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  • An insulator with barrier-element activity promotes alpha-spectrin gene expression in erythroid cells. 19008453

    Understanding mechanisms controlling expression of the alpha-spectrin gene is important for understanding erythropoiesis, membrane biogenesis, and spectrin-linked hemolytic anemia. We showed previously that a minimal alpha-spectrin promoter directed low levels of expression only in early erythroid development, indicating elements outside the promoter are required for expression in adult erythrocytes. Addition of noncoding exon 1' and intron 1' conferred a 10-fold increase in activity in reporter gene assays. In this report, we used a transgenic mouse model to show that addition of exon 1' and intron 1' to the alpha-spectrin promoter conferred tissue-specific expression of a linked (A)gamma-globin gene in erythroid cells at all developmental stages. Expression was nearly position-independent, as 21 of 23 lines expressed the transgene, and gamma-globin protein was present in 100% of erythrocytes, indicating uniform expression. Additional in vivo studies revealed that exon 1' functions as an insulator with barrier-element activity. Chromatin immunoprecipitation assays demonstrated that this region was occupied by the upstream stimulatory factors 1/2 (USF1/USF2), similar to the well-characterized chicken HS4 insulator. These data identify the first barrier element described in an erythrocyte membrane protein gene and indicate that exon 1' and intron 1' are excellent candidate regions for mutations in patients with spectrin-linked hemolytic anemia.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-729
    Nombre del producto:
    Anti-CTCF Antibody

  • Epigenetic switches of tobacco transgenes associate with transient redistribution of histone marks in callus culture. 23770973

    In plants, silencing is usually accompanied by DNA methylation and heterochromatic histone marks. We studied these epigenetic modifications in different epialleles of 35S promoter (P35S)-driven tobacco transgenes. In locus 1, the T-DNA was organized as an inverted repeat, and the residing neomycin phosphotransferase II reporter gene (P35S-nptII) was silenced at the posttranscriptional (PTGS) level. Transcriptionally silenced (TGS) epialleles were generated by trans-acting RNA signals in hybrids or in a callus culture. PTGS to TGS conversion in callus culture was accompanied by loss of the euchromatic H3K4me3 mark in the transcribed region of locus 1, but this change was not transmitted to the regenerated plants from these calli. In contrast, cytosine methylation that spread from the transcribed region into the promoter was maintained in regenerants. Also, the TGS epialleles generated by trans-acting siRNAs did not change their active histone modifications. Thus, both TGS and PTGS epialleles exhibit euchromatic (H3K4me3 and H3K9ac) histone modifications despite heavy DNA methylation in the promoter and transcribed region, respectively. However, in the TGS locus (271), abundant heterochromatic H3K9me2 marks and DNA methylation were present on P35S. Heterochromatic histone modifications are not automatically installed on transcriptionally silenced loci in tobacco, suggesting that repressive histone marks and cytosine methylation may be uncoupled. However, transient loss of euchromatic modifications may guide de novo DNA methylation leading to formation of stable repressed epialleles with recovered eukaryotic marks. Compilation of available data on epigenetic modification of inactivated P35S in different systems is provided.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-212
    Nombre del producto:
    Anti-dimethyl-Histone H3 (Lys9) Antibody

  • Binding of the radiolabeled glycine site antagonist [3H]MDL 105,519 to homomeric NMDA-NR1a receptors. 8894619

    We have characterized the binding of [3H]MDL 105,519 ((E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1 H-indole-2-carboxylic acid), a NMDA receptor glycine recognition site antagonist, to homomeric NMDA subunit 1a (NR 1a) receptors. Chinese hamster ovary cells (CHO-K1) were transfected with the rat NR 1a gene and cell lines stably expressing the receptor were isolated from amongst clones resistant to the neomycin analog G418. Saturation analysis indicated that the radioligand bound to the homomeric receptor with a similar high affinity (Kd = 1.8 nM) to that reported for the native receptor. The binding capacity (Bmax) was 370 fmol/mg protein reflecting approximately 110000 receptors per cell. The radioligand interacted with a single class of binding sites as indicated by linear Scatchard transformation of the saturation data and a unitary Hill slope in competition experiments. Thus, the MDL 105,519 recognition site is present on the NR 1a subunit and has similar radioligand binding properties to the native brain-derived receptor. However, pharmacologic characterization of [3H]MDL 105,519 binding indicated that agonists were weaker competitors at the homometric receptor relative to the native receptors. In contrast, representative of three distinct chemical classes of glycine site antagonists exhibited similar potencies at both types of binding sites.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AP132A
    Nombre del producto:
    Goat Anti-Rabbit IgG Antibody, Alkaline Phosphatase conjugate

  • Knockout mice reveal a role for protein tyrosine phosphatase H1 in cognition. 18700002

    The present study has investigated the protein tyrosine phosphatase H1 (PTPH1) expression pattern in mouse brain and its impact on CNS functions.We have previously described a PTPH1-KO mouse, generated by replacing the PTP catalytic and the PDZ domain with a LacZ neomycin cassette. PTPH1 expression pattern was evaluated by LacZ staining in the brain and PTPH1-KO and WT mice (n = 10 per gender per genotype) were also behaviorally tested for CNS functions.In CNS, PTPH1 is expressed during development and in adulthood and mainly localized in hippocampus, thalamus, cortex and cerebellum neurons. The behavioral tests performed on the PTPH1-KO mice showed an impact on working memory in male mice and an impaired learning performance at rotarod in females.These results demonstrate for the first time a neuronal expression of PTPH1 and its functionality at the level of cognition.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB377
    Nombre del producto:
    Anti-NeuN Antibody, clone A60

  • Caspase activation in hair cells of the mouse utricle exposed to neomycin. 12351727

    Aminoglycoside exposure results in the apoptotic destruction of auditory and vestibular hair cells. This ototoxic hair cell death is prevented by broad-spectrum caspase inhibition. We have used in situ substrate detection, immunohistochemistry, and specific caspase inhibitors to determine which caspases are activated in the hair cells of the adult mouse utricle in response to neomycin exposure in vitro. In addition, we have examined the hierarchy of caspase activation. Our data indicate that both upstream caspase-8 and upstream caspase-9, as well as downstream caspase-3 are activated in hair cells exposed to neomycin. The inhibition of caspase-9-like activity provided significant protection of hair cells exposed to neomycin, whereas the inhibition of caspase-8-like activity was not effective in preventing neomycin-induced hair cell death. In addition, caspase-9 inhibition prevented the activation of downstream caspase-3, whereas the inhibition of caspase-8 did not. These data indicate that caspase-9 is the primary upstream caspase mediating neomycin-induced hair cell death in this preparation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Retroviral gene therapy vectors for prevention of excimer laser-induced corneal haze. 11923236

    PURPOSE: To determine the in vivo efficacy and safety of a retroviral vector bearing an antiproliferative dominant negative mutant cyclin G1 (dnG1) construct, when used for the prevention of corneal haze after phototherapeutic keratectomy (PTK). METHODS: For in vivo efficacy studies, a 6-mm-diameter, 150-microm-deep transepithelial PTK, performed with a clinical 193-nm ArF excimer laser (VISX Star2, Santa Clara, CA) was performed on the left eyes of 20 adult New Zealand White rabbits. The surgically altered eyes were subsequently treated with eye drops containing: a retroviral vector bearing a dnG1 construct (dnG1; n = 7), a control retroviral vector (null vector) bearing only the neomycin resistance, neo(r), gene (n = 7), or a retroviral vector bearing an antisense cyclin G1 (aG1) construct (n = 6). The time of closure of the corneal epithelial defect was monitored daily with fluorescein staining. Corneal haze was evaluated before surgery and at 2, 3, and 4 weeks after surgery, with a digital imaging system. Biodistribution studies for detection of potential vector dissemination to nontarget organs were conducted by PCR-based assay. RESULTS: The re-epithelialization rate was similar among treatment groups, with complete closure of the corneal epithelial defect at 72 hours (P > 0.05). Significant corneal haze developed in the null and aG1 vector-treated groups (P or= 0.05) at 3 to 4 weeks after PTK. In contrast, development of corneal haze was inhibited in the dnG1 vector-treated group when compared with the null vector-treated group (P 0.05). In parallel, a dramatic reduction to complete abrogation of abnormal extracellular matrix production was noted in the dnG1 vector-treated corneas when compared with the null and aG1 vector-treated groups. Biodistribution studies showed no evidence of vector dissemination in neighboring and distant organs. CONCLUSIONS: At therapeutic doses, eye drop application of the dnG1 retroviral vector is safe and effective in inhibiting development of corneal haze after PTK in rabbits.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1940
    Nombre del producto:
    Anti-Fibronectin Antibody, cellular, clone DH1

  • Stimulation by cochlear implant in unilaterally deaf rats reverses the decrease of inhibitory transmission in the inferior colliculus. 18973578

    In the last decade, numerous studies have investigated synaptic transmission changes in various auditory nuclei after unilateral cochlear injury. However, few data are available concerning the potential effect of electrical stimulation of the deafferented auditory nerve on the inhibitory neurotransmission in these nuclei. We report here for the first time the effect of chronic electrical stimulation of the deafferented auditory nerve on alpha1 subunit of the glycinergic receptor (GlyRalpha1) and glutamic acid decarboxylase (GAD)67 expression in the central nucleus of inferior colliculus (CIC). Adult rats were unilaterally cochleectomized by intracochlear neomycin sulphate injection. Fifteen days later, the ipsilateral auditory nerve was chronically stimulated either 4, 8 or 22 h daily, for 5 days using intracochlear bipolar electrodes. GlyRalpha1 and GAD67 mRNA and protein were quantified in the CIC using in situ hybridization and immunohistofluorescence methods. Our data showed that as after surgical ablation, GlyRalpha1 and GAD67 expression were strongly decreased in the contralateral CIC after unilateral chemical cochleectomy. Most importantly, these postlesional down-modulations were significantly reversed by chronic electrical stimulation of the deafferented auditory nerve. This recovery, however, did not persist for more than 5 days after the cessation of the deafferented auditory nerve electrical stimulation. Thus, downregulations of GlyRalpha1 and GAD67 may be involved both in the increased excitability observed in the CIC after unilateral deafness and consequently in the tinnitus frequently observed in unilateral adult deaf patients. Electrical stimulation of the deafferented auditory nerve in patients may be a potential new approach for treating tinnitus with unilateral hearing loss.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB5406
    Nombre del producto:
    Anti-GAD67 Antibody, clone 1G10.2

  • JNK signaling in neomycin-induced vestibular hair cell death. 17005344

    Mechanosensory hair cells are susceptible to apoptotic death in response to exposure to ototoxic drugs, including aminoglycoside antibiotics. The c-Jun n-terminal kinase (JNK) is a stress-activated protein kinase that can promote apoptotic cell death in a variety of systems. Inhibition of the JNK signaling pathway can prevent aminoglycoside-induced death of cochlear and vestibular sensory hair cells. We used an in vitro preparation of utricles from adult mice to examine the role of JNK activation in aminoglycoside-induced hair cell death. CEP-11004 was used as an indirect inhibitor of JNK signaling. Immunohistochemistry showed that both JNK and its downstream target c-Jun are phosphorylated in hair cells of utricles exposed to neomycin. CEP-11004 inhibited neomycin-induced phosphorylation of both JNK and c-Jun. CEP-11004 inhibited hair cell death in utricles exposed to moderate doses of neomycin. However, the results were not uniform across the dose-response function; CEP-11004 did not inhibit hair cell death in utricles exposed to high-dose neomycin. The CEP-11004-induced protective effect was not due to inhibition of PKC or p38, since neither Chelerythrine nor SB203580 could mimic the protective effect of CEP-11004. In addition, inhibition of JNK inhibited the activation of caspase-9 in hair cells. These results indicate that JNK plays an important role in neomycin-induced vestibular hair cell death and caspase-9 activation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB1778

  • Evaluation of ITX 5061, a scavenger receptor B1 antagonist: resistance selection and activity in combination with other hepatitis C virus antivirals. 22279172

    ITX 5061 is a scavenger receptor B1 antagonist that has entered phase 1 clinical trials in hepatitis C virus (HCV)-infected humans. We evaluated ITX 5061 in combination with interferon-α, ribavirin, and HCV protease and polymerase inhibitors in a genotype 2a infectious virus system. ITX 5061 is a potent inhibitor of HCV replication and is additive to synergistic with interferon-α, ribavirin, BILN2061, VX950, VX1, and 2'-C-methyladenosine. Resistance selection experiments were performed using a Jc1-FEO virus co-culture system and intermittent ITX 5061 exposure under neomycin selection. We identified a mutant virus with a substitution of aspartic acid for asparagine at the highly conserved position 415 in E2 (N415D). Introduction of this mutation into wild-type virus conferred high-level resistance to ITX 5061. There was no cross-resistance between ITX 5061 and HCV protease inhibitors or interferon-α. These results suggest that ITX 5061 is a promising compound for study in combination with other HCV inhibitors.
    Tipo de documento:
    Referencia
    Referencia del producto:
    12-349
    Nombre del producto:
    Goat Anti-Mouse IgG Antibody, HRP conjugate

  • Stable transfection of the diplomonad parasite Spironucleus salmonicida. 22983987

    Eukaryotic microbes are highly diverse, and many lineages remain poorly studied. One such lineage, the diplomonads, a group of binucleate heterotrophic flagellates, has been studied mainly due to the impact of Giardia intestinalis, an intestinal, diarrhea-causing parasite in humans and animals. Here we describe the development of a stable transfection system for use in Spironucleus salmonicida, a diplomonad that causes systemic spironucleosis in salmonid fish. We designed vectors in cassette format carrying epitope tags for localization (3×HA [where HA is hemagglutinin], 2× Escherichia coli OmpF linker and mouse langerin fusion sequence [2×OLLAS], 3×MYC) and purification of proteins (2× Strep-Tag II-FLAG tandem-affinity purification tag or streptavidin binding peptide-glutathione S-transferase [SBP-GST]) under the control of native or constitutive promoters. Three selectable gene markers, puromycin acetyltransferase (pac), blasticidin S-deaminase (bsr), and neomycin phosphotransferase (nptII), were successfully applied for the generation of stable transfectants. Site-specific integration on the S. salmonicida chromosome was shown to be possible using the bsr resistance gene. We epitope tagged six proteins and confirmed their expression by Western blotting. Next, we demonstrated the utility of these vectors by recording the subcellular localizations of the six proteins by laser scanning confocal microscopy. Finally, we described the creation of an S. salmonicida double transfectant suitable for colocalization studies. The transfection system described herein and the imminent completion of the S. salmonicida genome will make it possible to use comparative genomics as an investigative tool to explore specific, as well as general, diplomonad traits, benefiting research on both Giardia and Spironucleus.
    Tipo de documento:
    Referencia
    Referencia del producto:
    04-1624
    Nombre del producto:
    Anti-Centrin Antibody, clone 20H5