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Find video protocols related to scientific articles indexed in Pubmed.
Activation of TRPV1 mediates thymic stromal lymphopoietin release via the Ca2+/NFAT pathway in airway epithelial cells.
FEBS Lett.
PUBLISHED: 03-11-2014
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The airway epithelium is exposed to a range of irritants in the environment that are known to trigger inflammatory response such as asthma. Transient receptor potential vanilloid 1 (TRPV1) is a Ca(2+)-permeable cation channel critical for detecting noxious stimuli by sensory neurons. Recently increasing evidence suggests TRPV1 is also crucially involved in the pathophysiology of asthma on airway epithelium in human. Here we report that in airway epithelial cells TRPV1 activation potently induces allergic cytokine thymic stromal lymphopoietin (TSLP) release. TSLP induction by protease-activated receptor (PAR)-2 activation is also partially mediated by TRPV1 channels.
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PTEN interacts with histone H1 and controls chromatin condensation.
Cell Rep
PUBLISHED: 02-14-2014
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Chromatin organization and dynamics are integral to global gene transcription. Histone modification influences chromatin status and gene expression. PTEN plays multiple roles in tumor suppression, development, and metabolism. Here, we report on the interplay of PTEN, histone H1, and chromatin. We show that loss of PTEN leads to dissociation of histone H1 from chromatin and decondensation of chromatin. PTEN deletion also results in elevation of histone H4 acetylation at lysine 16, an epigenetic marker for chromatin activation. We found that PTEN and histone H1 physically interact through their C-terminal domains. Disruption of the PTEN C terminus promotes the chromatin association of MOF acetyltransferase and induces H4K16 acetylation. Hyperacetylation of H4K16 impairs the association of PTEN with histone H1, which constitutes regulatory feedback that may reduce chromatin stability. Our results demonstrate that PTEN controls chromatin condensation, thus influencing gene expression. We propose that PTEN regulates global gene transcription profiling through histones and chromatin remodeling.
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PTEN C-terminal deletion causes genomic instability and tumor development.
Cell Rep
PUBLISHED: 01-23-2014
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Tumor suppressor PTEN controls genomic stability and inhibits tumorigenesis. The N-terminal phosphatase domain of PTEN antagonizes the PI3K/AKT pathway, but its C-terminal function is less defined. Here, we describe a knockin mouse model of a nonsense mutation that results in the deletion of the entire Pten C-terminal region, referred to as Pten(?C). Mice heterozygous for Pten(?C) develop multiple spontaneous tumors, including cancers and B cell lymphoma. Heterozygous deletion of the Pten C-terminal domain also causes genomic instability and common fragile site rearrangement. We found that Pten C-terminal disruption induces p53 and its downstream targets. Simultaneous depletion of p53 promotes metastasis without influencing the initiation of tumors, suggesting that p53 mainly suppresses tumor progression. Our data highlight the essential role of the PTEN C terminus in the maintenance of genomic stability and suppression of tumorigenesis.
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PTEN?, a PTEN isoform translated through alternative initiation, regulates mitochondrial function and energy metabolism.
Cell Metab.
PUBLISHED: 01-07-2014
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PTEN is one of the most frequently mutated genes in human cancer. It is known that PTEN has a wide range of biological functions beyond tumor suppression. Here, we report that PTEN?, an N-terminally extended form of PTEN, functions in mitochondrial metabolism. Translation of PTEN? is initiated from a CUG codon upstream of and in-frame with the coding region of canonical PTEN. Eukaryotic translation initiation factor 2A (eIF2A) controls PTEN? translation, which requires a CUG-centered palindromic motif. We show that PTEN? induces cytochrome c oxidase activity and ATP production in mitochondria. TALEN-mediated somatic deletion of PTEN? impairs mitochondrial respiratory chain function. PTEN? interacts with canonical PTEN to increase PINK1 protein levels and promote energy production. Our studies demonstrate the importance of eIF2A-mediated alternative translation for generation of protein diversity in eukaryotic systems and provide insights into the mechanism by which the PTEN family is involved in multiple cellular processes.
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A transcriptomic study of the red palm weevil Rhynchophorus ferrugineus embryogenesis.
Insect Sci.
PUBLISHED: 09-23-2013
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The red palm weevil (RPW), Rhynchophorus ferrugineus (Coleoptera: Curculionidae), is an invasive, concealed, and destructive tissue borer, and it becomes a lethal pest of the palm family plants and has been reported to attack 20 palm species around the globe. Here we report a systematic transcriptomic study on embryogenesis of RPW, where we analyze the transcriptomes across five developmental stages of RPW embryogenesis, involving four embryonic stages (E1, E2, E3, and E4) and one larval stage (L1). Using the RNA-seq and next-generation platforms, we generate 80 to 91 million reads for each library and assemble 22,532 genes that are expressed at different embryonic stages. Among the total transcripts from the five embryonic development stages, we find that 30.45% are differentially expressed, 10.10% show stage-specificity, and even a larger fraction, 62.88%, exhibit constitutive expression in all the stages. We also analyze the expression dynamics of several conserved signaling pathways (such as Hedgehog, JAK-STAT, Notch, TGF-?, Ras/MAPK, and Wnt), as well as key developmental genes including those related to apoptosis, axis formation, Hox complex, neurogenesis, and segmentation. The datasets provide an essential resource for gene annotation and RPW functional genomics including studies by using tools and concepts from multiple disciplines, such as development, physiology, biochemistry, molecular biology, and genetics. This article is protected by copyright. All rights reserved.
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Genome sequence of the date palm Phoenix dactylifera L.
Nat Commun
PUBLISHED: 03-26-2013
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Date palm (Phoenix dactylifera L.) is a cultivated woody plant species with agricultural and economic importance. Here we report a genome assembly for an elite variety (Khalas), which is 605.4?Mb in size and covers >90% of the genome (~671?Mb) and >96% of its genes (~41,660 genes). Genomic sequence analysis demonstrates that P. dactylifera experienced a clear genome-wide duplication after either ancient whole genome duplications or massive segmental duplications. Genetic diversity analysis indicates that its stress resistance and sugar metabolism-related genes tend to be enriched in the chromosomal regions where the density of single-nucleotide polymorphisms is relatively low. Using transcriptomic data, we also illustrate the date palms unique sugar metabolism that underlies fruit development and ripening. Our large-scale genomic and transcriptomic data pave the way for further genomic studies not only on P. dactylifera but also other Arecaceae plants.
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Identification of UHRF1/2 as new N-methylpurine DNA glycosylase-interacting proteins.
Biochem. Biophys. Res. Commun.
PUBLISHED: 02-19-2013
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N-methylpurine DNA glycosylase (MPG), a DNA repair enzyme, functions in the DNA base excision repair (BER) pathway. Aberrant over-expression of MPG in various cancers suggests an important role of MPG in carcinogenesis. Identification of MPG-interacting proteins will help to dissect the molecular link between MPG and cancer development. In the present study, using immunoprecipitation coupled with mass spectrometry (IP/MS), we screened ubiquitin-like, containing PHD and RING finger domains 1 (UHRF1), an essential protein required for the maintenance of DNA methylation, as a MPG-interacting protein. Endogenous co-immunoprecipitation assay in cancer cells confirmed that UHRF1 interacted with MPG in a p53 status-independent manner. Confocal microscopy showed that endogenous MPG and UHRF1 were co-localized in the nucleoplasm. Furthermore, co-immunoprecipitation assay indicated that UHRF2, the homolog of UHRF1, could also interact with MPG. These results show that MPG and the UHRF family of proteins interact, thus providing a functional linkage between MPG and UHRF1/2.
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Effects on gene expression in rat liver after administration of RXR agonists: UAB30, 4-methyl-UAB30, and Targretin (Bexarotene).
Mol. Pharmacol.
PUBLISHED: 01-04-2013
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Examination of three retinoid X receptor (RXR) agonists [Targretin (TRG), UAB30, and 4-methyl-UAB30 (4-Me-UAB30)] showed that all inhibited mammary cancer in rodents and two (TRG and 4-Me-UAB30) strikingly increased serum triglyceride levels. Agents were administered in diets to female Sprague-Dawley rats. Liver RNA was isolated and microarrayed on the Affymetrix GeneChip Rat Exon 1.0 ST array. Statistical tests identified genes that exhibited differential expression and fell into groups, or modules, with differential expression among agonists. Genes in specific modules were changed by one, two, or all three agonists. An interactome analysis assessed the effects on genes that heterodimerize with known nuclear receptors. For proliferator-activated receptor ?/RXR-activated genes, the strongest response was TRG > 4-Me-UAB30 > UAB30. Many liver X receptor/RXR-related genes (e.g., Scd-1 and Srebf1, which are associated with increased triglycerides) were highly expressed in TRG and 4-Me-UAB30- but not UAB30-treated livers. Minimal expression changes were associated with retinoic acid receptor or vitamin D receptor heterodimers by any of the agonists. UAB30 unexpectedly and uniquely activated genes associated with the aryl hydrocarbon hydroxylase (Ah) receptor (Cyp1a1, Cyp1a2, Cyp1b1, and Nqo1). Based on the Ah receptor activation, UAB30 was tested for its ability to prevent dimethylbenzanthracene (DMBA)-induced mammary cancers, presumably by inhibiting DMBA activation, and was highly effective. Gene expression changes were determined by reverse transcriptase-polymerase chain reaction in rat livers treated with Targretin for 2.3, 7, and 21 days. These showed similar gene expression changes at all three time points, arguing some steady-state effect. Different patterns of gene expression among the agonists provided insight into molecular differences and allowed one to predict certain physiologic consequences of agonist treatment.
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A versatile water soluble fluorescent probe for ratiometric sensing of Hg2+ and bovine serum albumin.
Dalton Trans
PUBLISHED: 08-22-2011
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A novel tetraazamacrocycle fluorescent sensor (6-(1-(dimethylamino)-5-naphthalene sulfonyl)-3,6,9,15-tetraazabicyclo[9.3.1] pentadeca-1(15),11,13-triene, 1) has been designed and prepared, which can be utilized for selective and ratiometric sensing of Hg(2+) and bovine serum albumin (BSA) with two different responsive modes in aqueous solution at physiological pH (50 mM Tris-HCl, pH 7.6). Above 0.5 ppb Hg(2+) can be discerned by coordination with 1 and the emission color changes enable 1 to be applied to a fast Hg(2+) test paper assay. Sensor 1 has also been demonstrated to be easily cell-penetrable and applicable for Hg(2+) imaging in living cells. Imaging of BSA in the gel using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) stained in the medium containing 1 verified that the binding of 1 and BSA was successful in the presence of nonprotein substances. The linear range of 1 towards BSA utilizing ratiometric fluorescent calibration via noncovalent interaction in solution is 0-100 ?g mL(-1) with a detection limit of 1 ?g mL(-1), and has been successfully employed to determine the albumin concentration in blood serum by means of ratiometric fluorescent measurements for the first time. Finally, sensor 1 behaves as a fluorescent molecular switch composed of triple logic gates upon chemical inputs of Hg(2+) and BSA, which potentially provides intelligent diagnostics for Hg(2+) contaminated serum on the nanoscale.
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The E3 ligase Smurf1 regulates Wolfram syndrome protein stability at the endoplasmic reticulum.
J. Biol. Chem.
PUBLISHED: 03-28-2011
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The HECT-type ubiquitin ligase (E3) Smad ubiquitination regulatory factor 1 (Smurf1) targets various substrates, including Smad1/5, RhoA, Prickle 1, MEKK2, and JunB for degradation and thereby regulates adult bone formation and embryonic development. Here, we identify the endoplasmic reticulum (ER)-localized Wolfram syndrome protein (WFS1) as a specific degradation substrate of Smurf1. Mutations in the WFS1 gene cause Wolfram syndrome, an autosomal recessive disorder characterized by diabetes mellitus and optic atrophy. WFS1 negatively regulates the ER stress response, and WFS1 deficiency in mice increases ER stress and triggers apoptosis. We show that Smurf1 interacts with WFS1 at the ER and promotes the ubiquitination and proteasomal degradation of WFS1. A C-terminal luminal region in WFS1, including residues 667-700, is involved in this degradation. Wild-type WFS1 as well as a subset of WFS1 mutants that include this degron region are susceptible to Smurf1-mediated degradation. By contrast, pathophysiological deletion mutants of WFS1 lacking the degron, such as W648X, Y660X, and Q667X, are resistant to degradation by Smurf1. Depletion of Smurf1 by RNA interference results in increased WFS1 and decreased ATF6? levels. Furthermore, we show that ER stress induces Smurf1 degradation and WFS1 up-regulation. These findings reveal for the first time that Smurf1 targets an ER-localized protein for degradation and that Smurf1 is regulated by ER stress.
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CREB is a novel nuclear target of PTEN phosphatase.
Cancer Res.
PUBLISHED: 03-08-2011
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PTEN phosphatase is a potent tumor suppressor that regulates multiple cellular functions. In the cytoplasm, PTEN dephosphorylates its primary lipid substrate, phosphatidylinositol 3,4,5-trisphosphate, to antagonize the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway. It has also become increasingly evident that PTEN functions in the nucleus and may play an important part in transcription regulation, but its nuclear targets remain elusive. In this report, we demonstrate the transcription factor cyclic AMP response element-binding protein (CREB) is a protein target of PTEN phosphatase and that PTEN deficiency leads to CREB phosphorylation independent of the PI3K/AKT pathway. Using confocal immunofluorescence and reciprocal immunoprecipitation, we further show that PTEN colocalizes with CREB and physically interacts with CREB. Moreover, we use both in vitro and in vivo experiments to show PTEN can dephosphorylate CREB in a phosphatase-dependent manner, suggesting that CREB is a substrate of PTEN nuclear phosphatase. Loss of Pten results in an elevated RNA level of multiple CREB transcriptional targets and increased cell proliferation, which can be reversed by a nonphosphorylatable CREB mutant or knockdown of CREB. These data reveal a mechanism for PTEN modulation of CREB-mediated gene transcription and cell growth. Our study thus characterizes PTEN as a nuclear phophatase of a transcription factor and identifies CREB as a novel protein target of PTEN phosphatase, which contributes to better understanding of PTEN function in the nucleus.
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Highly specific capture and direct MALDI-MS analysis of phosphorylated peptides using novel multifunctional chitosan-GMA-IDA-Fe (III) nanosphere.
Anal Bioanal Chem
PUBLISHED: 03-07-2011
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In this study, we describe a method for highly specific enrichment of phosphopeptides with multifunctional chitosan-glycidyl methacrylate (GMA)-iminodiacetic acid (IDA)-Fe (III) nanospheres for direct analysis by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This is the first time that chitosan has been used to create nanospheres support material for selective enrichment of phosphopeptides by modification with GMA, derivatization with IDA, and loading with Fe (III) ions. Chitosan-GMA-IDA-Fe (III) nanospheres with a diameter of 20 to 100 nm have multifunctional chemical moieties which confer unique properties, good dispersibility in highly acidic binding buffers, as well as good biocompatibility and chemical stability which improves their specific interaction with phosphopeptides using various types of acid binding buffers. The process of enrichment is very simple, quick, efficient, and specific. Its high specificity and efficiency for purification of phosphopeptides is reflected in the very low and substoichiometric amounts of phosphopeptides which can be detected, in quantities as low as 1:3,000 M ratios. Compared with other state-of the-art technologies such as the use of conventional Fe(3+)-IMAC and TiO(2), these chitosan nanosphere techniques show superior specificity and sensitivity. Moreover, the resultant chitosan-GMA-IDA-Fe(3+) nanosphere-absorbed phosphopeptides can be either directly analyzed by MALDI-TOF MS analysis or eluted and further analyzed by nano-LC-MS/MS.
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Smurf1 ubiquitin ligase targets Kruppel-like factor KLF2 for ubiquitination and degradation in human lung cancer H1299 cells.
Biochem. Biophys. Res. Commun.
PUBLISHED: 02-27-2011
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Krüppel-like factor 2 (KLF2) has been demonstrated to be essential for normal lung development, erythroid differentiation, T-cell differentiation, migration and homing. However, the mechanisms underlying the regulation of KLF2, in particular its responsible E3 ligase is still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets KLF2 for poly-ubiquitination and proteasomal degradation specifically in lung cancer H1299 cells. The catalytic ligase activity of Smurf1 is required for it to regulate KLF2. Consequently, Smurf1 represses the transcriptional factor activity of KLF2 and regulates the expression its downstream genes such as CD62L and Wee1. This study provided the first evidence that Smurf1 functions as an E3 ligase to promote the ubiquitination and proteasomal degradation of KLF2.
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Novel multifunctional chitosan-GMA-IDA-Cu(II) nanospheres for high dynamic range characterization of the human plasma proteome.
Anal Bioanal Chem
PUBLISHED: 02-14-2011
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In this study, we describe characterization of the human plasma proteome based on analysis with multifunctional chitosan-GMA-IDA-Cu(II) nanospheres. Chitosan-GMA-IDA-Cu(II) nanospheres with diameters of 20 to 100 nm have unique properties due to multifunctional chemical moieties, high surface area, high capacity, good dispersibility in buffer solution as well as good biocompatibility and chemical stability which improves their specific interaction with peptides and proteins of the human plasma using different binding buffers. Combining these chitosan-GMA-IDA-Cu(II) nanospheres with MS spectrometry results in a novel strategy which should make it possible to characterize the plasma proteome in a single test. Peptides and proteins adsorbed on the nanosphere can be directly detected by MALDI-TOF-MS. The eluted lower molecular weight peptides and proteins are identified by nano-LC-ESI-MS/MS. A total of 842 unique LMW peptides and 1,682 human unredundant proteins IDs were identified in two different binding buffers, which included relatively low-level proteins (e.g., pg/mL of IL3 Interleukin-3) co-distributed with high-abundance proteins (e.g., 35-55 mg/mL level serum albumin). As such, this nanosphere technique selectively enabled the identification of proteins over a dynamic range of greater than 8 orders of magnitude. Considering this capacity for selective enrichment of peptides and proteins in human plasma, and the large number of LMW peptides and proteins which can be identified, this method promises to accelerate discovery of biomarkers for clinical application.
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A Zn2+-specific turn-on fluorescent probe for ratiometric sensing of pyrophosphate in both water and blood serum.
Dalton Trans
PUBLISHED: 12-17-2010
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A novel fluorescent sensor composed of a naphthalene functionalized tetraazamacrocycle ligand 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene-3-methyl naphthalene (1) and Zn(2+) has been designed and prepared, which can be utilized for selective and ratiometric sensing of pyrophosphate (PPi) over other phosphate-containing anions in aqueous solution at physiological pH. Notably, the water soluble 1 itself also exhibits a selective enhanced fluorescent response to Zn(2+), and the complex 1-Zn(2+) thus formed eventually fulfils the synergic Zn(2+) coordination-altered strategy with PPi. Furthermore, the ratiometric sensing of 1-Zn(2+) towards PPi performed well even in blood serum milieu. Finally, the sensor 1-Zn(2+) was successfully employed to monitor a real-time assay of inorganic pyrophosphatase (PPase) by means of ratiometric fluorescent measurements for the first time.
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ATM-mediated NuSAP phosphorylation induces mitotic arrest.
Biochem. Biophys. Res. Commun.
PUBLISHED: 11-23-2010
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NuSAP is a microtubule-associated protein that plays an important role in spindle assembly. NuSAP deficiency in mice leads to early embryonic lethality. Spindle assembly in NuSAP-deficient cells is highly inefficient and chromosomes remain dispersed in the mitotic cytoplasm. ATM is a key kinase that phosphorylates a series of substrates to mediate G1/S control. However, the role of ATM at the G2/M phase is not well understood. Here we demonstrate that ectopic expression of NuSAP lead to mitotic arrest observably dependent on the kinase activity of ATM. When endogenous ATM was depleted or its kinase activity was inhibited, NuSAP could not cause mitotic arrest. We further show ATM interacts with NuSAP and phosphorylates NuSAP on Ser124. The phosphorylation and interaction occur specifically at G2/M-phase. Collectively, our work has uncovered an ATM-dependent checkpoint pathway that prevents mitotic progression by targeting a microtubule-associated protein, NuSAP.
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The complete chloroplast genome sequence of date palm (Phoenix dactylifera L.).
PLoS ONE
PUBLISHED: 07-07-2010
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Date palm (Phoenix dactylifera L.), a member of Arecaceae family, is one of the three major economically important woody palms--the two other palms being oil palm and coconut tree--and its fruit is a staple food among Middle East and North African nations, as well as many other tropical and subtropical regions. Here we report a complete sequence of the data palm chloroplast (cp) genome based on pyrosequencing.
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Human microRNA oncogenes and tumor suppressors show significantly different biological patterns: from functions to targets.
PLoS ONE
PUBLISHED: 06-03-2010
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MicroRNAs (miRNAs) are small noncoding RNAs which play essential roles in many important biological processes. Therefore, their dysfunction is associated with a variety of human diseases, including cancer. Increasing evidence shows that miRNAs can act as oncogenes or tumor suppressors, and although there is great interest in research into these cancer-associated miRNAs, little is known about them. In this study, we performed a comprehensive analysis of putative human miRNA oncogenes and tumor suppressors. We found that miRNA oncogenes and tumor suppressors clearly show different patterns in function, evolutionary rate, expression, chromosome distribution, molecule size, free energy, transcription factors, and targets. For example, miRNA oncogenes are located mainly in the amplified regions in human cancers, whereas miRNA tumor suppressors are located mainly in the deleted regions. miRNA oncogenes tend to cleave target mRNAs more frequently than miRNA tumor suppressors. These results indicate that these two types of cancer-associated miRNAs play different roles in cancer formation and development. Moreover, the patterns identified here can discriminate novel miRNA oncogenes and tumor suppressors with a high degree of accuracy. This study represents the first large-scale bioinformatic analysis of human miRNA oncogenes and tumor suppressors. Our findings provide help for not only understanding of miRNAs in cancer but also for the specific identification of novel miRNAs as miRNA oncogenes and tumor suppressors. In addition, the data presented in this study will be valuable for the study of both miRNAs and cancer.
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ARF-dependent regulation of ATM and p53 associated KZNF (Apak) protein activity in response to oncogenic stress.
FEBS Lett.
PUBLISHED: 05-27-2010
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The KRAB-type zinc-finger protein Apak (ATM and p53 associated KZNF protein) specifically suppresses p53-mediated apoptosis. Upon DNA damage, Apak is phosphorylated and inhibited by ATM kinase, resulting in p53 activation. However, how Apak is regulated in response to oncogenic stress remains unknown. Here we show that upon oncogene activation, Apak is inhibited in the tumor suppressor ARF-dependent but ATM-independent manner. Oncogene-induced ARF protein directly interacts with Apak and competes with p53 to bind to Apak, resulting in Apak dissociation from p53. Thus, Apak is differentially regulated in the ARF and ATM-dependent manner in response to oncogenic stress and DNA damage, respectively.
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Smad ubiquitylation regulatory factor 1/2 (Smurf1/2) promotes p53 degradation by stabilizing the E3 ligase MDM2.
J. Biol. Chem.
PUBLISHED: 05-18-2010
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The tumor suppressor p53 protein is tightly regulated by a ubiquitin-proteasomal degradation mechanism. Several E3 ubiquitin ligases, including MDM2 (mouse double minute 2), have been reported to play an essential role in the regulation of p53 stability. However, it remains unclear how the activity of these E3 ligases is regulated. Here, we show that the HECT-type E3 ligase Smurf1/2 (Smad ubiquitylation regulatory factor 1/2) promotes p53 degradation by enhancing the activity of the E3 ligase MDM2. We provide evidence that the role of Smurf1/2 on the p53 stability is not dependent on the E3 activity of Smurf1/2 but rather is dependent on the activity of MDM2. We find that Smurf1/2 stabilizes MDM2 by enhancing the heterodimerization of MDM2 with MDMX, during which Smurf1/2 interacts with MDM2 and MDMX. We finally provide evidence that Smurf1/2 regulates apoptosis through p53. To our knowledge, this is the first report to demonstrate that Smurf1/2 functions as a factor to stabilize MDM2 protein rather than as a direct E3 ligase in regulation of p53 degradation.
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HECT ubiquitin ligase Smurf1 targets the tumor suppressor ING2 for ubiquitination and degradation.
FEBS Lett.
PUBLISHED: 03-27-2010
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Inhibitor of growth 2 (ING2) gene encodes a candidate tumor suppressor and is frequently reduced in many tumors. However, the mechanisms underlying the regulation of ING2, in particular its protein stability, are still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets ING2 for poly-ubiquitination and proteasomal degradation. Intriguingly, the ING2 binding domain in Smurf1 was mapped to the catalytic HECT domain. Furthermore, the C-terminal PHD domain of ING2 was required for Smurf1-mediated degradation. This study provided the first evidence that the stability of ING2 could be regulated by ubiquitin-mediated degradation.
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Phosphatase-dependent regulation of epithelial mitogen-activated protein kinase responses to toxin-induced membrane pores.
PLoS ONE
PUBLISHED: 10-29-2009
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Diverse bacterial species produce pore-forming toxins (PFT) that can puncture eukaryotic cell membranes. Host cells respond to sublytic concentrations of PFT through conserved intracellular signaling pathways, including activation of mitogen-activated protein kinases (MAPK), which are critical to cell survival. Here we demonstrate that in respiratory epithelial cells p38 and JNK MAPK were phosphorylated within 30 min of exposure to pneumolysin, the PFT from Streptococcus pneumoniae. This activation was tightly regulated, and dephosphorylation of both MAPK occurred within 60 min following exposure. Pretreatment of epithelial cells with inhibitors of cellular phosphatases, including sodium orthovanadate, calyculin A, and okadaic acid, prolonged and intensified MAPK activation. Specific inhibition of MAPK phosphatase-1 did not affect the kinetics of MAPK activation in PFT-exposed epithelial cells, but siRNA-mediated knockdown of serine/threonine phosphatases PP1 and PP2A were potent inhibitors of MAPK dephosphorylation. These results indicate an important role for PP1 and PP2A in termination of epithelial responses to PFT and only a minor contribution of dual-specificity phosphatases, such as MAPK phosphatase-1, which are the major regulators of MAPK signals in other cell types. Epithelial regulation of MAPK signaling in response to membrane disruption involves distinct pathways and may require different strategies for therapeutic interventions.
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Identification of genes involved in immune response, microsatellite, and SNP markers from expressed sequence tags generated from hemocytes of freshwater pearl mussel (Hyriopsis cumingii).
Mar. Biotechnol.
PUBLISHED: 07-31-2009
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Triangle sail mussel (Hyriopsis cumingii) is the most important mussel species commercially exploited for freshwater pearl production in China. However, its genome research is still at the infantry. Genomic resources for this species are largely not available. The objectives of this study was to generate expressed sequence tags from a hemocyte cDNA library, to identify genes involved in defense mechanisms, and to identify polymorphic markers from the expressed sequence tag (EST) resources for genetic analysis. A total of 5,290 ESTs were sequenced, obtaining 481 contigs and 1,165 singletons. BLAST similarity analysis indicated almost half (46.5%) of these ESTs were homologs of known genes while 53.5% were transcripts of unknown identities. Based on sequence similarities, 50 genes were identified as putative genes involved in immune and defense functions such as hemocyte immune process, stress proteins, adhesive proteins, proteases and protease regulators, antimicrobial peptides, lysosomal enzymes, cell apoptosis, and cell cycle proteins. A total of 201 microsatellites were identified from these ESTs, with 31 having sufficient flanking sequences for primer design. Polymerase chain reaction amplification was successful for 18 primer pairs and 14 of them were polymorphic. A total of 987 putative single nucleotide polymorphisms were identified including 204 transitions, 611 transversions, and 172 indels; 12 of them were involved in nine genes of defense mechanisms. These resources provide the material basis for future marker validation and genetic linkage and quantitative trait loci analysis in the freshwater pearl mussel.
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Chasing relationships between nutrition and reproduction: A comparative transcriptome analysis of hepatopancreas and testis from Eriocheir sinensis.
Comp. Biochem. Physiol. Part D Genomics Proteomics
PUBLISHED: 04-15-2009
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There is a delicate relationship between nutrition and reproduction of mitten crab (Eriocheir sinensis). The crabs store significant amounts of energy in hepatopancreas, which is prepared for significant energy output and expenditure during reproduction, but the internal molecular mechanism has never been known. Here we present the first relationship between hepatopancreas and testis of E. sinensis. We acquired 6287 high quality expressed sequence tags (EST), representing 3829 unigenes totally, from healthy male mitten crabs of first grade. We investigated the Gene Ontology and the main metabolism processes of hepatopancreas and testis from E. sinensis. Genes most likely expressed more frequently and localized in hepatopancreas, and abundant genes from testis for multiple functions. Many genes important for the nutrition regulation are in the EST resource, including arginine kinase, leptin receptor-like protein, seminal plasma glycoprotein 120, and many kinds of zinc finger proteins. The EST data also revealed genes such as heat shock protein 70, testis enhanced gene transcript (TEGT), Cyclin K, etc. predicted to play important roles in regulation of reproduction mechanisms. Among these genes, alignment of leptin receptor-like protein and vasa-like protein from E. sinensis and other species showed even more genomic information on E. sinensis. We identified seventeen genes relevant to control of nutrition mechanisms and eleven genes involved in regulation of reproduction. And this study provides insights into the genetic and molecular mechanisms of nutrition and reproduction in the crab. Such information would facilitate the optimization of breeding in the aquaculture of mitten crabs.
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Whole genome sequencing of lamb rotavirus and comparative analysis with other mammalian rotaviruses.
Virus Genes
PUBLISHED: 01-26-2009
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Rotavirus (RV) epidemiological surveys with molecular analysis of various strains are required for gastroenteritis control and prevention. The lamb rotavirus strain NT, isolated from a diarrhea lamb in China, is considered as a promising vaccine strain. The whole genome of the lamb-NT strain was determined by sequence analysis. Sequence identity and phylogenetic analysis defined the lamb-NT strain as group A, genotype G10P[15]/NSP4[A]/SG1 strain. Comparative genomic analysis of the lamb-NT strain and 17 reference strains reveals that gene reassortments between rotaviruses circulating in different species occurred. Alignment of protein sequences of the genes shows some variations in the important functional regions of VP3 and VP4. These variations are related to host range restriction, virulence, and other potential characters of rotaviruses. Besides, this study also makes a significant foundation for the study of genetic classification, epidemiology, and antigenic diversity of rotaviruses on the molecular level.
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Identification and characterization of CAC1 as a novel CDK2-associated cullin.
Cell Cycle
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Cell cycle progression is tightly controlled by cyclins and cyclin-dependent kinases (CDKs). CDK2 plays a crucial role in regulating cell cycle progression, but how CDK2 is regulated is still incompletely understood. In this study, we report the identification and characterization of a novel gene CAC1 that regulates CDK2 activity. The open reading frame sequence of this gene encodes a protein of 369 amino acids which contains a Cullin domain, and this protein is physically associated with CDK2. As such, we have designated it Cdk-Associated Cullin1, or CAC1. CAC1 is highly expressed in cancer tissues and cancer cell lines. Interestingly, CAC1 is expressed in a cell cycle-dependent manner and its expression is high in late G(1) to S phase. Knockdown of CAC1 by RNAi inhibits cell proliferation and induces G(1)/S arrest. Since CAC1 interacts with CDK2 and promotes the kinase activity of CDK2 protein, we propose that CAC1 is a novel cell cycle associated protein capable of promoting cell proliferation. Our data provide insight into the mechanism by which CDK2 is regulated and the molecular basis of cell cycle progression in cancer.
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hSSB1 regulates both the stability and the transcriptional activity of p53.
Cell Res.
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The tumor suppressor p53 is essential for several cellular processes that are involved in the response to diverse genotoxic stress, including cell cycle arrest, DNA repair, apoptosis and senescence. Studies of the regulation of p53 have mostly focused on its stability and transactivation; however, new regulatory molecules for p53 have also been frequently identified. Here, we report that human ssDNA binding protein SSB1 (hSSB1), a novel DNA damage-associated protein, can interact with p53 and protect p53 from ubiquitin-mediated degradation. Furthermore, hSSB1 also associates with the acetyltransferase p300 and is required for efficient transcriptional activation of the p53 target gene p21 by affecting the acetylation of p53 at lysine382. Functionally, the hSSB1 knockdown-induced abrogation of the G2/M checkpoint is partially dependent on p53 or p300. Collectively, our results indicate that hSSB1 may regulate DNA damage checkpoints by positively modulating p53 and its downstream target p21.
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FOXO3 induces FOXO1-dependent autophagy by activating the AKT1 signaling pathway.
Autophagy
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Forkhead box O (FOXO) transcriptional protein family members, including FOXO1 and FOXO3, are involved in the modulation of autophagy. However, whether there is redundancy between FOXO1 and FOXO3 in the ability to induce autophagy remains unclear. In this study, we showed that FOXO3 induced a transcription-dependent autophagy, and FOXO1 was required for this process. Overexpression of wild-type FOXO3 (WT) or FOXO3 (3A), which harbors alanine mutations at residues Thr32, Ser253 and Ser315, but not transcription-inactive FOXO3 (?DB3A), significantly induced autophagy in the human embryonic kidney cell line HEK293T and mouse embryonic fibroblast (MEF) cell lines. Interestingly, depletion of FOXO1 by siRNA attenuated FOXO3-induced autophagy. Our data also showed that FOXO3 overexpression did not increase the expression of FOXO1 at the protein level, although FOXO3 was capable of binding the promoter region of FOXO1 and inducing an increase in the transcription of FOXO1 mRNA. Furthermore, our results showed that FOXO3 promoted the translocation of FOXO1 from the nucleus to the cytoplasm, resulting in an increase in FOXO1-induced autophagy. Moreover, our results supported a mechanism whereby FOXO3 dramatically increased the expression of the class I PtdIns3K catalytic subunit PIK3CA, leading to an increase in AKT1 activity, which resulted in the phosphorylation and nuclear export of FOXO1. To the best of our knowledge, our data are the first to suggest that FOXO1 plays a central role in FOXO3-induced autophagy.
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Synthesis and characterization of a novel boronic acid-functionalized chitosan polymeric nanosphere for highly specific enrichment of glycopeptides.
Carbohydr Polym
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In this study we describe a method for highly specific enrichment of glycopeptides with boronic acid-functionalized chitosan polymeric nanospheres and matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS). This is the first time chitosan has been used to create nanosphere support material for selective enrichment of glycopeptides by modification with glycidyl methacrylate (GMA) and derivatization with 3-aminophenylboronic acid (APB). Due to their multifunctional chemical moieties, these 20-100 nm chitosan-GMA-APB nanospheres have unique properties, such as good dispersibility, good biocompatibility and chemical stability, as well as augmented specificity with glycopeptides. Enrichment conditions were optimized by using trypsin digested glycoprotein horseradish peroxidase. The high specificity of chitosan-GMA-APB nanospheres was demonstrated by effectively enriching glycopeptides from a digest mixture of horseradish peroxidase and nonglycoproteins (bovine serum albumin (BSA)).
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N-methylpurine DNA glycosylase inhibits p53-mediated cell cycle arrest and coordinates with p53 to determine sensitivity to alkylating agents.
Cell Res.
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Alkylating agents induce genome-wide base damage, which is repaired mainly by N-methylpurine DNA glycosylase (MPG). An elevated expression of MPG in certain types of tumor cells confers higher sensitivity to alkylation agents because MPG-induced apurinic/apyrimidic (AP) sites trigger more strand breaks. However, the determinant of drug sensitivity or insensitivity still remains unclear. Here, we report that the p53 status coordinates with MPG to play a pivotal role in such process. MPG expression is positive in breast, lung and colon cancers (38.7%, 43.4% and 25.3%, respectively) but negative in all adjacent normal tissues. MPG directly binds to the tumor suppressor p53 and represses p53 activity in unstressed cells. The overexpression of MPG reduced, whereas depletion of MPG increased, the expression levels of pro-arrest gene downstream of p53 including p21, 14-3-3? and Gadd45 but not proapoptotic ones. The N-terminal region of MPG was specifically required for the interaction with the DNA binding domain of p53. Upon DNA alkylation stress, in p53 wild-type tumor cells, p53 dissociated from MPG and induced cell growth arrest. Then, AP sites were repaired efficiently, which led to insensitivity to alkylating agents. By contrast, in p53-mutated cells, the AP sites were repaired with low efficacy. To our knowledge, this is the first direct evidence to show that a DNA repair enzyme functions as a selective regulator of p53, and these findings provide new insights into the functional linkage between MPG and p53 in cancer therapy.
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Large-scale collection and annotation of gene models for date palm (Phoenix dactylifera, L.).
Plant Mol. Biol.
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The date palm (Phoenix dactylifera L.), famed for its sugar-rich fruits (dates) and cultivated by humans since 4,000 B.C., is an economically important crop in the Middle East, Northern Africa, and increasingly other places where climates are suitable. Despite a long history of human cultivation, the understanding of P. dactylifera genetics and molecular biology are rather limited, hindered by lack of basic data in high quality from genomics and transcriptomics. Here we report a large-scale effort in generating gene models (assembled expressed sequence tags or ESTs and mapped to a genome assembly) for P. dactylifera, using the long-read pyrosequencing platform (Roche/454 GS FLX Titanium) in high coverage. We built fourteen cDNA libraries from different P. dactylifera tissues (cultivar Khalas) and acquired 15,778,993 raw sequencing reads-about one million sequencing reads per library-and the pooled sequences were assembled into 67,651 non-redundant contigs and 301,978 singletons. We annotated 52,725 contigs based on the plant databases and 45 contigs based on functional domains referencing to the Pfam database. From the annotated contigs, we assigned GO (Gene Ontology) terms to 36,086 contigs and KEGG pathways to 7,032 contigs. Our comparative analysis showed that 70.6 % (47,930), 69.4 % (47,089), 68.4 % (46,441), and 69.3 % (47,048) of the P. dactylifera gene models are shared with rice, sorghum, Arabidopsis, and grapevine, respectively. We also assigned our gene models into house-keeping and tissue-specific genes based on their tissue specificity.
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Exome sequencing identifies MXRA5 as a novel cancer gene frequently mutated in non-small cell lung carcinoma from Chinese patients.
Carcinogenesis
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Lung cancer has become the top killer among malignant tumors in China and is significantly associated with somatic genetic alterations. We performed exome sequencing of 14 non-small cell lung carcinomas (NSCLCs) with matched adjacent normal lung tissues extracted from Chinese patients. In addition to the lung cancer-related genes (TP53, EGFR, KRAS, PIK3CA, and ROS1), this study revealed "novel" genes not previously implicated in NSCLC. Especially, matrix-remodeling associated 5 was the second most frequently mutated gene in NSCLC (first is TP53). Subsequent Sanger sequencing of matrix-remodeling associated 5 in an additional sample set consisting of 52 paired tumor-normal DNA samples revealed that 15% of Chinese NSCLCs contained somatic mutations in matrix-remodeling associated 5. These findings, together with the results from pathway analysis, strongly indicate that altered extracellular matrix-remodeling may be involved in the etiology of NSCLC.
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A complete sequence and transcriptomic analyses of date palm (Phoenix dactylifera L.) mitochondrial genome.
PLoS ONE
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Based on next-generation sequencing data, we assembled the mitochondrial (mt) genome of date palm (Phoenix dactylifera L.) into a circular molecule of 715,001 bp in length. The mt genome of P. dactylifera encodes 38 proteins, 30 tRNAs, and 3 ribosomal RNAs, which constitute a gene content of 6.5% (46,770 bp) over the full length. The rest, 93.5% of the genome sequence, is comprised of cp (chloroplast)-derived (10.3% with respect to the whole genome length) and non-coding sequences. In the non-coding regions, there are 0.33% tandem and 2.3% long repeats. Our transcriptomic data from eight tissues (root, seed, bud, fruit, green leaf, yellow leaf, female flower, and male flower) showed higher gene expression levels in male flower, root, bud, and female flower, as compared to four other tissues. We identified 120 potential SNPs among three date palm cultivars (Khalas, Fahal, and Sukry), and successfully found seven SNPs in the coding sequences. A phylogenetic analysis, based on 22 conserved genes of 15 representative plant mitochondria, showed that P. dactylifera positions at the root of all sequenced monocot mt genomes. In addition, consistent with previous discoveries, there are three co-transcribed gene clusters-18S-5S rRNA, rps3-rpl16 and nad3-rps12-in P. dactylifera, which are highly conserved among all known mitochondrial genomes of angiosperms.
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Diacylglycerol acyl transferase 1 overexpression detoxifies cardiac lipids in PPAR? transgenic mice.
J. Lipid Res.
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Accumulation of excess lipids is associated with heart failure. The effects of transgenic expression of diacylglycerol acyl transferase 1 (DGAT1) in cardiomyocytes is controversial. We explored whether mice expressing DGAT1 via the myosin heavy chain (MHC) promoter develop heart dysfunction with aging or after crossing with mice over expressing peroxisome proliferator-activated receptor ? (PPAR?) in the heart. MHC-DGAT1 transgenic mice had increased heart triglyceride but no evidence of heart dysfunction, even up to age 12 months. The MHC-DGAT1 transgene improved heart dysfunction and survival of MHC-PPAR?-expressing transgenic mice. Both diacylglycerol and ceramide levels in the heart were reduced by this cross, as were the levels of several mRNAs of genes involved in lipid metabolism. There were fewer large lipid droplets in MHC-DGAT1×MHC-PPAR? mice compared with MHC-PPAR?, but total lipid content was not changed. Therefore, overexpression of DGAT1 is not toxic to the heart but reduces levels of toxic lipids and improves lipotoxic cardiomyopathy. Moreover, the beneficial effects of DGAT1 illustrate the interrelationship of several lipid metabolic pathways and the difficulty of assigning benefit to an isolated change in one potentially toxic lipid species.
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Evidence for inhibition of HIF-1? prolyl hydroxylase 3 activity by four biologically active tetraazamacrocycles.
Org. Biomol. Chem.
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Hypoxia inducible factor 1 (HIF-1) is central to the hypoxic response in mammals. HIF-1? prolyl hydroxylase 3 (PHD3) degrades HIF through the hydroxylation of HIF-1?. Inhibition of PHD3 activity is crucial for up-regulating HIF-1? levels, thereby acting as HIF-dependent diseases therapy. Macrocyclic polyamines which display high stability on iron-chelating may well inhibit the enzyme activity. Thus inhibition and interaction on catalytic PHD3 by four biologically active tetraazamacrocycles (1-4), which have two types of parent rings to chelate iron(ii) dissimilarly, were studied. The apparent IC(50) values of 2.56, 1.91, 5.29 and 2.44 ?M, respectively, showed good inhibition potency of the four compounds. K(I) values were 7.86, 3.69, 1.59 and 2.92 ?M for 1-4, respectively. Different inhibition actions of the two groups of compounds were identified. Circular dichroism (CD) and fluorescence spectrometries proved that one type of compound has significant effects on protein conformation while another type does not. Computational methodology was constructed to employ the equilibrium geometry of enzyme active site with the presence of substrate competitive inhibitor. Iron(ii) coordination in the active site by inhibitors of this kind induces conformational change of the enzyme and blocks substrate binding.
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High-throughput sequencing-based gene profiling on multi-staged fruit development of date palm (Phoenix dactylifera, L.).
Plant Mol. Biol.
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Date palm provides both staple food and gardening for the Middle East and North African countries for thousands of years. Its fruits have diversified significantly, such as nutritional content, size, length, weight color, and ripping process. Dates palm represent an excellent model system for the study of fruit development and diversity of fruit-bearing palm species that produce the most versatile fruit types as compared to other plant families. Using Roche/454 GS FLX instrument, we acquired 7.6 million sequence tags from seven fruiting stages (F1-F7). Over 99% of the raw reads are assembled, and the numbers of isotigs (equivalent to transcription units or unigenes) range from 30,684 to 40,378 during different fruiting stages. We annotated isotigs using BLASTX and BLASTN, and mapped 74% of the isotigs to known functional sequences or genes. Based on gene ontology categorization and pathway analysis, we have identified 10 core cell division genes, 18 ripening related genes, and 7 starch metabolic enzymes, which are involved as nutrition storage and sugar/starch metabolisms. We noticed that many metabolic pathways vary significantly during fruit development, and carbohydrate metabolism (especially sugar synthesis) is particularly prominent during fruit ripening. Transcriptomics study on various fruiting stages of date palm shows complicated metabolic activities during fruit development, ripening, synthesis and accumulation of starch enzymes and other related sugars. Most Genes are highly expressed in early stages of development, while late developmental stages are critical for fruit ripening including most of the metabolism associated ones.
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Apak competes with p53 for direct binding to intron 1 of p53AIP1 to regulate apoptosis.
EMBO Rep.
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The KRAB-type zinc-finger protein Apak was recently identified as a negative regulator of p53-mediated apoptosis. However, the mechanism of this selective regulation is not fully understood. Here, we show that Apak recognizes the TCTTN2?30TTGT consensus sequence through its zinc-fingers. This sequence is specifically found in intron 1 of the proapoptotic p53 target gene p53AIP1 and largely overlaps with the p53-binding sequence. Apak competes with p53 for binding to this site to inhibit p53AIP1 expression. Upon DNA damage, Apak dissociates from the DNA, which abolishes its inhibitory effect on p53-mediated apoptosis.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.