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M-MLV (Moloney Murine Leukemia Virus) Reverse Transcriptase (RT) is a recombinant DNA polymerase that synthesizes a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA hybrid.
2012
Authors: Y Li, Y Zhang, R Li, W Chen, M Howell, R Zhang, G Chen
The roles of vitamin A (VA) in the development of metabolic diseases remain unanswered. We have reported that retinoids synergized with insulin to induce the expression of sterol-regulatory element-binding protein 1c gene (Srebp-1c) expression in primary rat hepatocytes. Additionally, the hepatic Srebp-1c expression is elevated in Zucker fatty (ZF) rats, and reduced in those fed a VA deficient diet. VA is metabolized to retinoic acid (RA) for regulating gene expression.
2008
Authors: JM Gonzalez, RD Dijkhuis, DD Johnson, JN Carter, SE Johnson
Ractopamine-HCl (RAC) is a beta-adrenergic agonist with variable effects on cattle performance and carcass variables. Cull cows fed RAC (200 mg . head(-1) . d(-1)) demonstrate an increased size of type I and II muscle fibers that does not translate into a larger ribeye area. The objective of this study was to examine the dose-dependent effects of RAC on cull cow muscle morphometrics. Eighty-eight cull beef cows representing 2 breed types (n = 44 each) were fed 0, 100, 200, and 300 mg . head(-1) . d(-1) of RAC for the last 28 d of a 54-d feeding period. On d 54, cows were slaughtered, and samples of the LM and semimembranosus muscle (SM) from 16 randomly selected carcasses (n = 4 per treatment) were taken for measurement of beta (2)-adrenergic receptors and type I, IIA, and IIX myosin heavy chain (MyHC) gene expression.
For the reverse transcription of RNA. The Ambion kit includes a M-MLV RT that maintains full RNase H activity for increased sensitivity of detection in RT-PCR experiments. Includes sufficient reagents for 50 reactions.
2012
Stable changes in CD4+ T lymphocyte miRNA expression after exposure to HIV-1
Authors: F Bignami, E Pilotti, L Bertoncelli, P Ronzi, M Gulli, N Marmiroli, G Magnani, M Pinti, L Lopalco, C Mussini, R Ruotolo, M Galli, A Cossarizza, C Casoli
MicroRNAs (miRNAs) inhibit HIV-1 expression by either modulating host innate immunity or by directly interfering with viral mRNAs. We evaluated the expression of 377 miRNAs in CD4+ T cells from HIV-1 élite long-term nonprogressors (éLTNPs), naive patients, and multiply exposed uninfected (MEU) patients, and we observed that the éLTNP patients clustered with naive patients, whereas all MEU subjects grouped together. The discriminatory power of miRNAs showed that 21 miRNAs significantly differentiated éLTNP from MEU patients and 23 miRNAs distinguished naive from MEU patients, whereas only 1 miRNA (miR-155) discriminated éLTNP from naive patients.
High Capacity cDNA Reverse Transcription Kit
The High Capacity cDNA Reverse Transcription Kit (formerly the High Capacity cDNA Archive Kit) delivers extremely high-quality, single-stranded cDNA from 0.02 to 2 µg input total RNA. Ideal for generating cDNA archives. The High Capacity cDNA Reverse Transcription Kit can convert up to 2 µg of input total RNA in a single 20 µl reaction. Reactions can be scaled up to 100 µl to generate 10 µg of cDNA from a single reaction The cDNA generated is ideal for a range of applications including real-time PCR, standard PCR, and microarrays.
2012
Authors: Y He, Y Yu, Y Zhang, J Song, A Mitra, Y Zhang, Y Wang, D Sun, S Zhang
Gene expression of lymphocytes was found to be influenced by histone methylation in mammals and trimethylation of lysine 27 on histone H3 (H3K27me3) normally represses genes expressions. Peripheral blood lymphocytes are the main source of somatic cells in the milk of dairy cows that vary frequently in response to the infection or injury of mammary gland and number of parities.
2012
Trim28 Is Required for Epigenetic Stability During Mouse Oocyte to Embryo Transition
Authors: DM Messerschmidt, W de Vries, M Ito, D Solter, A Ferguson-Smith, BB Knowles
Phenotypic variability in genetic disease is usually attributed to genetic background variation or environmental influence. Here, we show that deletion of a single gene, Trim28 (Kap1 or Tif1β), from the maternal germ line alone, on an otherwise identical genetic background, results in severe phenotypic and epigenetic variability that leads to embryonic lethality. We identify early and minute epigenetic variations in blastomeres of the preimplantation embryo of these animals, suggesting that the embryonic lethality may result from the misregulation of genomic imprinting in mice lacking maternal Trim28. Our results reveal the long-range effects of a maternal gene deletion on epigenetic memory and illustrate the delicate equilibrium of maternal and zygotic factors during nuclear reprogramming.
Novel transcription reaction conditions and Ambion's patented, high-yield technology allow the synthesis of 10–50 times the amount of RNA produced by conventional transcription reactions. Each Ambion kit includes sufficient reagents for 200 reactions.
2012
Authors: VG Kramer, SM Schader, M Oliveira, SP Colby-Germinario, DA Donahue, DN Singhroy, R Tressler, RD Sloan, MA Wainberg
HIV entry inhibitors, such as maraviroc (MVC), prevent cell-free viruses from entering the cells. In clinical trials, patients who were treated with MVC often displayed viral loads that were above the limit of conventional viral load detection compared to efavirenz-based regimens. We hypothesize that viruses blocked by entry inhibitors may be redistributed to plasma, where they artificially increase viral load measurements compared to those with the use of antiretroviral drugs (ARVs) that act intracellularly.
2012
Development and characterization of a stable eGFP enterovirus 71 for antiviral screening
Authors: B Shang, C Deng, H Ye, W Xu, Z Yuan, P-Y Shi, B Zhang
Enterovirus 71 (EV71) is one of the major causative agents for hand, foot, and mouth disease. There is currently no clinically approved vaccine or antiviral treatment for EV71 infection. To facilitate antiviral drug discovery, we developed an infectious cDNA clone of an epidemic strain of EV71 and a stable eGFP reporter EV71. The reporter virus was generated by engineering the eGFP gene between the 5′ untranslated region and VP4 gene of the EV71 genome.
2012
Authors: Zhi-Rui Liu, Jie Tao, Bang-Qian Dong, Gang Ding, Zhi-Jun Cheng, Hui-Qiong He, Yong-Hua Ji
Chitin synthase (CHS) is an enzyme that is required for chitin formation in insect cuticles and other tissues. In this study, CHS genes from two destructive rice insect pests, the brown planthopper Nilaparvata lugens and the small brown planthopper Laodelphax striatellus, were cloned. Phylogenetic analysis showed that these genes belonged to class CHS1 of the CHS gene family.
2012
Authors: IE Catrina, SAE Marras, DP Bratu
New approaches for imaging dynamic processes involving RNAs in living cells are continuously being developed and optimized. The use of molecular beacons synthesized from 2′-O-methylribonucleotides (which are resistant to cellular nucleases) is an established approach for visualizing native mRNAs in real time.
2012
Authors: Y Liu, K Timani, X Ou, HE Broxmeyer, JJ He
We reported previously that HIV-1 the Tat-interacting protein of 110 kDa (TIP110; P110(NRB)/SART3/p110) is important in regulation of hematopoiesis, and in maintaining pluripotent factor (NANOG, OCT4, and SOX2) expression in and pluripotency of human embryonic stem cells (hESCs). Here we show that TIP110 expression in hESCs line H9 and embryonal carcinoma cell line NT-2 is regulated by C-MYC expression in ESCs through an E box present in the TIP110 promoter region. Through up- and down- modulation of expression, TIP110 induces OCT4 exon 1a inclusion and exon 1b skipping in our OCT4 minigene model. Thus, TIP110 expression in ESCs regulates alternative splicing of OCT4, an event required for pluripotency of ESCs.
Novel transcription reaction conditions and Ambion's patented, high-yield technology allow the synthesis of large mass amounts of RNA produced from small templates (<300 bases). Each Ambion kit includes sufficient reagents for 25 reactions.
2013
Mechanism of chiral proofreading during translation of the genetic code
Authors: C Ufer, CC Wang, M Fähling, H Schiebel, BJ Thiele, EE Billett, H Kuhn, A Borchert
S Ahmad, SB Routh, V Kamarthapu, J Chalissery, S Muthukumar, T Hussain, SP Kruparani, MV Deshmukh, R Sankaranarayanan.
The biological macromolecular world is homochiral and effective enforcement and perpetuation of this homochirality is essential for cell survival. In this study, we present the mechanistic basis of a configuration-specific enzyme that selectively removes D-amino acids erroneously coupled to tRNAs.
2013
Authors: S Ahmad, SB Routh, V Kamarthapu, J Chalissery, S Muthukumar, T Hussain, SP Kruparani, MV Deshmukh, R Sankaranarayanan
The biological macromolecular world is homochiral and effective enforcement and perpetuation of this homochirality is essential for cell survival. In this study, we present the mechanistic basis of a configuration-specific enzyme that selectively removes D-amino acids erroneously coupled to tRNAs. The crystal structure of dimeric D-aminoacyl-tRNA deacylase (DTD) from Plasmodium falciparum in complex with a substrate-mimicking analog shows how it uses an invariant ‘cross-subunit’ Gly-cisPro dipeptide to capture the chiral centre of incoming D-aminoacyl-tRNA. While no protein residues are directly involved in catalysis, the unique side chain-independent mode of substrate recognition provides a clear explanation for DTD’s ability to act on multiple D-amino acids.
2008
Authors: C Ufer, CC Wang, M Fähling, H Schiebel, BJ Thiele, EE Billett, H Kuhn, A Borchert
Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a moonlighting selenoprotein, which has been implicated in basic cell functions such as anti-oxidative defense, apoptosis, and gene expression regulation. GPx4-null mice die in utero at midgestation, and developmental retardation of the brain appears to play a major role.
The Ambion MEGAscript RNAi Kit is for the synthesis of large mass amounts of dsRNA for RNAi experiments in nonmammalian systems. The MEGAscript RNAi Kit uses Ambion's patented high-yield transcription technology and includes reagents for transcription, nuclease digestion, and clean up. The kit includes sufficient reagents for 20 reactions.
2012
Authors: PN Paradkara, L Trinidada, R Voyseya, J-B Ducheminb, PJ Walker
Although West Nile virus (WNV) and other arthropod-borne viruses are a major public health problem, the mechanisms of antiviral immunity in mosquitoes are poorly understood. Dicer-2, responsible for the RNAi-mediated response through the C-terminal RNase-III domain, also contains an N-terminal DExD/H-box helicase domain similar to mammalian RIG-I/MDA5 which, in Drosophila, was found to be required for activation of an antiviral gene, Vago. Here we show that the Culex orthologue of Vago (CxVago) is up-regulated in response to WNV infection in a Dicer-2–dependent manner. Further, our data show that CxVago is a secreted peptide that restricts WNV infection by activation of the Jak-STAT pathway. Thus, Vago appears to function as an IFN-like antiviral cytokine in mosquitoes.
2012
RNAi silencing of the Meloidogyne incognita Rpn7 gene reduces nematode parasitic success
Authors: J Niu, H Jian, J Xu, C Chen, Q Guo, Q Liu, Y Guo
RNA interference (RNAi) techniques provide a major breakthrough in functional analysis for plant parasitic nematodes (PPNs). It offers the possibility of identifying new essential targets and consequently developing new resistance transgenes.
MessageAmp Premier RNA Amplification Kit
The MessageAmp Premier RNA Amplification Kit is the latest innovation for the preparation of RNA samples for microarray analysis. The kit builds upon the linear RNA amplification method and reagents developed for MessageAmp II Kits with enhancements that create a simplified workflow, improved performance, and more flexible RNA input requirements.
2012
Authors: G Verma, H Bhatia, M Datta
The pro-inflammatory cytokine, IL-1β, is a critical component of the persistent inflammatory milieu that pancreatic cancer cells frequently encounter. Although several studies report diverse mechanisms responsible for this association, yet a comprehensive global analysis of the effect of IL-1β in these cells is not clearly evident. In this study, we performed whole genome transcriptome analysis of control and IL-1β treated human pancreatic MIA PaCa-2 cells, validated the most targeted pathway and evaluated the role of JNK therein. 225 Genes were up-regulated and 1215 were down-regulated and these were categorized into biological processes and cellular pathways using the PANTHER classification system.
MessageAmp II aRNA Amplification Kit
The Ambion MessageAmp II aRNA Amplification Kit is for the amplification of as little as 100 pg of input RNA for GeneChip analysis. The kit includes sufficient reagents for 20 aRNA amplifications.
2011
Authors: D Li, P Chen, X-Y Li, L-Y Zhang, W Xiong, M Zhou, L Xiao, F Zeng, X-L Li, M-H Wu, G-Y Li
Although several miRNAs have been identified to be involved in glioblastoma tumorigenesis, little is known about the global expression profiles of miRNAs and their functional targets in astrocytomas at earlier stages of malignancy. In this study the global expression of miRNAs and mRNAs in normal brain tissue samples and grade I–III astrocytomas were analyzed parallelly using microarrays, and the grade-specific expression profiles of them were obtained by unsupervised hierarchical clustering.
2012
Authors: IE Catrina, SAE Marras, DP Bratu
New approaches for imaging dynamic processes involving RNAs in living cells are continuously being developed and optimized. The use of molecular beacons synthesized from 2′-O-methylribonucleotides (which are resistant to cellular nucleases) is an established approach for visualizing native mRNAs in real time.
2012
Authors: Y Liu, K Timani, X Ou, HE Broxmeyer, JJ He
We reported previously that HIV-1 the Tat-interacting protein of 110 kDa (TIP110; P110(NRB)/SART3/p110) is important in regulation of hematopoiesis, and in maintaining pluripotent factor (NANOG, OCT4, and SOX2) expression in and pluripotency of human embryonic stem cells (hESCs). Here we show that TIP110 expression in hESCs line H9 and embryonal carcinoma cell line NT-2 is regulated by C-MYC expression in ESCs through an E box present in the TIP110 promoter region. Through up- and down- modulation of expression, TIP110 induces OCT4 exon 1a inclusion and exon 1b skipping in our OCT4 minigene model. Thus, TIP110 expression in ESCs regulates alternative splicing of OCT4, an event required for pluripotency of ESCs.
mirVana miRNA Probe Construction Kit
The Ambion mirVana miRNA Probe Construction Kit uses in vitro transcription to generate short (<100 nt) radiolabeled or nonisotopically labeled RNA probes in less than 1 hour. The mirVana miRNA Probe Construction Kit includes reagents for preparing 30 in vitro transcription templates as well as reagents for 30 in vitro transcription reactions.
2013
MicroRNAs 221 and 222 regulate the undifferentiated state in mammalian male germ cells
Authors: QE Yang, KE Racicot, AV Kaucher, MJ Oatley, JM Oatley
Continuity of cycling cell lineages relies on the activities of undifferentiated stem cell-containing subpopulations. Transition to a differentiating state must occur periodically in a fraction of the population to supply mature cells, coincident with maintenance of the undifferentiated state in others to sustain a foundational stem cell pool. At present, molecular mechanisms regulating these activities are poorly defined for most cell lineages. Spermatogenesis is a model process that is supported by an undifferentiated spermatogonial population and transition to a differentiating state involves attained expression of the KIT receptor.
The Ambion MAXIscript T7 In Vitro Transcription Kit synthesizes RNA probes in just 10 minutes for use in ribonuclease protection assays, in situ hybridization, and blot hybridizations. Each kit contains sufficient reagents for 30 reactions.
2013
Identification of purple sea urchin telomerase RNA using a next-generation sequencing based approach
Authors: Y Li, JD Podlevsky, M Marz, X Qi, S Hoffmann, PF Stadler, JJL Chen
Telomerase is a ribonucleoprotein (RNP) enzyme essential for telomere maintenance and chromosome stability. While the catalytic telomerase reverse transcriptase (TERT) protein is well conserved across eukaryotes, telomerase RNA (TR) is extensively divergent in size, sequence, and structure. This diversity prohibits TR identification from many important organisms.
For the polyadenylation of in vitro transcribed RNA to enhance translation initiation efficiency. Sufficient reagents are included for 25 reactions.
2012
Expression profile of miRNAs in Populus cathayana L. and Salix matsudana Koidz under salt stress
Authors: J Zhou, M Liu, J Jiang, G Qiao, S Lin, H Li, L Xie, R Zhuo
Soil salinization can lead to environmental and ecological problems worldwide. Abiotic stressors, including salinity, are suspected to regulate microRNA (miRNA) expression. Plants exposed to such abiotic stressors express specific miRNAs, which are genes encoding small non-coding RNAs of 20–24 nucleotides. miRNAs are known to exist widely in plant genomes, and are endogenous.
2011
Label-Free MicroRNA Profiling Not Biased by 3' End 2'-O-Methylation
Authors: Y Shen, K-x Zheng, D Duan, L Jiang, J Li
Accurate quantification of miRNA expression level is essential to the study of its biology, and many cutting-edge technologies have been developed to accommodate this need. Yet most of them were designed primarily for the “regular” RNAs such as animal miRNAs and may overlook the fact that plant miRNAs and many other small noncoding RNAs are 2′-O-methylated at the 3′ end nucleotide. According to our experimental data and previous reports, this structural variation is detrimental to the effectiveness of the commonly used enzymatic labeling methods, leading to strongly biased results (24-fold difference).
The Ambion BrightStar BioDetect Kit is a low-background, high-sensitivity, nonisotopic detection system compatible with northern, Southern, dot blot, and nuclease protection assays. Includes sufficient reagents for the processing of 1000 cm2 membrane.
2012
The Novel Cis-Encoded Small RNA h2cR Is a Negative Regulator of hfq2 in Burkholderia cenocepacia
Authors: CG Ramos, PJP da Costa, G Döring, JH Leitão
Small non-coding regulatory RNAs (sRNAs) post-transcriptionally affect multiple phenotypes in prokaryotes and eukaryotes, yet most of the underlying regulatory mechanisms and the nature of the target mRNAs remain unclear. Here we report the identification and functional analysis of the novel cis-encoded sRNA h2cR, from the human opportunistic pathogen Burkholderia cenocepacia J2315.
Silencer siRNA Construction Kit
The Ambion Silencer siRNA Construction Kit is for the synthesis of siRNAs by in vitro transcription of DNA templates and costs less per siRNA than chemical synthesis. The kit includes sufficient reagents to synthesize 15 siRNAs.
2011
N6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO
Authors: G Jia, Y Fu, X Zhao, Q Dai, G Zheng, Y Yang, C Yi, T Lindahl, T Pan, Y-G Yang, C He
We report here that fat mass and obesity-associated protein (FTO) has efficient oxidative demethylation activity targeting the abundant N6-methyladenosine (m6A) residues in RNA in vitro. FTO knockdown with siRNA led to increased amounts of m6A in mRNA, whereas overexpression of FTO resulted in decreased amounts of m6A in human cells. We further show the partial colocalization of FTO with nuclear speckles, which supports the notion that m6A in nuclear RNA is a major physiological substrate of FTO.
RNA interference (RNAi) is the best way to effectively knock down gene expression to study protein function in a wide range of cell types.
2013
Authors: A Solini, S Menini, C Rossi, C Ricci, E Santini, C Blasetti Fantauzzi, C Iacobini, G Pugliese
Renal disease associated with type 2 diabetes and the metabolic syndrome is characterized by a distinct inflammatory phenotype. The purinergic 2X7 receptor (P2X7 R) and the nucleotide-binding and oligomerization domain-like receptor containing a pyrin domain 3 (NLRP3) inflammasome have been separately shown to play a role in two models of non-metabolic chronic kidney disease.
2013
Authors: AR Rodrigues, H Almeida, AM Gouveia
The melanocortin system has a clear effect on the mobilisation of stored lipids in adipocytes. The aim of the current study was to investigate the role of melanocortin 5 receptor (MC5R) on α-melanocyte-stimulating hormone (α-MSH)-induced lipolysis in 3T3-L1 adipocytes. To this end, MC5R expression was decreased by small interfering RNA (siRNA), which significantly impaired the α-MSH stimulation of lipolysis, as determined by glycerol and nonesterified fatty-acid (NEFA) quantification. The functional role of α-MSH/MC5R on triglyceride (TG) hydrolysis was mediated by hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), perilipin 1 (PLIN1) and acetyl-CoA carboxylase (ACC). Immunofluorescence microscopy revealed that phosphorylated HSL clearly surrounded lipid droplets in α-MSH-stimulated adipocytes, whereas PLIN1 left the immediate periphery of lipids. These observations were lost when the expression of MC5R was suppressed.
2013
Authors: A Brozovic, L Vukovic, D Stupin Polanc, I Arany, B Köberle, G Fritz, Z Fiket, D Majhen, A Ambriovic-Ristov, M Osmak
The major obstacle of successful tumor treatment with carboplatin (CBP) is the development of drug resistance. In the present study, we found that following treatment with CBP the amount of platinum which enters the human laryngeal carcinoma (HEp2)-derived CBP-resistant (7T) cells is reduced relative to the parental HEp2.
2013
Authors: S Niland, B Ditkowski, D Parrandier, L Roth, H Augustin, JA Eble
Objective—The snake venom component rhodocetin-αβ (RCαβ) stimulates endothelial cell motility in an α2β1 integrin–independent manner. We aimed to elucidate its cellular and molecular mechanisms.
2012
MicroRNA-203 enhances Coxsackievirus B3 replication through targeting zinc finger protein-148
Authors: MG Hemida, X Ye, HM Zhang, PJ Hanson, Z Liu, BM McManus, D Yang
Coxsackievirus B3 (CVB3) is the primary causal agent of viral myocarditis. During infection, it hijacks host genes to favour its own replication. However, the underlying mechanism is still unclear. Although the viral receptor is an important factor for viral infectivity, other factors such as microRNAs (miRNA) may also play an essential role in its replication after host cell entry
2012
Authors: H-Y Zhen, Q-H He, Y Li, J Zhou, C Yao, Y-N Liu, L-J Ma
Lidamycin is a potential anti-cancer drug, which is widely used in a variety of human cancer types. It has been reported that lidamycin inhibited mouse embryonic carcinoma (EC) cells growth through down-regulation of embryonic stem (ES) cell-like genes. In this study, whether 0.01 nM lidamycin induces neuronal differentiation of mouse EC cells was investigated.
2012
Cellular delivery of polynucleotides by cationic cyclodextrin polyrotaxanes
Authors: P Dandekar, R Jain, M Keil, B Loretz, Leon Muijs, M Schneider, D Auerbach, G Jung, C-M Lehr, G Wenz
Cationic polyrotaxanes, obtained by temperature activated threading of cationic cyclodextrin derivatives onto water‐soluble cationic polymers (ionenes), form metastable nanometric polyplexes with pDNA and combinations of siRNA with pDNA. Because of their low toxicity, the polyrotaxane polyplexes constitute a very interesting system for the transfection of polynucleotides into mammalian cells. The complexation of Cy3-labeled siRNA within the polyplexes was demonstrated by fluorescence correlation spectroscopy. The uptake of the polyplexes (red) was imaged by confocal fluorescence microscopy using the A549 cell line as a model (blue: nuclei, green: membranes). The results prove the potential of polyrotaxanes for further investigations involving knocking down genes of therapeutic interest.
2012
MicroRNA-328 May Influence Myopia Development by Mediating the PAX6 Gene
Authors: K-C Chen, E Hsi1, C-Y Hu, W-W Chou, C-L Liang, S-H H Juo
We showed previously that single nucleotide polymorphism (SNP) rs662702 in PAX6 may be located in a microRNA-328 binding site that causes susceptibility to high myopia. Our study was done to elucidate the role of PAX6 and its relationship with microRNA-328 in myopia.
2012
A novel cell-penetrating peptide TAT-A1 delivers siRNA into tumor cells selectively
Authors: B Fang, L Jiang, M Zhang, FZ Ren
siRNA is promising in anti-tumor therapy. The main challenge is lack of tumor-specific intracellular delivery. In this study, a 6 amino acids peptide (A1) with high affinity for vascular endothelial growth factor receptor-1 (VEGFR1) was conjugated with a cell penetrating peptide (CPP) TAT to form a tumor-selective CPP. To evaluate the tumor-targeted penetrate property of TAT-A1, the uptake of TAT-A1 was measured by flow cytometry. The selectivity in vitro was tested in co-cultured tumor cells and normal cells by laser confocal microscope. The internalization efficiency of TAT-A1 was significantly higher than that of TAT (p < 0.05). TAT-A1 penetrated into tumor cells selectively when added to co-cultured tumor cells and normal cells due to the recognition of VEGFR1 which is over-expressed on tumor cells. Furthermore, siRNA was successfully transferred by TAT-A1 into tumor cells in a similar way of Lipofectamine 2000, which was proved to be an efficient vector. The knockout effect of siRNA transferred by TAT-A1 was obtained at both mRNA and protein level. These results indicated that the tumor-targeted TAT-A1 can act as an excellent vehicle for specific delivery of anti-cancer agents.
2012
MicroRNA-181b and microRNA-9 mediate arsenic-induced angiogenesis via NRP1
Authors: Y Cui, Z Han, Y Hu, G Song, C Hao, H Xia, X Ma
Environmental exposure to inorganic arsenic compounds has been reported to have serious health effects on humans. Recent studies reported that arsenic targets endothelial cells lining blood vessels, and endothelial cell activation or dysfunction, may underlie the pathogenesis of arsenic-induced diseases and developmental toxicity. It has been reported that microRNAs (miRNAs) may act as an angiogenic switch by regulating related genes. The present study was designed to test the hypothesis that arsenite-regulated miRNAs play pivotal roles in arsenic-induced toxicity.
2012
Authors: H ten Freyhaus, ES Calay, A Yalcin, SN Vallerie, L Yang, ZZ Calay, F Saatcioglu, GS Hotamisligil
The six-transmembrane protein Stamp2 plays an important role in metabolically triggered inflammation and insulin action. We report that Stamp2 is expressed in human and mouse macrophages, is regulated upon differentiation or activation, acts as an anti-inflammatory protein, and regulates foam cell formation. Absence of Stamp2 results in significant increases in cellular NADPH levels, and both NADPH homeostasis and the exaggerated inflammatory response of Stamp2−/− macrophages are rescued by exogenous wild-type but not by a reductase-deficient Stamp2 molecule.
2012
Authors: G Swaminathan, F Rossi, L-J Sierra, A Gupta, S Navas-Martín, J Martín-García
HIV-1 infection of macrophages plays a key role in viral pathogenesis and progression to AIDS. Polyinosine-polycytidylic acid (poly(I:C); a synthetic analog of dsRNA) and bacterial lipopolysaccharide (LPS), the ligands for Toll-like receptors (TLR) TLR3 and TLR4, respectively, are known to decrease HIV-1 infection in monocyte-derived macrophages (MDMs), but the mechanism(s) are incompletely understood.
2012
Authors: LM Litchfield, KA Riggs, AM Hockenberry, LD Oliver, KG Barnhart, J Cai, WM Pierce Jr, MM Ivanova, PJ Bates, SN Appana, S Datta, P Kulesza, J McBryan, CM Klinge
The orphan nuclear receptor COUP-TFII plays an < role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine- resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer.
2011
Authors: CY Hsieh, MJ Hsu, G Hsiao, YH Wang, CW Huang, SW Chen, T Jayakumar, PT Chiu, YH Chiu, JR Sheu
Recent studies have demonstrated that transcription factor nuclear factor (NF)-κB inhibition may contribute to the protective anti-inflammatory actions of andrographolide, an abundant component of plants of the genus Andrographis. However, the precise mechanism by which andrographolide inhibits NF-κB signaling remains unclear. We thus investigated the mechanism involved in andrographolide suppression of NF-κB signaling in rat vascular smooth muscle cells (VSMCs) exposed to proinflammatory stimuli, LPS, and IFN-γ. Andrographolide was shown to suppress LPS/IFN-γ-induced inducible nitric-oxide synthase and matrix metalloprotease 9 expression in rat VSMCs.
2011
Authors: J Antoine-Bertrand, A Ghogha, V Luangrath, FK Bedford, N Lamarche-Vane
The receptor Deleted in Colorectal Cancer (DCC) mediates the attractive response of axons to the guidance cue netrin-1 during development. On netrin-1 stimulation, DCC is phosphorylated and induces the assembly of signaling complexes within the growth cone, leading to activation of cytoskeleton regulators, namely the GTPases Rac1 and Cdc42.
2011
Authors: P Tarapore, Y Shu, P Guo, S-M Ho
Ovarian cancer is a highly metastatic and lethal disease, making it imperative to find treatments that target late-stage malignant tumors. The packaging RNA (pRNA) of bacteriophage phi29 DNA-packaging motor has been reported to function as a highly versatile vehicle to carry small interference RNA (siRNA) for silencing of survivin. In this article, we explore the potential of pRNA as a vehicle to carry siRNA specifically targeted to metallothionein-IIa (MT-IIA) messenger RNA (mRNA), and compare it to survivin targeting pRNA.
2011
Col V siRNA Engineered Tenocytes for Tendon Tissue Engineering
Authors: P Lu, GR Zhang, XH Song, XH Zou, LL Wang, HW Ouyang
The presence of uniformly small collagen fibrils in tendon repair is believed to play a major role in suboptimal tendon healing. Collagen V is significantly elevated in healing tendons and plays an important role in fibrillogenesis. The objective of this study was to investigate the effect of a particular chain of collagen V on the fibrillogenesis of Sprague-Dawley rat tenocytes, as well as the efficacy of Col V siRNA engineered tenocytes for tendon tissue engineering.
2011
Wnt Signaling Mediates Pathological Vascular Growth in Proliferative Retinopathy
Authors: RB Chalamalasetty, W C Dunty Jr, KK Biris, R Ajima, M Iacovino, A Beisaw, L Feigenbaum, DL Chapman, JK Yoon, M Kyba, TP Yamaguchi
Ischemic proliferative retinopathy, characterized by pathological retinal neovascularization, is a major cause of blindness in working-age adults and children. Defining the molecular pathways distinguishing pathological neovascularization from normal vessels is critical to controlling these blinding diseases with targeted therapy. Because mutations in Wnt signaling cause defective retinal vasculature in humans with some characteristics of the pathological vessels in retinopathy, we investigated the potential role of Wnt signaling in pathological retinal vascular growth in proliferative retinopathy.
2010
Authors: Z Yang, W Xiaohua, J Lei, T Ruoyun, X Mingxia, H Weichun, F Li, W Ping, Y Junwei
Urate is produced as the major end product of purine metabolism. In the last decade, the incidence of hyperuricemia increased markedly, and similar trends in the epidemiology of metabolic syndrome have been observed. Hyperuricemia is associated with renal disease, and recent studies have reported that mild hyperuricemia results in hypertension, intrarenal vascular disease, and renal injury. This has led to the hypothesis that uric acid may contribute to renal fibrosis and progressive renal disease.
2010
Evaluation of the effects of small interfering RNAs on in vitro replication of feline herpesvirus-1
Authors: RP Wilkes, SA Kania
To evaluate the ability of 5 small interfering RNAs (siRNAs) targeting mRNA of the feline herpesvirus-1 (FHV-1) DNA polymerase gene to reduce in vitro viral replication and gene expression of FHV-1, to evaluate combinations of these siRNAs with siRNAs that target the glycoprotein D gene of FHV-1, and to determine the combination or combinations of siRNAs that yield the greatest inhibition of in vitro viral replication.
2007
P-selectin primes leukocyte integrin activation during inflammation
Authors: H-B Wang, J-T Wang, L Zhang, ZH Geng, W-L Xu, T Xu, Y Huo, X Zhu, EF Plow, M Chen, J-G Geng
Selectins mediate leukocyte rolling and prime leukocytes for integrin-mediated leukocyte adhesion. However, neither the in vivo importance of nor the signaling pathway by which selectin-mediated integrin activation occurs has been determined. We report here that P-selectin-deficient mice manifested impaired leukocyte adhesion, which was 'rescued' by soluble P-selectin. Mechanistically, the cytoplasmic domain of P-selectin glycoprotein ligand 1 formed a constitutive complex with Nef-associated factor 1. After binding of P-selectin, Src kinases phosphorylated Nef-associated factor 1, which recruited the phosphoinositide-3-OH kinase p85-p110 heterodimer and resulted in activation of leukocyte integrins. Inhibition of this signal-transduction pathway diminished the adhesion of leukocytes to capillary venules and suppressed peritoneal infiltration of leukocytes. Our data demonstrate the functional importance of this newly identified signaling pathway mediated by P-selectin glycoprotein ligand 1.
2007
Authors: M Echevarrìa, AM Munõz-Cabello, R Sánchez-Silva, JJ Toledo-Aral, J López-Barneo
O2 is essential for aerobic life, and the classic view is that it diffuses freely across the plasma membrane. However, measurements of O2 permeability of lipid bilayers have indicated that it is much lower than previously thought, and therefore, the existence of membrane O2 channels has been suggested. We hypothesized that, besides its role as a water channel, aquaporin-1 (AQP-1) could also work as an O2 transporter, because this transmembrane protein appears to be CO2-permeable and is highly expressed in cells with rapid O2 turnover (erythrocytes and microvessel endothelium).
2006
Authors: JM Holaska, S Rais-Bahrami, KL Wilson
X-linked Emery-Dreifuss muscular dystrophy (X-EDMD) is inherited through mutations in emerin, a nuclear membrane protein. Emerin has proposed roles in nuclear architecture and gene regulation, but direct molecular links to disease were unknown. We report that Lim-domain only 7 (Lmo7) binds emerin directly with 125 nM affinity; the C-terminal half of human Lmo7 (hLmo7C) was sufficient to bind emerin in vitro. Lmo7 appeared relevant to EDMD because a deletion that removes Lmo7 (plus eight exons of a neighboring gene) in mice causes dystrophic muscles (1). Lmo7 localizes in the nucleus, cytoplasm and cell surface, particularly adhesion junctions (2). Our data suggest endogenous Lmo7 is a nucleocytoplasmic shuttling protein, and might also localize at focal adhesions in HeLa cells.
Ambion mMESSAGE mMACHINE High Yield Capped RNA Transcription Kits use Ambion's patented high-yield transcription technology for the routine synthesis of 15–35 μg of 7-methyl guanosine capped RNA from 1 μg of template DNA. Each kit includes sufficient reagents for 25 reactions.
2007
Prometaphase APC[cdh1] activity prevents non-disjunction in mammalian oocytes
Authors: A Reis, S Madgwick, H-Y Chang, I Nabti, M Levasseur, KT Jones
The first female meiotic division (meiosis I, MI) is uniquely prone to chromosome segregation errors through non-disjunction, resulting in trisomies and early pregnancy loss1. Here, we show a fundamental difference in the control of mammalian meiosis that may underlie such susceptibility.
Ambion mMESSAGE mMACHINE High Yield Capped RNA Transcription Kits use Ambion's patented high-yield transcription technology for the routine synthesis of 15–35 μg of 7-methyl guanosine capped RNA from 1 μg of template DNA. Each kit includes sufficient reagents for 25 reactions.
2012
The forkhead transcription factor FoxY regulates Nanos
Authors: Jia L. Song,Gary M. Wessel
FoxY is a member of the forkhead transcription factor family that appeared enriched in the presumptive germ line of sea urchins (Ransick et al. Dev Biol 2002;246:132). Here, we test the hypothesis that FoxY is involved in germ line determination in this animal. We found two splice forms of FoxY that share the same DNA-binding domain, but vary in the carboxy-terminal trans-activation/repression domain. Both forms of the FoxY protein are present in the egg and in the early embryo, and their mRNAs accumulate to their highest levels in the small micromeres and adjacent non-skeletogenic mesoderm. Knockdown of FoxY resulted in a dramatic decrease in Nanos mRNA and protein levels as well as a loss of coelomic pouches in 2-week-old larvae. Our results indicate that FoxY positively regulates Nanos at the transcriptional level and is essential for reproductive potential in this organism. Mol. Reprod. Dev. 79: 680–688, 2012. © 2012 Wiley Periodicals, Inc.
2012
Authors: WA Munoz, M Kloc, K Cho, M Lee, I Hofmann, A Sater, K Vleminckx, PD McCrea
The p120-catenin family has undergone a significant expansion during the evolution of vertebrates, resulting in varied functions that have yet to be discerned or fully characterized. Likewise, members of the plakophilins, a related catenin subfamily, are found throughout the cell with little known about their functions outside the desmosomal plaque.
Ambion mMESSAGE mMACHINE High Yield Capped RNA Transcription Kits use Ambion's patented high-yield transcription technology for the routine synthesis of 15–35 μg of 7-methyl guanosine capped RNA from 1 μg of template DNA.
2012
Authors: J Wan, M Yourshaw, H Mamsa et al
RNA exosomes are multi-subunit complexes conserved throughout evolution1 and are emerging as the major cellular machinery for processing, surveillance and turnover of a diverse spectrum of coding and noncoding RNA substrates essential for viability2.
2012
Pharmacological kinetics of BmK AS, a sodium channel site 4-specific modulator on Nav1.3
Authors: Zhi-Rui Liu, Jie Tao, Bang-Qian Dong, Gang Ding, Zhi-Jun Cheng, Hui-Qiong He, Yong-Hua Ji
In this study, the pharmacological kinetics of Buthus martensi Karsch (BmK) AS, a specific modulator of voltage-gated sodium channel site 4, was investigated on Nav1.3 expressed in Xenopus oocytes.
2012
Modulation of Gain-of-function α6*-Nicotinic Acetylcholine Receptor by β3 Subunits
Authors: B Dash, RJ Lukas
We previously have shown that β3 subunits either eliminate (e.g. for all-human (h) or all-mouse (m) α6β4β3-nAChR) or potentiate (e.g. for hybrid mα6hβ4hβ3- or mα6mβ4hβ3-nAChR containing subunits from different species) function of α6*-nAChR expressed in Xenopus oocytes, and that nAChR hα6 subunit residues Asn-143 and Met-145 in N-terminal domain loop E are important for dominant-negative effects of nAChR hβ3 subunits on hα6*-nAChR function. Here, we tested the hypothesis that these effects of β3 subunits would be preserved even if nAChR α6 subunits harbored gain-of-function, leucine- or valine-to-serine mutations at 9′ or 13′ positions (L9′S or V13′S) in their second transmembrane domains, yielding receptors with heightened functional activity and more amenable to assessment of effects of β3 subunit incorporation.
2011
Authors: P Silveyra, M Raval, B Simmons, S DiAngelo, G Wang, J Floros
Two human genes, SFTPA1 (SP-A1) and SFTPA2 (SP-A2), encode surfactant protein A, a molecule of innate immunity and surfactant-related functions. Several genetic variants have been identified for both genes. These include nucleolymorphisms, as well as alternative splicing patterns at the 5′ untranslated region (5′UTR).
mMESSAGE mMACHINE T7 ULTRA Kit
The Ambion mMESSAGE mMACHINE T7 Ultra Kit combines a new cap analog—Anti-Reverse Cap Analog (ARCA)—with Ambion's patented high-yield transcription technology, to generate RNA transcripts that produce higher protein yields compared to other transcripts upon translation. The kit includes sufficient reagents for 50 reactions.
2012
Replacement of H1 linker histone during bovine somatic cell nuclear transfer
Authors: Y Yun, GM Zhao, SJ Wu, W Li, AM Lei
Linker histone variants are involved in regulation of chromosome organization and gene transcription; several subtypes are expressed in the maturing oocyte and developing embryo. In Xenopus and mice, the transition between linker histone variants occurred following nuclear transfer, and apparently contributed to donor nuclear reprogramming. To determine whether such linker histone replacement occurred after bovine nuclear transfer, red fluorescent protein (RFP) tagged H1e (somatic linker histone H1e) donor cells and Venus tagged H1foo eggs were created, enucleated eggs were injected with donor cells, and embryos were created by fusion.
2008
Authors: Irina Y Tcherepanova, Melissa D Adams, Xiaorong Feng, Atsushi Hinohara, Joe Horvatinovich, David Calderhead, Don Healey and Charles A Nicolette
RNA transfection into dendritic cells (DCs) is widely used to achieve antigen expression as well as to modify DC properties. CD40L is expressed by activated T cells and interacts with CD40 receptors expressed on the surface of the DCs leading to Th1 polarization. Previous studies demonstrated that ectopic CD40L expression via DNA transfection into DCs can activate the CD40 receptor signal transduction cascade. In contrast to previous reports, this study demonstrates that the same effect can be achieved when RNA encoding CD40L is electroporated into DCs as evidenced by secretion of IL-12.
Amino Allyl MessageAmp II aRNA Amplification Kit
The Ambion Amino Allyl II MessageAmp aRNA Amplification Kit includes reagents for aRNA amplification with amimo allyl NTP incorporation. The kit includes sufficient reagents for 20 reactions. Cy dyes are not included.
2013
Authors: Y Chen, M Oba, LL Guan
Calf starters are usually offered to dairy calves to facilitate the weaning process, however, the effect of solid feed consumption on gut health has not been well studied. This study aimed to investigate the effect of calf starter feeding on the gut bacterial community and mucosal immune functions in dairy calves during weaning transition. Mucosal tissue and digesta samples were collected from rumen, jejunum, ileum, cecum, and colon upon slaughtering of calves (n = 8) after feeding the experimental diets [milk replacer (MR) or milk replacer + calf starter (MR+S)] for 6 wk.
2012
Authors: Y Chen, M Oba, LL Guan
In order to determine differences in the ruminal bacterial community and host Toll-like receptor (TLR) gene expression of beef cattle with different susceptibility to acidosis, rumen papillae and content were collected from acidosis-susceptible (AS, n = 3) and acidosis-resistant (AR, n = 3) steers. The ruminal bacterial community was characterized using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real time PCR (qRT-PCR) analysis.
2011
Authors: B Pouvreau, S Baud, V Vernoud, V Morin, C Py, G Gendrot, J-P Pichon, J Rouster, W Paul,PM Rogowsky
WRINKLED1 (WRI1), a key regulator of seed oil biosynthesis in Arabidopsis (Arabidopsis thaliana), was duplicated during the genome amplification of the cereal ancestor genome 90 million years ago. Both maize (Zea mays) coorthologs ZmWri1a and ZmWri1b show a strong transcriptional induction during the early filling stage of the embryo and complement the reduced fatty acid content of Arabidopsis wri1-4 seeds, suggesting conservation of molecular function.
Illumina® TotalPrep RNA Amplification Kit
Ambion's Illumina® TotalPrep RNA Amplification Kit (patent pending) is a complete system for generating biotinylated, amplified RNA for hybridization with Illumina Sentrix® arrays. The kit have significantly increased sensitivity, providing increased Percent Present calls and single round amplification with just 50 ng of input RNA.
2011
Anthrax Lethal Toxin-Induced Gene Expression Changes in Mouse Lung
Authors: EK Dumas, PM Cox, C O’Connor Fullenwider, M Nguyen, M Centola, MB Frank, I Dozmorov, JA James, AD Farris
A major virulence factor of Bacillus anthracis is the anthrax Lethal Toxin (LeTx), a bipartite toxin composed of Protective Antigen and Lethal Factor. Systemic administration of LeTx to laboratory animals leads to death associated with vascular leakage and pulmonary edema. In this study, we investigated whether systemic exposure of mice to LeTx would induce gene expression changes associated with vascular/capillary leakage in lung tissue. We observed enhanced susceptibility of A/J mice to death by systemic LeTx administration compared to the C57BL/6 strain. LeTx-induced groups of both up- and down-regulated genes were observed in mouse lungs 6 h after systemic administration of wild type toxin compared to lungs of mice exposed to an inactive mutant form of the toxin. Lungs of the less susceptible C57BL/6 strain showed 80% fewer differentially expressed genes compared to lungs of the more sensitive A/J strain.
This kit is a major improvement over the basic rapid amplification of cDNA ends (RACE) protocol. The RLM-RACE procedure selects only full-length mRNA — no rRNA, tRNA or degraded RNA — and facilitates the cloning of sequences from the 5' ends of messages.
2013
Authors: S Rana, SK Lattoo, N Dhar, S Razdan, WW Bhat, RS Dhar, R Vishwakarma
Withania somnifera (L.) Dunal, a highly reputed medicinal plant, synthesizes a large array of steroidal lactone triterpenoids called withanolides. Although its chemical profile and pharmacological activities have been studied extensively during the last two decades, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis.
2012
Authors: H Lou, H Li, M Yeager, K Im, B Gold, TD Schneider, JF Fraumeni Jr, SJ Chanock, SK Anderson, M Dean
Beta-microseminoprotein (MSP)/MSMB is an immunoglobulin superfamily protein synthesized by prostate epithelial cells and secreted into seminal plasma. Variants in the promoter of the MSMB gene have been associated with the risk of prostate cancer (PCa) in several independent genome-wide association studies. Both MSMB and an adjacent gene, NCOA4, are subjected to transcriptional control via androgen response elements. The gene product of NCOA4 interacts directly with the androgen receptor as a co-activator to enhance AR transcriptional activity. Here, we provide evidence for the expression of full-length MSMB-NCOA4 fusion transcripts regulated by the MSMB promoter.
2012
Authors: L Zhai, Z Liu, X Zou, Y Jiang, F Qiu, Y Zheng, Z Zhang
MicroRNAs (miRNAs) are critical post-transcriptional modulators of gene expression involving in plant responses to abiotic stress. However, the regulation of miRNA in the morphological response to waterlogging is poorly understood in maize. In this study, we detected miRNAs and their targets that expressed in waterlogged crown roots of maize seedlings in two inbred lines (Hz32 and Mo17) by RNA sequencing.
2011
Anthrax Lethal Toxin-Induced Gene Expression Changes in Mouse Lung
Authors: EK Dumas, PM Cox, C O’Connor Fullenwider, M Nguyen, M Centola, MB Frank, I Dozmorov, JA James, AD Farris
A major virulence factor of Bacillus anthracis is the anthrax Lethal Toxin (LeTx), a bipartite toxin composed of Protective Antigen and Lethal Factor. Systemic administration of LeTx to laboratory animals leads to death associated with vascular leakage and pulmonary edema. In this study, we investigated whether systemic exposure of mice to LeTx would induce gene expression changes associated with vascular/capillary leakage in lung tissue. We observed enhanced susceptibility of A/J mice to death by systemic LeTx administration compared to the C57BL/6 strain. LeTx-induced groups of both up- and down-regulated genes were observed in mouse lungs 6 h after systemic administration of wild type toxin compared to lungs of mice exposed to an inactive mutant form of the toxin. Lungs of the less susceptible C57BL/6 strain showed 80% fewer differentially expressed genes compared to lungs of the more sensitive A/J strain.
2009
Authors: M Knight, N Raghavan, C Goodall, C Cousin, W Ittiprasert, A Sayed, A Miller, DL Williams, CJ Bayne
To identify gene(s) that may be associated with resistance/susceptibility in the intermediate snail host Biomphalaria glabrata to Schistosoma mansoni infection, a snail albumen gland cDNA library was differentially screened and a partial cDNA encoding an antioxidant enzyme thioredoxin peroxidase (Tpx), or peroxiredoxin (Prx), was identified. The 753 bp full-length, single-copy, constitutively expressed gene now referred to as BgPrx4 was later isolated.
For Research Use Only. Not for use in diagnostic procedures.