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Zhou J.,Huazhong University of Science and Technology | Wang H.,ingenious targeting laboratory | Xiong Y.,Huazhong University of Science and Technology | Li Z.,Huazhong University of Science and Technology | And 2 more authors.
Journal of Ethnopharmacology | Year: 2010

Aim of the study: Puerarin (Pur) is a primary component of the most functional extracts of Pueraria lobata used in traditional Chinese medicine for centuries. Since it has been postulated that Pur protects the brain against glutamate (Glu) neurotoxicity, we investigated the effects of Pur on Glu-induced axonal transport impairment in primary hippocampal neurons in this study. Materials and methods: Primary hippocampal cultures were prepared from 2-day-old Sprague-Dawley rats. Intracellular calcium concentration [Ca2+]i, neurofilament (NF) phosphorylation and protein kinase activity for Cdk5 were measured. Time-lapse imaging technology was used to capture the NF axonal transport in the cultured neurons with transiently transfected fluorescence protein linked to the N-terminus of NF-M (EGFP-NFM). Results: The results showed that Pur significantly diminished the Glu-induced elevation of [Ca2+]i in dose-dependent manner and antagonized the Glu-evoked increases in NF phosphorylation at protein levels. The neurons under the Glu treatment displayed the accumulation of immobile NF clusters in the cell body and the reduced rates of axonal transport of NFs by 72.8% compared to the control neurons. Intriguingly, Pur reversed the slowed rate of the axonal transport by 35.6%. Pur also remarkably attenuated Glu-evoked activation of Cdk5. Conclusions: Pur may play a role in protecting against Glu-induced NF axonal transport impairment in rat primary hippocampal neurons by inhibiting the increased [Ca2+]i and by impeding the activation of Cdk5. © 2010 Elsevier Ireland Ltd.


PubMed | University of Calgary, Cornell University, Scripps Research Institute, New York Medical College and ingenious targeting laboratory
Type: | Journal: Nature communications | Year: 2015

Cross-species studies enable rapid translational discovery and produce the broadest impact when both mechanism and phenotype are consistent across organisms. We developed a knock-in mouse that biologically recapitulates a common human mutation in the gene for fatty acid amide hydrolase (FAAH) (C385A; rs324420), the primary catabolic enzyme for the endocannabinoid anandamide. This common polymorphism impacts the expression and activity of FAAH, thereby increasing anandamide levels. Here, we show that the genetic knock-in mouse and human variant allele carriers exhibit parallel alterations in biochemisty, neurocircuitry and behaviour. Specifically, there is reduced FAAH expression associated with the variant allele that selectively enhances fronto-amygdala connectivity and fear extinction learning, and decreases anxiety-like behaviours. These results suggest a gain of function in fear regulation and may indicate for whom and for what anxiety symptoms FAAH inhibitors or exposure-based therapies will be most efficacious, bridging an important translational gap between the mouse and human.


Rezende N.C.,Cornell University | Lee M.-Y.,Cornell University | Monette S.,Cornell University | Mark W.,Sloan Kettering Institute | And 3 more authors.
Developmental Biology | Year: 2011

Rex1 (Zfp42), GeneID 132625, is a gene whose expression is closely associated with pluripotency/multipotency in both mouse and human embryonic stem cells. To study the function of the murine Rex1 gene in vivo, we have used cre/lox technology to create Rex1(floxed) mice and mice deficient in Rex1 gene function. Rex1-/-males are characterized by an age-associated decrease in sperm counts, abnormal sperm morphology, and mild testicular atrophy. We characterized global patterns of gene expression in primary germ cells by microarray and identified the growth hormone responsive gene, GRTP1, as a transcript present at a 4.5 fold higher level in wild type (WT) compared to Rex1-/- mice. We analyzed immature germ cell (Dazl), proliferating (PCNA), and Sertoli cell populations, and quantitated levels of apoptosis in Rex1-/- as compared to WT testes. We evaluated the expression of proteins previously reported to correlate with Rex1 expression, such as STAT3, phospho-STAT3, p38, and phospho-p38 in the testis. We report a distinct cellular localization of total STAT3 protein in Rex1-/- affected testes. Our data suggest that loss of Rex1 leads to impaired testicular function. © 2011 Elsevier B.V.


Lee M.-Y.,Cornell University | Lu A.,Cornell University | Lu A.,ingenious targeting laboratory | Gudas L.J.,Cornell University
Journal of Cellular Physiology | Year: 2010

Rex1 (zfp42) was identified by our laboratory because of its reduced expression in F9 teratocarcinoma stem cells after retinoic acid (RA) treatment. The Rex1 (Zfp42) gene is currently widely used as a marker of embryonic stem cells. We compared the transcriptional regulation of the human Rex1 gene in NTera-2 (NT-2) human teratocarcinoma, normal human prostate epithelial cells (PrEC), and prostate cancer cells (PC-3) by promoter/luciferase analyses. Oct4, Sox2, Nanog, and Dax1 transcripts are expressed at higher levels in NT-2 and PrEC cells than in PC-3 cells. Co-transfection analyses showed that YY1 and Rex1 are positive regulators of hRex1 transcription in NT-2 and PrEC cells, whereas Nanog is not. Serial deletion constructs of the hRex1 promoter were created and analyzed, by which we identified a potential negative regulatory site that is located between -1 and -0.4 kb of the hRex1 promoter. We also delineated regions of the hRex1 promoter between -0.4 kb and the TSS that, when mutated, reduced transcriptional activation; these are putative Rex1 binding sites. Mutation of a putative Rex1 binding site in electrophoretic mobility shift assays (EMSA) resulted in reduced protein binding. Taken together, our results indicate that hRex1 binds to the hRex1 promoter region at -298 bp and positively regulates hRex1 transcription, but that this regulation is lost in PC-3 human prostate cancer cells. This lack of positive transcriptional regulation by the hRex1 protein may be responsible for the lack of Rex1 expression in PC-3 prostate cancer cells. © 2010 Wiley-Liss, Inc.


Dincheva I.,Cornell University | Drysdale A.T.,Cornell University | Hartley C.A.,Cornell University | Johnson D.C.,Cornell University | And 12 more authors.
Nature Communications | Year: 2015

Cross-species studies enable rapid translational discovery and produce the broadest impact when both mechanism and phenotype are consistent across organisms. We developed a knock-in mouse that biologically recapitulates a common human mutation in the gene for fatty acid amide hydrolase (FAAH) (C385A; rs324420), the primary catabolic enzyme for the endocannabinoid anandamide. This common polymorphism impacts the expression and activity of FAAH, thereby increasing anandamide levels. Here, we show that the genetic knock-in mouse and human variant allele carriers exhibit parallel alterations in biochemisty, neurocircuitry and behaviour. Specifically, there is reduced FAAH expression associated with the variant allele that selectively enhances fronto-amygdala connectivity and fear extinction learning, and decreases anxiety-like behaviours. These results suggest a gain of function in fear regulation and may indicate for whom and for what anxiety symptoms FAAH inhibitors or exposure-based therapies will be most efficacious, bridging an important translational gap between the mouse and human. © 2015 Macmillan Publishers Limited. All rights reserved.


Dewing A.S.T.,University of Hawaii at Manoa | Rueli R.H.,University of Hawaii at Manoa | Robles M.J.,University of Hawaii at Manoa | Nguyen-Wu E.D.,University of Hawaii at Manoa | And 3 more authors.
RNA Biology | Year: 2012

Selenoprotein P (Sepp1), a glycoprotein rich in selenium, is thought to function in selenium transport throughout the body. The sepp1 gene locus potentially produces three alternative transcripts that differ only in their 5' untranslated regions (5'UTRs) and not in their protein coding regions, as indicated by transcript information in genomic databases. Here we investigated the distribution, relative expression, and biological significance of these transcript variants. We confirmed the expression of Sepp1 transcript variants using PC R and sequencing. Using 5'-RACE, we identified multiple 5'-termini upstream from three different splice donor sites, and a single splice acceptor site for exon 2. We found regional and temporal changes in variant expression in select adult and neonate murine tissue and brain regions. Distribution of variants in heart and kidney varied with stage of development. Notably, the Sepp1b variant was localized specifically to the hippocampus in brain. Targeted silencing of individual variants using RNAi demonstrated the biological importance for all transcript variants in cell viability. Additionally, we determined that the Sepp1b variant is a specific target for the miR-7 microRNA by means of its unique 5'UTR structure. Our results emphasize the importance of non-coding transcript variations as a regulatory means for Sepp1 expression in different tissues and stages of development. The presence of a variant localized in the hippocampus and regulated by a microRNA may have implications for the known deficits in synaptic function caused by genetic deletion of Sepp1. © 2012 Landes Bioscience.


Budhwar R.,Mount Sinai School of Medicine | Lu A.,Mount Sinai School of Medicine | Lu A.,ingenious targeting laboratory | Hirsch J.P.,Mount Sinai School of Medicine
Molecular Biology of the Cell | Year: 2010

GPB1 and GPB2 encode kelch repeat-containing proteins that regulate protein kinase A (PKA) in yeast by a cAMP-independent process. Here we show that Gpb1 and Gpb2 stimulate phosphorylation of PKA regulatory subunit Bcy1 in low glucose concentrations, thereby promoting the inhibitory function of Bcy1 when nutrients are scarce and PKA activity is expected to be low. Gpb1 and Gpb2 stimulate Bcy1 phosphorylation at an unknown site, and this modification stabilizes Bcy1 that has been phosphorylated by PKA catalytic subunits at serine-145. The BCY1 S145A mutation eliminates the effect of gpb1Δ gpb2Δ on Bcy1 stability but maintains their effect on phosphorylation and signaling, indicating that modulation of PKA activity by Gpb1 and Gpb2 is not solely due to increased levels of Bcy1. Inhibition of PKA catalytic subunits that are ATP analog-sensitive causes increased Bcy1 phosphorylation at the unknown site in high glucose. When PKA is inhibited, gpb1Δ gpb2Δ mutations have no effect on Bcy1 phosphorylation. Therefore, Gpb1 and Gpb2 oppose PKA activity by blocking the ability of PKA to inhibit Bcy1 phosphorylation at a site other than serine-145. Stimulation of Bcy1 phosphorylation by Gpb1 and Gpb2 produces a form of Bcy1 that is more stable and is a more effective PKA inhibitor. © 2010 R. Budhwar et al.


Zhou J.,Huazhong University of Science and Technology | Wang Y.,Huazhong University of Science and Technology | Xiong Y.,Huazhong University of Science and Technology | Wang H.,ingenious targeting laboratory | And 2 more authors.
Ultrasound in Medicine and Biology | Year: 2010

Here we report a new, simple and efficient method by using ultrasound and a microbubble agent (SonoVue) for delivering a gene to balloon-injured carotid arteries for restenosis prophylaxis. The tissue factor pathway inhibitor-2 (TFPI-2) has been shown to inhibit the postinjury intimae hyperplasia in atherosclerotic vessels. New Zealand white rabbits were divided into 4 groups with 14 in each, a treatment control for balloon injury, a gene vehicle control, a gene delivery of TFPI-2 without using ultrasound and a gene delivery of TFPI-2 using ultrasound. After four weeks, the injured artery neointimal proliferation was significantly lower in the TFPI-2 group with ultrasound than the control groups (p < 0.01) according to the measurement of the mean luminal diameters by B-mode ultrasonography. The ratio of intimal/media area and the stenosis rate in the gene delivery facilitated by ultrasound were significantly lower than those of the nonultrasound gene delivering method (p < 0.01). © 2010 World Federation for Ultrasound in Medicine & Biology.


Chen D.,Huazhong University of Science and Technology | Liu Y.,Huazhong University of Science and Technology | He W.,Compulsory Detoxification Treatment Center | Wang H.,ingenious targeting laboratory | Wang Z.,Huazhong University of Science and Technology
Journal of Huazhong University of Science and Technology - Medical Science | Year: 2012

This study examined the effects of combined administration of tyrosine, lecithin, L-glutamine and L-5-hydroxytryptophan (5-HTP) on heroin withdrawal syndromes and mental symptoms in detoxified heroin addicts. In the cluster-randomized placebo-controlled trial, 83 detoxified heroin addicts were recruited from a detoxification treatment center in Wuhan, China. Patients in the intervention group (n=41) were given the combined treatment with tyrosine, lecithin, L-glutamine and 5-HTP and those in the control group (n=42) were administered the placebo. The sleep status and the withdrawal symptoms were observed daily throughout the study, and the mood states were monitored pre- and post-intervention. The results showed that the insomnia and withdrawal scores were significantly improved over time in participants in the intervention group as compared with those in the control group. A greater reduction in tension-anxiety, depression-dejection, anger-hostility, fatigue-inertia and total mood disturbance, and a greater increase in their vigor-activity symptoms were found at day 6 in the intervention group than in the control group (all P<0.05). It was concluded that the neurotransmitter- precursor-supplement intervention is effective in alleviating the withdrawal and mood symptoms and it may become a supplementary method for patients' recovery from heroin addiction. © Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2012.


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