki.se/. Conclusions The SVM approach is well suited to the AO prediction problem, and yields a prediction accuracy superior to previous methods. The profiled SVM was found to perform better than the standard SVM, suggesting that it could lead to improvements in other prediction problems as well.

Use of versican variant V3 and versican antisense expression to engineer cultured human skin containing increased content of insoluble elastin.

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Merrilees, Mervyn J; Falk, Ben A; Zuo, Ning; Dickinson, Michelle E; May, Barnaby C H; Wight, Thomas N

2017-01-01

Skin substitutes for repair of dermal wounds are deficient in functional elastic fibres. We report that the content of insoluble elastin in the dermis of cultured human skin can be increased though the use of two approaches that enhance elastogenesis by dermal fibroblasts, forced expression of versican variant V3, which lacks glycosaminoglycan (GAG) chains, and forced expression of versican antisense to decrease levels of versican variant V1 with GAG chains. Human dermal fibroblasts transduced with V3 or anti-versican were cultured under standard conditions over a period of 4 weeks to produce dermal sheets, with growth enhanced though multiple seedings for the first 3 weeks. Human keratinocytes, cultured in supplemented media, were added to the 4-week dermal sheets and the skin layer cultured for a further week. At 5 weeks, keratinocytes were multilayered and differentiated, with desmosome junctions thoughout and keratin deposits in the upper squamous layers. The dermal layer was composed of layered fibroblasts surrounded by extracellular matrix of collagen bundles and, in control cultures, small scattered elastin deposits. Forced expression of V3 and versican antisense slowed growth, decreased versican V1 expression, increased tropoelastin expression and/or the deposition of large aggregates of insoluble elastin in the dermal layer, and increased tissue stiffness, as measured by nano-indentation. Skin sheets were also cultured on Endoform Dermal Template™, the biodegradable wound dressing made from the lamina propria of sheep foregut. Skin structure and the enhanced deposition of elastin by forced expression of V3 and anti-versican were preserved on this supportive substrate. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

Down-regulation of the Antisense Mitochondrial Non-coding RNAs (ncRNAs) Is a Unique Vulnerability of Cancer Cells and a Potential Target for Cancer Therapy*

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Vidaurre, Soledad; Fitzpatrick, Christopher; Burzio, Verónica A.; Briones, Macarena; Villota, Claudio; Villegas, Jaime; Echenique, Javiera; Oliveira-Cruz, Luciana; Araya, Mariela; Borgna, Vincenzo; Socías, Teresa; Lopez, Constanza; Avila, Rodolfo; Burzio, Luis O.

2014-01-01

Hallmarks of cancer are fundamental principles involved in cancer progression. We propose an additional generalized hallmark of malignant transformation corresponding to the differential expression of a family of mitochondrial ncRNAs (ncmtRNAs) that comprises sense and antisense members, all of which contain stem-loop structures. Normal proliferating cells express sense (SncmtRNA) and antisense (ASncmtRNA) transcripts. In contrast, the ASncmtRNAs are down-regulated in tumor cells regardless of tissue of origin. Here we show that knockdown of the low copy number of the ASncmtRNAs in several tumor cell lines induces cell death by apoptosis without affecting the viability of normal cells. In addition, knockdown of ASncmtRNAs potentiates apoptotic cell death by inhibiting survivin expression, a member of the inhibitor of apoptosis (IAP) family. Down-regulation of survivin is at the translational level and is probably mediated by microRNAs generated by dicing of the double-stranded stem of the ASncmtRNAs, as suggested by evidence presented here, in which the ASncmtRNAs are bound to Dicer and knockdown of the ASncmtRNAs reduces reporter luciferase activity in a vector carrying the 3′-UTR of survivin mRNA. Taken together, down-regulation of the ASncmtRNAs constitutes a vulnerability or Achilles' heel of cancer cells, suggesting that the ASncmtRNAs are promising targets for cancer therapy. PMID:25100722

Thiolated chitosan nanoparticles as a delivery system for antisense therapy: evaluation against EGFR in T47D breast cancer cells

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Talaei F

2011-09-01

Full Text Available Fatemeh Talaei1, Ebrahim Azizi2, Rassoul Dinarvand3, Fatemeh Atyabi31Novel Drug Delivery Systems Lab, 2Molecular Research Lab, Department of Pharmacology and Toxicology, 3Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, IranAbstract: Thiolated chitosan has high transfection and mucoadhesive properties. We investigated the potential of two recently synthesized polymers: NAC-C (N-acetyl cysteine-chitosan and NAP-C (N-acetyl penicillamine-chitosan in anticancer drug delivery targeting epidermal growth factor receptor (EGFR. Doxorubicin (DOX and antisense oligonucleotide (ASOND-loaded polymer nanoparticles were prepared in water by a gelation process. Particle characterization, drug loading, and drug release were evaluated. To verify drug delivery efficiency in vitro experiments on a breast cancer cell line (T47D were performed. EGFR gene and protein expression was analyzed by real time quantitative polymerase chain reaction and Western blotting, respectively. A loading percentage of 63% ± 5% for ASOND and 70% ± 5% for DOX was achieved. Drug release data after 15 hours showed that ASOND and DOX were completely released from chitosan-based particles while a lower and more sustained release of only 22% ± 8% was measured for thiolated particles. In a cytosol simulated release medium/reducing environment, such as found intracellularly, polymer-based nanoparticles dissociated, liberating approximately 50% of both active substances within 7 hours. ASOND-loaded polymer nanoparticles had higher stability and high mucoadhesive properties. The ASOND-loaded thiolated particles significantly suppressed EGFR gene expression in T47D cells compared with ASOND-loaded chitosan particles and downregulated EGFR protein expression in cells. This study could facilitate future investigations into the functionality of NAP-C and NAC-C polymers as an efficient ASOND delivery system in vitro and in vivo

Maternal DHA supplementation protects rat offspring against impairment of learning and memory following prenatal exposure to valproic acid.

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Gao, Jingquan; Wu, Hongmei; Cao, Yonggang; Liang, Shuang; Sun, Caihong; Wang, Peng; Wang, Ji; Sun, Hongli; Wu, Lijie

2016-09-01

Docosahexaenoic acid (22:6n-3; DHA) is known to play a critical role in postnatal brain development. However, there have been no studies investigating the preventive effect of DHA on prenatal valproic acid (VPA)-induced behavioral and molecular alterations in offspring. The present study was to evaluate the neuroprotective effects in offspring using maternal feeding of DHA to rats exposed to VPA in pregnancy. In the present study, rats were exposed to VPA on day 12.5 of pregnancy; DHA was administered at the dosages of 100, 300 and 500 mg/kg/day for 3 weeks from day 1 to 21 of pregnancy. The results showed that maternal feeding of DHA to the prenatal exposed to VPA (1) prevented VPA-induced learning and memory impairment but did not change social-related behavior, (2) increased total DHA content in offspring plasma and hippocampus, (3) rescued VPA-induced neuronal loss and apoptosis of pyramidal cells in hippocampal CA1, (4) influenced the content of malondialdehyde and glutathione and the activities of superoxide dismutase and glutathione in the hippocampus, (5) altered levels of apoptosis-related proteins (Bcl-2, Bax and caspase-3) and inhibited the activity of caspase-3 in offspring hippocampus and (6) enhanced relative levels of p-CaMKII and p-CREB proteins in the hippocampus. These findings suggest that maternal feeding with DHA may prevent prenatal VPA-induced impairment of learning and memory, normalize several different molecules associated with oxidative stress and apoptosis in the hippocampus of offspring, and exert preventive effects on prenatal VPA-induced brain dysfunction. Copyright © 2016 Elsevier Inc. All rights reserved.

Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain

DEFF Research Database (Denmark)

Koppelhus, Uffe; Shiraishi, Takehiko; Zachar, Vladimir

2008-01-01

Conjugation to cationic cell penetrating peptides (such as Tat, Penetratin, or oligo arginines) efficiently improves the cellular uptake of large hydrophilic molecules such as oligonucleotides and peptide nucleic acids, but the cellular uptake is predominantly via an unproductive endosomal pathway...... for future in vivo applications. We find that simply conjugating a lipid domain (fatty acid) to the cationic peptide (a CatLip conjugate) increases the biological effect of the corresponding PNA (CatLip) conjugates in a luciferase cellular antisense assay up to 2 orders of magnitude. The effect increases...... with increasing length of the fatty acid (C8-C16) but in parallel also results in increased cellular toxicity, with decanoic acid being optimal. Furthermore, the relative enhancement is significantly higher for Tat peptide compared to oligoarginine. Confocal microscopy and chloroquine enhancement indicates...

Age-Related Sensory Impairments and Risk of Cognitive Impairment

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Fischer, Mary E; Cruickshanks, Karen J.; Schubert, Carla R; Pinto, Alex A; Carlsson, Cynthia M; Klein, Barbara EK; Klein, Ronald; Tweed, Ted S.

2016-01-01

Background/Objectives To evaluate the associations of sensory impairments with the 10-year risk of cognitive impairment. Previous work has primarily focused on the relationship between a single sensory system and cognition. Design The Epidemiology of Hearing Loss Study (EHLS) is a longitudinal, population-based study of aging in the Beaver Dam, WI community. Baseline examinations were conducted in 1993 and follow-up exams have been conducted every 5 years. Setting General community Participants EHLS members without cognitive impairment at EHLS-2 (1998–2000). There were 1,884 participants (mean age = 66.7 years) with complete EHLS-2 sensory data and follow-up information. Measurements Cognitive impairment was a Mini-Mental State Examination score of impairment was a pure-tone average of hearing thresholds (0.5, 1, 2 and 4 kHz) of > 25 decibel Hearing Level in either ear. Visual impairment was Pelli-Robson contrast sensitivity of impairment was a San Diego Odor Identification Test score of impairment were independently associated with cognitive impairment risk [Hearing: Hazard Ratio (HR) = 1.90, 95% Confidence Interval (C.I.) = 1.11, 3.26; Vision: HR = 2.05, 95% C.I. = 1.24, 3.38; Olfaction: HR = 3.92, 95% C.I. = 2.45, 6.26]. However, 85% with hearing impairment, 81% with visual impairment, and 76% with olfactory impairment did not develop cognitive impairment during follow-up. Conclusion The relationship between sensory impairment and cognitive impairment was not unique to one sensory system suggesting sensorineural health may be a marker of brain aging. The development of a combined sensorineurocognitive measure may be useful in uncovering mechanisms of healthy brain aging. PMID:27611845

PI3K/Akt/GSK3β induced CREB activation ameliorates arsenic mediated alterations in NMDA receptors and associated signaling in rat hippocampus: Neuroprotective role of curcumin.

Science.gov (United States)

Srivastava, Pranay; Dhuriya, Yogesh K; Kumar, Vivek; Srivastava, Akriti; Gupta, Richa; Shukla, Rajendra K; Yadav, Rajesh S; Dwivedi, Hari N; Pant, Aditya B; Khanna, Vinay K

2018-04-30

Protective efficacy of curcumin in arsenic induced NMDA receptor dysfunctions and PI3K/Akt/ GSK3β signalling in hippocampus has been investigated in vivo and in vitro. Exposure to sodium arsenite (in vivo - 20 mg/kg, body weight p.o. for 28 days; in vitro - 10 μM for 24 h) and curcumin (in vivo - 100 mg/kg body weight p.o. for 28 days; in vitro - 20 μM for 24 h) was carried out alone or simultaneously. Treatment with curcumin ameliorated sodium arsenite induced alterations in the levels of NMDA receptors, its receptor subunits and synaptic proteins - pCaMKIIα, PSD-95 and SynGAP both in vivo and in vitro. Decreased levels of BDNF, pAkt, pERK1/2, pGSK3β and pCREB on sodium arsenite exposure were also protected by curcumin. Curcumin was found to decrease sodium arsenite induced changes in hippocampus by modulating PI3K/Akt/GSK3β neuronal survival pathway, known to regulate various cellular events. Treatment of hippocampal cultures with pharmacological inhibitors for ERK1/2, GSK3β and Akt individually inhibited levels of CREB and proteins associated with PI3K/Akt/GSK3β pathway. Simultaneous treatment with curcumin was found to improve sodium arsenite induced learning and memory deficits in rats assessed by water maze and Y-maze. The results provide evidence that curcumin exercises its neuroprotective effect involving PI3K/Akt pathway which may affect NMDA receptors and downstream signalling through TrKβ and BDNF in arsenic induced cognitive deficits in hippocampus. Copyright © 2018 Elsevier B.V. All rights reserved.

Neuroplastic Correlates in the mPFC Underlying the Impairment of Stress-Coping Ability and Cognitive Flexibility in Adult Rats Exposed to Chronic Mild Stress during Adolescence

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Yu Zhang

2017-01-01

Full Text Available Using a valid chronic mild stress (CMS model of depression, we found that adolescent (postnatal days [PND] 28–41 CMS induced transient alterations in anhedonia that did not persist into adulthood after a 3-week recovery period. Previously stressed adult rats exhibited more immobility/despair behaviors in the forced swimming test and a greater number of trials to reach criterion in the set-shifting task, suggesting the impaired ability to cope with stressors and the cognitive flexibility that allows adaptation to dynamic environments during adulthood. In addition, adult rat exposure to adolescent CMS had a relatively inhibited activation in ERK signaling and downstream protein expression of phosphorylated cAMP-response element-binding protein (CREB and brain-derived neurotrophic factor (BDNF in the medial prefrontal cortex. Further correlation analysis demonstrated that immobility and set-shifting performance were positively correlated with the inhibition of ERK signaling. These results indicated adolescent CMS can be used as an effective stressor to model an increased predisposition to adult depression.

Specific regulation of point-mutated K-ras-immortalized cell proliferation by a photodynamic antisense strategy.

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Higuchi, Maiko; Yamayoshi, Asako; Kato, Kiyoko; Kobori, Akio; Wake, Norio; Murakami, Akira

2010-02-01

It has been reported that point mutations in genes are responsible for various cancers, and the selective regulation of gene expression is an important factor in developing new types of anticancer drugs. To develop effective drugs for the regulation of point-mutated genes, we focused on photoreactive antisense oligonucleotides. Previously, we reported that photoreactive oligonucleotides containing 2'-O-psoralenylmethoxyethyl adenosine (2'-Ps-eom) showed drastic photoreactivity in a strictly sequence-specific manner. Here, we demonstrated the specific gene regulatory effects of 2'-Ps-eom on [(12)Val]K-ras mutant (GGT --> GTT). Photo-cross-linking between target mRNAs and 2'-Ps-eom was sequence-specific, and the effect was UVA irradiation-dependent. Furthermore, 2'-Ps-eom was able to inhibit K-ras-immortalized cell proliferation (K12V) but not Vco cells that have the wild-type K-ras gene. These results suggest that the 2'-Ps-eom will be a powerful nucleic acid drug to inhibit the expression of disease-causing point mutation genes, and has great therapeutic potential in the treatment of cancer.

Changes in Oleic Acid Content of Transgenic Soybeans by Antisense RNA Mediated Posttranscriptional Gene Silencing

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Ling Zhang

2014-01-01

Full Text Available The Delta-12 oleate desaturase gene (FAD2-1, which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of seed oil. In this study, we inhibited the expression of endogenous Delta-12 oleate desaturase GmFad2-1b gene by using antisense RNA in soybean Williams 82. By employing the soybean cotyledonary-node method, a part of the cDNA of soybean GmFad2-1b 801 bp was cloned for the construction of a pCAMBIA3300 vector under the soybean seed promoter BCSP. Leaf painting, LibertyLink strip, PCR, Southern blot, qRT-PCR, and fatty acid analysis were used to detect the insertion and expression of GmFad2-1b in the transgenic soybean lines. The results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 51.71% and a reduction in palmitic acid (to <3% in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and the nontransgenic oil extracts.

RNA synthetic biology inspired from bacteria: construction of transcription attenuators under antisense regulation.

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Dawid, Alexandre; Cayrol, Bastien; Isambert, Hervé

2009-07-01

Among all biopolymers, ribonucleic acids or RNA have unique functional versatility, which led to the early suggestion that RNA alone (or a closely related biopolymer) might have once sustained a primitive form of life based on a single type of biopolymer. This has been supported by the demonstration of processive RNA-based replication and the discovery of 'riboswitches' or RNA switches, which directly sense their metabolic environment. In this paper, we further explore the plausibility of this 'RNA world' scenario and show, through synthetic molecular design guided by advanced RNA simulations, that RNA can also perform elementary regulation tasks on its own. We demonstrate that RNA synthetic regulatory modules directly inspired from bacterial transcription attenuators can efficiently activate or repress the expression of other RNA by merely controlling their folding paths 'on the fly' during transcription through simple RNA-RNA antisense interaction. Factors, such as NTP concentration and RNA synthesis rate, affecting the efficiency of this kinetic regulation mechanism are also studied and discussed in the light of evolutionary constraints. Overall, this suggests that direct coupling among synthesis, folding and regulation of RNAs may have enabled the early emergence of autonomous RNA-based regulation networks in absence of both DNA and protein partners.

RNA synthetic biology inspired from bacteria: construction of transcription attenuators under antisense regulation

International Nuclear Information System (INIS)

Dawid, Alexandre; Cayrol, Bastien; Isambert, Hervé

2009-01-01

Among all biopolymers, ribonucleic acids or RNA have unique functional versatility, which led to the early suggestion that RNA alone (or a closely related biopolymer) might have once sustained a primitive form of life based on a single type of biopolymer. This has been supported by the demonstration of processive RNA-based replication and the discovery of 'riboswitches' or RNA switches, which directly sense their metabolic environment. In this paper, we further explore the plausibility of this 'RNA world' scenario and show, through synthetic molecular design guided by advanced RNA simulations, that RNA can also perform elementary regulation tasks on its own. We demonstrate that RNA synthetic regulatory modules directly inspired from bacterial transcription attenuators can efficiently activate or repress the expression of other RNA by merely controlling their folding paths 'on the fly' during transcription through simple RNA–RNA antisense interaction. Factors, such as NTP concentration and RNA synthesis rate, affecting the efficiency of this kinetic regulation mechanism are also studied and discussed in the light of evolutionary constraints. Overall, this suggests that direct coupling among synthesis, folding and regulation of RNAs may have enabled the early emergence of autonomous RNA-based regulation networks in absence of both DNA and protein partners

Glycogen Reduction in Myotubes of Late-Onset Pompe Disease Patients Using Antisense Technology.

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Goina, Elisa; Peruzzo, Paolo; Bembi, Bruno; Dardis, Andrea; Buratti, Emanuele

2017-09-06

Glycogen storage disease type II (GSDII) is a lysosomal disorder caused by the deficient activity of acid alpha-glucosidase (GAA) enzyme, leading to the accumulation of glycogen within the lysosomes. The disease has been classified in infantile and late-onset forms. Most late-onset patients share a splicing mutation c.-32-13T > G in intron 1 of the GAA gene that prevents efficient recognition of exon 2 by the spliceosome. In this study, we have mapped the splicing silencers of GAA exon 2 and developed antisense morpholino oligonucleotides (AMOs) to inhibit those regions and rescue normal splicing in the presence of the c.-32-13T > G mutation. Using a minigene approach and patient fibroblasts, we successfully increased inclusion of exon 2 in the mRNA and GAA enzyme production by targeting a specific silencer with a combination of AMOs. Most importantly, the use of these AMOs in patient myotubes results in a decreased accumulation of glycogen. To our knowledge, this is the only therapeutic approach resulting in a decrease of glycogen accumulation in patient tissues beside enzyme replacement therapy (ERT) and TFEB overexpression. As a result, it may represent a highly novel and promising therapeutic line for GSDII. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice

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Dominic Jauvin

2017-06-01

Full Text Available Myotonic dystrophy type 1 (DM1, a dominant hereditary muscular dystrophy, is caused by an abnormal expansion of a (CTGn trinucleotide repeat in the 3′ UTR of the human dystrophia myotonica protein kinase (DMPK gene. As a consequence, mutant transcripts containing expanded CUG repeats are retained in nuclear foci and alter the function of splicing regulatory factors members of the MBNL and CELF families, resulting in alternative splicing misregulation of specific transcripts in affected DM1 tissues. In the present study, we treated DMSXL mice systemically with a 2′-4′-constrained, ethyl-modified (ISIS 486178 antisense oligonucleotide (ASO targeted to the 3′ UTR of the DMPK gene, which led to a 70% reduction in CUGexp RNA abundance and foci in different skeletal muscles and a 30% reduction in the heart. Furthermore, treatment with ISIS 486178 ASO improved body weight, muscle strength, and muscle histology, whereas no overt toxicity was detected. This is evidence that the reduction of CUGexp RNA improves muscle strength in DM1, suggesting that muscle weakness in DM1 patients may be improved following elimination of toxic RNAs.

Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice.

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Jauvin, Dominic; Chrétien, Jessina; Pandey, Sanjay K; Martineau, Laurie; Revillod, Lucille; Bassez, Guillaume; Lachon, Aline; MacLeod, A Robert; Gourdon, Geneviève; Wheeler, Thurman M; Thornton, Charles A; Bennett, C Frank; Puymirat, Jack

2017-06-16

Myotonic dystrophy type 1 (DM1), a dominant hereditary muscular dystrophy, is caused by an abnormal expansion of a (CTG) n trinucleotide repeat in the 3' UTR of the human dystrophia myotonica protein kinase (DMPK) gene. As a consequence, mutant transcripts containing expanded CUG repeats are retained in nuclear foci and alter the function of splicing regulatory factors members of the MBNL and CELF families, resulting in alternative splicing misregulation of specific transcripts in affected DM1 tissues. In the present study, we treated DMSXL mice systemically with a 2'-4'-constrained, ethyl-modified (ISIS 486178) antisense oligonucleotide (ASO) targeted to the 3' UTR of the DMPK gene, which led to a 70% reduction in CUG exp RNA abundance and foci in different skeletal muscles and a 30% reduction in the heart. Furthermore, treatment with ISIS 486178 ASO improved body weight, muscle strength, and muscle histology, whereas no overt toxicity was detected. This is evidence that the reduction of CUG exp RNA improves muscle strength in DM1, suggesting that muscle weakness in DM1 patients may be improved following elimination of toxic RNAs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Activation of Gαq Signaling Enhances Memory Consolidation and Slows Cognitive Decline.

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Arey, Rachel N; Stein, Geneva M; Kaletsky, Rachel; Kauffman, Amanda; Murphy, Coleen T

2018-05-02

Perhaps the most devastating decline with age is the loss of memory. Therefore, identifying mechanisms to restore memory function with age is critical. Using C. elegans associative learning and memory assays, we identified a gain-of-function G αq signaling pathway mutant that forms a long-term (cAMP response element binding protein [CREB]-dependent) memory following one conditioned stimulus-unconditioned stimulus (CS-US) pairing, which usually requires seven CS-US pairings. Increased CREB activity in AIM interneurons reduces the threshold for memory consolidation through transcription of a set of previously identified "long-term memory" genes. Enhanced G αq signaling in the AWC sensory neuron is both necessary and sufficient for improved memory and increased AIM CREB activity, and activation of G αq specifically in aged animals rescues the ability to form memory. Activation of G αq in AWC sensory neurons non-cell autonomously induces consolidation after one CS-US pairing, enabling both cognitive function maintenance with age and restoration of memory function in animals with impaired memory performance without decreased longevity. Copyright © 2018 Elsevier Inc. All rights reserved.

Genetic manipulation of RPS5 gene expression modulates the initiation of commitment of MEL cells to erythroid maturation: Implications in understanding ribosomopathies.

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Vizirianakis, Ioannis S; Papachristou, Eleni T; Andreadis, Panagiotis; Zopounidou, Elena; Matragkou, Christina N; Tsiftsoglou, Asterios S

2015-07-01

Impairment of ribosome biogenesis contributes to the molecular pathophysiology of ribosomopathies by deregulating cell-lineage specific proliferation, differentiation and apoptosis decisions of haematopoietic progenitor cells. Here, using pro-erythroblast-like murine erythroleukemia (MEL) cells, a model system of erythroid maturation, we aimed to investigate whether genetic manipulation of RPS5 expression affects the capacity of cells to grow and differentiate in culture. Parental MEL cells stably transfected with full length RPS5 cDNA in sense (MEL-C14 culture) or antisense (MEL-antisenseRPS5 culture) orientation, as well as MEL cells transiently transfected with siRNAs specific for RPS5 gene silencing (MEL-RPS5siRNA culture) were assessed for their ability to fully execute their erythroid maturation program in culture. The data obtained thus far indicate that: a) MEL-antisenseRPS5 exhibit a pronounced delay in the initiation of differentiation, as well as an impairment of commitment, since the continuous presence of the inducer in culture is required for the cells to fully execute their erythroid maturation program. b) RNAi-mediating silencing of RPS5 gene expression resulted in the inability of MEL cells to differentiate; however, when these cells were allowed to recapitulate normal RPS5 gene expression levels they regained their differentiation capacity by accumulating high proportion of erythroid mature cells. c) Interestingly the latter, is accompanied by morphological changes of cells and an impairment of their proliferation and apoptosis potential. Such data for the first time correlate the RPS5 gene expression levels with the differentiation capacity of MEL cells in vitro, a fact that might also have implications in understanding ribosomopathies.

Inhibition of p38/CREB phosphorylation and COX-2 expression by olive oil polyphenols underlies their anti-proliferative effects

International Nuclear Information System (INIS)

Corona, Giulia; Deiana, Monica; Incani, Alessandra; Vauzour, David; Assunta Dessi, M.; Spencer, Jeremy P.E.

2007-01-01

We investigated the anti-proliferative effects of an olive oil polyphenolic extract on human colon adenocarcinoma cells. Analysis indicated that the extract contained hydroxytyrosol, tyrosol and the various secoiridoid derivatives, including oleuropein. This extract exerted a strong inhibitory effect on cancer cell proliferation, which was linked to the induction of a G2/M phase cell cycle block. Following treatment with the extract (50 μg/ml) the number of cells in the G2/M phase increased to 51.82 ± 2.69% relative to control cells (15.1 ± 2.5%). This G2/M block was mediated by the ability of olive oil polyphenols (50 μg/ml) to exert rapid inhibition of p38 (38.7 ± 4.7%) and CREB (28.6 ± 5.5%) phosphorylation which led to a downstream reduction in COX-2 expression (56.9 ± 9.3%). Our data suggest that olive oil polyphenols may exert chemopreventative effects in the large intestine by interacting with signalling pathways responsible for colorectal cancer development

Down-regulation of the antisense mitochondrial non-coding RNAs (ncRNAs) is a unique vulnerability of cancer cells and a potential target for cancer therapy.

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Vidaurre, Soledad; Fitzpatrick, Christopher; Burzio, Verónica A; Briones, Macarena; Villota, Claudio; Villegas, Jaime; Echenique, Javiera; Oliveira-Cruz, Luciana; Araya, Mariela; Borgna, Vincenzo; Socías, Teresa; Lopez, Constanza; Avila, Rodolfo; Burzio, Luis O

2014-09-26

Hallmarks of cancer are fundamental principles involved in cancer progression. We propose an additional generalized hallmark of malignant transformation corresponding to the differential expression of a family of mitochondrial ncRNAs (ncmtRNAs) that comprises sense and antisense members, all of which contain stem-loop structures. Normal proliferating cells express sense (SncmtRNA) and antisense (ASncmtRNA) transcripts. In contrast, the ASncmtRNAs are down-regulated in tumor cells regardless of tissue of origin. Here we show that knockdown of the low copy number of the ASncmtRNAs in several tumor cell lines induces cell death by apoptosis without affecting the viability of normal cells. In addition, knockdown of ASncmtRNAs potentiates apoptotic cell death by inhibiting survivin expression, a member of the inhibitor of apoptosis (IAP) family. Down-regulation of survivin is at the translational level and is probably mediated by microRNAs generated by dicing of the double-stranded stem of the ASncmtRNAs, as suggested by evidence presented here, in which the ASncmtRNAs are bound to Dicer and knockdown of the ASncmtRNAs reduces reporter luciferase activity in a vector carrying the 3'-UTR of survivin mRNA. Taken together, down-regulation of the ASncmtRNAs constitutes a vulnerability or Achilles' heel of cancer cells, suggesting that the ASncmtRNAs are promising targets for cancer therapy. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The zebrafish progranulin gene family and antisense transcripts

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Baranowski David

2005-11-01

Full Text Available Abstract Background Progranulin is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor that has been implicated in development, wound healing and in the progression of many cancers. The single mammalian progranulin gene encodes a glycoprotein precursor consisting of seven and one half tandemly repeated non-identical copies of the cystine-rich granulin motif. A genome-wide duplication event hypothesized to have occurred at the base of the teleost radiation predicts that mammalian progranulin may be represented by two co-orthologues in zebrafish. Results The cDNAs encoding two zebrafish granulin precursors, progranulins-A and -B, were characterized and found to contain 10 and 9 copies of the granulin motif respectively. The cDNAs and genes encoding the two forms of granulin, progranulins-1 and -2, were also cloned and sequenced. Both latter peptides were found to be encoded by precursors with a simplified architecture consisting of one and one half copies of the granulin motif. A cDNA encoding a chimeric progranulin which likely arises through the mechanism of trans-splicing between grn1 and grn2 was also characterized. A non-coding RNA gene with antisense complementarity to both grn1 and grn2 was identified which may have functional implications with respect to gene dosage, as well as in restricting the formation of the chimeric form of progranulin. Chromosomal localization of the four progranulin (grn genes reveals syntenic conservation for grna only, suggesting that it is the true orthologue of mammalian grn. RT-PCR and whole-mount in situ hybridization analysis of zebrafish grns during development reveals that combined expression of grna and grnb, but not grn1 and grn2, recapitulate many of the expression patterns observed for the murine counterpart. This includes maternal deposition, widespread central nervous system distribution and specific localization within the epithelial

A potent inhibitor of SIK2, 3, 3', 7-trihydroxy-4'-methoxyflavon (4'-O-methylfisetin, promotes melanogenesis in B16F10 melanoma cells.

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Ayako Kumagai

Full Text Available Flavonoids, which are plant polyphenols, are now widely used in supplements and cosmetics. Here, we report that 4'-methylflavonoids are potent inducers of melanogenesis in B16F10 melanoma cells and in mice. We recently identified salt inducible kinase 2 (SIK2 as an inhibitor of melanogenesis via the suppression of the cAMP-response element binding protein (CREB-specific coactivator 1 (TORC1. Using an in vitro kinase assay targeting SIK2, we identified fisetin as a candidate inhibitor, possibly being capable of promoting melanogenesis. However, fisetin neither inhibited the CREB-inhibitory activity of SIK2 nor promoted melanogenesis in B16F10 melanoma cells. Conversely, mono-methyl-flavonoids, such as diosmetin (4'-O-metlylluteolin, efficiently inhibited SIK2 and promoted melanogenesis in this cell line. The cAMP-CREB system is impaired in A(y/a mice and these mice have yellow hair as a result of pheomelanogenesis, while Sik2(+/-; A(y/a mice also have yellow hair, but activate eumelanogenesis when they are exposed to CREB stimulators. Feeding Sik2(+/-; A(y/a mice with diets supplemented with fisetin resulted in their hair color changing to brown, and metabolite analysis suggested the presence of mono-methylfisetin in their feces. Thus, we decided to synthesize 4'-O-methylfisetin (4'MF and found that 4'MF strongly induced melanogenesis in B16F10 melanoma cells, which was accompanied by the nuclear translocation of TORC1, and the 4'-O-methylfisetin-induced melanogenic programs were inhibited by the overexpression of dominant negative TORC1. In conclusion, compounds that modulate SIK2 cascades are helpful to regulate melanogenesis via TORC1 without affecting cAMP levels, and the combined analysis of Sik2(+/- mice and metabolites from these mice is an effective strategy to identify beneficial compounds to regulate CREB activity in vivo.

Ultraviolet B (UVB) induction of the c-fos promoter is mediated by phospho-cAMP response element binding protein (CREB) binding to CRE and c-fos activator protein 1 site (FAP1) cis elements.

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Gonzales, Melissa; Bowden, G Tim

2002-06-26

The ultraviolet B (UVB) portion (280-320 nm) of the ultraviolet spectrum has been shown to contribute to the development of non-melanoma skin cancer in humans. Research in the human keratinocyte cell line, HaCaT, revealed that UVB irradiation caused the upregulation of the transcription factor activator protein-1 (AP-1). The AP-1 complex formed in UVB-irradiated HaCaT cells is specifically composed of c-fos and Jun D. c-Fos expression was induced in a manner that correlated with the UVB-induced activation of AP-1. To investigate how c-fos expression is regulated by UVB irradiation, the role of each of four cis elements within the c-fos promoter was evaluated. Clustered point mutations at the sis inducible element (SIE), serum response element (SRE), c-fos AP-1 site (FAP1), or cyclic AMP response elements (CRE) significantly inhibited UVB induction of the c-fos promoter. This indicated that all four cis elements are required for maximum promoter activity. The CRE and FAP1 elements were the two most active cis elements that mediate the UVB transactivation of c-fos. Homodimers of phosphorylated cAMP response element binding protein (CREB) were induced by UVB irradiation to bind to each of these elements. Therefore, CREB may function as an important regulatory protein in the UVB-induced expression of c-fos.