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Sample records for mrna blotting fluorescence

  1. Changes in rRNA levels during stress invalidates results from mRNA blotting: Fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels

    DEFF Research Database (Denmark)

    Hansen, M.C.; Nielsen, A.K.; Molin, Søren

    2001-01-01

    obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting...... the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell...... decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes...

  2. Fluorescence-based Western blotting for quantitation of protein biomarkers in clinical samples.

    Science.gov (United States)

    Zellner, Maria; Babeluk, Rita; Diestinger, Michael; Pirchegger, Petra; Skeledzic, Senada; Oehler, Rudolf

    2008-09-01

    Since most high throughput techniques used in biomarker discovery are very time and cost intensive, highly specific and quantitative analytical alternative application methods are needed for the routine analysis. Conventional Western blotting allows detection of specific proteins to the level of single isotypes while its quantitative accuracy is rather limited. We report a novel and improved quantitative Western blotting method. The use of fluorescently labelled secondary antibodies strongly extends the dynamic range of the quantitation and improves the correlation with the protein amount (r=0.997). By an additional fluorescent staining of all proteins immediately after their transfer to the blot membrane, it is possible to visualise simultaneously the antibody binding and the total protein profile. This allows for an accurate correction for protein load. Applying this normalisation it could be demonstrated that fluorescence-based Western blotting is able to reproduce a quantitative analysis of two specific proteins in blood platelet samples from 44 subjects with different diseases as initially conducted by 2D-DIGE. These results show that the proposed fluorescence-based Western blotting is an adequate application technique for biomarker quantitation and suggest possibilities of employment that go far beyond.

  3. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.

    Directory of Open Access Journals (Sweden)

    Samantha L Eaton

    Full Text Available Western blotting has been a key technique for determining the relative expression of proteins within complex biological samples since the first publications in 1979. Recent developments in sensitive fluorescent labels, with truly quantifiable linear ranges and greater limits of detection, have allowed biologists to probe tissue specific pathways and processes with higher resolution than ever before. However, the application of quantitative Western blotting (QWB to a range of healthy tissues and those from degenerative models has highlighted a problem with significant consequences for quantitative protein analysis: how can researchers conduct comparative expression analyses when many of the commonly used reference proteins (e.g. loading controls are differentially expressed? Here we demonstrate that common controls, including actin and tubulin, are differentially expressed in tissues from a wide range of animal models of neurodegeneration. We highlight the prevalence of such alterations through examination of published "-omics" data, and demonstrate similar responses in sensitive QWB experiments. For example, QWB analysis of spinal cord from a murine model of Spinal Muscular Atrophy using an Odyssey scanner revealed that beta-actin expression was decreased by 19.3±2% compared to healthy littermate controls. Thus, normalising QWB data to β-actin in these circumstances could result in 'skewing' of all data by ∼20%. We further demonstrate that differential expression of commonly used loading controls was not restricted to the nervous system, but was also detectable across multiple tissues, including bone, fat and internal organs. Moreover, expression of these "control" proteins was not consistent between different portions of the same tissue, highlighting the importance of careful and consistent tissue sampling for QWB experiments. Finally, having illustrated the problem of selecting appropriate single protein loading controls, we demonstrate

  4. Western blotting.

    Science.gov (United States)

    Kurien, Biji T; Scofield, R Hal

    2006-04-01

    Western blotting (protein blotting or immunoblotting) is a powerful and important procedure for the immunodetection of proteins post-electrophoresis, particularly proteins that are of low abundance. Since the inception of the protocol for protein transfer from an electrophoresed gel to a membrane in 1979, protein blotting has evolved greatly. The scientific community is now confronted with a variety of ways and means to carry out this transfer. This review describes the various procedures that have been used to transfer proteins from a gel to a membrane based on the principles of simple diffusion, vacuum-assisted solvent flow and electrophoretic elution. Finally, a brief description of methods generally used to detect antigens on blots is also described.

  5. Southern blotting.

    Science.gov (United States)

    Brown, T

    2001-05-01

    Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support (the properties and advantages of the different types of membrane, transfer buffer, and transfer method are discussed in detail), resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. This appendix describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, using a high-salt transfer buffer to promote binding of DNA to the membrane. With the high-salt buffer, the DNA becomes bound to the membrane during transfer but not permanently immobilized. Immobilization is achieved by UV irradiation (for nylon) or baking (for nitrocellulose). A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. The advantage of this combination is that no post-transfer immobilization step is required, as the positively charged membrane binds DNA irreversibly under alkaline transfer conditions. The method can also be used with neutral nylon membranes but less DNA will be retained. A second alternate protocol describes a transfer method based on a different transfer-stack setup. The traditional method of upward capillary transfer of DNA from gel to membrane described in the first basic and alternate protocols has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The downward capillary method described in

  6. IgG western blot for confirmatory diagnosis of equivocal cases of toxoplasmosis by EIA-IgG and fluorescent antibody test.

    Science.gov (United States)

    Khammari, Imen; Saghrouni, Fatma; Yaacoub, Alia; Gaied Meksi, Sondoss; Ach, Hinda; Garma, Lamia; Fathallah, Akila; Ben Saïd, Moncef

    2013-08-01

    The performance values of available techniques used in serodiagnosis of toxoplasmosis are satisfactory but they raise problems of equivocal and discordant results for very low IgG titers. Recently marketed, LDBio-Toxo II IgG Western blot (IB) showed an excellent correlation with the dye test. We estimated the proportion of equivocal and discordant results between the enzyme immunoassay Platelia Toxo IgG (EIA-IgG) and fluorescent antibody test (FAT) and assessed the usefulness of the IB as a confirmatory test. Out of 2,136 sera collected from pregnant women, 1,644 (77.0%) tested unequivocally positive and 407 (19.0%) were negative in both EIA-IgG and FAT. The remaining 85 (4%) sera showed equivocal or discordant results. Among them, 73 (85.9%) were positive and 12 (14.1%) were negative in IB. Forty-one (89.1%) equivocal sera in EIA-IgG and 46 (86.8%) equivocal sera in FAT were positive in IB. Reducing the cut-off values of both screening techniques improved significantly their sensitivity in detecting very low IgG titers at the expense of their specificity. In conclusion, equivocal results in routine-used techniques and their discordance in determination of the immune status in pregnancy women were not uncommon. IB test appeard to be highly useful in these situations as a confirmatory technique.

  7. Lectin-Array Blotting.

    Science.gov (United States)

    Pazos, Raquel; Echevarria, Juan; Hernandez, Alvaro; Reichardt, Niels-Christian

    2017-09-01

    Aberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and rapid protocol for qualitative analysis of protein glycosylation in complex biological mixtures. While traditional lectin arrays only provide an average signal for the glycans in the mixture, which is usually dominated by the most abundant proteins, our method provides individual lectin binding profiles for all proteins separated in the gel electrophoresis step. Proteins do not have to be excised from the gel for subsequent analysis via the lectin array but are transferred by contact diffusion from the gel to a glass slide presenting multiple copies of printed lectin arrays. Fluorescently marked glycoproteins are trapped by the printed lectins via specific carbohydrate-lectin interactions and after a washing step their binding profile with up to 20 lectin probes is analyzed with a fluorescent scanner. The method produces the equivalent of 20 lectin blots in a single experiment, giving detailed insight into the binding epitopes present in the fractionated proteins. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  8. Testing UK blood donors for exposure to human parvovirus 4 using a time-resolved fluorescence immunoassay to screen sera and Western blot to confirm reactive samples.

    Science.gov (United States)

    Maple, Peter A C; Beard, Stuart; Parry, Ruth P; Brown, Kevin E

    2013-10-01

    Human parvovirus 4 (ParV4), a newly described member of the family Parvoviridae, like B19V, has been found in pooled plasma preparations. The extent, and significance, of ParV4 exposure in UK blood donors remain to be determined and reliable detection of ParV4 immunoglobulin (Ig)G, using validated methods, is needed. With ParV4 virus-like particles a ParV4 IgG time-resolved fluorescence immunoassay (TRFIA) was developed. There is no gold standard or reference assay for measuring ParV4 IgG and the utility of the TRFIA was first examined using a panel of sera from people who inject drugs (PWIDS)--a high-prevalence population for ParV4 infection. Western blotting was used to confirm the specificity of TRFIA-reactive sera. Two cohorts of UK blood donor sera comprising 452 sera collected in 1999 and 156 sera collected in 2009 were tested for ParV4 IgG. Additional testing for B19V IgG, hepatitis C virus antibodies (anti-HCV), and ParV4 DNA was also undertaken. The rate of ParV4 IgG seroprevalence in PWIDS was 20.7% and ParV4 IgG was positively associated with the presence of anti-HCV with 68.4% ParV4 IgG-positive sera testing anti-HCV-positive versus 17.1% ParV4 IgG-negative sera. Overall seropositivity for ParV4 IgG, in 608 UK blood donors was 4.76%. The ParV4 IgG seropositivity for sera collected in 1999 was 5.08%, compared to 3.84% for sera collected in 2009. No ParV4 IgG-positive blood donor sera had detectable ParV4 DNA. ParV4 IgG has been found in UK blood donors and this finding needs further investigation. © 2013 American Association of Blood Banks.

  9. Introduction to protein blotting.

    Science.gov (United States)

    Kurien, Biji T; Scofield, R Hal

    2009-01-01

    Protein blotting is a powerful and important procedure for the immunodetection of proteins following electrophoresis, particularly proteins that are of low abundance. Since the inception of the protocol for protein transfer from an electrophoresed gel to a membrane in 1979, protein blotting has evolved greatly. The scientific community is now confronted with a variety of ways and means to carry out this transfer.

  10. Northern blotting analysis

    DEFF Research Database (Denmark)

    Josefsen, Knud; Nielsen, Henrik

    2011-01-01

    Northern blotting analysis is a classical method for analysis of the size and steady-state level of a specific RNA in a complex sample. In short, the RNA is size-fractionated by gel electrophoresis and transferred by blotting onto a membrane to which the RNA is covalently bound. Then, the membrane...... is analysed by hybridization to one or more specific probes that are labelled for subsequent detection. Northern blotting is relatively simple to perform, inexpensive, and not plagued by artefacts. Recent developments of hybridization membranes and buffers have resulted in increased sensitivity closing...

  11. Western blotting: an introduction.

    Science.gov (United States)

    Kurien, Biji T; Scofield, R Hal

    2015-01-01

    Western blotting is an important procedure for the immunodetection of proteins, particularly proteins that are of low abundance. This process involves the transfer of protein patterns from gel to microporous membrane. Electrophoretic as well as non-electrophoretic transfer of proteins to membranes was first described in 1979. Protein blotting has evolved greatly since the inception of this protocol, allowing protein transfer to be accomplished in a variety of ways.

  12. Northern blotting analysis

    DEFF Research Database (Denmark)

    Josefsen, Knud; Nielsen, Henrik

    2011-01-01

    is analysed by hybridization to one or more specific probes that are labelled for subsequent detection. Northern blotting is relatively simple to perform, inexpensive, and not plagued by artefacts. Recent developments of hybridization membranes and buffers have resulted in increased sensitivity closing...

  13. The early days of blotting.

    Science.gov (United States)

    Southern, Edwin

    2015-01-01

    The history of the development of DNA blotting is described in this chapter. DNA blotting, involving the transfer of electrophoretically separated DNA fragments to a membrane support through capillary action, is also known as Southern blotting. This procedure enables the detection of a specific DNA sequence by hybridization with probes. The term Southern blotting led to a "geographic" naming tradition, with RNA blotting bearing the name Northern blotting and protein transfer to membranes becoming known as Western blotting.

  14. Western Blotting using Capillary Electrophoresis

    OpenAIRE

    Anderson, Gwendolyn J.; Cipolla, Cynthia; Kennedy, Robert T.

    2011-01-01

    A microscale Western blotting system based on separating sodium-dodecyl sulfate protein complexes by capillary gel electrophoresis followed by deposition onto a blotting membrane for immunoassay is described. In the system, the separation capillary is grounded through a sheath capillary to a mobile X-Y translation stage which moves a blotting membrane past the capillary outlet for protein deposition. The blotting membrane is moistened with a methanol and buffer mixture to facilitate protein a...

  15. Evaluation of an mRNA lipofection procedure for human dendritic cells and induction of cytotoxic T lymphocytes against enhanced green fluorescence protein.

    Science.gov (United States)

    Okano, Kozue; Fukui, Mikiko; Suehiro, Yutaka; Hamanaka, Yuichiro; Imai, Kohzoh; Hinoda, Yuji

    2003-01-01

    We utilized an mRNA lipofection procedure in human dendritic cells (DCs) and attempted to induce cytotoxic T lymphocytes (CTLs) against enhanced green fluorescence protein (EGFP). EGFP mRNA was transfected into phytohemagglutinin (PHA)-stimulated lymphocytes or adherent peripheral blood mononuclear cell-derived DCs using a liposomal reagent. Lipofection efficiency was measured by flow cytometry. In PHA-stimulated lymphocytes, increasing concentrations of liposome or mRNA increased EGFP expression levels by up to 64.4%, but caused a decrease in cell viability. A similar trend was also observed in DCs. For 70% DC viability, the concentration of liposomes was 24 microl/ml, and the mRNA concentration was 6 microg/ml. Under these conditions, ELISPOT and (51)Cr release assays were performed on CD8+ T cells stimulated twice with EGFP mRNA-transfected DCs. The number of interferon-gamma-producing cells was increased when the CD8+ T cells were cocultured for 24 h with PHA-stimulated lymphocytes transfected with EGFP mRNA. The level of specific lysis of EGFP mRNA-transfected DCs also increased to approximately 80%, with an effector to target ratio of 40:1. These data suggest that EGFP is immunogenic for human T cells, confirming that our lipofection procedure may be of use for inducing specific CTLs. Copyright 2003 S. Karger AG, Basel

  16. Protein blotting protocol for beginners.

    Science.gov (United States)

    Petrasovits, Lars A

    2014-01-01

    The transfer and immobilization of biological macromolecules onto solid nitrocellulose or nylon (polyvinylidene difluoride (PVDF)) membranes subsequently followed by specific detection is referred to as blotting. DNA blots are called Southerns after the inventor of the technique, Edwin Southern. By analogy, RNA blots are referred to as northerns and protein blots as westerns (Burnette, Anal Biochem 112:195-203, 1981). With few exceptions, western blotting involves five steps, namely, sample collection, preparation, separation, immobilization, and detection. In this chapter, protocols for the entire process from sample collection to detection are described.

  17. TLC blot (far-eastern blot) and its applications.

    Science.gov (United States)

    Taki, Takao; Gonzalez, Tania Valdes; Goto-Inoue, Naoko; Hayasaka, Takahiro; Setou, Mitsutoshi

    2009-01-01

    A simple method for transfer of lipids including phospholipids, glycolipids, and neutral lipids from a high-performance thin-layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane, called TLC blot (far-eastern blot), is presented. Lipids separated on a HPTLC plate are blotted quantitatively. This procedure made it possible to purify individual lipids from a blotted membrane in a short time. Binding study, immunodetection, and mass spectrometric analysis are available for PVDF membrane. Furthermore, the world of molecular species imaging is opened by a scanning analysis with a combination of TLC blot and matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometer (TLC-Blot/MALDI-TOF MS).

  18. Western blotting using capillary electrophoresis.

    Science.gov (United States)

    Anderson, Gwendolyn J; M Cipolla, Cynthia; Kennedy, Robert T

    2011-02-15

    A microscale Western blotting system based on separating sodium-dodecyl sulfate protein complexes by capillary gel electrophoresis followed by deposition onto a blotting membrane for immunoassay is described. In the system, the separation capillary is grounded through a sheath capillary to a mobile X-Y translation stage which moves a blotting membrane past the capillary outlet for protein deposition. The blotting membrane is moistened with a methanol and buffer mixture to facilitate protein adsorption. Although discrete protein zones could be detected, bands were broadened by ∼1.7-fold by transfer to membrane. A complete Western blot for lysozyme was completed in about one hour with 50 pg mass detection limit from low microgram per milliliter samples. These results demonstrate substantial reduction in time requirements and improvement in mass sensitivity compared to conventional Western blots. Western blotting using capillary electrophoresis shows promise to analyze low volume samples with reduced reagents and time, while retaining the information content of a typical Western blot.

  19. Molecular beacon-decorated polymethylmethacrylate core-shell fluorescent nanoparticles for the detection of survivin mRNA in human cancer cells.

    Science.gov (United States)

    Adinolfi, Barbara; Pellegrino, Mario; Giannetti, Ambra; Tombelli, Sara; Trono, Cosimo; Sotgiu, Giovanna; Varchi, Greta; Ballestri, Marco; Posati, Tamara; Carpi, Sara; Nieri, Paola; Baldini, Francesco

    2017-02-15

    One of the main goals of nanomedicine in cancer is the development of effective drug delivery systems, primarily nanoparticles. Survivin, an overexpressed anti-apoptotic protein in cancer, represents a pharmacological target for therapy and a Molecular Beacon (MB) specific for survivin mRNA is available. In this study, the ability of polymethylmethacrylate nanoparticles (PMMA-NPs) to promote survivin MB uptake in human A549 cells was investigated. Fluorescent and positively charged core PMMA-NPs of nearly 60nm, obtained through an emulsion co-polymerization reaction, and the MB alone were evaluated in solution, for their analytical characterization; then, the MB specificity and functionality were verified after adsorption onto the PMMA-NPs. The carrier ability of PMMA-NPs in A549 was examined by confocal microscopy. With the optimized protocol, a hardly detectable fluorescent signal was obtained after incubation of the cells with the MB alone (fluorescent spots per cell of 1.90±0.40 with a mean area of 1.04±0.20µm 2 ), while bright fluorescent spots inside the cells were evident by using the MB loaded onto the PMMA-NPs. (27.50±2.30 fluorescent spots per cell with a mean area of 2.35±0.16µm 2 ). These results demonstrate the ability of the PMMA-NPs to promote the survivin-MB internalization, suggesting that this complex might represent a promising strategy for intracellular sensing and for the reduction of cancer cell proliferation. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Western blotting using chemiluminescent substrates.

    Science.gov (United States)

    Alegria-Schaffer, Alice

    2014-01-01

    Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture (Towbin et al., 1979). The technique enables indirect detection of protein samples immobilized on a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. The Design of a Quantitative Western Blot Experiment

    Directory of Open Access Journals (Sweden)

    Sean C. Taylor

    2014-01-01

    Full Text Available Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013 and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

  2. Ultrasensitive Detection of Proteins on Western Blots with Semiconducting Polymer Dots

    OpenAIRE

    Ye, Fangmao; Smith, Polina B.; Wu, Changfeng; Chiu, Daniel T.

    2013-01-01

    We demonstrate ultrasensitive fluorescence imaging of proteins on Western blots using a bright, compact, and orange-emitting semiconducting polymer dot (CN-PPV). We achieved a detection limit at the single-picogram level in dot blots; with conventional Western blotting, we detected 50 pg of transferrin and trypsin inhibitor after SDS-PAGE and transfer onto a PVDF membrane. Our method does not require any additional equipment or time compared to the conventional procedure with traditional fluo...

  3. Detection of proteins on blot transfer membranes.

    Science.gov (United States)

    Sasse, Joachim; Gallagher, Sean R

    2003-11-01

    In the basic and alternate protocols of this unit, proteins are stained after electroblotting from polyacrylamide gels to blot transfer membranes. If the samples of interest are electrophoresed in duplicate and transferred to a blot transfer membrane, half of the membrane can be stained to determine the efficiency of transfer to the membrane and the other half can be used for immunoblotting (i.e., western blotting). Detection limits of each staining method are given along with a list of compatible blot transfer membranes and gels. A support protocol describes a method for alkali treatment that enhances subsequent staining of bound proteins.

  4. Visualization of mcr mRNA in a methanogen by fluorescence in situ hybridization with an oligonucleotide probe and two-pass tyramide signal amplification (two-pass TSA-FISH).

    Science.gov (United States)

    Kubota, Kengo; Ohashi, Akiyoshi; Imachi, Hiroyuki; Harada, Hideki

    2006-09-01

    Two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a horseradish peroxidase (HRP)-labeled oligonucleotide probe was applied to detect prokaryotic mRNA. In this study, mRNA of a key enzyme for methanogenesis, methyl coenzyme M reductase (mcr), in Methanococcus vannielii was targeted. Applicability of mRNA-targeted probes to in situ hybridization was verified by Clone-FISH. It was observed that sensitivity of two-pass TSA-FISH was significantly higher than that of TSA-FISH, which was further increased by the addition of dextran sulphate in TSA working solution. Signals from two-pass TSA-FISH were more reliable compared to the weak, spotty signals yielded by TSA-FISH.

  5. Western Blotting of the Endocannabinoid System.

    Science.gov (United States)

    Wager-Miller, Jim; Mackie, Ken

    2016-01-01

    Measuring expression levels of G protein-coupled receptors (GPCRs) is an important step for understanding the distribution, function, and regulation of these receptors. A common approach for detecting proteins from complex biological systems is Western blotting. In this chapter, we describe a general approach to Western blotting protein components of the endocannabinoid system using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose membranes, with a focus on detecting type 1 cannabinoid (CB1) receptors. When this technique is carefully used, specifically with validation of the primary antibodies, it can provide quantitative information on protein expression levels. Additional information can also be inferred from Western blotting such as potential posttranslational modifications that can be further evaluated by specific analytical techniques.

  6. Multiplexed Western Blotting Using Microchip Electrophoresis.

    Science.gov (United States)

    Jin, Shi; Furtaw, Michael D; Chen, Huaxian; Lamb, Don T; Ferguson, Stephen A; Arvin, Natalie E; Dawod, Mohamed; Kennedy, Robert T

    2016-07-05

    Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps.

  7. TLC-Blot (Far-Eastern Blot) and Its Application to Functional Lipidomics.

    Science.gov (United States)

    Taki, Takao

    2015-01-01

    A simple method for transfer of lipids-including phospholipids, glycolipids, and neutral lipids-from a high performance thin layer chromatography (HPTLC) plate to a polyvinylidene difluoride (PVDF) membrane, TLC-Blot (Far-Eastern Blot), and its biochemical applications are presented. This chapter presents the conventional procedures for separating lipid from tissue samples, cultured cells, and serum and the subsequent development of TLC. Individual lipids separated on an HPTLC plate can be transferred to the PVDF membrane quantitatively and also isolated from the lipid-blotted membrane by a one-step purification procedure. Immunodetection with monoclonal antibodies and treatment with lipid-metabolizing enzymes on the lipid-blotted membrane are possible. The method for identification of individual lipids transferred on the PVDF membrane using matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry (TLC-Blot/MALDI-TOF MS) is shown as a functional lipidomics application.

  8. Automated design of genomic Southern blot probes

    Directory of Open Access Journals (Sweden)

    Komiyama Noboru H

    2010-01-01

    Full Text Available Abstract Background Sothern blotting is a DNA analysis technique that has found widespread application in molecular biology. It has been used for gene discovery and mapping and has diagnostic and forensic applications, including mutation detection in patient samples and DNA fingerprinting in criminal investigations. Southern blotting has been employed as the definitive method for detecting transgene integration, and successful homologous recombination in gene targeting experiments. The technique employs a labeled DNA probe to detect a specific DNA sequence in a complex DNA sample that has been separated by restriction-digest and gel electrophoresis. Critically for the technique to succeed the probe must be unique to the target locus so as not to cross-hybridize to other endogenous DNA within the sample. Investigators routinely employ a manual approach to probe design. A genome browser is used to extract DNA sequence from the locus of interest, which is searched against the target genome using a BLAST-like tool. Ideally a single perfect match is obtained to the target, with little cross-reactivity caused by homologous DNA sequence present in the genome and/or repetitive and low-complexity elements in the candidate probe. This is a labor intensive process often requiring several attempts to find a suitable probe for laboratory testing. Results We have written an informatic pipeline to automatically design genomic Sothern blot probes that specifically attempts to optimize the resultant probe, employing a brute-force strategy of generating many candidate probes of acceptable length in the user-specified design window, searching all against the target genome, then scoring and ranking the candidates by uniqueness and repetitive DNA element content. Using these in silico measures we can automatically design probes that we predict to perform as well, or better, than our previous manual designs, while considerably reducing design time. We went on to

  9. [Better performance of Western blotting: quick vs slow protein transfer, blotting membranes and the visualization methods].

    Science.gov (United States)

    Kong, Ling-Quan; Pu, Ying-Hui; Ma, Shi-Kun

    2008-01-01

    To study how the choices of the quick vs slow protein transfer, the blotting membranes and the visualization methods influence the performance of Western blotting. The cellular proteins were abstracted from human breast cell line MDA-MB-231 for analysis with Western blotting using quick (2 h) and slow (overnight) protein transfer, different blotting membranes (nitrocellulose, PVDF and nylon membranes) and different visualization methods (ECL and DAB). In Western blotting with slow and quick protein transfer, the prestained marker presented more distinct bands on nitrocellulose membrane than on the nylon and PVDF membranes, and the latter also showed clear bands on the back of the membrane to very likely cause confusion, which did not occur with nitrocellulose membrane. PVDF membrane allowed slightly clearer visualization of the proteins with DAB method as compared with nitrocellulose and nylon membranes, and on the latter two membranes, quick protein transfer was likely to result in somehow irregular bands in comparison with slow protein transfer. With slow protein transfer and chemiluminescence for visualization, all the 3 membranes showed clear background, while with quick protein transfer, nylon membrane gave rise to obvious background noise but the other two membranes did not. Different membranes should be selected for immunoblotting according to the actual needs of the experiment. Slow transfer of the proteins onto the membranes often has better effect than quick transfer, and enhanced chemiluminescence is superior to DAB for protein visualization and allows highly specific and sensitive analysis of the protein expressions.

  10. BLOTS AND ALL: A HISTORY OF THE RORSCHACH INK BLOT TEST IN BRITAIN.

    Science.gov (United States)

    Hubbard, Katherine; Hegarty, Peter

    2016-01-01

    Despite the easily recognizable nature of the Rorschach ink blot test very little is known about the history of the test in Britain. We attend to the oft-ignored history of the Rorschach test in Britain and compare it to its history in the US. Prior to the Second World War, Rorschach testing in Britain had attracted advocates and critiques. Afterward, the British Rorschach Forum, a network with a high proportion of women, developed around the Tavistock Institute in London and The Rorschach Newsletter. In 1968, the International Rorschach Congress was held in London but soon after the group became less exclusive, and fell into decline. A comparative account of the Rorschach in Britain demonstrates how different national institutions invested in the 'projective hypothesis' according to the influence of psychoanalysis, the adoption of a nationalized health system, and the social positioning of 'others' throughout the twentieth century. In comparing and contrasting the history of the Rorschach in Britain and the US, we decentralize and particularize the history of North American Psychology. © 2016 Wiley Periodicals, Inc.

  11. Multistrip western blotting to increase quantitative data output.

    Science.gov (United States)

    Kiyatkin, Anatoly; Aksamitiene, Edita

    2009-01-01

    The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip western blotting increases the data output per single blotting cycle up to tenfold, allows concurrent monitoring of up to nine different proteins from the same loading of the sample, and substantially improves the data accuracy by reducing immunoblotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data, and therefore is beneficial to apply in biomedical diagnostics, systems biology, and cell signaling research.

  12. Post-staining electroblotting for efficient and reliable peptide blotting.

    Science.gov (United States)

    Lee, Der-Yen; Chang, Geen-Dong

    2015-01-01

    Post-staining electroblotting has been previously described to transfer Coomassie blue-stained proteins from polyacrylamide gel onto polyvinylidene difluoride (PVDF) membranes. Actually, stained peptides can also be efficiently and reliably transferred. Because of selective staining procedures for peptides and increased retention of stained peptides on the membrane, even peptides with molecular masses less than 2 kDa such as bacitracin and granuliberin R are transferred with satisfactory results. For comparison, post-staining electroblotting is about 16-fold more sensitive than the conventional electroblotting for visualization of insulin on the membrane. Therefore, the peptide blots become practicable and more accessible to further applications, e.g., blot overlay detection or immunoblotting analysis. In addition, the efficiency of peptide transfer is favorable for N-terminal sequence analysis. With this method, peptide blotting can be normalized for further analysis such as blot overlay assay, immunoblotting, and N-terminal sequencing for identification of peptide in crude or partially purified samples.

  13. Multistrip Western blotting to increase quantitative data output

    OpenAIRE

    Kiyatkin, Anatoly; Aksamitiene, Edita

    2009-01-01

    The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical Western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip Western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single mem...

  14. Genetic relatedness of orbiviruses by RNA-RNA blot hybridization

    International Nuclear Information System (INIS)

    Bodkin, D.K.

    1985-01-01

    RNA-RNA blot hybridization was developed in order to identify type-specific genes among double-stranded (ds) RNA viruses, to assess the genetic relatedness of dsRNA viruses and to classify new strains. Viral dsRNA segments were electrophoresed through 10% polyacrylamide gels, transferred to membranes, and hybridized to [5' 32 P]-pCp labeled genomic RNA from a related strain. Hybridization was performed at 52 0 C, 50% formamide, 5X SSC. Under these conditions heterologous RNA species must share ≥ 74% sequence homology in order to form stable dsRNA hybrids. Cognate genes of nine members of the Palyam serogroup of orbiviruses were identified and their sequence relatedness to the prototype. Palyam virus, was determined. Reciprocal blot hybridizations were performed using radiolabeled genomic RNA of all members of the Palyam serogroup. Unique and variant genes were identified by lack of cross-homology or by weak homology between segments. Since genes 2 and 6 exhibited the highest degree of sequence variability, response to the vertebrate immune system may be a major cause of sequence divergence among members of a single serogroup. Changuinola serogroup isolates were compared by dot-blot hybridization, while Colorado tick fever (CTF) serogroup isolates were compared by the RNA-RNA blot hybridization procedure described for reovirus and Palyam serogroup isolates. Preliminary blot hybridization data were also obtained on the relatedness of members of different Orbivirus serogroups

  15. Western Blot of Stained Proteins from Dried Polyacrylamide Gels

    Science.gov (United States)

    Gruber, Claudia; Stan-Lotter, Helga

    1996-01-01

    Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.

  16. An alternative method for processing northern blots after capillary transfer.

    Science.gov (United States)

    Nilsen, Timothy W

    2015-03-02

    Different laboratories use different methods for the prehybridization, hybridization, and washing steps of the northern blotting procedure. In this protocol, a northern blot is pretreated with Church and Gilbert hybridization buffer to block nonspecific probe-binding sites. The immobilized RNA is then hybridized to a DNA probe specific for the RNA of interest. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. The solutions and conditions described here may be ideal for those who prefer to use fewer ingredients in their solutions. This protocol is designed to achieve the same goals as other northern blotting approaches. It minimizes background (nonspecific adherence of probe to membrane and nonspecific hybridization) and maximizes specific hybridization to RNAs immobilized on a membrane. © 2015 Cold Spring Harbor Laboratory Press.

  17. Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines

    Directory of Open Access Journals (Sweden)

    Barredo Julio C

    2002-03-01

    Full Text Available Abstract Background We describe an alternative method to determine mRNA half-life (t1/2 based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. Results Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen® fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp. To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR® Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h. Conclusions We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate.

  18. Molecular Cloning, mRNA Expression, and Localization of the G-protein Subunit Galphaq in Sheep Testis and Epididymis

    Directory of Open Access Journals (Sweden)

    Zhen Li

    2016-12-01

    Full Text Available The reproductive function of G-protein subunit Galphaq (GNAQ, a member of the G protein alpha subunit family, has been extensively studied in humans and rats. However, no data is available on its status in ruminants. The objectives of this study were to evaluate the expression pattern of the GNAQ in the testis and epididymis of sheep by polymerase chain reaction (PCR. The mRNA expression levels were detected by real-time fluorescent quantitative PCR, and cellular localization of GNAQ in the testis and epididymis was examined by immunohistochemistry. Additionally, GNAQ protein was qualitatively evaluated via western blot, with the results indicating that similarities between GNAQ mRNA levels from sheep was highly conserved with those observed in Bos taurus and Sus scrofa. Our results also indicated that GNAQ exists in the caput and cauda epididymis of sheep, while GNAQ in the testis and epididymis was localized to Leydig cells, spermatogonial stem cells, spermatocytes, Sertoli cells, spermatid, principal cells, and epididymis interstitial cells. The concentrations of GNAQ mRNA and protein in the caput and cauda epididymis were significantly greater than those observed in the corpus epididymis (p<0.01 and testis (p<0.05. Our results indicated that GNAQ exists at high concentrations in the caput and cauda epididymis of sheep, suggesting that GNAQ may play an important role in gonad development and sperm maturation.

  19. Silver and gold nanoparticle coated membranes applied to protein dot blots

    International Nuclear Information System (INIS)

    Xie, F.; Drozdowicz-Tomsia, K.; Shtoyko, T.; Goldys, E. M.

    2011-01-01

    Detection and identification of low abundance biomarker proteins is frequently based on various types of membrane-based devices. Lowering of the protein detection limits is vital in commercial applications such as lateral flow assays and in Western blots widely used in proteomics. These currently suffer from insufficient detection sensitivity and low retention for small 2–5 kDa proteins. In this study, we report the deposition of two types of metal nanoparticles: gold colloids (50–95 nm diameter) and silver fractals onto a range of commonly used types of membranes including polyvinylidene fluoride (PVDF). Due to strong affinity of proteins to noble metals, such modified membranes have the potential to effectively capture trace proteins preventing their loss. The membranes modified by metal particles were characterized optically and by SEM. The membrane performance in protein dot blots was evaluated using the protein—fluorophore conjugates Deep Purple-bovine serum albumin and fluorescein—human serum albumin. We found that the metal nanoparticles increase light extinction by metals, which is balanced by increased fluorescence, so that the effective fluorescence signal is unchanged. This feature combined with the capture of proteins by the nanoparticles embedded in the membrane increases the detection limit of membrane assays.

  20. Routine Western blot to check autophagic flux : Cautions and recommendations

    NARCIS (Netherlands)

    Gomez-Sanchez, Ruben; Pizarro-Estrella, Elisa; Yakhine-Diop, Sokhna M. S.; Rodriguez-Arribas, Mario; Bravo-San Pedro, Jose M.; Fuentes, Jose M.; Gonzalez-Polo, Rosa A.

    2015-01-01

    At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible

  1. A brief review of other notable protein blotting methods.

    Science.gov (United States)

    Kurien, Biji T; Scofield, R Hal

    2009-01-01

    A plethora of methods have been used for transferring proteins from the gel to the membrane. These include centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low and high molecular weight proteins, blotting of Coomassie Brilliant Blue (CBB)-stained proteins from polyacrylamide gels to transparencies, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before MALDI tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

  2. Other notable protein blotting methods: a brief review.

    Science.gov (United States)

    Kurien, Biji T; Scofield, R Hal

    2015-01-01

    Proteins have been transferred from the gel to the membrane by a variety of methods. These include vacuum blotting, centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low- and high-molecular-weight proteins, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization mass spectrometric-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before matrix-assisted laser desorption/ionization tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes. All these methods are briefly reviewed in this chapter.

  3. In Situ Blotting : A Novel Method for Direct Transfer of Native Proteins from Sectioned Tissue to Blotting Membrane

    NARCIS (Netherlands)

    Okabe, Masashi; Nyakas, Csaba; Buwalda, Bauke; Luiten, Paul G.M.

    1993-01-01

    We describe a novel technique for direct transfer of native proteins from unfixed frozen tissue sections to an immobilizing matrix, e.g., nitrocellulose, polyvinyliden difluoride, or positively charged nylon membranes. Proteins are directly blotted onto the membrane, providing optimal accessibility

  4. Blotting from PhastGel to Membranes by Ultrasound.

    Science.gov (United States)

    Kost, Joseph; Azagury, Aharon

    2015-01-01

    Ultrasound based approach for enhanced protein blotting is proposed. Three minutes of ultrasound exposure (1 MHz, 2.5 W/cm(2)) was sufficient for a clear transfer of proteins from a polyacrylamide gel (PhastGel) to nitrocellulose or Nylon 66 Biotrans membrane. The proteins evaluated were prestained sodium dodecyl sulfate-polyacrylamide standards (18,500-106,000 Da) and 14C-labeled Rainbow protein molecular weight markers (14,300-200,000 Da).

  5. Multi-strip Western blotting to increase quantitative data output

    OpenAIRE

    Aksamitiene, Edita; Hoek, Jan B.; Kholodenko, Boris; Kiyatkin, Anatoly

    2007-01-01

    The qualitative and quantitative measurement of protein abundance and protein modification states are essential in understanding their role in diverse cellular processes. Traditional Western blotting technique, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. We propose a modified immunoblotting procedure, which is based on simultaneous transfer of proteins from multiple gel-strips onto the same membrane, and is compatible wi...

  6. Determination of Diagnostic Antigens in Cattle Amphistomiasis Using Western Blotting

    Directory of Open Access Journals (Sweden)

    A Halajian

    2009-05-01

    Full Text Available "nBackground: Mixed infection with amphistomes seems common in native cattle of Iran. The aim of this study was to determine diagnostic antigens in cattle mixed amphistomiasis."nMethods: Specific antigens of Cotylophoron cotylophorum, Gastrothylax crumenifer and Paramphisto­mum cervi (mixed infection, the most common species, were collected from cattle was deter­mined. Adult trematodes were collected from the rumen of naturally infected cattle at meat inspec­tion. After their homogenization and centrifugation, somatic antigens were prepared and ana­lyzed by SDS-PAGE. Specific antigens were determinated by western blot with homologous and heterolo­gous sera. SDS-PAGE of whole worms extract was performed at different concentrations and subse­quent gels staining. Immunoblotting analysis using sera from cattle naturally infected with am­phistomes, Dicrocoelium dendriticum, Fasciola spp. and hydatid cyst was performed."nResults: Electrophorese analysis of somatic antigens revealed the presence of 10 and 21 protein bands at 4 µgr/ml and 8 µgr/ml with molecular weights ranging from 25-120 and 25-150 kDa, respectively. The best result was taken at 8 mg/ml concentration. Although western blot of these proteins demon­strate 5 major antigenic polypeptides ranging from 50 to 100 kDa which were recognized by serum of cat­tle naturally infected with mixed amphistomes.

  7. Acid-Urea Gel Electrophoresis and Western Blotting of Histones.

    Science.gov (United States)

    Hazzalin, Catherine A; Mahadevan, Louis C

    2017-01-01

    Acid-urea gel electrophoresis offers significant advantages over SDS-PAGE for analysis of post-translational protein modifications, being capable of resolving proteins of similar size but varying in charge. Hence, it can be used to separate protein variants with small charge-altering differences in primary sequence, and is particularly useful in the analysis of histones whose charge variation arises from post-translational modification, such as phosphorylation or acetylation. On acid-urea gels, histones that carry multiple modifications, each with a characteristic charge, are resolved into distinct bands, the so-called "histone ladder." Thus, the extent and distribution of different modification states of histones can be visualized. Here, we describe the analysis of histone H3 by acid-urea gel electrophoresis and western blotting.

  8. Mammalian α-polymerase: cloning of partial complementary DNA and immunobinding of catalytic subunit in crude homogenate protein blots

    International Nuclear Information System (INIS)

    SenGupta, D.N.; Kumar, P.; Zmudzka, B.Z.; Coughlin, S.; Vishwanatha, J.K.; Robey, F.A.; Parrott, C.; Wilson, S.H.

    1987-01-01

    A new polyclonal antibody against the α-polymerase catalytic polypeptide was prepared by using homogeneous HeLa cellα-polymerase. The antibody neutralized α-polymerase activity and was strong and specific for the α-polymerase catalytic polypeptide (M/sub r/ 183,000) in Western blot analysis of crude extracts of HeLa cells. The antibody was used to screen a cDNA library of newborn rat brain poly(A+) RNA in λgt11. A positive phage was identified and plaque purified. This phage, designated λpolα1.2, also was found to be positive with an antibody against Drosophila α-polymerase. The insert in λpolα1.2 (1183 base pairs) contained a poly(A) sequence at the 3' terminus and a short in-phase open reading frame at the 5' terminus. A synthetic oligopeptide (eight amino acids) corresponding to the open reading frame was used to raise antiserum in rabbits. Antibody affinity purified from this serum was found to be immunoreactive against purified α-polymerase by enzyme-linked immunosorbent assay and was capable of immunoprecipitating α-polymerase. This indicated the λpolα1.2 insert encoded an α-polymerase epitope and suggested that the cDNA corresponded to an α-polymerase mRNA. This was confirmed in hybrid selection experiments using pUC9 containing the cDNA insert and poly(A+) RNA from newborn rat brain; the insert hybridized to mRNA capable of encoding α-polymerase catalytic polypeptides. Northern blot analysis of rat brain poly(A+) RNA revealed that this mRNA is ∼5.4 kilobases

  9. Characterization of Nora Virus Structural Proteins via Western Blot Analysis.

    Science.gov (United States)

    Ericson, Brad L; Carlson, Darby J; Carlson, Kimberly A

    2016-01-01

    Nora virus is a single stranded RNA picorna-like virus with four open reading frames (ORFs). The coding potentials of the ORFs are not fully characterized, but ORF3 and ORF4 are believed to encode the capsid proteins (VP3, VP4a, VP4b, and VP4c) comprising the virion. To determine the polypeptide composition of Nora virus virions, polypeptides from purified virus were compared to polypeptides detected in Nora virus infected Drosophila melanogaster. Nora virus was purified from infected flies and used to challenge mice for the production of antisera. ORF3, ORF4a, ORF4b, and ORF4c were individually cloned and expressed in E. coli; resultant recombinant proteins purified and were used to make monospecific antisera. Antisera were evaluated via Western blot against whole virus particles and Nora virus infected fly lysates. Viral purification yielded two particle types with densities of ~1.31 g/mL (empty particles) and ~1.33 g/mL (complete virions). Comparison of purified virus polypeptide composition to Nora virus infected D. melanogaster lysate showed the number of proteins in infected cell lysates is less than purified virus. Our results suggest the virion is composed of 6 polypeptides, VP3, VP4a, two forms of VP4b, and two forms of VP4c. This polypeptide composition is similar to other small RNA insect viruses.

  10. Cy5 total protein normalization in Western blot analysis.

    Science.gov (United States)

    Hagner-McWhirter, Åsa; Laurin, Ylva; Larsson, Anita; Bjerneld, Erik J; Rönn, Ola

    2015-10-01

    Western blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control. We used a prelabeling protocol based on Cy5 N-hydroxysuccinimide ester labeling that produces a linear signal response. We obtained a low coefficient of variation (CV) of 7% between the ratio of extracellular signal-regulated kinase (ERK1/2) target to Cy5 total protein control signals over the whole loading range from 2.5 to 20.0μg of Chinese hamster ovary cell lysate protein. Corresponding experiments using actin or tubulin as controls for normalization resulted in CVs of 13 and 18%, respectively. Glyceraldehyde-3-phosphate dehydrogenase did not produce a proportional signal and was not suitable for normalization in these cells. A comparison of ERK1/2 signals from labeled and unlabeled samples showed that Cy5 prelabeling did not affect antibody binding. By using total protein normalization we analyzed PP2A and Smad2/3 levels with high confidence. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. All-in-one detector of circulating mRNA based on a smartphone

    Science.gov (United States)

    Cmiel, Vratislav; Gumulec, Jaromir; Svoboda, Ondrej; Raudenska, Martina; Hudcova, Kristyna; Sekora, Jiri; Balogh, Jaroslav; Masarik, Michal; Provaznik, Ivo

    2016-03-01

    Metallothionein is significantly elevated in various tumors, notably in prostate cancer on both mRNA and protein level. We demonstrated a strong predictive potential of free circulating metallothionein 2A isoform mRNA for patients with this cancer. Circulating mRNA detection relies on expensive equipment and requires high level of expertise. In this work we developed compact "all-in-one" laboratory system which replace microvolume spectrophotometer, thermocycler and realtime PCR machines. We managed to design and construct a microprocessor controlled heating/cooling chamber that ensures required temperature gradient. The chamber includes implemented optical system to enable fluorescence excitation and fluorescence analysis using a smart-phone.

  12. Northern blot analysis to investigate the abundance of microorganisms

    International Nuclear Information System (INIS)

    Krause, D.O.

    2005-01-01

    areas known as hyper-variable regions which have a high degree of sequence variation. As a result of this structure, it is possible to design signature oligonucleotide probes varying in length from about 15 to 30 nucleotides that are diagnostic of microorganisms at the kingdom, domain, genus and even species level. These signature sequences can be used in a variety of applications such as PCR analysis, construction of clone libraries or direct probing of bulk rRNA. In this chapter, I provide detailed protocols for the analysis of extracted rRNA and give detailed procedures that must be followed to do northern blot analysis of bulk RNA extracted from the rumen

  13. Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening.

    Science.gov (United States)

    Ng, Khong Y; Machida, Kazuya

    2017-01-01

    With a growing number of high-throughput studies, structural analyses, and availability of protein-protein interaction databases, it is now possible to apply web-based prediction tools to SH2 domain-interactions. However, in silico prediction is not always reliable and requires experimental validation. Rosette assay is a dot blot-based reverse-phase assay developed for the assessment of binding between SH2 domains and their ligands. It is conveniently customizable, allowing for low- to high-throughput analysis of interactions between various numbers of SH2 domains and their ligands, e.g., short peptides, purified proteins, and cell lysates. The binding assay is performed in a 96-well plate (MBA or MWA apparatus) in which a sample spotted membrane is incubated with up to 96 labeled SH2 domains. Bound domains are detected and quantified using a chemiluminescence or near-infrared fluorescence (IR) imaging system. In this chapter, we describe a practical protocol for rosette assay to assess interactions between synthesized tyrosine phosphorylated peptides and a library of GST-tagged SH2 domains. Since the methodology is not confined to assessment of SH2-pTyr interactions, rosette assay can be broadly utilized for ligand and drug screening using different protein interaction domains or antibodies.

  14. Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.

    Directory of Open Access Journals (Sweden)

    Eikan Mishima

    Full Text Available The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A, N6-methyladenosine (m6A, pseudouridine, and 5-methylcytidine (m5C showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.

  15. Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.

    Science.gov (United States)

    Mishima, Eikan; Jinno, Daisuke; Akiyama, Yasutoshi; Itoh, Kunihiko; Nankumo, Shinnosuke; Shima, Hisato; Kikuchi, Koichi; Takeuchi, Yoichi; Elkordy, Alaa; Suzuki, Takehiro; Niizuma, Kuniyasu; Ito, Sadayoshi; Tomioka, Yoshihisa; Abe, Takaaki

    2015-01-01

    The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.

  16. Rift Valley fever virus NSS gene expression correlates with a defect in nuclear mRNA export.

    Science.gov (United States)

    Copeland, Anna Maria; Van Deusen, Nicole M; Schmaljohn, Connie S

    2015-12-01

    We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NSS gene, but not the N, GN or NSM genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NSS, confirming that expression of NSS is likely responsible for this phenomenon. Published by Elsevier Inc.

  17. Evaluation of mRNA expression levels and electrophysiological function of neuron-like cells derived from canine bone marrow stromal cells.

    Science.gov (United States)

    Nakano, Rei; Edamura, Kazuya; Sugiya, Hiroshi; Narita, Takanori; Okabayashi, Ken; Moritomo, Tadaaki; Teshima, Kenji; Asano, Kazushi; Nakayama, Tomohiro

    2013-10-01

    To investigate the in vitro differentiation of canine bone marrow stromal cells (BMSCs) into functional, mature neurons. Bone marrow from 6 adult dogs. BMSCs were isolated from bone marrow and chemically induced to develop into neurons. The morphology of the BMSCs during neuronal induction was monitored, and immunocytochemical analyses for neuron markers were performed after the induction. Real-time PCR methods were used to evaluate the mRNA expression levels of markers for neural stem or progenitor cells, neurons, and ion channels, and western blotting was used to assess the expression of neuronal proteins before and after neuronal induction. The electrophysiological properties of the neuron-like cells induced from canine BMSCs were evaluated with fluorescent dye to monitor Ca(2)+ influx. Canine BMSCs developed a neuron-like morphology after neuronal induction. Immunocytochemical analysis revealed that these neuron-like cells were positive for neuron markers. After induction, the cells' mRNA expression levels of almost all neuron and ion channel markers increased, and the protein expression levels of nestin and neurofilament-L increased significantly. However, the neuron-like cells derived from canine BMSCs did not have the Ca(2)+ influx characteristic of spiking neurons. Although canine BMSCs had neuron-like morphological and biochemical properties after induction, they did not develop the electrophysiological characteristics of neurons. Thus, these results have suggested that canine BMSCs could have the capacity to differentiate into a neuronal lineage, but the differentiation protocol used may have been insufficient to induce development into functional neurons.

  18. The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

    OpenAIRE

    Silva, Jillian M.; McMahon, Martin

    2014-01-01

    The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the convent...

  19. Development of a dot blot assay using gene probes for the detection of enteroviruses in water

    International Nuclear Information System (INIS)

    Margolin, A.B.

    1986-01-01

    Enteric viruses are viruses which replicate in the intestinal tract of man and animals. One mode of transmission for enteric viruses is the fecal-oral route. Drinking water which has been contaminated with sewage or sewage effluent has been implicated as a means for the spread of enteric viruses. Current methods for the detection of enteric viruses in water requires the use of animal cell culture. This technique has several drawbacks. More rapid techniques, such as fluorescent antibody or radioimmunoassay do not have the needed sensitivity to detect the low levels of virus found in contaminated water. An alternative technique for the detection of viruses in water was sought. Recent advances in recombinant DNA technology now makes it possible to detect viruses without the use of cell culture or antibodies. Gene probes that hybridize to the RNA of poliovirus and hepatitis A virus were tested for their ability to detect different enteric viruses. The probes were labeled with 32 P dCTP and 32 P dATP to a specific activity greater then 1.0 x 10 9 cpm/ug DNA. One infectious unit of poliovirus and hepatitis A virus was detected using labeled cDNA probes. Upon comparison, the dot blot assay was as sensitive as tissue culture for the detection of poliovirus in beef extract, secondary effluent, and tap water. Environmental samples, such as secondary effluent, reclaimed wastewater and unchlorinated drinking water were also assayed for poliovirus and hepatitis A virus with the use of gene probes. The results presented here offer an alternative method for screening water samples for the presence of enteric viruses

  20. Fluorescence spectroscopy

    DEFF Research Database (Denmark)

    Bagatolli, Luis

    2016-01-01

    Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses the foundati......Fluorescence spectroscopy is a powerful experimental tool used by scientists from many disciplines. During the last decades there have been important developments on distinct fluorescence methods, particularly those related to the study of biological phenomena. This chapter discusses...

  1. Quantum dot bio-conjugate: as a western blot probe for highly sensitive detection of cellular proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kale, Sonia [Agharkar Research Institute (India); Kale, Anup [University of Alabama, Center for Materials for Information Technology (United States); Gholap, Haribhau; Rana, Abhimanyu [National Chemical Laboratory, Physical and Materials Chemistry Division (India); Desai, Rama [National Centre for Cell Science (India); Banpurkar, Arun [University of Pune, Department of Physics (India); Ogale, Satishchandra, E-mail: sb.ogale@ncl.res.in [National Chemical Laboratory, Physical and Materials Chemistry Division (India); Shastry, Padma, E-mail: padma@nccs.res.in [National Centre for Cell Science (India)

    2012-03-15

    In the present study, we report a quantum dot (QD)-tailored western blot analysis for a sensitive, rapid and flexible detection of the nuclear and cytoplasmic proteins. Highly luminescent CdTe and (CdTe)ZnS QDs are synthesized by aqueous method. High resolution transmission electron microscopy, Raman spectroscopy, fourier transform infrared spectroscopy, fluorescence spectroscopy and X-ray diffraction are used to characterize the properties of the quantum dots. The QDs are functionalized with antibodies of prostate apoptosis response-4 (Par-4), poly(ADP-ribose) polymerases and {beta} actin to specifically bind with the proteins localized in the nucleus and cytoplasm of the cells, respectively. The QD-conjugated antibodies are used to overcome the limitations of conventional western blot technique. The sensitivity and rapidity of protein detection in QD-based approach is very high, with detection limits up to 10 pg of protein. In addition, these labels provide the capability of enhanced identification and localization of marker proteins in intact cells by confocal laser scanning microscopy.

  2. A Laboratory Exercise Illustrating the Sensitivity and Specificity of Western Blot Analysis

    Science.gov (United States)

    Chang, Ming-Mei; Lovett, Janice

    2011-01-01

    Western blot analysis, commonly known as "Western blotting," is a standard tool in every laboratory where proteins are analyzed. It involves the separation of polypeptides in polyacrylamide gels followed by the electrophoretic transfer of the separated polypeptides onto a nitrocellulose or polyvinylidene fluoride membrane. A replica of the…

  3. Zinc blotting assay for detection of zinc binding prolamin in barley (Hordeum vulgare) grain

    DEFF Research Database (Denmark)

    Uddin, Mohammad Nasir; Nielsen, Ane Langkilde-Lauesen; Vincze, Eva

    2014-01-01

    In plants, zinc is commonly found bound to proteins. In barley (Hordeum vulgare), major storage proteins are alcohol-soluble prolamins known as hordeins, and some of them have the potential to bind or store zinc. 65Zn overlay and blotting techniques have been widely used for detecting zinc......-binding protein. However, to our knowledge so far this zinc blotting assay has never been applied to detect a prolamin fraction in barley grains. A radioactive zinc (65ZnCl2) blotting technique was optimized to detect zinc-binding prolamins, followed by development of an easy-to-follow nonradioactive colorimetric...... zinc blotting method with a zinc-sensing dye, dithizone. Hordeins were extracted from mature barley grain, separated by SDS-PAGE, blotted on a membrane, renatured, overlaid, and probed with zinc; subsequently, zinc-binding specificity of certain proteins was detected either by autoradiography or color...

  4. Region specific regulation of glutamic acid decarboxylase mRNA expression by dopamine neurons in rat brain.

    Science.gov (United States)

    Lindefors, N; Brene, S; Herrera-Marschitz, M; Persson, H

    1989-01-01

    In situ hybridization histochemistry and RNA blots were used to study the expression of glutamic acid decarboxylase (GAD) mRNA in rats with or without a unilateral lesion of midbrain dopamine neurons. Two populations of GAD mRNA positive neurons were found in the intact caudate-putamen, substantia nigra and fronto-parietal cortex. In caudate-putamen, only one out of ten of the GAD mRNA positive neurons expressed high levels, while in substantia nigra every second of the positive neurons expressed high levels of GAD mRNA. Relatively few, but intensively labelled neurons were found in the intact fronto-parietal cerebral cortex. In addition, one out of six of the GAD mRNA positive neurons in the fronto-parietal cortex showed a low labeling. On the ipsilateral side, the forebrain dopamine deafferentation induced an increase in the number of neurons expressing high levels of GAD mRNA in caudate-putamen, and a decrease in fronto-parietal cortex. A smaller decrease was also seen in substantia nigra. However, the total number of GAD mRNA positive neurons were not significantly changed in any of these brain regions. The changes in the levels of GAD mRNA after the dopamine lesion were confirmed by RNA blot analysis. Hence, midbrain dopamine neurons appear to control neuronal expression of GAD mRNA by a tonic down-regulation in a fraction of GAD mRNA positive neurons in caudate-putamen, and a tonic up-regulation in a fraction of GAD mRNA positive neurons in fronto-parietal cortex and substantia nigra.

  5. [Impacts of the formula of Suoquanwan(SQW) on expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency].

    Science.gov (United States)

    Cao, Hong-Ying; Wu, Qing-He; Huang, Ping; He, Jin-Yang

    2009-06-01

    To observe the impacts of the formula of Suoquanwan (SQW) on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency. The model rats were induced by adenine (250 mg/kg) for 4 weeks, then treated respectively with SQW or dDAVP. The expression of AQP-2 mRNA and AVPR-V2 mRNA in kidney of Yang-deficiency model by realtime fluorescence quantitative PCR method were investigated. In model rats, the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney decreased, dDAVP and SQW high dose could increased the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. The others had no influence on the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney. SQW can increase the expression of AQP-2 mRNA and AVPR-V2 mRNA in the kidney of rat polyuria model of Yang-deficiency.

  6. High ALK mRNA expression has a negative prognostic significance in rhabdomyosarcoma

    Science.gov (United States)

    Bonvini, P; Zin, A; Alaggio, R; Pawel, B; Bisogno, G; Rosolen, A

    2013-01-01

    Background: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in cancer, but its clinical and functional importance remain controversial. Mutation or amplification of ALK, as well as its expression levels assessed by conventional immunohistochemistry methods, has been linked to prognosis in cancer, although with potential bias because of the semi-quantitative approaches. Herein, we measured ALK mRNA expression in rhabdomyosarcoma (RMS) and determined its clinical impact on patients' stratification and outcome. Methods: Specimens were obtained from RMS patients and cell lines, and ALK expression was analysed by quantitative RT–PCR, western blotting, IHC, and copy number analysis. Results: High ALK mRNA expression was detected in the vast majority of PAX3/7-FOXO1-positive tumours, whereas PAX3/7-FOXO1-negative RMS displayed considerably lower amounts of both mRNA and protein. Notably, ALK mRNA distinguished unfavourable PAX3/7-FOXO1-positive tumours from PAX3/7-FOXO1-negative RMS (Ptumour size (PALK mRNA levels were of prognostic relevance by Cox univariate regression analysis and correlated with increased risk of relapse (P=0.001) and survival (P=0.01), whereas by multivariate analysis elevated ALK mRNA expression resulted a negative prognostic marker when clinical stage was not included. Conclusion: Quantitative assessment of ALK mRNA expression helps to improve risk stratification of RMS patients and identifies tumours with adverse biological characteristics and aggressive behaviour. PMID:24149177

  7. Exciton-controlled fluorescence: application to hybridization-sensitive fluorescent DNA probe.

    Science.gov (United States)

    Okamoto, Akimitsu; Ikeda, Shuji; Kubota, Takeshi; Yuki, Mizue; Yanagisawa, Hiroyuki

    2009-01-01

    A hybridization-sensitive fluorescent probe has been designed for nucleic acid detection, using the concept of fluorescence quenching caused by the intramolecular excitonic interaction of fluorescence dyes. We synthesized a doubly thiazole orange-labeled nucleotide showing high fluorescence intensity for a hybrid with the target nucleic acid and effective quenching for the single-stranded state. This exciton-controlled fluorescent probe was applied to living HeLa cells using microinjection to visualize intracellular mRNA localization. Immediately after injection of the probe into the cell, fluorescence was observed from the probe hybridizing with the target RNA. This fluorescence rapidly decreased upon addition of a competitor DNA. Multicoloring of this probe resulted in the simple simultaneous detection of plural target nucleic acid sequences. This probe realized a large, rapid, reversible change in fluorescence intensity in sensitive response to the amount of target nucleic acid, and facilitated spatiotemporal monitoring of the behavior of intracellular RNA.

  8. INSITU BLOTTING - A NOVEL METHOD FOR DIRECT TRANSFER OF NATIVE PROTEINS FROM SECTIONED TISSUE TO BLOTTING MEMBRANE - PROCEDURE AND SOME APPLICATIONS

    NARCIS (Netherlands)

    OKABE, M; NYAKAS, C; BUWALDA, B; LUITEN, PGM

    We describe a novel technique for direct transfer of native proteins from unfixed frozen tissue sections to an immobilizing matrix, e.g., nitrocellulose, polyvinyliden difluoride, or positively charged nylon membranes. Proteins are directly blotted onto the membrane, providing optimal accessibility

  9. Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots.

    Science.gov (United States)

    Abeyrathne, Priyanka D; Lam, Joseph S

    2007-04-01

    A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.

  10. SDS-Polyacrylamide Electrophoresis and Western Blotting Applied to the Study of Asthma.

    Science.gov (United States)

    García-Solaesa, Virginia; Abad, Sara Ciria

    2016-01-01

    Western blotting is used to analyze proteins after being separated by electrophoresis and subsequently electro-transferred to a membrane. Once immobilized, a specific protein can be identified through its reaction with a labeled antibody or antigen. It is a methodology commonly used in biomedical research such as asthma studies, to assess the pathways of inflammatory mediators involved in the disease.Here, we describe an example of western blotting to determine the factors involved in asthma. In this chapter, the methodology of western blotting is reviewed, paying attention on potential problems and giving interesting recommendations.

  11. Effect of transportation stress on heat shock protein 70 concentration and mRNA expression in heart and kidney tissues and serum enzyme activities and hormone concentrations of pigs.

    Science.gov (United States)

    Yu, Hong; Bao, En-Dong; Zhao, Ru-Qian; Lv, Qiong-Xia

    2007-11-01

    To determine the enzymatic and hormonal responses, heat shock protein 70 (Hsp70) production, and Hsp70 mRNA expression in heart and kidney tissues of transport-stressed pigs. 24 pigs (mean weight, 20 +/- 1 kg). Pigs were randomly placed into groups of 12 each. One group was transported for 2 hours. The other group was kept under normal conditions and used as control pigs. Sera were used to detect triiodothyronine, thyroxine, and cortisol concentrations and alanine aminotransferase, aspartate aminotransferase, and creatine kinase activities. The heart and kidneys of anesthetized pigs were harvested and frozen in liquid nitrogen for quantification of Hsp70 and Hsp70 mRNA. No significant differences were detected in serum alanine aminotransferase activity and triiodothyronine and cortisol concentrations between groups; however, the serum creatine kinase and aspartate aminotransferase activities and thyroxine concentrations were higher in transported pigs. Densitometric readings of western blots revealed that the amount of Hsp70 in heart and kidney tissues was significantly higher in transported pigs, compared with control pigs. Results of fluorescence quantitative real-time PCR assay revealed that the Hsp70 mRNA transcription in heart tissue, but not kidney tissue, was significantly higher in transported pigs, compared with control pigs. Transportation imposed a severe stress on pigs that was manifested as increased serum activities of aspartate aminotransferase and creatine kinase and increased amounts of Hsp70 and Hsp70 mRNA expression in heart and kidney tissues. Changes in serum enzyme activities were related to the tissue damage of transport-stressed pigs.

  12. Multistrip Western blotting: a tool for comparative quantitative analysis of multiple proteins.

    Science.gov (United States)

    Aksamitiene, Edita; Hoek, Jan B; Kiyatkin, Anatoly

    2015-01-01

    The qualitative and quantitative measurements of protein abundance and modification states are essential in understanding their functions in diverse cellular processes. Typical Western blotting, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. Multistrip Western blotting is a modified immunoblotting procedure based on simultaneous electrophoretic transfer of proteins from multiple strips of polyacrylamide gels to a single membrane sheet. In comparison with the conventional technique, Multistrip Western blotting increases data output per single blotting cycle up to tenfold; allows concurrent measurement of up to nine different total and/or posttranslationally modified protein expression obtained from the same loading of the sample; and substantially improves the data accuracy by reducing immunoblotting-derived signal errors. This approach enables statistically reliable comparison of different or repeated sets of data and therefore is advantageous to apply in biomedical diagnostics, systems biology, and cell signaling research.

  13. Histone gene expression in early development of Xenopus laevis. Analysis of histone mRNA in oocytes and embryos by blot-hybridization and cell-free translation

    NARCIS (Netherlands)

    van Dongen, W. M.; Moorman, A. F.; Destrée, O. H.

    1983-01-01

    This study comprises the hybridization analysis of electrophoretically separated histone mRNAs from oocytes and embryos of Xenopus laevis, and analysis of in vitro translation products of these mRNAs on polyacrylamide gels containing sodium dodecyl sulfate (SDS) or Triton X-100. In oocytes and

  14. Creatine kinase and alpha-actin mRNA levels decrease in diabetic rat hearts

    International Nuclear Information System (INIS)

    Popovich, B.; Barrieux, A.; Dillmann, W.H.

    1987-01-01

    Diabetic cardiomyopathy is associated with cardiac atrophy and isoenzyme redistribution. To determine if tissue specific changes occur in mRNAs coding for α-actin and creatine kinase (CK), they performed RNA blot analysis. Total ventricular RNA from control (C) and 4 wk old diabetic (D) rats were hybridized with 32 P cDNA probes for α-actin and CK. A tissue independent cDNA probe, CHOA was also used. Signal intensity was quantified by photodensitometry. D CK mRNA was 47 +/- 16% lower in D vs C. Insulin increases CK mRNA by 20% at 1.5 hs, and completely reverses the deficit after 4 wks. D α-actin mRNA is 66 +/- 18% lower in D vs C. Insulin normalized α-actin mRNA by 5 hs. CHOA mRNA is unchanged in D vs C, but D + insulin CHOA mRNA is 30 +/- 2% lower than C. In rats with diabetic cardiomyopathy, muscle specific CK and α-actin mRNAs are decreased. Insulin treatment reverses these changes

  15. Improvement of western blot test specificity for detecting equine serum antibodies to Sarcocystis neurona.

    Science.gov (United States)

    Rossano, M G; Mansfield, L S; Kaneene, J B; Murphy, A J; Brown, C M; Schott, H C; Fox, J C

    2000-01-01

    Equine protozoal myeloencephalitis (EPM) is a neurological disease of horses and ponies caused by the apicomplexan protozoan parasite Sarcocystis neurona. The purposes of this study were to develop the most stringent criteria possible for a positive test result, to estimate the sensitivity and specificity of the EPM Western blot antibody test, and to assess the ability of bovine antibodies to Sarcocystis cruzi to act as a blocking agent to minimize false-positive results in the western blot test for S. neurona. Sarcocystis neurona merozoites harvested from equine dermal cell culture were heat denatured, and the proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a 12-20% linear gradient gel. Separated proteins were electrophoretically transferred to polyvinylidene fluoride membranes and blocked in 1% bovine serum albumin and 0.5% Tween-Tris-buffered saline. Serum samples from 6 horses with S. neurona infections (confirmed by culture from neural tissue) and 57 horses without infections (horses from the Eastern Hemisphere, where S. neurona does not exist) were tested by Western blot. Horses from both groups had reactivity to the 62-, 30-, 16-, 13-, 11-, 10.5-, and 10-kD bands. Testing was repeated with another step. Blots were treated with bovine S. cruzi antibodies prior to loading the equine samples. After this modification of the Western blot test, positive infection status was significantly associated with reactivity to the 30- and 16-kD bands (Pblot had a sample sensitivity of 100% and sample specificity of 98%. It is concluded that the specificity of the Western blot test is improved by blocking proteins not specific to S. neurona and using reactivity to the 30- and 16-kD bands as the criterion for a positive test.

  16. Evaluation of two commercial systems for automated processing, reading, and interpretation of Lyme borreliosis Western blots.

    Science.gov (United States)

    Binnicker, M J; Jespersen, D J; Harring, J A; Rollins, L O; Bryant, S C; Beito, E M

    2008-07-01

    The diagnosis of Lyme borreliosis (LB) is commonly made by serologic testing with Western blot (WB) analysis serving as an important supplemental assay. Although specific, the interpretation of WBs for diagnosis of LB (i.e., Lyme WBs) is subjective, with considerable variability in results. In addition, the processing, reading, and interpretation of Lyme WBs are laborious and time-consuming procedures. With the need for rapid processing and more objective interpretation of Lyme WBs, we evaluated the performances of two automated interpretive systems, TrinBlot/BLOTrix (Trinity Biotech, Carlsbad, CA) and BeeBlot/ViraScan (Viramed Biotech AG, Munich, Germany), using 518 serum specimens submitted to our laboratory for Lyme WB analysis. The results of routine testing with visual interpretation were compared to those obtained by BLOTrix analysis of MarBlot immunoglobulin M (IgM) and IgG and by ViraScan analysis of ViraBlot and ViraStripe IgM and IgG assays. BLOTrix analysis demonstrated an agreement of 84.7% for IgM and 87.3% for IgG compared to visual reading and interpretation. ViraScan analysis of the ViraBlot assays demonstrated agreements of 85.7% for IgM and 94.2% for IgG, while ViraScan analysis of the ViraStripe IgM and IgG assays showed agreements of 87.1 and 93.1%, respectively. Testing by the automated systems yielded an average time savings of 64 min/run compared to processing, reading, and interpretation by our current procedure. Our findings demonstrated that automated processing and interpretive systems yield results comparable to those of visual interpretation, while reducing the subjectivity and time required for Lyme WB analysis.

  17. Single-cell mRNA transfection studies: delivery, kinetics and statistics by numbers.

    Science.gov (United States)

    Leonhardt, Carolin; Schwake, Gerlinde; Stögbauer, Tobias R; Rappl, Susanne; Kuhr, Jan-Timm; Ligon, Thomas S; Rädler, Joachim O

    2014-05-01

    In artificial gene delivery, messenger RNA (mRNA) is an attractive alternative to plasmid DNA (pDNA) since it does not require transfer into the cell nucleus. Here we show that, unlike for pDNA transfection, the delivery statistics and dynamics of mRNA-mediated expression are generic and predictable in terms of mathematical modeling. We measured the single-cell expression time-courses and levels of enhanced green fluorescent protein (eGFP) using time-lapse microscopy and flow cytometry (FC). The single-cell analysis provides direct access to the distribution of onset times, life times and expression rates of mRNA and eGFP. We introduce a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby the dose-response relation. Our results establish a statistical framework for mRNA transfection and as such should advance the development of RNA carriers and small interfering/micro RNA-based drugs. This team of authors established a statistical framework for mRNA transfection by using a two-step stochastic delivery model that reproduces the number distribution of successfully delivered and translated mRNA molecules and thereby their dose-response relation. This study establishes a nice connection between theory and experimental planning and will aid the cellular delivery of mRNA molecules. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  18. Northern and Southern blot analysis of human RNA and DNA in autopsy material

    DEFF Research Database (Denmark)

    Larsen, S; Rygaard, K; Asnaes, S

    1992-01-01

    was obtained less than two days postmortem. Histological examination showing slight or no autolysis and the presence of ribosomal bands after gel electrophoresis were both indicative parameters of RNA preservation. DNA was appropriate for Southern blotting when the tissue was obtained less than three to five...

  19. Effects of Reusing Gel Electrophoresis and Electrotransfer Buffers on Western Blotting.

    Science.gov (United States)

    Heda, Ghanshyam D; Omotola, Oluwabukola B; Heda, Rajiv P; Avery, Jamie

    2016-09-01

    SDS-PAGE and Western blotting are 2 of the most commonly used biochemical methods for protein analysis. Proteins are electrophoretically separated based on their MWs by SDS-PAGE and then electrotransferred to a solid membrane surface for subsequent protein-specific analysis by immunoblotting, a procedure commonly known as Western blotting. Both of these procedures use a salt-based buffer, with the latter procedure consisting of methanol as an additive known for its toxicity. Previous reports present a contradictory view in favor or against reusing electrotransfer buffer, also known as Towbin's transfer buffer (TTB), with an aim to reduce the toxic waste. In this report, we present a detailed analysis of not only reusing TTB but also gel electrophoresis buffer (EB) on proteins of low to high MW range. Our results suggest that EB can be reused for at least 5 times without compromising the electrophoretic separation of mixture of proteins in an MW standard, BSA, and crude cell lysates. Additionally, reuse of EB did not affect the quality of subsequent Western blots. Successive reuse of TTB, on the other hand, diminished the signal of proteins of different MWs in a protein standard and a high MW membrane protein cystic fibrosis transmembrane-conductance regulator (CFTR) in Western blotting.

  20. A simple DNA recombination screening method by RT-PCR as an alternative to Southern blot

    DEFF Research Database (Denmark)

    Albers, Eliene; Sbroggiò, Mauro; Martin Gonzalez, Javier

    2017-01-01

    The generation of genetically engineered mouse models (GEMMs), including knock-out (KO) and knock-in (KI) models, often requires genomic screening of many mouse ES cell (mESC) clones by Southern blot. The use of large targeting constructs facilitates the recombination of exogenous DNA in a specific...

  1. COMPARISONS OF ELISA AND WESTERN BLOT ASSAYS FOR DETECTION OF CRYPTOSPORIDIUM ANTIBODY

    Science.gov (United States)

    A seroprevalence survey was conducted using ELISA and Western blot (WB) assays for antibody to three Cryptosporidium antigens on 380 blood donors in Jackson County, Oregon. The purpose was to determine if either assay could detect serological evidence of an outbreak which occurre...

  2. [Clinical manifestation of Lyme borreliosis in children with positive and negatiwe western blot results].

    Science.gov (United States)

    Ołdak, Elzbieta; Rozkiewicz, Doroto; Sulik, Artur

    2008-01-01

    In the afforested area of North-Eastern Poland the risk of Borrelia burgdorferi infection seems to be higher compared to the other regions. Because of unspecific clinical manifestation of Lyme borreliosis in children the positive ELISA IgM results should be confirmed with Western blot IgM tests. Retrospective analysis of clinical signs and symptoms of Lyme borreliosis in children with positive ELISA IgM and positive Western blot IgM results and in children with positive ELISA IgM and negative Western blot IgM results. The study included 20 children reactive with ELISA IgM (Bellco Biomedica, Austria), hospitalized in Pediatric Infectious Diseases Clinic in 2007 due to probable diagnosis of Lyme disease. All children were tested with B. burgdorferi Western blot IgM and/or IgG assay (DRG, Diagnostics, Germany) as a second-step diagnosis. In 10 (50% females, 50% males) out of 20 children the results were positive (borreliosis) and in other 10 (80% females, 20% males) the results were negative (controls). In both groups of patients the retrospective analysis of signs and symptoms was done. The most often clinical manifestation of Lyme borreliosis in children was neuroborreliosis. Children presented Lyme meningitis (30%), facial nerve palsy (10%) and chronic or recurrent headaches (40%), associated with vertigo (20%), weakness (30%), fever (40%), and fatigue syndrome (30%). One patient presented Lyme arthritis. Children of control group presented with unspecific symptoms like isolated headaches (40%), arthralgias (70%), myalgias (10%) and abdomen pain (20%) (1) The most frequent clinical presentation of Lyme borreliosis in analyzed children was neuroborreliosis; (2) Isolated arthralgias in children reactive with B. burgdorferi ELISA IgM need to be confirmed with Western blot assay before implementing the antibiotic therapy.

  3. Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines

    DEFF Research Database (Denmark)

    Nørgaard, P; Spang-Thomsen, M; Poulsen, H S

    1996-01-01

    In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII...... and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II m......RNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF...

  4. Fluorescence microscopy.

    Science.gov (United States)

    Sanderson, Michael J; Smith, Ian; Parker, Ian; Bootman, Martin D

    2014-10-01

    Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation. © 2014 Cold Spring Harbor Laboratory Press.

  5. Analysis of differentially expressed genes in two immunologically distinct strains of Eimeria maxima using suppression subtractive hybridization and dot-blot hybridization

    Science.gov (United States)

    2014-01-01

    Background It is well known that different Eimeria maxima strains exhibit significant antigenic variation. However, the genetic basis of these phenotypes remains unclear. Methods Total RNA and mRNA were isolated from unsporulated oocysts of E. maxima strains SH and NT, which were found to have significant differences in immunogenicity in our previous research. Two subtractive cDNA libraries were constructed using suppression subtractive hybridization (SSH) and specific genes were further analyzed by dot-blot hybridization and qRT-PCR analysis. Results A total of 561 clones were selected from both cDNA libraries and the length of the inserted fragments was 0.25–1.0 kb. Dot-blot hybridization revealed a total of 86 differentially expressed clones (63 from strain SH and 23 from strain NT). Nucleotide sequencing analysis of these clones revealed ten specific contigs (six from strain SH and four from strain NT). Further analysis found that six contigs from strain SH and three from strain NT shared significant identities with previously reported proteins, and one contig was presumed to be novel. The specific differentially expressed genes were finally verified by RT-PCR and qRT-PCR analyses. Conclusions The data presented here suggest that specific genes identified between the two strains may be important molecules in the immunogenicity of E. maxima that may present potential new drug targets or vaccine candidates for coccidiosis. PMID:24894832

  6. Optimization of northern analysis by vacuum-blotting, RNA-transfer visualization, and ultraviolet fixation

    International Nuclear Information System (INIS)

    Kroczek, R.A.; Siebert, E.

    1990-01-01

    We have optimized Northern analysis at several steps. Overnight electrophoresis was replaced by short gel runs and overnight capillary transfer by rapid vacuum-blotting adapted to Northern analysis. Short uv irradiation was used as a substitute for the usual RNA fixation by baking. Direct staining of RNA before electrophoresis made it possible to check RNA integrity and to evaluate the quality of the size separation immediately after electrophoresis. In this system, RNA transfer onto the membrane support could also be quickly assessed after the blotting step. The net result of all modifications was a doubling of the autoradiography signal compared with that obtained by modern Northern protocols. At the same time, the duration of the procedure was shortened drastically, allowing an autoradiography signal to be obtained within 24 h

  7. Solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters

    International Nuclear Information System (INIS)

    Valtorta, F.; Schiebler, W.; Jahn, R.; Ceccarelli, B.; Greengard, P.

    1986-01-01

    A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters, and the blotted polypeptides are phyosphorylated with the catalytic subunit of cyclic AMP (adenosine 3':5'-monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides

  8. DISKRIMINASI KELAMIN PADA IKAN TUNA SIRIP KUNING, Yellowfin tuna MENGGUNAKAN ANALISIS DOT BLOT DAN ELISA

    Directory of Open Access Journals (Sweden)

    Gusti Ngurah Permana

    2014-08-01

    Full Text Available Pemahaman tentang penentuan jenis kelamin dalam populasi induk merupakan hal yang sangat penting bagi keberhasilan program pembenihan. Pengukuran reaksi antibodi dan aktivitas hormon testosterone, serta estradiol adalah metode dengan potensi yang secara akurat dapat menentukan jenis kelamin ikan tanpa mematikan ikan. Tujuan penelitian ini adalah untuk mengetahui akurasi metode dot blot dan ELISA dengan 11-ketotestorsterone (11-KT yang tersedia secara komersial EIA-kit untuk membedakan jenis kelamin ikan tuna sirip kuning. Hasil analisis menunjukkan bahwa metode dot blot menghasilkan ekspresi vitelogenin tampak jelas pada individu betina dan efek plasma terlihat transparan, jika dibandingkan dengan individu jantan. Interpretasi dari metode ini memerlukan pengalaman dan keahlian dalam akurasi pembacaan hasil. Aktivitas hormon 11-KT dengan sampel klip sirip dan plasma memberikan hasil yang baik dengan aktivitas hormon terlihat jelas.

  9. Three-Dimensional Mapping of mRNA Export through the Nuclear Pore Complex

    Directory of Open Access Journals (Sweden)

    Steven J. Schnell

    2014-11-01

    Full Text Available The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE. Plenty of nuclear pore complexes (NPCs embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms.

  10. Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

    OpenAIRE

    Penna, Aubin; Cahalan, Michael

    2007-01-01

    Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After tr...

  11. Antibody responses to Borrelia burgdorferi detected by western blot vary geographically in Canada

    OpenAIRE

    Ogden, Nicholas H.; Arsenault, Julie; Hatchette, Todd F.; Mechai, Samir; Lindsay, L. Robbin

    2017-01-01

    Lyme disease is emerging in eastern and central Canada, and most cases are diagnosed using the two-tier serological test (Enzyme Immuno Assay [EIA] followed by Western blot [WB]). Simplification of this algorithm would be advantageous unless it impacts test performance. In this study, accuracy of individual proteins of the IgG WB algorithm in predicting the overall test result in samples from Canadians was assessed. Because Borrelia burgdorferi strains vary geographically in Canada, geographi...

  12. Characterization of the structure of the erythropoietin receptor by ligand blotting

    International Nuclear Information System (INIS)

    Atkins, H.L.; Broudy, V.C.; Papayannopoulou, T.

    1991-01-01

    Erythropoietin (Epo) regulates the growth and differentiation of erythroid cells by binding to a specific receptor. We characterized the native Epo receptor on erythroleukemia cell lines by ligand blotting. Solubilized cell membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and probed with 125I-Epo. Specificity was demonstrated by inhibition of 125I-Epo binding by unlabeled excess Epo but not other peptide growth factors and by the cellular distribution of the Epo binding protein. A single membrane protein of 61 Kd ± 4 Kd was sufficient to bind 125I Epo in both human (OCIM2, K562) and murine (GM979, Rauscher, DA-1) cell lines. This finding is consistent with the predicted size of the Epo receptor from the murine cDNA clone. However, chemical crosslinking of 125I-Epo to its receptor has identified two Epo binding proteins of 105 Kd and 85 Kd. This difference may occur because the receptor is size fractionated before Epo binding in the ligand blot, but after Epo binding in crosslinking studies. Ligand blotting demonstrates that the native Epo receptor is composed of a single 61-Kd Epo binding protein, and suggests the presence of additional proteins of 20 to 25 Kd that associate with the receptor after Epo binding

  13. Inheritance of resistance to barley yellow dwarf virus detected by northern blot analysis

    International Nuclear Information System (INIS)

    Lorens, G.F.; Falk, B.W.; Qualset, C.O.

    1989-01-01

    Development of wheat (Triticum aestivum L.) cultivars tolerant to the barley yellow dwarf virus disease (BYD) has been limited by lack of precision in rating plants for response to infection, usually done by visual scoring of plant symptoms under field conditions. Other methodologies have been developed to study the host/pathogen relationship and to assess resistance or susceptibility. In this study northern dot blot analysis was used to determine barley yellow dwarf virus (BYDV) RNA concentrations of six wheat cultivars that differed in visual BYD symptom expression. Plants were infected with the NYPAV (PAV) isolate of BYDV in the greenhouse. At several dates after inoculation crude plant extracts were blotted on nitrocellulose and hybridized with a 32 P-labeled probe of the pPA8 cDNA clone of BYDV. The distribution of PRC for the F 2 population was compared to the distribution of BYD visual symptom scores for 403 F 2 plants of a similar F 2 population of NS 879/4 x Seri 82 under field conditions. The results were qualitatively similar, suggesting that northern dot blot analysis to measure PRC may be useful in understanding the genetics of resistance to BYD. This technique, when incorporated into breeding programs, could be important in the development of highly tolerant wheat cultivars with reduced losses to BYD

  14. Banding pattern indicative of echinococcosis in a commercial cysticercosis western blot

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    Tappe D

    2009-09-01

    Full Text Available Abstract Objective A commercial cysticercosis Western blot was evaluated for serological cross-reactivity of sera from patients with alveolar (AE and cystic echinococcosis (CE. Methods A total of 161 sera were examined, including 31 sera from AE-patients, 11 sera from CE-patients, 9 sera from patients with other parasitic diseases and 109 sera from patients with unrelated medical conditions. All AE-and CE-sera were also examined by the echinococcosis Western blot. Results More sera from patients with AE than with CE showed cross-reactivity in the form of ladder-like patterns ("Mikado aspect" and untypical bands at 6-8 kDa (71% and 77.4% versus 27.3% and 45.5%, respectively. In contrast, triplets of bands in the area above 50 kDa and between 24 and 39-42 kDa were more frequent in CE than in AE sera. The fuzzy band at 50-55 kDa typical for cysticercosis was absent in all AE and CE sera. Conclusions Atypical banding patterns in the cysticercosis Western blot should raise the suspicion of a metacestode infection different from Taenia solium, i.e. Echinococcus multilocularis or E. granulosus, especially when the Mikado aspect and an altered 6-8 kDa band is visible in the absence of a fuzzy 50-55 kDa band.

  15. A streamlined Western blot exercise: An efficient and greener approach in the laboratory classroom.

    Science.gov (United States)

    Ness, Traci L; Robinson, Rebekah L; Mojadedi, Wais; Peavy, Lydia; Weiland, Mitch H

    2015-01-01

    SDS-PAGE and western blotting are two commonly taught protein detection techniques in biochemistry and molecular biology laboratory classrooms. A pitfall associated with incorporating these techniques into the laboratory is the significant wait times that do not allow students to obtain timely results. The waiting associated with SDS-PAGE comes from staining and destaining, whereas with western blotting it is the times required for antibody incubations and the numerous wash steps. This laboratory exercise incorporates 2,2,2-trichloroethanol (TCE) into the SDS-PAGE gel allowing for visualization of migrated proteins in a matter of minutes, saving both the time and chemical waste associated with traditional Coomassie staining. Additionally, TCE staining does not affect protein transfer eliminating the requirement for duplicated gels for total protein and western analyses. Protein transfer can be confirmed immediately without the use of Ponceau S staining. Lastly, this western blot procedure has been further shortened by using an HRP-conjugated primary antibody, which eliminates the secondary antibody incubation and washes, and uses a colorimetric detection to allow for visualization by students without the need for specialized equipment. © 2015 The International Union of Biochemistry and Molecular Biology.

  16. Standardization of Licorice and TCM Formulations Using Eastern Blot Fingerprinting Analysis

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    Yukihiro Shoyama

    2013-01-01

    Full Text Available To prepare the antiglycyrrhizin (GC monoclonal antibody (MAb, GC was treated with NaIO4 resulting in aldehyde which can be combined with carrier protein. An antigen conjugate was performed by a matrix-assisted laser desorption/ionization TOF mass spectrometry to determine the hapten numbers in the conjugate. Anti-GC MAb was prepared from a hybridoma which was fixed from the spleen cells producing anti-GC MAb and the myeloma cells after immunization. The TCM and licorice extract were developed by TLC and blotted to a polyvinylidene difluoride (PVDF membrane. The membrane was treated by NaIO4 and protein, enzyme labeled secondary MAb, and finally substrate was added. Clear spot appeared on PVDF membrane identifying GC against a background containing large amount of impurities. In eastern blotting, the GC molecule was divided into two functions. The aglycone part is recognized as an epitope and the sugar moiety can be combined to membrane. The specific reactivity of sugar moiety in the GC molecule against anti-GC MAb might be modified by the NaIO4 treatment on the membrane because glycyrrhetic acid 3-O-glucuronide can be stained although the cross-reactivity is only 4.3%. Eastern blotting for GC can not only apply for the standardization of licorice and TCM, but also it can open for the other bioactive products.

  17. Positive IgG Western Blot for Borrelia burgdorferi in Colombia

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    Palacios Ricardo

    1999-01-01

    Full Text Available In order to evaluate the presence of specific IgG antibodies to Borrelia burgdorferi in patients with clinical manifestations associated with Lyme borreliosis in Cali, Colombia, 20 serum samples from patients with dermatologic signs, one cerebrospinal fluid (CSF sample from a patient with chronic neurologic and arthritic manifestations, and twelve serum samples from individuals without clinical signs associated with Lyme borreliosis were analyzed by IgG Western blot. The results were interpreted following the recommendations of the Centers for Diseases Control and Prevention (CDC for IgG Western blots. Four samples fulfilled the CDC criteria: two serum specimens from patients with morphea (localized scleroderma, the CSF from the patient with neurologic and arthritic manifestations, and one of the controls. Interpretation of positive serology for Lyme disease in non-endemic countries must be cautious. However these results suggest that the putative "Lyme-like" disease may correlate with positivity on Western blots, thus raising the possibility that a spirochete genospecies distinct from B. burgdorferi sensu stricto, or a Borrelia species other than B. burgdorferi sensu lato is the causative agent. Future work will focus on a survey of the local tick and rodent population for evidence of spirochete species that could be incriminated as the etiologic agent.

  18. Use of a Western blot technique for the serodiagnosis of glanders

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    de Souza Marcilia MA

    2011-01-01

    Full Text Available Abstract Background The in vivo diagnosis of glanders relies on the highly sensitive complement fixation test (CFT. Frequently observed false positive results are troublesome for veterinary authorities and cause financial losses to animal owners. Consequently, there is an urgent need to develop a test with high specificity. Hence, a Western blot assay making use of a partly purified lipopolysaccaride (LPS containing antigen of three Burkholderia mallei strains was developed. The test was validated investigating a comprehensive set of positive and negative sera obtained from horses and mules from endemic and non endemic areas. Results The developed Western blot assay showed a markedly higher diagnostic specificity when compared to the prescribed CFT and therefore can be used as a confirmatory test. However, the CFT remains the test of choice for routine testing of glanders due to its high sensitivity, its feasibility using standard laboratory equipment and its worldwide distribution in diagnostic laboratories. Conclusions The CFT should be amended by the newly validated Western blot to increase the positive likelihood ratio of glanders serodiagnosis in non endemic areas or areas with low glanders prevalence. Its use for international trade of horses and mules should be implemented by the OIE.

  19. Estrogen receptor mRNA in mineralized tissues of rainbow trout: calcium mobilization by estrogen.

    Science.gov (United States)

    Armour, K J; Lehane, D B; Pakdel, F; Valotaire, Y; Graham, R; Russell, R G; Henderson, I W

    1997-07-07

    RT-PCR was undertaken on total RNA extracts from bone and scales of the rainbow trout, Oncorhynchus mykiss. The rainbow trout estrogen receptor (ER)-specific primers used amplified a single product of expected size from each tissue which, using Southern blotting, strongly hybridized with a 32P-labelled rtER probe under stringent conditions. These data provide the first in vivo evidence of ER mRNA in bone and scale tissues of rainbow trout and suggest that the effects of estrogen observed in this study (increased bone mineral and decreased scale mineral contents, respectively) may be mediated directly through ER.

  20. Patterns of Limnohabitans Microdiversity across a Large Set of Freshwater Habitats as Revealed by Reverse Line Blot Hybridization

    Science.gov (United States)

    Jezbera, Jan; Jezberová, Jitka; Kasalický, Vojtěch; Šimek, Karel; Hahn, Martin W.

    2013-01-01

    Among abundant freshwater Betaproteobacteria, only few groups are considered to be of central ecological importance. One of them is the well-studied genus Limnohabitans and mainly its R-BT subcluster, investigated previously mainly by fluorescence in situ hybridization methods. We designed, based on sequences from a large Limnohabitans culture collection, 18 RLBH (Reverse Line Blot Hybridization) probes specific for different groups within the genus Limnohabitans by targeting diagnostic sequences on their 16 S–23 S rRNA ITS regions. The developed probes covered in sum 92% of the available isolates. This set of probes was applied to environmental DNA originating from 161 different European standing freshwater habitats to reveal the microdiversity (intra-genus) patterns of the Limnohabitans genus along a pH gradient. Investigated habitats differed in various physicochemical parameters, and represented a very broad range of standing freshwater habitats. The Limnohabitans microdiversity, assessed as number of RLBH-defined groups detected, increased significantly along the gradient of rising pH of habitats. 14 out of 18 probes returned detection signals that allowed predictions on the distribution of distinct Limnohabitans groups. Most probe-defined Limnohabitans groups showed preferences for alkaline habitats, one for acidic, and some seemed to lack preferences. Complete niche-separation was indicated for some of the probe-targeted groups. Moreover, bimodal distributions observed for some groups of Limnohabitans, suggested further niche separation between genotypes within the same probe-defined group. Statistical analyses suggested that different environmental parameters such as pH, conductivity, oxygen and altitude influenced the distribution of distinct groups. The results of our study do not support the hypothesis that the wide ecological distribution of Limnohabitans bacteria in standing freshwater habitats results from generalist adaptations of these bacteria

  1. Patterns of Limnohabitans microdiversity across a large set of freshwater habitats as revealed by Reverse Line Blot Hybridization.

    Directory of Open Access Journals (Sweden)

    Jan Jezbera

    Full Text Available Among abundant freshwater Betaproteobacteria, only few groups are considered to be of central ecological importance. One of them is the well-studied genus Limnohabitans and mainly its R-BT subcluster, investigated previously mainly by fluorescence in situ hybridization methods. We designed, based on sequences from a large Limnohabitans culture collection, 18 RLBH (Reverse Line Blot Hybridization probes specific for different groups within the genus Limnohabitans by targeting diagnostic sequences on their 16 S-23 S rRNA ITS regions. The developed probes covered in sum 92% of the available isolates. This set of probes was applied to environmental DNA originating from 161 different European standing freshwater habitats to reveal the microdiversity (intra-genus patterns of the Limnohabitans genus along a pH gradient. Investigated habitats differed in various physicochemical parameters, and represented a very broad range of standing freshwater habitats. The Limnohabitans microdiversity, assessed as number of RLBH-defined groups detected, increased significantly along the gradient of rising pH of habitats. 14 out of 18 probes returned detection signals that allowed predictions on the distribution of distinct Limnohabitans groups. Most probe-defined Limnohabitans groups showed preferences for alkaline habitats, one for acidic, and some seemed to lack preferences. Complete niche-separation was indicated for some of the probe-targeted groups. Moreover, bimodal distributions observed for some groups of Limnohabitans, suggested further niche separation between genotypes within the same probe-defined group. Statistical analyses suggested that different environmental parameters such as pH, conductivity, oxygen and altitude influenced the distribution of distinct groups. The results of our study do not support the hypothesis that the wide ecological distribution of Limnohabitans bacteria in standing freshwater habitats results from generalist adaptations of

  2. Effects of electro-acupuncture on ovarian P450arom, P450c17α and mRNA expression induced by letrozole in PCOS rats.

    Science.gov (United States)

    Sun, Jie; Jin, Chunlan; Wu, Huangan; Zhao, Jimeng; Cui, Yunhua; Liu, Huirong; Wu, Lingxiang; Shi, Yin; Zhu, Bing

    2013-01-01

    Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS). Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA) in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD) rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA) consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg(-1) of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA) showed significantly decreased androgens (i.e., androstenedione and testosterone) with significantly increased estrogens (i.e., estrone, estradiol) and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive and

  3. Effects of Electro-Acupuncture on Ovarian P450arom, P450c17α and mRNA Expression Induced by Letrozole in PCOS Rats

    Science.gov (United States)

    Wu, Huangan; Zhao, Jimeng; Cui, Yunhua; Liu, Huirong; Wu, Lingxiang; Shi, Yin; Zhu, Bing

    2013-01-01

    Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS). Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA) in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD) rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA) consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg−1 of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA) showed significantly decreased androgens (i.e., androstenedione and testosterone) with significantly increased estrogens (i.e., estrone, estradiol) and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive and

  4. Effects of electro-acupuncture on ovarian P450arom, P450c17α and mRNA expression induced by letrozole in PCOS rats.

    Directory of Open Access Journals (Sweden)

    Jie Sun

    Full Text Available Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS. Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg(-1 of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA showed significantly decreased androgens (i.e., androstenedione and testosterone with significantly increased estrogens (i.e., estrone, estradiol and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive

  5. Expression of IL-1β mRNA in mice after whole body X-irradiation

    International Nuclear Information System (INIS)

    Nemoto, Kumie; Ishihara, Hiroshi; Tanaka, Izumi; Suzuki, Gen; Tsuneoka, Kazuko; Yoshida, Kazuko; Ohtsu, Hiroshi

    1995-01-01

    IL-1β is a stimulator of hematopoietic and inflammatory systems, and also acts as a radioprotector. After whole-body exposure to sublethal doses of ionizing radiation, the IL-1β mRNA level in spleen cells increases for a short time prior to regeneration of the spleen. We analyzed spleen cells of C3H/He mice after whole-body irradiation with 3 Gy x-rays to determine the cause of this short-term increase in the transcription level. An increase in the level of the message in spleen cells, found by Northern blot hybridization, reached its peak 5 to 7 days after irradiation. There was a low correlation between the curves of the mRNA level and the ratio of monocyte/macrophage lineage cells; a typical source of the message. Spleen macrophages that produce a large amount of the message were found 7 days after irradiation in an in situ hybridization experiment in which heterogeneous spleen cell populations were used. In contrast, spleen cells had no detectable levels of macrophages rich in IL-1β mRNA before and 17 days after irradiation. Additionally, the population of message-rich cells was 9.4% of the total number of monocytes/macrophages in the spleen. These results suggest that the short-term increase in IL-1β mRNA is a result of the heterogeneous differentiation of a subpopulation of spleen macrophages before regeneration of the spleen. (author)

  6. mRNA related to insulin family in human placenta

    International Nuclear Information System (INIS)

    Younes, M.A.; D'Agostino, J.B.; Frazier, M.L.; Besch, P.K.

    1986-01-01

    The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A + ) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A + ) RNA templates. Five hundred transformants were initially screened by colony hybridization using a 32 P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II

  7. mRNA related to insulin family in human placenta

    Energy Technology Data Exchange (ETDEWEB)

    Younes, M.A.; D' Agostino, J.B.; Frazier, M.L.; Besch, P.K.

    1986-03-01

    The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A/sup +/) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A/sup +/) RNA templates. Five hundred transformants were initially screened by colony hybridization using a /sup 32/P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II.

  8. Rift Valley fever virus NS{sub S} gene expression correlates with a defect in nuclear mRNA export

    Energy Technology Data Exchange (ETDEWEB)

    Copeland, Anna Maria; Van Deusen, Nicole M.; Schmaljohn, Connie S., E-mail: Connie.s.schmaljohn.civ@mail.mil

    2015-12-15

    We investigated the localization of host mRNA during Rift Valley fever virus (RVFV) infection. Fluorescence in situ hybridization revealed that infection with RVFV altered the localization of host mRNA. mRNA accumulated in the nuclei of RVFV-infected but not mock-infected cells. Further, overexpression of the NS{sub S} gene, but not the N, G{sub N} or NS{sub M} genes correlated with mRNA nuclear accumulation. Nuclear accumulation of host mRNA was not observed in cells infected with a strain of RVFV lacking the gene encoding NS{sub S}, confirming that expression of NS{sub S} is likely responsible for this phenomenon. - Highlights: • Rift Valley fever virus (RVFV) infection alters the localization of host mRNA. • mRNA accumulates in the nuclei of RVFV-infected but not mock-infected cells. • NS{sub S} is likely responsible for mRNA relocalization to the nucleus.

  9. Glycosaminoglycan blotting on nitrocellulose membranes treated with cetylpyridinium chloride after agarose-gel electrophoretic separation.

    Science.gov (United States)

    Maccari, Francesca; Volpi, Nicola

    2002-09-01

    We describe a method for blotting and immobilizing several nonsulfated and sulfated complex polysaccharides on membranes made hydrophilic and positively charged by a cationic detergent after their separation by conventional agarose gel electrophoresis. Nitrocellulose membranes were derivatized with the cationic detergent cetylpyridinium chloride (CPC) and mixtures of glycosaminoglycans (GAGs) were capillary-blotted after their separation in agarose gel electrophoresis in barium acetate/1,2-diaminopropane. Single purified species of variously sulfated polysaccharides were transferred onto the derivatized membranes after electrophoresis with an efficiency of 100% and stained with alcian blue (irreversible staining) and toluidine blue (reversible staining) permitting about 0.1 nug threshold of detection. Nonsulfated polyanions, hyaluronic acid, a fructose-containing polysaccharide with a chondroitin backbone purified from Escherichia coli U1-41, and its defructosylated product, were also electrophoretically separated and transferred onto membranes. The limit of detection for desulfated GAGs was about 0.1-0.5 nug after irreversible or reversible staining. GAG extracts from bovine, lung and aorta, and human aorta and urine were separated by agarose gel electrophoresis and blotted on CPC-treated nitrocellulose membranes. The polysaccharide composition of these extracts was determined. The membrane stained with toluidine blue (reversible staining) was destained and the same lanes used for immunological detection or other applications. Reversible staining was also applied to recover single species of polysaccharides after electrophoretic separation of mixtures of GAGs and their transfer onto membranes. Single bands were released from the membrane with an efficiency of 70-100% for further biochemical characterization.

  10. FANCD2 Western blot as a diagnostic tool for Brazilian patients with Fanconi anemia

    Directory of Open Access Journals (Sweden)

    D.V. Pilonetto

    2009-03-01

    Full Text Available Fanconi anemia is a rare hereditary disease showing genetic heterogeneity due to a variety of mutations in genes involved in DNA repair pathways, which may lead to different clinical manifestations. Phenotypic variability makes diagnosis difficult based only on clinical manifestations, therefore laboratory tests are necessary. New advances in molecular pathogenesis of this disease led researchers to develop a diagnostic test based on Western blot for FANCD2. The objective of the present study was to determine the efficacy of this method for the diagnosis of 84 Brazilian patients with Fanconi anemia, all of whom tested positive for the diepoxybutane test, and 98 healthy controls. The FANCD2 monoubiquitinated isoform (FANCDS+/FANCD2L- was not detected in 77 patients (91.7%. In 2 patients (2.4%, there was an absence of both the monoubiquitinated and the non-ubiquitinated proteins (FANCD2S-/FANCD2L- and 5 patients (5.9% had both isoforms (FANCD2S+/FANCD2L+. This last phenotype suggests downstream subtypes or mosaicism. All controls were diepoxybutane negative and were also negative on the FANCD2 Western blot. The Western blot for FANCD2 presented a sensitivity of 94% (79/84 and specificity of 100% (98/98. This method was confirmed as an efficient approach to screen Brazilian patients with deleterious mutations on FANCD2 (FANCD2S-/FANCD2L- or other upstream genes of the FA/BRCA pathway (FANCDS+/FANCD2L-, to confirm the chromosome breakage test and to classify patients according to the level of FA/BRCA pathway defects. However, patients showing both FANCD2 isoforms (FANCD2S+/FANCD2L+ require additional studies to confirm mutations on downstream Fanconi anemia genes or the presence of mosaicism.

  11. Northern blots: capillary transfer of RNA from agarose gels and filter hybridization using standard stringency conditions.

    Science.gov (United States)

    Rio, Donald C

    2015-03-02

    In this protocol, an RNA sample, fractionated by gel electrophoresis, is transferred from the gel onto a membrane by capillary transfer. Short-wave UV light is used to fix the transferred RNA to the membrane. The membrane is then pretreated to block nonspecific probe-binding sites, and hybridization of the immobilized RNA to a (32)P-labeled DNA or RNA probe specific for the mRNA of interest is performed. Finally, the membrane is washed and subjected to autoradiography or phosphorimaging. Because exposure to UV cross-links the RNA to the membrane, the membrane can be stripped and hybridized with other probes. The procedure is suitable for detecting poly(A)(+)-selected mRNA or mRNA in total cellular RNA if the target transcript is relatively abundant. Using DNA or RNA probes labeled to 1 × 10(8)-10 × 10(8) cpm/µg, it should be possible to detect ∼5 pg of a specific RNA. © 2015 Cold Spring Harbor Laboratory Press.

  12. Blotting Assisted by Heating and Solvent Extraction for DESI-MS Imaging

    Science.gov (United States)

    Cabral, Elaine C.; Mirabelli, Mario F.; Perez, Consuelo J.; Ifa, Demian R.

    2013-06-01

    Imprints of potato sprout ( Solanum tuberosum L.), gingko leaves (Gingko biloba L. ) and strawberries (Fragaria x ananassa Duch. ) were successfully imaged by desorption electrospray ionization mass spectrometry (DESI-MS) on TLC plates through blotting assisted by heating and/or solvent extraction. Ion images showing the distribution of significant compounds such as glycoalkaloid toxins in potato sprout, ginkgolic acids and flavonoids in ginkgo leaves, and sugars and anthocyanidin in strawberry were obtained. Practical implications of this work include analysis of a wide range of irregular or soft materials by different imprinting conditions without requiring the addition of matrices or use of specific kinds of surfaces.

  13. Proteínas inmunodominantes de Brucella Melitensis evaluadas por Western Blot

    Directory of Open Access Journals (Sweden)

    Elizabeth Anaya

    1997-01-01

    Full Text Available Se separaron extractos de proteínas totales de Brucella melitensis en gel 15% SDS-PAGE. Su seroreactividad fue analizada por Western Blot con resultados satisfactorios. Para éste propósito sueros controles negativos (n=03, sueros de pacientes con brucelosis (n=34, cólera (n=12, tifoidea (n=02 y tuberculosis (n=02 fueron usados. Esta prueba inmunodiagnóstica detectó bandas seroreactivas altamente específicas (100% correspondientes a 8,14,18, un complejo de 25-48 y 58kDa. La sensibilidad del test fue del 90% usando los sueros antes mencionados.

  14. Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

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    Daniela Toro-Ascuy

    2016-11-01

    Full Text Available The human immunodeficiency virus type-1 (HIV-1 unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1, Staufen double-stranded RNA binding protein 1/2 (STAU1/2, or components of miRNA-induced silencing complex (miRISC and processing bodies (PBs. More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A, allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2, an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries.

  15. Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

    Directory of Open Access Journals (Sweden)

    N.S. Sato

    2004-07-01

    Full Text Available Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples, from healthy blood donors (50 samples, individuals with sexually transmitted disease other than syphilis (3 samples, and from individuals with other spirochetal diseases such as Lyme disease (20 samples and leptospirosis (3 samples. The assay had sensitivity of 95.1% (95% CI, 86.1 to 98.7% and a specificity of 94.7% (95% CI, 87.0 to 98.7%; a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

  16. Analysis of sperm antigens by sodium dodecyl sulfate gel/protein blot radioimmunobinding method

    International Nuclear Information System (INIS)

    Lee, C.Y.G.; Huang, Y.S.; Hu, P.C.; Gomel, V.; Menge, A.C.

    1982-01-01

    A radioimmunobinding method based on the blotting of renatured proteins from sodium dodecyl sulfate gels on to nitrocellulose filter papers was developed to analyze the sperm antigens that elicit serum anti-sperm antibodies. In rabbits, serum anti-sperm antibodies were raised by immunization with homologous epididymal spermatozoa mixed with complete Freund's adjuvant. The raised antisera from either male or female rabbits were shown to react with three major sperm protein bands on sodium dodecyl sulfate gels with the corresponding molecular weights of about 70,000 +/- 5000, 14,000, and 13,000, respectively. In humans, the monoclonal antibodies against human sperm were raised by a hybridoma technique. Out of six independent hybrid cell lines that were generated, three of them were shown to secrete immunoglobulins that react with the same two protein bands on sodium dodecyl sulfate gels, which have the approximate molecular weight of 10,000. The same procedure was also used to analyze human serum samples that were shown to contain anti-sperm antibodies by the known techniques. Unique sperm antigens that elicit anti-sperm antibodies in humans were identified and correlated. The results of this study suggest that sodium dodecyl sulfate gel/protein blot radioimmunobinding method may be a sensitive and useful tool for the study of sperm antigens that elicit autoimmune responses and their association with human infertility

  17. Immunodiagnosis of Echinococcus Infections: Confirmatory Testing and Species Differentiation by a New Commercial Western Blot

    Science.gov (United States)

    Liance, Martine; Janin, Veronique; Bresson-Hadni, Solange; Vuitton, Dominique-Angele; Houin, Rene; Piarroux, Renaud

    2000-01-01

    The Echinococcus Western Blot IgG (LDBIO Diagnostics, Lyon, France), using a whole larval antigen from Echinococcus multilocularis, was evaluated for serodiagnosis and differentiation between two human parasitic infections of worldwide importance: cystic echinococcosis, due to Echinococcus granulosus, and alveolar echinococcosis, due to E. multilocularis. Fifty and 61 serum samples from patients with cystic and alveolar echinococcosis, respectively, were used for assessing diagnostic sensitivity. The sensitivity of the assay was compared with those of screening tests used for these applications. Sera used for assessing cross-reactivities were from 154 patients with other diseases, either parasitic or not. The assay allowed the detection of serum immunoglobulin G antibodies in 97% of Echinococcus-infected patients. It had a higher sensitivity than screening assays for the detection for each echinococcosis. The assay allowed us to correctly distinguish between E. granulosus- and E. multilocularis-infected patients in 76% of cases. It did not allow us to distinguish active from inactive forms of both echinococcoses. The occurrence of cross-reactivities with neurocysticercosis indicates the necessity for retesting sera with species-specific antigens, for rare patients with neurologic disorders. This study shows the usefulness of the commercially available Echinococcus Western Blot IgG for the serological confirmation of human echinococcosis. PMID:11015390

  18. Checking transfer efficiency and equal loading via qualitative optical way in western blotting.

    Science.gov (United States)

    Gong, Jun-Hua; Gong, Jian-Ping; Zheng, Kai-Wen

    2017-11-01

    The ability to determine that successful transfer and equal loading occur prior to using primary antibodies is important. And total protein staining is commonly used to check transfer efficiency and normalization, which play a crucial role in western blotting. Ponceau S and coomassie blue are commonly used, but there are disadvantages reported in recent years. Therefore, we are interested in finding another method, which is cheap, easy and fast. As we know, protein binding region of PVDF membrane is still hydrophilic when carbinol volatilizes, however, the non-protein binding region of PVDF membrane became hydrophobic again. And this different wettability between non-protein binding region and protein binding region of Polyvinylidene difluoride membrane may be used to check transfer efficiency and equal loading in western blotting. Based on the principle above, we describe an optical approach where an experimenter can observe that the proteins have been transferred to the membrane without any staining within minutes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Detection of alien genetic introgressions in bread wheat using dot-blot genomic hybridisation.

    Science.gov (United States)

    Rey, María-Dolores; Prieto, Pilar

    2017-01-01

    Simple, reliable methods for the identification of alien genetic introgressions are required in plant breeding programmes. The use of genomic dot-blot hybridisation allows the detection of small Hordeum chilense genomic introgressions in the descendants of genetic crosses between wheat and H. chilense addition or substitution lines in wheat when molecular markers are difficult to use. Based on genomic in situ hybridisation, DNA samples from wheat lines carrying putatively H. chilense introgressions were immobilised on a membrane, blocked with wheat genomic DNA and hybridised with biotin-labelled H. chilense genomic DNA as a probe. This dot-blot screening reduced the number of plants necessary to be analysed by molecular markers or in situ hybridisation, saving time and money. The technique was sensitive enough to detect a minimum of 5 ng of total genomic DNA immobilised on the membrane or about 1/420 dilution of H. chilense genomic DNA in the wheat background. The robustness of the technique was verified by in situ hybridisation. In addition, the detection of other wheat relative species such as Hordeum vulgare , Secale cereale and Agropyron cristatum in the wheat background was also reported .

  20. Self-amplifying mRNA vaccines.

    Science.gov (United States)

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

    2015-01-01

    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Clinical significance of LUNX mRNA, CK19 mRNA, CEA mRNA expression in detecting micrometastasis from lung cancer

    International Nuclear Information System (INIS)

    Zhu Guangying; Liu Delin; Chen Jie

    2003-01-01

    Objective: To evaluate the sensitivity, specificity and clinical significance of CK19 mRNA, CEA mRNA and LUNX mRNA for detecting micrometastasis by sampling the peripheral blood and regional lymph nodes of lung cancer patients. Methods: Reverse transcriptase chain reaction (RT-PCR) was used to detect LUNX mRNA, CK19 mRNA, CEA mRNA for micrometastasis by sampling the peripheral blood of 48 lung cancer patients and 44 regional lymph nodes of such patients treated by curative resection. Peripheral blood of 30 patients with pulmonary benign lesions and 10 normal healthy volunteers and lymph nodes of 6 patients with benign pulmonary diseases served as control. Results: 1) LUNX mRNA, CK19 mRNA, CEA mRNA were expressed in all (35/35) lung cancer tissues. 2) In the peripheral blood from 48 lung cancer patients, 30 (62.5%) were positive for LUNX mRNA, 24 (50.0%) positive for CK19 mRNA and 32(66.7%) positive for CEA mRNA. The positive detection rates of micrometastasis in 44 lymph nodes from lung cancer patients were 36.4% (16 out of 44) for LUNX mRNA, 27.3% (12 out of 44) for CK19 mRNA and 40.9% (18 out of 44) for CEA mRNA. 3) In the 30 blood samples from patients with pulmonary benign diseases, 2 (6.7%) expressed CK19 mRNA, but none expressed LUNX mRNA or CEA mRNA. All the 3 molecular markers were negative in the 10 blood samples from healthy volunteers. In 11 lymph nodes from patients with pulmonary benign lesions, none was positive for any of the three markers. 4) In 44 regional lymph nodes from lung cancer patients, 6 (13.6%) were positive for metastasis by histopathological examination, with a positive rate significantly lower than that of the RT-PCR (P<0.05). 5) The micrometastatic positive rate in the peripheral blood of 40 non-small cell lung cancer (NSCLC) patients was significantly related to TNM stage (P=0.01). Conclusions: LUNX mRNA, CK19 MRNA, CEA mRNA are all appropriate target genes for the detection of micrometastasis from lung cancer. LUNX mRNA and CEA mRNA

  2. Arc mRNA induction in striatal efferent neurons associated with response learning.

    Science.gov (United States)

    Daberkow, D P; Riedy, M D; Kesner, R P; Keefe, K A

    2007-07-01

    The dorsal striatum is involved in motor-response learning, but the extent to which distinct populations of striatal efferent neurons are differentially involved in such learning is unknown. Activity-regulated, cytoskeleton-associated (Arc) protein is an effector immediate-early gene implicated in synaptic plasticity. We examined arc mRNA expression in striatopallidal vs. striatonigral efferent neurons in dorsomedial and dorsolateral striatum of rats engaged in reversal learning on a T-maze motor-response task. Male Sprague-Dawley rats learned to turn right or left for 3 days. Half of the rats then underwent reversal training. The remaining rats were yoked to rats undergoing reversal training, such that they ran the same number of trials but ran them as continued-acquisition trials. Brains were removed and processed using double-label fluorescent in situ hybridization for arc and preproenkephalin (PPE) mRNA. In the reversal, but not the continued-acquisition, group there was a significant relation between the overall arc mRNA signal in dorsomedial striatum and the number of trials run, with rats reaching criterion in fewer trials having higher levels of arc mRNA expression. A similar relation was seen between the numbers of PPE(+) and PPE(-) neurons in dorsomedial striatum with cytoplasmic arc mRNA expression. Interestingly, in behaviourally activated animals significantly more PPE(-) neurons had cytoplasmic arc mRNA expression. These data suggest that Arc in both striatonigral and striatopallidal efferent neurons is involved in striatal synaptic plasticity mediating motor-response learning in the T-maze and that there is differential processing of arc mRNA in distinct subpopulations of striatal efferent neurons.

  3. Should we ignore western blots when selecting antibodies for other applications?

    DEFF Research Database (Denmark)

    Uhlén, Mathias

    2017-01-01

    .In the Human Protein Atlas (HPA) program, we have validated more than 24,000 in-house-generated antibodies directed to 17,000 human target proteins2. Although there is often a correlation between performance in different applications, we have observed many examples of antibodies that show strong support...... applications and that this influences the epitopes exposed on the target protein, which might have profound consequences for the ability of a given antibody to bind specifically to its target. As an example, proteins that are analyzed by immunohistochemistry (IHC) are normally first cross-linked with formalin.......In conclusion, western blot and protein array analyses can indeed be useful tools when selecting specific antibodies for other applications. The use of these methods is encouraged both for antibody providers and users, and antibodies with signs of cross-reactivity in these applications should be treated...

  4. Electrospun nitrocellulose and nylon: Design and fabrication of novel high performance platforms for protein blotting applications

    Directory of Open Access Journals (Sweden)

    Bowlin Gary L

    2007-10-01

    Full Text Available Abstract Background Electrospinning is a non-mechanical processing strategy that can be used to process a variety of native and synthetic polymers into highly porous materials composed of nano-scale to micron-scale diameter fibers. By nature, electrospun materials exhibit an extensive surface area and highly interconnected pore spaces. In this study we adopted a biological engineering approach to ask how the specific unique advantages of the electrospinning process might be exploited to produce a new class of research/diagnostic tools. Methods The electrospinning properties of nitrocellulose, charged nylon and blends of these materials are characterized. Results Nitrocellulose electrospun from a starting concentration of Conclusion The flexibility afforded by electrospinning process makes it possible to tailor blotting membranes to specific applications. Electrospinning has a variety of potential applications in the clinical diagnostic field of use.

  5. HIV‑2 antibody detection after indeterminate or negative HIV‑1 Western blot in Cuba, 2005-2008.

    Science.gov (United States)

    Díaz, Dervel F; Ortiz, Eva; Martín, Dayamí; Nibot, Carmen; Rizo, Adis; Silva, Eladio

    2012-01-01

    INTRODUCTION Differentiating between HIV-1 and HIV-2 infection is the first step to understanding HIV transmission, epidemiology and pathogenesis in geographical areas where both viruses circulate. In Cuba, positive results in mixed HIV-1/2 screening assays are confirmed by HIV-1 Western blot. Indeterminate results constitute the main limitation of this test and HIV-2 infection is among their possible causes; hence the importance of second-stage screening and confirmatory tests for HIV-2 infection. OBJECTIVE Investigate the contribution of HIV-2 antibodies to negative or indeterminate HIV-1 Western blot results in serum samples from 2005 through 2008 in Cuba. METHODS HIV-2 reactivity was studied using the ELISA DAVIH-VIH-2 diagnostic kit (Cuba) in 1723 serum samples with negative or indeterminate results for HIV-1 Western blot from January 2005 through December 2008. Duplicate sera reactive by ELISA were confirmed by HIV-2 Western blot, results interpreted according to WHO criteria. The epidemiological interview established by Cuba's National Program for Prevention and Control Sexually-Transmitted Diseases and HIV/AIDS was applied to HIV-2 Western blot-positive patients. RESULTS Among all sera studied, HIV-2 ELISA identified 12 reactive serum samples (0.70%) and 1711 non-reactive (99.30%). Western blot analysis of the 12 ELISA-reactive samples confirmed two positive samples (16.67%), 4 negative (33.33%) and 6 indeterminate (50%). Positive samples reacted against the p16, p26, gp36, p53, p56, p68 and gp105 proteins. All 12 ELISA-reactive samples belonged to the HIV-1 Western blot indeterminate group. The two HIV-2-positive samples showed well defined reactivity to gp160, p53, p55 and p34 of HIV-1. HIV-1 seroconversion was observed in all 10 remaining samples during serological followup. CONCLUSIONS Two new HIV-2 seropositive cases were diagnosed using DAVIH-VIH-2 and HIV-2 Western blot in indeterminate HIV-1 Western blot samples. Results support the recommendation

  6. TSE strain differentiation in mice by immunohistochemical PrP(Sc) profiles and triplex Western blot.

    Science.gov (United States)

    van Keulen, Lucien J M; Langeveld, Jan P M; Dolstra, Corry H; Jacobs, Jorg; Bossers, Alex; van Zijderveld, Fred G

    2015-10-01

    TSE strains are routinely identified by their incubation period and vacuolation profile in the brain after intracerebral inoculation and serial passaging in inbred mouse lines. There are some major drawbacks to this method that are related to the variation in vacuolation that exists in the brains of mice infected with the same TSE strain and to variation between observers and laboratories in scoring vacuolation and determining the final incubation period. We investigated the potential of PrP(Sc) immunohistochemistry and triplex Western blotting as possible alternative methods to differentiate between TSE strains. TSE reference strains ME7, 87A/87V, 22A/22C, 79A/79V and 301C/301V were intracerebrally inoculated in RIII or VM inbred mice that differ in their PrP genotype. Immunohistochemical PrP(Sc) profiles were drawn up by scanning light microscopy both on coronal and sagittal sections. On the basis of the localization of PrP(Sc) in the cerebral cortex, hippocampus, and cerebellar cortex and the overall type of PrP(Sc) staining, all TSE strains could be well differentiated from each other through their typical strain dependent characteristics. In addition, Western blot showed that the combination of glycosylation profile and 12B2 epitope content of PrP(Sc) allowed to distinguish between all reference strains except for ME7 and 22A in VM mice. TSE strains in mice can be identified on the basis of their PrP(Sc) profile alone. The potential to identify TSE strains in ruminants with these PrP(Sc) profiles after a single primary passage in mice will be the topic of future studies. © 2014 British Neuropathological Society.

  7. Increase of prolactin mRNA in the rat hypothalamus after intracerebroventricular injection of VIP or PACAP.

    Science.gov (United States)

    Bredow, S; Kacsóh, B; Obál, F; Fang, J; Krueger, J M

    1994-10-17

    Vasoactive intestinal peptide (VIP), the structurally homologous pituitary adenylate cyclase-activating peptide (PACAP) and the pituitary hormone, prolactin (PRL) enhance rapid eye movement sleep (REMS). VIP and PACAP are both inducers of PRL gene expression and release in the pituitary gland. Little is known about PRL regulation in the brain although it is hypothesized that the REMS-promoting activity of i.c.v. administered VIP may be mediated via the activation of cerebral PRL. To test whether VIP or PACAP in fact increase intracerebral mRNA, the peptides (VIP: 30 or 300 pmol; PACAP: 220 pmol) were injected i.c.v. into rats at dark onset. 1 h later, cDNA was synthesized from purified hypothalamic mRNA. Standardized amounts were analysed for PRL using the polymerase chain reaction followed by Southern blotting and hybridization. Compared with beta-actin mRNA levels, both VIP and PACAP increased PRL mRNA levels in a dose-dependent fashion though VIP was more effective on a molar basis. The previously reported alternatively spliced PRL mRNA (lacking exon 4) was not detected. The data support the hypothesis that the REMS-promoting activity of central VIP and PACAP might be mediated by cerebral PRL.

  8. Pressure overload stimulated cardiac hypertrophy leads to a rapid decrease in the mRNA for creatine kinase

    International Nuclear Information System (INIS)

    Boheler, K.; Popovich, B.; Dillmann, W.H.

    1987-01-01

    Cardiac hypertrophy (CH) leads to a decrease in creatine kinase (CK) enzymatic activity. To determine if the mRNA for CK also decreases with CH, they performed the following studies. Cardiac RNA was isolated from rats subjected to either abdominal aortic stenosis (AS) or sham surgery. Through Northern blot analysis, total cardiac RNA was quantitated with a CK specific 32 P-labelled cDNA clone. At 3 and 8 days post-constriction, the mRNA for CK decreases by 54.6 +/- 7% and 65.3 +/- 18% respectively, whereas the heart weight increases by 19% and 37% relative to controls. Further studies indicate that CK mRNA also decreases by 41.8% in hypothyroid rats (Tx) but decreases by a total of 68.1% in Tx rats subjected to 8 days of AS. Pressure overload stimulated CH leads to a rapid decrease in CK mRNA in normal and Tx rats. This CK mRNA decrease may account for the decreased efficiency of contraction seen in CH

  9. Effect of electroacupuncture on the mRNA and protein expression of Rho-A and Rho-associated kinase II in spinal cord injury rats

    Directory of Open Access Journals (Sweden)

    You-jiang Min

    2017-01-01

    Full Text Available Electroacupuncture is beneficial for the recovery of spinal cord injury, but the underlying mechanism is unclear. The Rho/Rho-associated kinase (ROCK signaling pathway regulates the actin cytoskeleton by controlling the adhesive and migratory behaviors of cells that could inhibit neurite regrowth after neural injury and consequently hinder the recovery from spinal cord injury. Therefore, we hypothesized electroacupuncture could affect the Rho/ROCK signaling pathway to promote the recovery of spinal cord injury. In our experiments, the spinal cord injury in adult Sprague-Dawley rats was caused by an impact device. Those rats were subjected to electroacupuncture at Yaoyangguan (GV3, Dazhui (GV14, Zusanli (ST36 and Ciliao (BL32 and/or monosialoganglioside treatment. Behavioral scores revealed that the hindlimb motor functions improved with those treatments. Real-time quantitative polymerase chain reaction, fluorescence in situ hybridization and western blot assay showed that electroacupuncture suppressed the mRNA and protein expression of Rho-A and Rho-associated kinase II (ROCKII of injured spinal cord. Although monosialoganglioside promoted the recovery of hindlimb motor function, monosialoganglioside did not affect the expression of Rho-A and ROCKII. However, electroacupuncture combined with monosialoganglioside did not further improve the motor function or suppress the expression of Rho-A and ROCKII. Our data suggested that the electroacupuncture could specifically inhibit the activation of the Rho/ROCK signaling pathway thus partially contributing to the repair of injured spinal cord. Monosialoganglioside could promote the motor function but did not suppress expression of RhoA and ROCKII. There was no synergistic effect of electroacupuncture combined with monosialoganglioside.

  10. Spectroscopic detection of fluorescent protein marker gene activity in genetically modified plants

    Science.gov (United States)

    Liew, O. W.; Chong, Jenny P. C.; Asundi, Anand K.

    2005-04-01

    This work focuses on developing a portable fibre optic fluorescence analyser for rapid identification of genetically modified plants tagged with a fluorescent marker gene. Independent transgenic tobacco plant lines expressing the enhanced green fluorescence protein (EGFP) gene were regenerated following Agrobacterium-mediated gene transfer. Molecular characterisation of these plant lines was carried out at the DNA level by PCR screening to confirm their transgenic status. Conventional transgene expression analysis was then carried out at the RNA level by RT-PCR and at the protein level by Western blotting using anti-GFP rabbit antiserum. The amount of plant-expressed EGFP on a Western blot was quantified against known amounts of purified EGFP by scanning densitometry. The expression level of EGFP in transformed plants was found to range from 0.1 - 0.6% of total extractable protein. A comparison between conventional western analysis of transformants and direct spectroscopic quantification using the fibre optic fluorescence analyser was made. The results showed that spectroscopic measurements of fluorescence emission from strong EGFP expressors correlated positively with Western blot data. However, the fluorescence analyser was also able to identify weakly expressing plant transformants below the detection limit of colorimetric Western blotting.

  11. Localization of glucocorticoid receptor mRNA in the male rat brain by in situ hybridization

    International Nuclear Information System (INIS)

    Aronsson, M.; Fuxe, K.; Dong, Y.; Agnati, L.F.; Okret, S.; Gustafsson, J.A.

    1988-01-01

    The localization and distribution of mRNA encoding the glucocorticoid receptor (GR) was investigated in tissue sections of the adult male rat brain by in situ hybridization and RNA blot analysis. GR mRNA levels were measured by quantitative autoradiography with 35S- and 32P-labeled RNA probes, respectively. Strong labeling was observed within the pyramidal nerve cells of the CA1 and CA2 areas of the hippocampal formation, in the granular cells of the dentate gyrus, in the parvocellular nerve cells of the paraventricular hypothalamic nucleus, and in the cells of the arcuate nucleus, especially the parvocellular part. Moderate labeling of a large number of nerve cells was observed within layers II, III, and VI of the neocortex and in many thalamic nuclei, especially the anterior and ventral nuclear groups as well as several midline nuclei. Within the cerebellar cortex, strong labeling was observed all over the granular layer. In the lower brainstem, strong labeling was found within the entire locus coeruleus and within the mesencephalic raphe nuclei rich in noradrenaline and 5-hydroxytryptamine cell bodies, respectively. A close correlation was found between the distribution of GR mRNA and the distribution of previously described GR immunoreactivity. These studies open the possibility of obtaining additional information on in vivo regulation of GR synthesis and how the brain may alter its sensitivity to circulating glucocorticoids

  12. Comparison of Multispot EIA with Western blot for confirmatory serodiagnosis of HIV.

    Science.gov (United States)

    Torian, Lucia V; Forgione, Lisa A; Punsalang, Amado E; Pirillo, Robert E; Oleszko, William R

    2011-12-01

    Recent improvements in the sensitivity of immunoassays (IA) used for HIV screening, coupled with increasing recognition of the importance of rapid point-of-care testing, have led to proposals to adjust the algorithm for serodiagnosis of HIV so that screening and confirmation can be performed using a dual or triple IA sequence that does not require Western blotting for confirmation. One IA that has been proposed as a second or confirmatory test is the Bio-Rad Multispot(®) Rapid HIV-1/HIV-2 Test. This test would have the added advantage of differentiating between HIV-1 and HIV-2 antibodies. To compare the sensitivity and type-specificity of an algorithm combining a 3rd generation enzyme immunoassay (EIA) followed by a confirmatory Multispot with the conventional algorithm that combines a 3rd generation EIA (Bio-Rad GS HIV-1/HIV-2 Plus O EIA) followed by confirmatory Western blot (Bio-Rad GS HIV-1 WB). 8760 serum specimens submitted for HIV testing to the New York City Public Health Laboratory between May 22, 2007, and April 30, 2010, tested repeatedly positive on 3rd generation HIV-1-2+O EIA screening and received parallel confirmatory testing by WB and Multispot (MS). 8678/8760 (99.1%) specimens tested WB-positive; 82 (0.9%) tested WB-negative or indeterminate (IND). 8690/8760 specimens (99.2%) tested MS-positive, of which 14 (17.1%) had been classified as negative or IND by WB. Among the HIV-1 WB-positive specimens, MS classified 26 (0.29%) as HIV-2. Among the HIV-1 WB negative and IND, MS detected 12 HIV-2. MS detected an additional 14 HIV-1 infections among WB negative or IND specimens, differentiated 26 HIV-1 WB positives as HIV-2, and detected 12 additional HIV-2 infections among WB negative/IND. A dual 3rd generation EIA algorithm incorporating MS had equivalent HIV-1 sensitivity to the 3rd generation EIA-WB algorithm and had the added advantage of detecting 12 HIV-2 specimens that were not HIV-1 WB cross-reactors. In this series an algorithm using EIA

  13. Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism.

    Science.gov (United States)

    Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K; Lehtonen, Jukka Y A

    2016-04-20

    As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3'-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

    Directory of Open Access Journals (Sweden)

    MORALES Olga Lucía

    2002-01-01

    Full Text Available Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB assay was performed using excretory/secretory antigens (E/S of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

  15. Performance of PCR-reverse blot hybridization assay for detection of rifampicin-resistant Mycobacterium leprae.

    Science.gov (United States)

    Wang, Hye-young; Kim, Hyunjung; Kim, Yeun; Bang, Hyeeun; Kim, Jong-Pill; Hwang, Joo Hwan; Cho, Sang-Nae; Kim, Tae Ue; Lee, Hyeyoung

    2015-10-01

    Drug resistance in Mycobacterium leprae is a significant problem in countries where leprosy is endemic. A sensitive, specific, and high-throughput reverse blot hybridization assay (REBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. It has been shown that resistance to RIF in M. leprae involves mutations in the rpoB gene encoding the -subunit of the RNA polymerase. The PCR-REBA simultaneously detects both 6 wild-type regions and 5 different mutations (507 AGC, 513 GTG, 516 TAT, 531 ATG, and 531 TTC) including the most prevalent mutations at positions 507 and 531. Thirty-one clinical isolates provided by Korea Institute of Hansen-s Disease were analyzed by PCR-REBA with RIF resistance of rpoB gene. As a result, missense mutations at codons 507 AGC and 531 ATG with 2-nucleotide substitutions were found in one sample, and a missense mutation at codon 516 TAT and ΔWT6 (deletion of 530-534) was found in another sample. These cases were confirmed by DNA sequence analysis. This rapid, simple, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in M. leprae.

  16. Direct tissue blot immunoassay for detection of Xylella fastidiosa in olive trees

    Directory of Open Access Journals (Sweden)

    Khaled DJELOUAH

    2015-01-01

    Full Text Available A direct tissue blot immunoassay (DTBIA technique has been compared with ELISA and PCR for detection of Xylella fastidiosa in olive trees from Apulia (southern Italy. Fresh cross-sections of young twigs and leaf petioles were printed onto nitrocellulose membranes and analyzed in the laboratory. Analyses of a first group of 61 samples gave similar efficiency for the three diagnostic techniques for detection the bacterium (24 positive and 36 negative samples, except for a single sample which was positive only with DTBIA and PCR. Similar results were obtained by separately analyzing suckers and twigs collected from different sectors of tree canopies of a second group of 20 olive trees (ten symptomatic and ten symptomless. In this second test the three diagnostic techniques confirmed the irregular distribution of the bacterium in the tree canopies and erratic detectability of the pathogen in the young suckers. It is therefore necessary to analyse composite samples per tree which should be prepared with twigs collected from different sides of the canopy. The efficiency comparable to ELISA and PCR, combined with the advantages of easier handling, speed and cost, make DTBIA a valid alternative to ELISA in large-scale surveys for occurrence of X. fastidiosa. Moreover, the printing of membranes directly in the field prevents infections spreading to Xylella-free areas, through movement of plant material with pathogen vectors for laboratory testing.

  17. Evaluation of an enzyme-linked immunoelectrotransfer blot test for the confirmatory serodiagnosis of human toxocariasis

    Directory of Open Access Journals (Sweden)

    William H Roldán

    2009-05-01

    Full Text Available To improve the serodiagnosis of human toxocariasis, a sensitive and specific enzyme-linked immunoelectrotransfer blot (EITB-IgG test was developed and evaluated using Toxocara canislarvae excretory-secretory antigens for detecting anti-Toxocara IgG antibodies. The EITB-IgG profile of toxocariasis was characterized by comparing 27 sera from patients with toxocariasis, 110 sera from healthy subjects and 186 sera from patients with other helminth diseases (ascariasis, ancylostomiasis, trichuriasis, enterobiasis, strongyloidiasis, hymenolepiasis, diphyllobothriasis, taeniasis, cysticercosis, hydatidosis and fascioliasis. Antigenic bands of 24, 28, 30, 35, 56, 117, 136 and 152 kDa were predominantly recognized in sera from all patients with toxocariasis. However, only bands of 24-35 kDa were highly specific for Toxocara infection (98.3%, whereas other antigenic bands observed displayed cross-reactivity. Additionally, when the results of the EITB-IgG test were compared to those of the ELISA-IgG test, a 100% concordance was observed for positive results in human toxocariasis cases. The concordance for negative results between the two tests for healthy subjects and patients with other helminth diseases were 96.3% and 53.7%, respectively, showing that the EITB-IgG test has a higher specificity than ELISA. In conclusion, the EITB-IgG test is a very useful tool to confirm the serological diagnosis of human toxocariasis.

  18. Use of nitrocellulose blotting for the study of hepatitis B surface antigen electrophoresed in agarose gels

    Energy Technology Data Exchange (ETDEWEB)

    McMichael, J C; Greisiger, L M; Millman, I [Institute for Cancer Research, Philadelphia, PA (USA). Fox Chase Cancer Center

    1981-08-28

    Nitrocellulose-protein blotting of serum electrophoresed in agarose gels has been adapted for the study of hepatitis B surface antigen (HBsAg). /sup 125/I-labeled anti-HBs was used as the antigen probe, and the electrophoretic migration was monitored by autoradiography. The method required 3 ..mu..l or less of serum and could detect as little as 1 pg of purified HBsAg. Typically, the authors observed two bands of HBsAg; a moving band which migrated about one-third the distance moved by human serum albumin and a non-migratory band which remained at the loading site. Some examples of the use of the method include: (1) empirical methods for correlating HBsAg concentration in serum to film darkness; (2) observations of mobility changes in serial sera from dialysis patients with chronic HBsAg antigenemia; and (3) detection of related antigens such as antigen from the PLC/PRF/5 hepatoma tissue culture line and the cross-reacting woodchuck hepatitis virus surface antigen (WHsAg).

  19. Retrospective study of hemoparasites in cattle in southern Italy by reverse line blot hybridization.

    Science.gov (United States)

    Ceci, Luigi; Iarussi, Fabrizio; Greco, Beatrice; Lacinio, Rosanna; Fornelli, Stefania; Carelli, Grazia

    2014-06-01

    Tick-borne diseases are widespread in tropical and temperate regions and are responsible for important economic losses in those areas. In order to assess the presence and prevalence of various pathogens in southern Italy, we retrospectively analyzed cattle blood samples collected for a previous study in 2000 using reverse line blot (RLB) hybridization. The study had been carried out in three regions of southern Italy on 1,500 randomly selected and apparently healthy adult cattle. RLB showed that 43.7% of the cattle were positive for nine different species of hemoparasites with either a single infection or a mixed infection. Theileria buffeli was the most common species found, being present in 27.3% of the animals, followed by Anaplasma marginale in 18.1%, Anaplasma centrale in 13.8%, Babesia bigemina and Anaplasma bovis in 4.2%, Anaplasma phagocytophilum in 1.7%, Babesia bovis in 1.6%, Babesia major in 0.2% and Babesia divergens in 0.1%. Complete blood counts showed different degrees of anemia in 363 animals (24.2%) and of these, 169 were RLB-positive for at least one pathogen. Among the ticks that were collected from the cattle, the following species were identified: Rhipicephalus bursa, Ixodes ricinus, Hyalomma marginatum, Boophilus annulatus, Dermacentor marginatus and Haemaphysalis (sulcata, parva, inermis and punctata). The results obtained confirmed the spread of endemic tick-borne pathogens in the regions studied.

  20. Indeterminate human immunodeficiency virus western blot results in Iranian patients with discordant screening assay results

    International Nuclear Information System (INIS)

    Ravanshad, M.; Sabahi, F.; Mahboudi, F.; Sabahi, F.

    2006-01-01

    The Western blot (WB) assay is the most widely accepted confirmatory assay for the detection and confirmation of antibodies to human immunodeficiency virus type 1 (HIV-1) and 2 (HIV-2). However, indeterminate WB reactivity to HIV-1 and HIV-2 proteins may occur in individuals who do not appear to be infected with HIV. In this study, we describe the results of indeterminate WB reactivity in Iranian patients with discordant screening assays. The samples were obtained from Iranian Blood Transfusion Center, Tehran, Iran and evaluated in the Biotechnology Process Development Center, Pasteur Institute of Iran, Tehran, Iran between 2003 and 2004. A total of 4707 were tested for the presence of HIV-1 antibodies. Six hundred and four (12.8%) patients tested for HIV were positive for HIV-1 antibody. Nine (1.49%) have discordant results among screening assays and indeterminate WB results as interpreted by Centers for Disease Control and Prevention (CDC) criteria. Most (66.7%) of these indeterminate WB results were due to p24 reactivity. However, 2(22.2%) display reactivity to both gp41 and gp120 proteins [Positive by World Health Organization (WHO) criteria]. Of 9 WB assays initially indeterminate by the CDC criteria and with follow-up samples 8(88.8%) became negative when retested subsequently while one (11.1%) remained indeterminate for more than a year and were thus considered negative. In addition all the indeterminate samples were negative when assessed by polymerase chain reaction assay. In general, there were was an 88.8% concordance between the CDC and WHO criteria for an indeterminate WB result. The CDC II criteria for an indeterminate WB result. The CDC II criteria best met the specified objectives for diagnosis in our setting. (author)

  1. Two-Phase Contiguous Supported Lipid Bilayer Model for Membrane Rafts via Polymer Blotting and Stenciling.

    Science.gov (United States)

    Richards, Mark J; Daniel, Susan

    2017-02-07

    The supported lipid bilayer has been portrayed as a useful model of the cell membrane compatible with many biophysical tools and techniques that demonstrate its appeal in learning about the basic features of the plasma membrane. However, some of its potential has yet to be realized, particularly in the area of bilayer patterning and phase/composition heterogeneity. In this work, we generate contiguous bilayer patterns as a model system that captures the general features of membrane domains and lipid rafts. Micropatterned polymer templates of two types are investigated for generating patterned bilayer formation: polymer blotting and polymer lift-off stenciling. While these approaches have been used previously to create bilayer arrays by corralling bilayers patches with various types of boundaries impenetrable to bilayer diffusion, unique to the methods presented here, there are no physical barriers to diffusion. In this work, interfaces between contiguous lipid phases define the pattern shapes, with continuity between them allowing transfer of membrane-bound biomolecules between the phases. We examine effectors of membrane domain stability including temperature and cholesterol content to investigate domain dynamics. Contiguous patterning of supported bilayers as a model of lipid rafts expands the application of the SLB to an area with current appeal and brings with it a useful toolset for characterization and analysis. These combined tools should be helpful to researchers investigating lipid raft dynamics and function and biomolecule partitioning studies. Additionally, this patterning technique may be useful for applications such as bioseparations that exploit differences in lipid phase partitioning or creation of membranes that bind species like viruses preferentially at lipid phase boundaries, to name a few.

  2. Antibody responses to Borrelia burgdorferi detected by western blot vary geographically in Canada.

    Science.gov (United States)

    Ogden, Nicholas H; Arsenault, Julie; Hatchette, Todd F; Mechai, Samir; Lindsay, L Robbin

    2017-01-01

    Lyme disease is emerging in eastern and central Canada, and most cases are diagnosed using the two-tier serological test (Enzyme Immuno Assay [EIA] followed by Western blot [WB]). Simplification of this algorithm would be advantageous unless it impacts test performance. In this study, accuracy of individual proteins of the IgG WB algorithm in predicting the overall test result in samples from Canadians was assessed. Because Borrelia burgdorferi strains vary geographically in Canada, geographic variations in serological responses were also explored. Metrics of relative sensitivity, specificity and the kappa statistic measure of concordance were used to assess the capacity of responses to individual proteins to predict the overall IgG WB result of 2524 EIA (C6)-positive samples from across Canada. Geographic and interannual variations in proportions of samples testing positive were explored by logistic regression. No one protein was highly concordant with the IgG WB result. Significant variations were found amongst years and geographic regions in the prevalence of samples testing positive using the overall IgG WB algorithm, and for individual proteins of the algorithm. In most cases the prevalence of samples testing positive were highest in Nova Scotia, and lower in samples from Manitoba westwards. These findings suggest that the current two tier test may not be simplified and continued use of the current two-tier test method and interpretation is recommended. Geographic and interannual variations in the prevalence of samples testing positive may be consistent with B. burgdorferi strain variation in Canada, and further studies are needed to explore this.

  3. Antibody responses to Borrelia burgdorferi detected by western blot vary geographically in Canada.

    Directory of Open Access Journals (Sweden)

    Nicholas H Ogden

    Full Text Available Lyme disease is emerging in eastern and central Canada, and most cases are diagnosed using the two-tier serological test (Enzyme Immuno Assay [EIA] followed by Western blot [WB]. Simplification of this algorithm would be advantageous unless it impacts test performance. In this study, accuracy of individual proteins of the IgG WB algorithm in predicting the overall test result in samples from Canadians was assessed. Because Borrelia burgdorferi strains vary geographically in Canada, geographic variations in serological responses were also explored. Metrics of relative sensitivity, specificity and the kappa statistic measure of concordance were used to assess the capacity of responses to individual proteins to predict the overall IgG WB result of 2524 EIA (C6-positive samples from across Canada. Geographic and interannual variations in proportions of samples testing positive were explored by logistic regression. No one protein was highly concordant with the IgG WB result. Significant variations were found amongst years and geographic regions in the prevalence of samples testing positive using the overall IgG WB algorithm, and for individual proteins of the algorithm. In most cases the prevalence of samples testing positive were highest in Nova Scotia, and lower in samples from Manitoba westwards. These findings suggest that the current two tier test may not be simplified and continued use of the current two-tier test method and interpretation is recommended. Geographic and interannual variations in the prevalence of samples testing positive may be consistent with B. burgdorferi strain variation in Canada, and further studies are needed to explore this.

  4. Simultaneous Detection of Bovine Theileria and Babesia Species by Reverse Line Blot Hybridization

    Science.gov (United States)

    Gubbels, J. M.; de Vos, A. P.; van der Weide, M.; Viseras, J.; Schouls, L. M.; de Vries, E.; Jongejan, F.

    1999-01-01

    A reverse line blot (RLB) assay was developed for the identification of cattle carrying different species of Theileria and Babesia simultaneously. We included Theileria annulata, T. parva, T. mutans, T. taurotragi, and T. velifera in the assay, as well as parasites belonging to the T. sergenti-T. buffeli-T. orientalis group. The Babesia species included were Babesia bovis, B. bigemina, and B. divergens. The assay employs one set of primers for specific amplification of the rRNA gene V4 hypervariable regions of all Theileria and Babesia species. PCR products obtained from blood samples were hybridized to a membrane onto which nine species-specific oligonucleotides were covalently linked. Cross-reactions were not observed between any of the tested species. No DNA sequences from Bos taurus or other hemoparasites (Trypanosoma species, Cowdria ruminantium, Anaplasma marginale, and Ehrlichia species) were amplified. The sensitivity of the assay was determined at 0.000001% parasitemia, enabling detection of the carrier state of most parasites. Mixed DNAs from five different parasites were correctly identified. Moreover, blood samples from cattle experimentally infected with two different parasites reacted only with the corresponding species-specific oligonucleotides. Finally, RLB was used to screen blood samples collected from carrier cattle in two regions of Spain. T. annulata, T. orientalis, and B. bigemina were identified in these samples. In conclusion, the RLB is a versatile technique for simultaneous detection of all bovine tick-borne protozoan parasites. We recommend its use for integrated epidemiological monitoring of tick-borne disease, since RLB can also be used for screening ticks and can easily be expanded to include additional hemoparasite species. PMID:10325324

  5. Epidemiology of Babesia, Anaplasma and Trypanosoma species using a new expanded reverse line blot hybridization assay.

    Science.gov (United States)

    Paoletta, Martina Soledad; López Arias, Ludmila; de la Fournière, Sofía; Guillemi, Eliana Carolina; Luciani, Carlos; Sarmiento, Néstor Fabián; Mosqueda, Juan; Farber, Marisa Diana; Wilkowsky, Silvina Elizabeth

    2018-02-01

    Vector-borne hemoparasitic infections are a major problem that affects livestock industries worldwide, particularly in tropical and subtropical regions. In this work, a reverse line blot (RLB) hybridization assay was developed for the simultaneous detection and identification of Anaplasma, Babesia and bovine trypanosomes, encompassing in this way the most relevant hemoparasites that affect cattle. A total of 186 bovine blood samples collected from two different ecoepidemiological regions of northeast Argentina, with and without tick control, were analyzed with this new RLB. High diversity of parasites, such as Babesia bovis, B. bigemina, Anaplasma marginale and three different Trypanosoma species, was found. High rates of coinfections were also detected, and significant differences were observed not only in the prevalence of parasites but also in the level of coinfections between the two analyzed areas. Regarding the Trypanosoma genus, we provide molecular evidence of the presence of T. vivax and T. theileri for the first time in Argentina. Besides, since the RLB is a prospective tool, it allowed the identification of a yet unknown bovine trypanosome which could not be assigned to any of the bovine species known so far. In the present study we provide new insights on the prevalence of several pathogens that directly impact on livestock production in Argentina. The RLB assay developed here allows to identify simultaneously numerous pathogenic species which can also be easily expanded to detect other blood borne pathogens. These characteristics make the RLB hybridization assay an essential tool for epidemiological survey of all vector-borne pathogens. Copyright © 2017 Elsevier GmbH. All rights reserved.

  6. Simultaneous detection of mRNA and protein stem cell markers in live cells

    Directory of Open Access Journals (Sweden)

    Bao Gang

    2009-04-01

    Full Text Available Abstract Background Biological studies and medical application of stem cells often require the isolation of stem cells from a mixed cell population, including the detection of cancer stem cells in tumor tissue, and isolation of induced pluripotent stem cells after eliciting the expression of specific genes in adult cells. Here we report the detection of Oct-4 mRNA and SSEA-1 protein in live carcinoma stem cells using respectively molecular beacon and dye-labeled antibody, aiming to establish a new method for stem cells detection and isolation. Results Quantification of Oct-4 mRNA and protein in P19 mouse carcinoma stem cells using respectively RT-PCR and immunocytochemistry confirmed that their levels drastically decreased after differentiation. To visualize Oct-4 mRNA in live stem cells, molecular beacons were designed, synthesized and validated, and the detection specificity was confirmed using control studies. We found that the fluorescence signal from Oct-4-targeting molecular beacons provides a clear discrimination between undifferentiated and retinoic acid-induced differentiated cells. Using deconvolution fluorescence microscopy, Oct-4 mRNAs were found to reside on one side of the cytosol. We demonstrated that, using a combination of Oct-4 mRNA-targeting molecular beacon with SSEA-1 antibody in flow cytometric analysis, undifferentiated stem cells can be clearly distinguished from differentiated cells. We revealed that Oct-4 targeting molecular beacons do not seem to affect stem cell biology. Conclusion Molecular beacons have the potential to provide a powerful tool for highly specific detection and isolation of stem cells, including cancer stem cells and induced pluripotent stem (iPS cells without disturbing cell physiology. It is advantageous to perform simultaneous detection of intracellular (mRNA and cell-surface (protein stem cell markers in flow cytometric analysis, which may lead to high detection sensitivity and efficiency.

  7. mRNA processing in yeast

    International Nuclear Information System (INIS)

    Stevens, A.

    1982-01-01

    Investigations in this laboratory center on basic enzymatic reactions of RNA. Still undefined are reactions involved in the conversion of precursors of mRA (pre-mRNA) to mRNA in eukaryotes. The pre-mRNA is called heterogeneous nuclear RNA and is 2 to 6 times larger than mRNA. The conversion, called splicing, involves a removal of internal sequences called introns by endoribonuclease action followed by a rejoining of the 3'- and 5'-end fragments, called exons, by ligating activity. It has not been possible yet to study the enzymes involved in vitro. Also undefined are reactions involved in the turnover or discarding of certain of the pre-mRNA molecules. Yeast is a simple eukaryote and may be expected to have the same, but perhaps simpler, processing reactions as the higher eukaryotes. Two enzymes involved in the processing of pre-mRNA and mRNA in yeast are under investigation. Both enzymes have been partially purified from ribonucleoprotein particles of yeast. The first is a unique decapping enzyme which cleaves [ 3 H]m 7 Gppp [ 14 C]RNA-poly (A) of yeast, yielding [ 3 H]m 7 GDP and is suggested by the finding that the diphosphate product, m 7 GpppA(G), and UDP-glucose are not hydrolyzed. The second enzyme is an endoribonuclease which converts both the [ 3 H] and [ 14 C] labels of [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) from an oligo(dT)-cellulose bound form to an unbound, acid-insoluble form. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA. The inhibition of the enzyme by ethidium bromide and its stimulation by small nuclear RNA suggest that it may be a processing ribonuclease, requiring specific double-stranded features in its substrate. The characterization of the unique decapping enzyme and endoribonuclease may help to understand reactions involved in the processing of pre-mRNA and mRNA in eukaryotes

  8. Identification of alternatively spliced TIMP-1 mRNA in cancer cell lines and colon cancer tissue

    DEFF Research Database (Denmark)

    Usher, Pernille Autzen; Sieuwerts, A.M.; Bartels, Annette

    2007-01-01

    TIMP-1 is a promising new candidate as a prognostic marker in colorectal and breast cancer. We now describe the discovery of two alternatively spliced variants of TIMP-1 mRNA. The two variants lacking exon 2 (del-2) and 5 (del-5), respectively, were identified in human cancer cell lines by RT......-PCR. The del-2 variant was, furthermore, detected in extracts from 12 colorectal cancer tissue samples. By western blotting additional bands of lower molecular mass than full-length TIMP-1 were identified in tumor tissue, but not in plasma samples obtained from cancer patients. The two splice variants of TIMP...

  9. Product-selective blot: a technique for measuring enzyme activities in large numbers of samples and in native electrophoresis gels

    International Nuclear Information System (INIS)

    Thompson, G.A.; Davies, H.M.; McDonald, N.

    1985-01-01

    A method termed product-selective blotting has been developed for screening large numbers of samples for enzyme activity. The technique is particularly well suited to detection of enzymes in native electrophoresis gels. The principle of the method was demonstrated by blotting samples from glutaminase or glutamate synthase reactions into an agarose gel embedded with ion-exchange resin under conditions favoring binding of product (glutamate) over substrates and other substances in the reaction mixture. After washes to remove these unbound substances, the product was measured using either fluorometric staining or radiometric techniques. Glutaminase activity in native electrophoresis gels was visualized by a related procedure in which substrates and products from reactions run in the electrophoresis gel were blotted directly into a resin-containing image gel. Considering the selective-binding materials available for use in the image gel, along with the possible detection systems, this method has potentially broad application

  10. Localization of mRNA in vertebrate axonal compartments by in situ hybridization.

    Science.gov (United States)

    Sotelo-Silveira, José Roberto; Calliari, Aldo; Kun, Alejandra; Elizondo, Victoria; Canclini, Lucía; Sotelo, José Roberto

    2011-01-01

    The conclusive demonstration of RNA in vertebrate axons by in situ hybridization (ISH) has been elusive. We review the most important reasons for difficulties, including low concentration of axonal RNAs, localization in specific cortical domains, and the need to isolate axons. We demonstrate the importance of axon micro-dissection to obtain a whole mount perspective of mRNA distribution in the axonal territory. We describe a protocol to perform fluorescent ISH in isolated axons and guidelines for the preservation of structural and molecular integrity of cortical RNA-containing domains (e.g., Periaxoplasmic Ribosomal Plaques, or PARPs) in isolated axoplasm.

  11. Imaging and high-sensitivity quantification of chemiluminescent labeled DNA-blots

    International Nuclear Information System (INIS)

    Dorner, G.

    1997-01-01

    The present thesis has for objective the development of both, methods of DNA labeling by chemiluminescence (via the catalytic activity of the enzyme alkaline phosphatase - AP) and an appropriate imaging system. Offering a competitive alternative to the detection of classical radio-labels in molecular-biological experiments of the blotting type, this technique should permit the realization of quantitative studies of gene expression at ultra-high sensitivity necessary in particular for differential-screening experiments. To reach our aim. we separated the project into three different parts. In a first step an imager based on a liquid-nitrogen-cooled CCD coupled to a standard optics (50 mm/fl.2) has been installed and characterized. This system offers a sensitive area of up to 625 cm 2 , a spatial resolution of 0.3-1 mm (depending on the field of view) and a sensitivity sufficient to detect 10 fg/mm 2 labeled DNA. In a second part, the chemiluminescent light-generation process in solution has been investigated to optimize the parameters temperature. pH and concentration of the substrate as well as the enzyme. The substrate offering the highest light yield (CDP-Star in addition with the enhancer EMERALD II) allows quantification of AP down to 10 -15 M within a dynamic range of 10 4 in solution. Finally. preparation, immobilization and detection of AP-labeled DNA probes (via a biotin-streptavidin-biotin-AP bridge) on nylon membranes has been optimized. A linear relation between the light intensities and the amount of DNA was observed in a range of 10 fg/mm 2 - 100 pg/mm 2 . Hybridization of the probes to bacterial cloned target-DNA has been addressed after examination of the best hybridization conditions. Our protocol includes the treatment of a proteinase, which resulted in a significantly lower background on the filter. The results of our investigations suggest that the main conditions for a reliable differential-screening experiment are fulfilled when using

  12. Testing for myositis specific autoantibodies: Comparison between line blot and immunoprecipitation assays in 57 myositis sera.

    Science.gov (United States)

    Cavazzana, Ilaria; Fredi, Micaela; Ceribelli, Angela; Mordenti, Cristina; Ferrari, Fabio; Carabellese, Nice; Tincani, Angela; Satoh, Minoru; Franceschini, Franco

    2016-06-01

    To analyze the performance of a line blot assay for the identification of autoantibodies in sera of patients affected by myositis, compared with immunoprecipitation (IP) as gold standard. 66 sera of patients with myositis (23 polymyositis, 8 anti-synthetase syndromes, 29 dermatomyositis and 6 overlap syndromes) were tested by commercial LB (Euroimmun, Lubeck, Germany); 57 sera were analyzed also by IP of K562 cell extract radiolabeled with (35)S-methionine. Inter-rater agreement was calculated with Cohen's k coefficient. Myositis-specific antibodies (MSA) were detected in 36/57 sera (63%) by IP and in 39/66 sera (59%) by LB. The most frequent MSA found by LB were anti-Jo1 and anti-Mi2 found in 15% (10/66) of sera, followed by anti-NXP2 and anti-SRP detected in 106% (7/66) of sera. Anti-TIF1gamma and anti-MDA5 were found in 6 (9%) and 5 sera (7.6%), respectively. A good agreement between methods was found only for anti-TIF1γ, anti-MDA5 and anti-NXP-2 antibodies, while a moderate agreement was estimated for anti-Mi2 and anti-EJ. By contrast, a high discordance rate for the detection of anti-Jo1 antibodies was evident (k: 0.3). Multiple positivity for MSA were found in 11/66 (17%) by LB and 0/57 by IP (p: 0001). Comparing the clinical features of these 11 sera, we found total discrepancies between assays in 3 sera (27.3%), a relative discrepancy due to the occurrence of one discordant autoantibody (not confirmed by IP) in 5 cases (45.5%) and a total discrepancy between LB and IP results, but with a relative concordance with clinical features were found in other 3 sera (27.3%). The semiquantitative results do not support the interpretation of the data. The use of LB assay allowed the detection of new MSA, such as anti-MDA5, anti-MJ and anti-TIF1gamma antibodies, previously not found with routine methods. However, the high prevalence of multiple positivities and the high discondant rate of anti-Jo1 antibodies could create some misinterpretation of the results from the

  13. Reviews in fluorescence 2010

    CERN Document Server

    Geddes, Chris D

    2011-01-01

    ""Reviews in Fluorescence 2010"", the seventh volume of the book serial from Springer, serves as a comprehensive collection of current trends and emerging hot topics in the field of fluorescence and closely related disciplines. It summarizes the year's progress in fluorescence and its applications, with authoritative analytical reviews specialized enough to be attractive to professional researchers, yet also appealing to the wider audience of scientists in related disciplines of fluorescence. ""Reviews in Fluorescence"" offers an essential reference material for any lab working in the fluoresc

  14. Principles of fluorescence techniques

    CERN Document Server

    2016-01-01

    Fluorescence techniques are being used and applied increasingly in academics and industry. The Principles of Fluorescence Techniques course will outline the basic concepts of fluorescence techniques and the successful utilization of the currently available commercial instrumentation. The course is designed for students who utilize fluorescence techniques and instrumentation and for researchers and industrial scientists who wish to deepen their knowledge of fluorescence applications. Key scientists in the field will deliver theoretical lectures. The lectures will be complemented by the direct utilization of steady-state and lifetime fluorescence instrumentation and confocal microscopy for FLIM and FRET applications provided by leading companies.

  15. Principles of mRNA transport in yeast.

    Science.gov (United States)

    Heym, Roland Gerhard; Niessing, Dierk

    2012-06-01

    mRNA localization and localized translation is a common mechanism by which cellular asymmetry is achieved. In higher eukaryotes the mRNA transport machinery is required for such diverse processes as stem cell division and neuronal plasticity. Because mRNA localization in metazoans is highly complex, studies at the molecular level have proven to be cumbersome. However, active mRNA transport has also been reported in fungi including Saccharomyces cerevisiae, Ustilago maydis and Candida albicans, in which these events are less difficult to study. Amongst them, budding yeast S. cerevisiae has yielded mechanistic insights that exceed our understanding of other mRNA localization events to date. In contrast to most reviews, we refrain here from summarizing mRNA localization events from different organisms. Instead we give an in-depth account of ASH1 mRNA localization in budding yeast. This approach is particularly suited to providing a more holistic view of the interconnection between the individual steps of mRNA localization, from transcriptional events to cytoplasmic mRNA transport and localized translation. Because of our advanced mechanistic understanding of mRNA localization in yeast, the present review may also be informative for scientists working, for example, on mRNA localization in embryogenesis or in neurons.

  16. Expression of a serine protease (motopsin PRSS12) mRNA in the mouse brain: in situ hybridization histochemical study.

    Science.gov (United States)

    Iijima, N; Tanaka, M; Mitsui, S; Yamamura, Y; Yamaguchi, N; Ibata, Y

    1999-03-20

    Serine proteases are considered to play several important roles in the brain. In an attempt to find novel brain-specific serine proteases (BSSPs), motopsin (PRSS-12) was cloned from a mouse brain cDNA library by polymerase chain reaction (PCR). Northern blot analysis demonstrated that the postnatal 10-day mouse brain contained the most amount of motopsin mRNA. At this developmental stage, in situ hybridization histochemistry showed that motopsin mRNA was specifically expressed in the following regions: cerebral cortical layers II/III, V and VIb, endopiriform cortex and the limbic system, particularly in the CA1 region of the hippocampal formation. In addition, in the brainstem, the oculomotor nucleus, trochlear nucleus, mecencephalic and motor nuclei of trigeminal nerve (N), abducens nucleus, facial nucleus, nucleus of the raphe pontis, dorsoral motor nucleus of vagal N, hypoglossal nucleus and ambiguus nucleus showed motopsin mRNA expression. Expression was also found in the anterior horn of the spinal cord. The above findings strongly suggest that neurons in almost all motor nuclei, particularly in the brainstem and spinal cord, express motopsin mRNA, and that motopsin seems to have a close relation to the functional role of efferent neurons. Copyright 1999 Elsevier Science B.V.

  17. Fluorescence Correlation Spectroscopy to find the critical balance between extracellular association and intracellular dissociation of mRNA-complexes.

    Science.gov (United States)

    Zhang, Heyang; De Smedt, Stefaan C; Remaut, Katrien

    2018-05-10

    Fluorescence Correlation Spectroscopy (FCS) is a promising tool to study interactions on a single molecule level. The diffusion of fluorescent molecules in and out of the excitation volume of a confocal microscope leads to the fluorescence fluctuations that give information on the average number of fluorescent molecules present in the excitation volume and their diffusion coefficients. In this context, we complexed mRNA into lipoplexes and polyplexes and explored the association/dissociation degree of complexes by using gel electrophoresis and FCS. FCS enabled us to measure the association and dissociation degree of mRNA-based complexes both in buffer and protein-rich biological fluids such as human serum and ascitic fluid, which is a clear advantage over gel electrophoresis that was only applicable in protein-free buffer solutions. Furthermore, following the complex stability in buffer and biological fluids by FCS assisted to understand how complex characteristics, such as charge ratio and strength of mRNA binding, correlated to the transfection efficiency. We found that linear polyethyleneimine prevented efficient translation of mRNA, most likely due to a too strong mRNA binding, whereas the lipid based carrier Lipofectamine ® messengerMAX did succeed in efficient release and subsequent translation of mRNA in the cytoplasm of the cells. Overall, FCS is a reliable tool for the in depth characterization of mRNA complexes and can help us to find the critical balance keeping mRNA bound in complexes in the extracellular environment and efficient intracellular mRNA release leading to protein production. The delivery of messenger RNA (mRNA) to cells is promising to treat a variety of diseases. Therefore, the mRNA is typically packed in small lipid particles or polymer particles that help the mRNA to reach the cytoplasm of the cells. These particles should bind and carry the mRNA in the extracellular environment (e.g. blood, peritoneal fluid, ...), but should release

  18. Reviews in fluorescence 2008

    CERN Document Server

    Geddes, Chris D

    2010-01-01

    This volume serves as a comprehensive collection of current trends and emerging hot topics in the field of fluorescence spectroscopy. It summarizes the year's progress in fluorescence and its applications as well as includes authoritative analytical reviews.

  19. Fluorescent optical position sensor

    Science.gov (United States)

    Weiss, Jonathan D.

    2005-11-15

    A fluorescent optical position sensor and method of operation. A small excitation source side-pumps a localized region of fluorescence at an unknown position along a fluorescent waveguide. As the fluorescent light travels down the waveguide, the intensity of fluorescent light decreases due to absorption. By measuring with one (or two) photodetectors the attenuated intensity of fluorescent light emitted from one (or both) ends of the waveguide, the position of the excitation source relative to the waveguide can be determined by comparing the measured light intensity to a calibrated response curve or mathematical model. Alternatively, excitation light can be pumped into an end of the waveguide, which generates an exponentially-decaying continuous source of fluorescent light along the length of the waveguide. The position of a photodetector oriented to view the side of the waveguide can be uniquely determined by measuring the intensity of the fluorescent light emitted radially at that location.

  20. Different domains of Bacillus thuringiensis delta-endotoxins can bind to insect midgut membrane proteins on ligand blots

    NARCIS (Netherlands)

    Maagd, de R.A.; Klei, van der H.; Bakker, P.L.; Stiekema, W.J.; Bosch, D.

    1996-01-01

    We investigated the role of the constituent domains of the CryIA(b) and CryIA(c) δ-endotoxins in binding to midgut epithelial cell membrane proteins of Spodoptera exigua and Manduca sexta on ligand blots. A collection of wild- type and CryIC-CryIA hybrid toxins was used for this purpose. As

  1. Safe biodegradable fluorescent particles

    Science.gov (United States)

    Martin, Sue I [Berkeley, CA; Fergenson, David P [Alamo, CA; Srivastava, Abneesh [Santa Clara, CA; Bogan, Michael J [Dublin, CA; Riot, Vincent J [Oakland, CA; Frank, Matthias [Oakland, CA

    2010-08-24

    A human-safe fluorescence particle that can be used for fluorescence detection instruments or act as a safe simulant for mimicking the fluorescence properties of microorganisms. The particle comprises a non-biological carrier and natural fluorophores encapsulated in the non-biological carrier. By doping biodegradable-polymer drug delivery microspheres with natural or synthetic fluorophores, the desired fluorescence can be attained or biological organisms can be simulated without the associated risks and logistical difficulties of live microorganisms.

  2. Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins.

    Science.gov (United States)

    Ramsey, Kathryn A; Rushton, Zachary L; Ehre, Camille

    2016-06-14

    Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

  3. Optimization of fluorescent proteins

    NARCIS (Netherlands)

    Bindels, D.S.; Goedhart, J.; Hink, M.A.; van Weeren, L.; Joosen, L.; Gadella (jr.), T.W.J.; Engelborghs, Y.; Visser, A.J.W.G.

    2014-01-01

    Nowadays, fluorescent protein (FP) variants have been engineered to fluoresce in all different colors; to display photoswitchable, or photochromic, behavior; or to show yet other beneficial properties that enable or enhance a still growing set of new fluorescence spectroscopy and microcopy

  4. Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen.

    Science.gov (United States)

    Deng, Dawei; Li, Yang; Xue, Jianpeng; Wang, Jie; Ai, Guanhua; Li, Xin; Gu, Yueqing

    2015-01-01

    Messenger RNA (mRNA), a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP) beacon containing a bare gold nanoparticle (AuNP) as fluorescence quencher and thiol-terminated fluorescently labeled stem-loop-stem oligonucleotide sequences attached by Au-S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b) mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.

  5. Altered expression of asparagine synthetase mRNA in human leukemic and carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Goodwin, L.O.; Guzowski, D.E.; Millan, C.A. [North Shore Univ. Hospital/Cornell Univ. Medical College, Manhasset, NY (United States)] [and others

    1994-09-01

    Asparagine synthetase (AS) is the enzyme responsible for the ATP-dependant conversion of aspartic acid to asparagine. The AS gene is expressed constitutively in most mammalian cells, including cells of the lymphoid lineage, as a 2 kb mRNA. In some leukemic phenotypes, AS expression is abrogated, resulting in no detectable enzyme activity. These cells are rendered sensitive to killing by L-asparaginase, which destroys extracellular asparagine. Prolonged treatment of leukemic cells with this agent can lead to resistance and the reappearance of AS activity, suggesting derepression of the AS gene, which has been shown to be regulated by intracellular levels of asparagine. Modulation of AS expression by asparagine employs cis and trans-acting elements involved in transcriptional and translational regulation. We have cloned and sequenced the human AS gene and surrounding sequence elements as well as the full-length cDNA. Using probes specific to the third and fourth exons of AS, we have identified an additional higher molecular weight mRNA (2.7 kb) in Northern blots derived from a chronic myelogenous leukemia and a colon carcinoma but not in normal lymphocytic or other human cell lines. We speculate that elements present in the cancer-derived mRNAs may be involved in the derepression of AS activity. This hypothesis is being evaluated by RNase protection assays using RNA isolated from a variety of human cell lines to characterize and elucidate the nature of this additional AS encoded message.

  6. Expression and clinicopathological significance of Mel-18 mRNA in colorectal cancer.

    Science.gov (United States)

    Tao, Ji; Liu, Yan-Long; Zhang, Gan; Ma, Yu-Yan; Cui, Bin-Bin; Yang, Yan-Mei

    2014-10-01

    Mel-18 is a member of the polycomb group (PcG) of proteins, which are chromatin regulatory factors that play an important role in oncogenesis. This study was designed to investigate the clinical and prognostic significance of Mel-18 in colorectal cancer (CRC) patients. For this purpose, expression of Mel-18 mRNA was evaluated in 82 primary CRC and paired noncancerous mucosa samples by qRT-PCR and Western blotting. We found that overall Mel-18 mRNA expression in the CRC tissue was significantly lower than in the noncancerous mucosal tissue (p = 0.007, Wilcoxon matched-pairs signed-ranks test). Mel-18 was conversely correlated with the pathological classifications (p = 0.003 for T, p Mel-18 showed prolonged disease-free survivals (DFS) (p Mel-18 expression may be a risk factor for the patients' 3-year DFS (HR = 1.895; 95 % CI 1.032, 3.477; p = 0.039). It was therefore concluded that the lower Mel-18 expression might contribute to the CRC development/progression.

  7. Keratin14 mRNA expression in human pneumocytes during quiescence, repair and disease.

    Directory of Open Access Journals (Sweden)

    Marco Confalonieri

    Full Text Available The lung alveoli slowly self-renew pneumocytes, but their facultative regeneration capacity is rapidly efficient after an injury, so fibrosis infrequently occurs. We recently observed Keratin 14 (KRT14 expression during diffuse alveolar damage (DAD, but not in controls. We wonder if KRT14 may be a marker of pneumocyte transition from quiescence to regeneration. Quantitative PCR and Western blot analyses highlighted the presence of KRT14 (mRNA and protein only in human lung samples with DAD or interstitial lung disease (ILD. In the exponentially growing cell lines A549 and H441, the mRNA and protein levels of KRT14 peaked at day one after cell seeding and decreased at day two, opposite to what observed for the proliferation marker E2F1. The inverse relation of KRT14 versus E2F1 expression holds true also for other proliferative markers, such as cyclin E1 and cyclin D1. Of interest, we also found that E2F1 silencing caused cell cycle arrest and increased KRT14 expression, whilst E2F1 stimulation induced cell cycle progression and decreased KRT14. KRT14 also increased in proliferative pneumocytes (HPAEpiC just before transdifferentiation. Overall, our results suggest that KRT14 is a viable biomarker of pneumocyte activation, and repair/regeneration. The involvement of KRT14 in regenerative process may suggest a novel pharmaceutical target to accelerate lung repair.

  8. Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells.

    Science.gov (United States)

    Van Tendeloo, V F; Ponsaerts, P; Lardon, F; Nijs, G; Lenjou, M; Van Broeckhoven, C; Van Bockstaele, D R; Berneman, Z N

    2001-07-01

    Designing effective strategies to load human dendritic cells (DCs) with tumor antigens is a challenging approach for DC-based tumor vaccines. Here, a cytoplasmic expression system based on mRNA electroporation to efficiently introduce tumor antigens into DCs is described. Preliminary experiments in K562 cells using an enhanced green fluorescent protein (EGFP) reporter gene revealed that mRNA electroporation as compared with plasmid DNA electroporation showed a markedly improved transfection efficiency (89% versus 40% EGFP(+) cells, respectively) and induced a strikingly lower cell toxicity (15% death rate with mRNA versus 51% with plasmid DNA). Next, mRNA electroporation was applied for nonviral transfection of different types of human DCs, including monocyte-derived DCs (Mo-DCs), CD34(+) progenitor-derived DCs (34-DCs) and Langerhans cells (34-LCs). High-level transgene expression by mRNA electroporation was obtained in more than 50% of all DC types. mRNA-electroporated DCs retained their phenotype and maturational potential. Importantly, DCs electroporated with mRNA-encoding Melan-A strongly activated a Melan-A-specific cytotoxic T lymphocyte (CTL) clone in an HLA-restricted manner and were superior to mRNA-lipofected or -pulsed DCs. Optimal stimulation of the CTL occurred when Mo-DCs underwent maturation following mRNA transfection. Strikingly, a nonspecific stimulation of CTL was observed when DCs were transfected with plasmid DNA. The data clearly demonstrate that Mo-DCs electroporated with mRNA efficiently present functional antigenic peptides to cytotoxic T cells. Therefore, electroporation of mRNA-encoding tumor antigens is a powerful technique to charge human dendritic cells with tumor antigens and could serve applications in future DC-based tumor vaccines.

  9. Analysis of the 3’ untranslated regions of α-tubulin and S-crystallin mRNA and the identification of CPEB in dark- and light-adapted octopus retinas

    Science.gov (United States)

    Kelly, Shannan; Yamamoto, Hideki

    2008-01-01

    Purpose We previously reported the differential expression and translation of mRNA and protein in dark- and light-adapted octopus retinas, which may result from cytoplasmic polyadenylation element (CPE)–dependent mRNA masking and unmasking. Here we investigate the presence of CPEs in α-tubulin and S-crystallin mRNA and report the identification of cytoplasmic polyadenylation element binding protein (CPEB) in light- and dark-adapted octopus retinas. Methods 3’-RACE and sequencing were used to isolate and analyze the 3’-UTRs of α-tubulin and S-crystallin mRNA. Total retinal protein isolated from light- and dark-adapted octopus retinas was subjected to western blot analysis followed by CPEB antibody detection, PEP-171 inhibition of CPEB, and dephosphorylation of CPEB. Results The following CPE-like sequence was detected in the 3’-UTR of isolated long S-crystallin mRNA variants: UUUAACA. No CPE or CPE-like sequences were detected in the 3’-UTRs of α-tubulin mRNA or of the short S-crystallin mRNA variants. Western blot analysis detected CPEB as two putative bands migrating between 60-80 kDa, while a third band migrated below 30 kDa in dark- and light-adapted retinas. Conclusions The detection of CPEB and the identification of the putative CPE-like sequences in the S-crystallin 3’-UTR suggest that CPEB may be involved in the activation of masked S-crystallin mRNA, but not in the regulation of α-tubulin mRNA, resulting in increased S-crystallin protein synthesis in dark-adapted octopus retinas. PMID:18682811

  10. Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75(NTR)

    DEFF Research Database (Denmark)

    Skeldal, Sune; Kjaergaard, Maj M; Alwasel, Saleh

    2015-01-01

    the vps10p domain receptor sortilin and the neurotrophin receptor p75(NTR). However, proNGF-induced receptor complex formation has been difficult to directly assess other than by western blotting. We here describe a fluorescence resonance energy transfer (FRET) based fluorescence plate reader assay...

  11. mRNA Cancer Vaccines-Messages that Prevail.

    Science.gov (United States)

    Grunwitz, Christian; Kranz, Lena M

    2017-01-01

    During the last decade, mRNA became increasingly recognized as a versatile tool for the development of new innovative therapeutics. Especially for vaccine development, mRNA is of outstanding interest and numerous clinical trials have been initiated. Strikingly, all of these studies have proven that large-scale GMP production of mRNA is feasible and concordantly report a favorable safety profile of mRNA vaccines. Induction of T-cell immunity is a multi-faceted process comprising antigen acquisition, antigen processing and presentation, as well as immune stimulation. The effectiveness of mRNA vaccines is critically dependent on making the antigen(s) of interest available to professional antigen-presenting cells, especially DCs. Efficient delivery of mRNA into DCs in vivo remains a major challenge in the mRNA vaccine field. This review summarizes the principles of mRNA vaccines and highlights the importance of in vivo mRNA delivery and recent advances in harnessing their therapeutic potential.

  12. CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

    Science.gov (United States)

    Busi, Florent; Cresteil, Thierry

    2005-09-01

    The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

  13. A single-step simultaneous protein staining procedure for polyacrylamide gels and nitrocellulose membranes by Alta during western blot analysis.

    Science.gov (United States)

    Pal, Jayanta K; Berwal, Sunil K; Soni, Rupali N

    2012-01-01

    A simple method for staining of proteins simultaneously on sodium dodecyl sulfate (SDS) polyacrylamide gels and nitrocellulose membranes by Alta during western blot analysis is described. A 5% solution of Alta, a commercially available cosmetic preparation, is added in the upper tank buffer during electrophoresis. On completion of electrophoresis, the gel is washed in distilled water and viewed on a white light plate and a transilluminator to photograph the protein profiles. The gel is processed for western blot transfer of proteins onto a nitrocellulose membrane, and upon completion, the protein profiles on the membrane are viewed and photographed as stated above. The membrane can then be processed for immunostaining as per the standard procedure. Thus, the staining procedure using Alta is simple, rapid (without any need of destaining), and cost-effective.

  14. Influenza A Virus NS1 Protein Promotes Efficient Nuclear Export of Unspliced Viral M1 mRNA.

    Science.gov (United States)

    Pereira, Carina F; Read, Eliot K C; Wise, Helen M; Amorim, Maria J; Digard, Paul

    2017-08-01

    Influenza A virus mRNAs are transcribed by the viral RNA-dependent RNA polymerase in the cell nucleus before being exported to the cytoplasm for translation. Segment 7 produces two major transcripts: an unspliced mRNA that encodes the M1 matrix protein and a spliced transcript that encodes the M2 ion channel. Export of both mRNAs is dependent on the cellular NXF1/TAP pathway, but it is unclear how they are recruited to the export machinery or how the intron-containing but unspliced M1 mRNA bypasses the normal quality-control checkpoints. Using fluorescent in situ hybridization to monitor segment 7 mRNA localization, we found that cytoplasmic accumulation of unspliced M1 mRNA was inefficient in the absence of NS1, both in the context of segment 7 RNPs reconstituted by plasmid transfection and in mutant virus-infected cells. This effect was independent of any major effect on steady-state levels of segment 7 mRNA or splicing but corresponded to a ∼5-fold reduction in the accumulation of M1. A similar defect in intronless hemagglutinin (HA) mRNA nuclear export was seen with an NS1 mutant virus. Efficient export of M1 mRNA required both an intact NS1 RNA-binding domain and effector domain. Furthermore, while wild-type NS1 interacted with cellular NXF1 and also increased the interaction of segment 7 mRNA with NXF1, mutant NS1 polypeptides unable to promote mRNA export did neither. Thus, we propose that NS1 facilitates late viral gene expression by acting as an adaptor between viral mRNAs and the cellular nuclear export machinery to promote their nuclear export. IMPORTANCE Influenza A virus is a major pathogen of a wide variety of mammalian and avian species that threatens public health and food security. A fuller understanding of the virus life cycle is important to aid control strategies. The virus has a small genome that encodes relatively few proteins that are often multifunctional. Here, we characterize a new function for the NS1 protein, showing that, as well as

  15. Detection of mutations related to drug resistance in M. tuberculosis by dot blot hybridization and spoligotyping using specific radiolabelled probes

    International Nuclear Information System (INIS)

    El-Maghraby, T.K.; Abdelazeim, O.

    2002-01-01

    The present work has been conducted to determine the mutations related to drug resistance in M. tuberculosis in 63 Egyptian isolates using dot blot hybridization and spoligotyping. The PCR was done for amplification rpoB and katG genes in isolates. Dot blot hybridization were done to PCR products by using specific radiolabelled probes. Moreover, spoligotyping was done to know about the different strains found in Egypt. The results revealed that 58% from isolates had drug resistance to one or more of antituberculosis drugs. The results of spoligotyping have revealed that some Egyptian isolates are identical with the international code while the rest has not been identified yet. DNA sequencing was done to identify the mutation that not clear in dot blot hybridization. Early diagnosis of geno typing resistance to antituberculosis drugs is important as well as allow appropriate early patients management with few days of TB diagnosis. Using such strategy for early diagnosis of TB drug resistance allow and fast and potent patient's management

  16. MiR-200a is involved in rat epididymal development by targeting β-catenin mRNA

    Institute of Scientific and Technical Information of China (English)

    Xiaojiang Wu; Botao Zhao; Wei Li; Yue Chen; Ruqiang Liang; Lin Li; Youxin Jin; Kangcheng Ruan

    2012-01-01

    The expression of 350 microRNAs (miRNAs) in epididymis of rat from postnatal development to adult (from postnatal days 7-70) was profiled with home-made miRNA microarray.Among them,48 miRNAs changed significantly, in which the expression of miR-200a increased obviously with time,in a good agreement with that obtained from northern blot analysis.The real-time quantitative-polymerase chain reaction result indicated that temporal expression of rat β-catenin was exactly inversed to that of miR-200a during rat epididymal development,implying that miR-200a might also target β-catenin mRNA in rat epididymis as reported by Saydam et al.in humans.The bioinformatic analysis indicated that 3' untranslated region of rat β-catenin mRNA did contain a putative binding site for miR-200a.Meanwhile,it was found that the sequence of this binding site was different from that of human β-catenin mRNA with a deletion of two adjacent nucleotides (U and C).But the results of luciferase targeting assay in HEK 293T cells and the overexpression of miR-200a in rat NRK cells demonstrated that miR-200a did target rat β-catenin mRNA and cause the suppression of its expression.All these results show that miR-200a should be involved in rat epididymal development by targeting β-catenin mRNA of rat and suppressing its expression.

  17. Tumor necrosis factor-alpha regulates the Hypocretin system via mRNA degradation and ubiquitination.

    Science.gov (United States)

    Zhan, Shuqin; Cai, Guo-Qiang; Zheng, Anni; Wang, Yuping; Jia, Jianping; Fang, Haotian; Yang, Youfeng; Hu, Meng; Ding, Qiang

    2011-04-01

    Recent studies recognize that Hypocretin system (also known as Orexin) plays a critical role in sleep/wake disorders and feeding behaviors. However, little is known about the regulation of the Hypocretin system. It is also known that tumor necrosis factor alpha (TNF-α) is involved in the regulation of sleep/wake cycle. Here, we test our hypothesis that the Hypocretin system is regulated by TNF-α. Prepro-Hypocretin and Hypocretin receptor 2 (HcrtR2) can be detected at a very low level in rat B35 neuroblastoma cells. In response to TNF-α, Prepro-Hypocretin mRNA and protein levels are down-regulated, and also HcrtR2 protein level is down-regulated in B35 cells. To investigate the mechanism, exogenous rat Prepro-Hypocretin and rat HcrtR2 were overexpressed in B35 cells. In response to TNF-α, protein and mRNA of Prepro-Hypocretin are significantly decreased (by 93% and 94%, respectively), and the half-life of Prepro-Hypocretin mRNA is decreased in a time- and dose-dependent manner. The level of HcrtR2 mRNA level is not affected by TNF-α treatment; however, HcrtR2 protein level is significantly decreased (by 86%) through ubiquitination in B35 cells treated with TNF-α. Downregulation of cellular inhibitor of apoptosis protein-1 and -2 (cIAP-1 and -2) abrogates the HcrtR2 ubiquitination induced by TNF-α. The control green fluorescent protein (GFP) expression is not affected by TNF-α treatment. These studies demonstrate that TNF-α can impair the function of the Hypocretin system by reducing the levels of both Prepro-Hypocretin and HcrtR2. Copyright © 2010 Elsevier B.V. All rights reserved.

  18. Presence of albumin mRNA precursors in nuclei of analbuminemic rat liver lacking cytoplasmic albumin mRNA.

    OpenAIRE

    Esumi, H; Takahashi, Y; Sekiya, T; Sato, S; Nagase, S; Sugimura, T

    1982-01-01

    Analbuminemic rats, which lack serum albumin, were previously found to have no albumin mRNA in the cytoplasm of the liver. In the present study, the existence of nuclear albumin mRNA precursors in the liver of analbuminemic rats was examined by RNA X cDNA hybridization kinetics. Albumin mRNA precursors were present in the nuclei of analbuminemic rat liver at almost normal levels, despite the absence of albumin mRNA from the cytoplasm. Nuclear RNA of analbuminemic rat liver was subjected to el...

  19. Atomic-fluorescence spectrophotometry

    International Nuclear Information System (INIS)

    Bakhturova, N.F.; Yudelevich, I.G.

    1975-01-01

    Atomic-fluorescence spectrophotometry, a comparatively new method for the analysis of trace quantities, has developed rapidly in the past ten years. Theoretical and experimental studies by many workers have shown that atomic-fluorescence spectrophotometry (AFS) is capable of achieving a better limit than atomic absorption for a large number of elements. The present review examines briefly the principles of atomic-fluorescence spectrophotometry and the types of fluorescent transition. The excitation sources, flame and nonflame atomizers, used in AFS are described. The limits of detection achieved up to the present, using flame and nonflame methods of atomization are given

  20. Fluorescence of irradiated hydrocarbons

    International Nuclear Information System (INIS)

    Gulis, I.G.; Evdokimenko, V.M.; Lapkovskij, M.P.; Petrov, P.T.; Gulis, I.M.; Markevich, S.V.

    1977-01-01

    A visible fluorescence has been found out in γ-irradiated aqueous of carbohydrates. Two bands have been distinguished in fluorescence spectra of the irradiated solution of dextran: a short-wave band lambdasub(max)=140 nm (where lambda is a wave length) at lambdasub(β)=380 nm and a long-wave band with lambdasub(max)=540 nm at lambdasub(β)=430 nm. A similar form of the spectrum has been obtained for irradiated solutions of starch, amylopectin, lowmolecular glucose. It has been concluded that a macromolecule of polysaccharides includes fluorescent centres. A relation between fluorescence and α-oxiketon groups formed under irradiation has been pointed out

  1. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis

    DEFF Research Database (Denmark)

    Krakauer, Martin; Sorensen, P; Khademi, M

    2008-01-01

    BACKGROUND: Interferon (IFN)-beta therapy in multiple sclerosis (MS) has been suggested to promote a deviation from T lymphocyte production of pathogenic Th1 cytokines to less detrimental Th2 cytokines, but this is still controversial. We studied patterns of in vivo blood mononuclear cell (MNC...... of any Th1 or Th2 cytokines. The largest changes in cytokine mRNA levels occurred early (~9-12 h) after an IFN-beta injection. CONCLUSION: We found no evidence of a Th1- or Th2-mRNA-promoting effect of IFN-beta therapy. The therapeutic effect of IFN-beta is more likely attributable to the induction...

  2. Cloning of cDNA sequences of a progestin-regulated mRNA from MCF7 human breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Chalbos, D; Westley, B; Alibert, C; Rochefort, H

    1986-01-24

    A cDNA clone corresponding to an mRNA regulated by the progestin R5020, has been isolated by differential screening of a cDNA library from the MCF7 breast cancer cell line, which contains estrogen and progesterone receptors. This probe hybridized with a single species of poly A + RNA of 8-kb molecular weight as shown by Northern blot analysis and could also be used to total RNA preparation. This recombinant cone hybridized specifically to an mRNA coding for a 250,000 daltons protein when translated in vitro. This protein was identical to the 250 kDa progestin-regulated protein that the authors previously described as shown by immunoprecipitation with specific rabbit polyclonal antibodies. Dose-response curve and specificity studies show that the accumulation of the Pg8 mRNA and that of the 250-kDa protein was increased by 5 to 30-fold following progestin treatment and that this effect was mediated by the progesterone receptor. Time course of induction indicated that the accumulation of mRNA was rapid and preceded that of the protein. This is the first report on a cloned cDNA probe of progestin-regulated mRNA in human cell lines.

  3. Neurotrophins and their receptors in the rat pituitary gland: regulation of BDNF and trkB mRNA levels by adrenal hormones.

    Science.gov (United States)

    Kononen, J; Soinila, S; Persson, H; Honkaniemi, J; Hökfelt, T; Pelto-Huikko, M

    1994-12-01

    We studied the expression of messenger ribonucleic acids (mRNAs) for neurotrophins and neurotrophin receptors in the rat pituitary gland and examined the influence of adrenal hormones on their mRNA levels, using in situ hybridization and Northern blot analysis. The only neurotrophin present at detectable levels in the pituitary was brain-derived neurotrophic factor (BDNF), which was observed in the anterior and intermediate lobes. Several transcripts of the putative receptor for BDNF, trkB, were present in the anterior and posterior lobes of the pituitary. A low amount of trkC mRNA was found in both the anterior and the intermediate lobe. Dexamethasone treatment decreased both BDNF and trkB mRNA levels in the anterior lobe of the pituitary. Adrenalectomy had no effect on trkB expression, but it decreased BDNF mRNA levels in comparison to the control animals. This effect could not be reversed by dexamethasone substitution, suggesting that BDNF, mRNA levels may be regulated not only by glucocorticoids but also by other adrenal hormones. These results demonstrate that BDNF, trkB and trkC are expressed in the pituitary gland and that glucocorticoids and possibly other adrenal hormones may modulate pituitary functions by regulating the expression of neurotrophic factors and their receptors. Whether BDNF acts as a secreted hormone, a trophic factor, or has autocrine/paracrine functions within the pituitary through its receptor, trkB, remains to be studied.

  4. Cloning and tissue distribution of rat hear fatty acid binding protein mRNA: identical forms in heart and skeletal muscle

    International Nuclear Information System (INIS)

    Claffey, K.P.; Herrera, V.L.; Brecher, P.; Ruiz-Opazo, N.

    1987-01-01

    A fatty acid binding protein (FABP) as been identified and characterized in rat heart, but the function and regulation of this protein are unclear. In this study the cDNA for rat heart FABP was cloned from a λ gt11 library. Sequencing of the cDNA showed an open reading frame coding for a protein with 133 amino acids and a calculated size of 14,776 daltons. Several differences were found between the sequence determined from the cDNA and that reported previously by protein sequencing techniques. Northern blot analysis using rat heart FABP cDNA as a probe established the presence of an abundant mRNA in rat heart about 0.85 kilobases in length. This mRNA was detected, but was not abundant, in fetal heart tissue. Tissue distribution studies showed a similar mRNA species in red, but not white, skeletal muscle. In general, the mRNA tissue distribution was similar to that of the protein detected by Western immunoblot analysis, suggesting that heart FABP expression may be regulated at the transcriptional level. S1 nuclease mapping studies confirmed that the mRNA hybridized to rat heart FABP cDNA was identical in heart and red skeletal muscle throughout the entire open reading frame. The structural differences between heart FABP and other members of this multigene family may be related to the functional requirements of oxidative muscle for fatty acids as a fuel source

  5. Cloning and tissue distribution of rat hear fatty acid binding protein mRNA: identical forms in heart and skeletal muscle

    Energy Technology Data Exchange (ETDEWEB)

    Claffey, K.P.; Herrera, V.L.; Brecher, P.; Ruiz-Opazo, N.

    1987-12-01

    A fatty acid binding protein (FABP) as been identified and characterized in rat heart, but the function and regulation of this protein are unclear. In this study the cDNA for rat heart FABP was cloned from a lambda gt11 library. Sequencing of the cDNA showed an open reading frame coding for a protein with 133 amino acids and a calculated size of 14,776 daltons. Several differences were found between the sequence determined from the cDNA and that reported previously by protein sequencing techniques. Northern blot analysis using rat heart FABP cDNA as a probe established the presence of an abundant mRNA in rat heart about 0.85 kilobases in length. This mRNA was detected, but was not abundant, in fetal heart tissue. Tissue distribution studies showed a similar mRNA species in red, but not white, skeletal muscle. In general, the mRNA tissue distribution was similar to that of the protein detected by Western immunoblot analysis, suggesting that heart FABP expression may be regulated at the transcriptional level. S1 nuclease mapping studies confirmed that the mRNA hybridized to rat heart FABP cDNA was identical in heart and red skeletal muscle throughout the entire open reading frame. The structural differences between heart FABP and other members of this multigene family may be related to the functional requirements of oxidative muscle for fatty acids as a fuel source.

  6. Definition of the complete Schistosoma mansoni hemoglobinase mRNA sequence and gene expression in developing parasites.

    Science.gov (United States)

    el Meanawy, M A; Aji, T; Phillips, N F; Davis, R E; Salata, R A; Malhotra, I; McClain, D; Aikawa, M; Davis, A H

    1990-07-01

    Schistosoma mansoni uses a variety of proteases termed hemoglobinases to obtain nutrition from host globin. Previous reports have characterized cDNAs encoding 1 of these enzymes. However, these sequences did not define the primary structures of the mRNA and protein. The complete sequence of the 1390 base mRNA has now been determined. It encodes a 50 kDa primary translation product. In vitro translations coupled with immunoprecipitations and Western blots of parasite lysates allowed visualization of the 50 kDa form. Production of the 31 kDa mature hemoglobinase from the 50 kDa species involves removal of both NH2 and COOH terminal residues from the primary translation product. Expression of hemoglobinase mRNA and protein was examined during larval parasite development. Low levels were observed in young schistosomula. After 6-9 days in culture, high hemoglobinase levels were seen which correlated with the onset of red blood cell feeding. Immunoelectron microscopy was employed to examine hemoglobinase location and function. In adult worms the enzyme was associated with the gut lumen and gut epithelium. In cercariae, the protease was observed in the head gland, suggesting new roles for the protease.

  7. Differences in correlation of mRNA gene expression in mice sensitive and resistant to radiation-induced pulmonary fibrosis

    International Nuclear Information System (INIS)

    Johnston, C.J.; Piedboeuf, B.; Finkelstein, J.N.; Baggs, R.; Rubin, P.

    1995-01-01

    Fibrosis, characterized by the accumulation of collagen, is a late result of thoracic irradiation. The purpose of this study was to determine if extracellular matrix protein and transforming growth factor β mRNA expression are altered late in the course of pulmonary fibrosis after irradiation, and then to determine if these changes differ between two strains of mice which vary in their sensitivity to radiation. Radiation-sensitive (C57BL/6) and radiation-resistant (C3H/HeJ) mice were irradiated with a single dose of 5 or 12.5 Gy to the thorax. Total lung RNA was prepared and immobilized by Northern and slot blotting and hybridized with radiolabeled cDNA probes for collagens I, III and IV, fibronectin, and transforming growth factor β 1 and β 3 . Autoradiographic data were quantified by video densitometry and results normalized to a control probe encoding for glyceralde-hyde-3-phosphate dehydrogenase. Alterations in mRNA abundance were observed in the sensitive mice at all times, while levels in the resistant mice were unaffected until 26 weeks after irradiation. The relationship between extracellular matrix protein per se and increased mRNA abundance suggests that late matrix protein accumulation may be a function of gene expression. Differences in levels of transforming growth factor βmRNA may lead to strain-dependent variation in fibrotic response and may also contribute to the radiation-induced component of pulmonary fibrosis. 32 refs., 5 figs

  8. Effects of low dose radiation on expressions of ICAM-1 mRNA and protein in kidney of diabetic mice

    International Nuclear Information System (INIS)

    Zhang Chi; Li Xiaokun; Gong Shouliang; Liu Xiaoju; Zhao Xue; Liu Xiaoju; Zhao Xue; Shen Wenjie; Li Cai; Cai Lu

    2010-01-01

    Objective: To study the effects of low dose radiation (LDR) on the expressions of intercellular adhesion molecule-1 (ICAM-1) mRNA and protein in kidney of diabetes mellitus (DM) mice and illuminate that anti-inflammation of LDR is a main mechanism for diabetic therapy. Methods: The healthy and right age C57BL/6J mice were divided into 4 groups including control, DM, LDR and DM/LDR. The mice in DM and DM/LDR groups were injected intraperitoneally with streptozocin (STZ) to set up DM models. The mice in DM/LDR and LDR groups were irradiated with 25 mGy every other day for 4 weeks. The expressions of ICAM-1 mRNA and protein in kidney were detected with RT-PCR and Western blotting 2, 4, 8, 12 and 16 weeks after irradiation. Results: The expressions of ICAM-1 mRNA and protein in kidney had no significant difference among 4 groups before LDR (P>0.05). The expressions of ICAM-1 mRNA and protein 2 weeks after irradiation with LDR were higher than those in the other 3 groups (P<0.05). The expressions of ICAM-1 mRNA and protein in the DM/LDR group 4 weeks after irradiation were also significantly higher than those in non-DM groups (P<0.05), but still significantly lower than those in DM group (P<0.05), and the significant differences were kept to 16 weeks after irradiation. But the expressions of ICAM-1 mRNA and protein in LDR group were significantly higher than those in control group (P<0.05). IHC assay showed that the glomerular and tubular in DM and DM/LDR groups were abnormal and the quantities of the positive staining cells were significantly increased compared with non-DM groups. However the damage of glomerular and tubular in DM/LDR was significantly supressed compared with DM group and the positive staining cells were also decreased. Conclusion: Under the circumstance of DM, LDR can significantly decrease the expressions of ICAM-1 mRNA and protein in mouse kidney to relief the inflammation reaction in kidney; but in normal condition, LDR can improve the immunity and

  9. Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis

    Energy Technology Data Exchange (ETDEWEB)

    Aravalli, Rajagopal N., E-mail: aravalli@umn.edu [Department of Radiology, University of Minnesota Medical School, MMC 292, 420 Delaware Street SE, Minneapolis, MN 55455 (United States); Park, Chang W. [Department of Medicine, University of Minnesota Medical School, MMC 36, 420 Delaware Street SE, Minneapolis, MN 55455 (United States); Steer, Clifford J., E-mail: steer001@umn.edu [Department of Medicine, University of Minnesota Medical School, MMC 36, 420 Delaware Street SE, Minneapolis, MN 55455 (United States); Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455 (United States)

    2016-08-26

    The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.

  10. Detection of Sleeping Beauty transposition in the genome of host cells by non-radioactive Southern blot analysis

    International Nuclear Information System (INIS)

    Aravalli, Rajagopal N.; Park, Chang W.; Steer, Clifford J.

    2016-01-01

    The Sleeping Beauty transposon (SB-Tn) system is being used widely as a DNA vector for the delivery of therapeutic transgenes, as well as a tool for the insertional mutagenesis in animal models. In order to accurately assess the insertional potential and properties related to the integration of SB it is essential to determine the copy number of SB-Tn in the host genome. Recently developed SB100X transposase has demonstrated an integration rate that was much higher than the original SB10 and that of other versions of hyperactive SB transposases, such as HSB3 or HSB17. In this study, we have constructed a series of SB vectors carrying either a DsRed or a human β-globin transgene that was encompassed by cHS4 insulator elements, and containing the SB100X transposase gene outside the SB-Tn unit within the same vector in cis configuration. These SB-Tn constructs were introduced into the K-562 erythroid cell line, and their presence in the genomes of host cells was analyzed by Southern blot analysis using non-radioactive probes. Many copies of SB-Tn insertions were detected in host cells regardless of transgene sequences or the presence of cHS4 insulator elements. Interestingly, the size difference of 2.4 kb between insulated SB and non-insulated controls did not reflect the proportional difference in copy numbers of inserted SB-Tns. We then attempted methylation-sensitive Southern blots to assess the potential influence of cHS4 insulator elements on the epigenetic modification of SB-Tn. Our results indicated that SB100X was able to integrate at multiple sites with the number of SB-Tn copies larger than 6 kb in size. In addition, the non-radioactive Southern blot protocols developed here will be useful to detect integrated SB-Tn copies in any mammalian cell type.

  11. Western blot banding pattern in early Lyme borreliosis among patients from an endemic region of north-eastern Poland.

    Science.gov (United States)

    Flisiak, R; Wierzbicka, I; Prokopowicz, D

    1998-01-01

    Aim of this study was evaluation of Western blot banding patterns in different clinical forms of early Lyme borreliosis diagnosed in patients from north-eastern Poland, recognized as endemic for tick-borne diseases. Study was performed on serum samples of 48 patients with Lyme borreliosis and 26 healthy volunteers, as controls. Samples tested routinely for total antibody with enzyme immunoassay were subsequently analysed for specific antibodies with Western blot based on antigen extract of European strain of Borrelia burgdorferi. In patients, IgM antibodies were the most frequently directed against 41 kDa and 58 kDa antigens, whereas in control group only antibodies against 45 kDa and 58 kDa were present. Similar response was observed in respect to IgG antibodies. Evaluation of banding pattern in respect to clinical form of the disease revealed the highest prevalence of IgM and IgG anti-41 kDa antibodies in patients with erythema migrans and Lyme arthritis, and anti-58 kDa in neuroborreliosis patients, who had no anti-21 kDa antibodies. Relatively high frequency of IgG antibodies against 21, 30 and 93 kDa antigens was typical for neuroborreliosis. Bands count was significantly higher in different clinical forms of the disease than in controls, and it was the highest in neuroborreliosis. Combined analysis of Western blot results (IgM/IgG) enabled to achieve higher sensitivity (84%) and specificity (100%) than available with the most recommended EIA kits.

  12. La técnica de Western Blot como criterio de identidad para la vacuna antimeningocócica Men B Western Blot technique as an identity criterion for Men B antimeningococcal vaccine

    Directory of Open Access Journals (Sweden)

    R. Rosario Diéguez Castro

    2009-12-01

    Full Text Available Se desarrolló y validó la técnica de Western Blot aplicada a la vacuna antimeningocócica Men B producida en el Instituto Finlay con el objetivo de demostrar un criterio de identidad. En el estudio de las proteínas antigénicas de la vacuna, P1.15 y P1.4 en vesícula de membrana externa,monograneles y producto final se emplearon en la identificación anticuerpos monoclonales específicos para estas proteínas. Los parámetros desarrollados en la validación de la técnica fueron: especificidad, límite de detección, repetibilidad, precisión intermedia, reproducibilidad y robustez. El método cumplió con los parámetros señalados, por lo que se consideró validado.Western Blot technique was developed and validated, applied to Men B meningococcal vaccine produced in "Carlos J, Finlay" Institute to demonstrate an identity criterion. In study of antigenic proteins of the vaccine, we used P1.15 y P1.4 in vesicle of external membrane, monogranels, and end product to identify the monoclonal antibodies specific of these proteins. Parameters developed in technique validation included: specificity, detection limit, repetition, average accuracy, reproduction, and strength. Method fulfilled with specified parameters, thus considering its validation.

  13. Membranes and Fluorescence microscopy

    DEFF Research Database (Denmark)

    Bagatolli, Luis

    2009-01-01

    Fluorescence spectroscopy-based techniques using conventional fluorimeters have been extensively applied since the late 1960s to study different aspects of membrane-related phenomena, i.e., mainly relating to lipid-lipid and lipid-protein (peptide) interactions. Even though fluorescence...

  14. Multimodal fluorescence imaging spectroscopy

    NARCIS (Netherlands)

    Stopel, Martijn H W; Blum, Christian; Subramaniam, Vinod; Engelborghs, Yves; Visser, Anthonie J.W.G.

    2014-01-01

    Multimodal fluorescence imaging is a versatile method that has a wide application range from biological studies to materials science. Typical observables in multimodal fluorescence imaging are intensity, lifetime, excitation, and emission spectra which are recorded at chosen locations at the sample.

  15. Mere end blot en bid af hverdagen- Måltidet i et leve- og bomiljø

    DEFF Research Database (Denmark)

    Bundgaard, Karen Marie

    2005-01-01

    . Datamaterialet bygger på deltagerobservationer og interviews. Undersøgelsen viste, at den måde måltiderne var organiseret på gav tid og rum til en hjemlig atmosfære, til et levende fællesskab, til det at være noget og at være sig selv og til at have værdifulde gøremål. Måltiderne var ikke blot en bid – men en...

  16. Detection of Rickettsia in Rhipicephalus sanguineus Ticks and Ctenocephalides felis Fleas from Southeastern Tunisia by Reverse Line Blot Assay

    Science.gov (United States)

    Khrouf, Fatma; M'Ghirbi, Youmna; Znazen, Abir; Ben Jemaa, Mounir; Hammami, Adnene

    2014-01-01

    Ticks (n = 663) and fleas (n = 470) collected from domestic animals from southeastern Tunisia were screened for Rickettsia infection using reverse line blot assay. Evidence of spotted fever group Rickettsia was obtained. We detected Rickettsia felis in fleas, Rickettsia massiliae Bar 29 and the Rickettsia conorii Israeli spotted fever strain in ticks, and Rickettsia conorii subsp. conorii and Rickettsia spp. in both arthropods. The sensitivity of the adopted technique allowed the identification of a new association between fleas and R. conorii subsp. conorii species. The presence of these vector-borne Rickettsia infections should be considered when diagnosing this disease in humans in Tunisia. PMID:24226919

  17. Gold nanoparticle-based beacon to detect STAT5b mRNA expression in living cells: a case optimized by bioinformatics screen

    Directory of Open Access Journals (Sweden)

    Deng D

    2015-04-01

    Full Text Available Dawei Deng,* Yang Li,* Jianpeng Xue, Jie Wang, Guanhua Ai, Xin Li, Yueqing GuDepartment of Biomedical Engineering, China Pharmaceutical University, Nanjing, People’s Republic of China*These authors contributed equally to this workAbstract: Messenger RNA (mRNA, a single-strand ribonucleic acid with functional gene information is usually abnormally expressed in cancer cells and has become a promising biomarker for the study of tumor progress. Hairpin DNA-coated gold nanoparticle (hDAuNP beacon containing a bare gold nanoparticle (AuNP as fluorescence quencher and thiol-terminated fluorescently labeled stem–loop–stem oligonucleotide sequences attached by Au–S bond is currently a new nanoscale biodiagnostic platform capable of mRNA detection, in which the design of the loop region sequence is crucial for hybridizing with the target mRNA. Hence, in this study, to improve the sensitivity and selectivity of hDAuNP beacon simultaneously, the loop region of hairpin DNA was screened by bioinformatics strategy. Here, signal transducer and activator of transcription 5b (STAT5b mRNA was selected and used as a practical example. The results from the combined characterizations using optical techniques, flow cytometry assay, and cell microscopic imaging showed that after optimization, the as-prepared hDAuNP beacon had higher selectivity and sensitivity for the detection of STAT5b mRNA in living cells, as compared with our previous beacon. Thus, the bioinformatics method may be a promising new strategy for assisting in the designing of the hDAuNP beacon, extending its application in the detection of mRNA expression and the resultant mRNA-based biological processes and disease pathogenesis.Keywords: molecular beacon, bioinformatics, gold nanoparticle, STAT5b mRNA, visual detection

  18. Prediction of the optimum hybridization conditions of dot-blot-SNP analysis using estimated melting temperature of oligonucleotide probes.

    Science.gov (United States)

    Shiokai, Sachiko; Kitashiba, Hiroyasu; Nishio, Takeshi

    2010-08-01

    Although the dot-blot-SNP technique is a simple cost-saving technique suitable for genotyping of many plant individuals, optimization of hybridization and washing conditions for each SNP marker requires much time and labor. For prediction of the optimum hybridization conditions for each probe, we compared T (m) values estimated from nucleotide sequences using the DINAMelt web server, measured T (m) values, and hybridization conditions yielding allele-specific signals. The estimated T (m) values were comparable to the measured T (m) values with small differences of less than 3 degrees C for most of the probes. There were differences of approximately 14 degrees C between the specific signal detection conditions and estimated T (m) values. Change of one level of SSC concentrations of 0.1, 0.2, 0.5, and 1.0x SSC corresponded to a difference of approximately 5 degrees C in optimum signal detection temperature. Increasing the sensitivity of signal detection by shortening the exposure time to X-ray film changed the optimum hybridization condition for specific signal detection. Addition of competitive oligonucleotides to the hybridization mixture increased the suitable hybridization conditions by 1.8. Based on these results, optimum hybridization conditions for newly produced dot-blot-SNP markers will become predictable.

  19. Label-free DNA quantification via a 'pipette, aggregate and blot' (PAB) approach with magnetic silica particles on filter paper.

    Science.gov (United States)

    Li, Jingyi; Liu, Qian; Alsamarri, Hussein; Lounsbury, Jenny A; Haversitick, Doris M; Landers, James P

    2013-03-07

    Reliable measurement of DNA concentration is essential for a broad range of applications in biology and molecular biology, and for many of these, quantifying the nucleic acid content is inextricably linked to obtaining optimal results. In its most simplistic form, quantitative analysis of nucleic acids can be accomplished by UV-Vis absorbance and, in more sophisticated format, by fluorimetry. A recently reported new concept, the 'pinwheel assay', involves a label-free approach for quantifying DNA through aggregation of paramagnetic beads in a rotating magnetic field. Here, we describe a simplified version of that assay adapted for execution using only a pipet and filter paper. The 'pipette, aggregate, and blot' (PAB) approach allows DNA to induce bead aggregation in a pipette tip through exposure to a magnetic field, followed by dispensing (blotting) onto filter paper. The filter paper immortalises the extent of aggregation, and digital images of the immortalized bead conformation, acquired with either a document scanner or a cell phone camera, allows for DNA quantification using a noncomplex algorithm. Human genomic DNA samples extracted from blood are quantified with the PAB approach and the results utilized to define the volume of sample used in a PCR reaction that is sensitive to input mass of template DNA. Integrating the PAB assay with paper-based DNA extraction and detection modalities has the potential to yield 'DNA quant-on-paper' devices that may be useful for point-of-care testing.

  20. Profiling EGFR activity in head and neck squamous cell carcinoma by using a novel layered membrane Western blot technology.

    Science.gov (United States)

    Patel, Vyomesh; Ramesh, Arun; Traicoff, June L; Baibakov, Galina; Emmert-Buck, Michael R; Gutkind, J Silvio; Knezevic, Vladimir

    2005-05-01

    Given the role of epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas (HNSCC), several rational approaches have now been utilized to abrogate tyrosine kinase activity and its disengagement from downstream signal transducers. Monitoring the activity of these molecules could potentially be useful to determine not only drug efficacy but also to identify HNSCC patients most likely to benefit from this type of therapy. In this study we have used a novel high throughput multi-layered Western blotting (MLWestern) method that allows the detection of multiple proteins from a single experiment in order to characterize key components in the EGFR signaling pathway in HNSCC cells. Total and activated forms of EGFR and the downstream effectors, Erk and Akt were readily detected in HNSCC cells, where in the control cells (HaCaT) these proteins could only be detected in EGF stimulated cells. Results from conventional Western blot and MLWestern were comparable. Clustering analysis of protein expression revealed similarities in cellular response between some of the cell lines indicative of similarities in their biological response. The data indicate that MLWestern can be potentially applied to identify molecular targets that could be used for rational therapeutic intervention strategies.

  1. mRNA and Protein Levels for GABA[subscript A][alpha]4, [alpha]5, [beta]1 and GABA[subscript B]R1 Receptors are Altered in Brains from Subjects with Autism

    Science.gov (United States)

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rooney, Robert J.; Patel, Diven H.; Thuras, Paul D.

    2010-01-01

    We have shown altered expression of gamma-aminobutyric acid A (GABA[subscript A]) and gamma-aminobutyric acid B (GABA[subscript B]) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3…

  2. Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing.

    Science.gov (United States)

    Anvar, Seyed Yahya; Allard, Guy; Tseng, Elizabeth; Sheynkman, Gloria M; de Klerk, Eleonora; Vermaat, Martijn; Yin, Raymund H; Johansson, Hans E; Ariyurek, Yavuz; den Dunnen, Johan T; Turner, Stephen W; 't Hoen, Peter A C

    2018-03-29

    The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells. Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

  3. Fluorescence and Spectral Imaging

    Directory of Open Access Journals (Sweden)

    Ralph S. DaCosta

    2007-01-01

    Full Text Available Early identification of dysplasia remains a critical goal for diagnostic endoscopy since early discovery directly improves patient survival because it allows endoscopic or surgical intervention with disease localized without lymph node involvement. Clinical studies have successfully used tissue autofluorescence with conventional white light endoscopy and biopsy for detecting adenomatous colonic polyps, differentiating benign hyperplastic from adenomas with acceptable sensitivity and specificity. In Barrett's esophagus, the detection of dysplasia remains problematic because of background inflammation, whereas in the squamous esophagus, autofluorescence imaging appears to be more dependable. Point fluorescence spectroscopy, although playing a crucial role in the pioneering mechanistic development of fluorescence endoscopic imaging, does not seem to have a current function in endoscopy because of its nontargeted sampling and suboptimal sensitivity and specificity. Other point spectroscopic modalities, such as Raman spectroscopy and elastic light scattering, continue to be evaluated in clinical studies, but still suffer the significant disadvantages of being random and nonimaging. A recent addition to the fluorescence endoscopic imaging arsenal is the use of confocal fluorescence endomicroscopy, which provides real-time optical biopsy for the first time. To improve detection of dysplasia in the gastrointestinal tract, a new and exciting development has been the use of exogenous fluorescence contrast probes that specifically target a variety of disease-related cellular biomarkers using conventional fluorescent dyes and novel potent fluorescent nanocrystals (i.e., quantum dots. This is an area of great promise, but still in its infancy, and preclinical studies are currently under way.

  4. Comparison of mRNA levels of three ethylene receptors in senescing flowers of carnation (Dianthus caryophyllus L.).

    Science.gov (United States)

    Shibuya, Kenichi; Nagata, Masayasu; Tanikawa, Natsu; Yoshioka, Toshihito; Hashiba, Teruyoshi; Satoh, Shigeru

    2002-03-01

    Three ethylene receptor genes, DC-ERS1, DC-ERS2 and DC-ETR1, were previously identified in carnation (Dianthus caryophyllus L.). Here, the presence of mRNAs for respective genes in flower tissues and their changes during flower senescence are investigated by Northern blot analysis. DC-ERS2 and DC-ETR1 mRNAs were present in considerable amounts in petals, ovaries and styles of the flower at the full-opening stage. In the petals the level of DC-ERS2 mRNA showed a decreasing trend toward the late stage of flower senescence, whereas it increased slightly in ovaries and was unchanged in styles throughout the senescence period. However, DC-ETR1 mRNA showed no or little changes in any of the tissues during senescence. Exogenously applied ethylene did not affect the levels of DC-ERS2 and DC-ETR1 mRNAs in petals. Ethylene production in the flowers was blocked by treatment with 1,1-dimethyl-4-(phenylsulphonyl)semicarbazide (DPSS), but the mRNA levels for DC-ERS2 and DC-ETR1 decreased in the petals. DC-ERS1 mRNA was not detected in any cases. These results indicate that DC-ERS2 and DC-ETR1 are ethylene receptor genes responsible for ethylene perception and that their expression is regulated in a tissue-specific manner and independently of ethylene in carnation flowers during senescence.

  5. Mere end blot pirringer

    DEFF Research Database (Denmark)

    Jantzen, Christian; Østergaard, Per

    2006-01-01

    Med udgangspunkt i en række cases på vellykkede oplevelsesøkonomiske forretningsmodeller argumenterer artiklen for, at oplevelsesprodukter skal bygge på et klart tema, som forbrugeren kan koble sig sanse- og følelsesmæssigt op på. Forbrugeren skal kunne omsætte produktets pirringer til egne erfar...

  6. Ej blot til lyst

    DEFF Research Database (Denmark)

    Leroyer, Patrick

    2010-01-01

    The purpose of this article is to reassert the crucial importance of access to data in lexicographic information tools and, expanding on this, to establish the existence of two distinct lexicographic access modes - consultation and navigation. It is explained how the tools can be decalibrated whe...... balance between user, access, and data is disturbed, and how access to data then is jeopardized. Taking online wine guides as a case in point, it is shown how such multifunctional information tools do benefit from a lexicographic design featuring both access modes....

  7. [Expression of heat shock protein 70 and its mRNA in career exposure to manganese].

    Science.gov (United States)

    Chen, Wenwen; Shao, Hua; Chi, Mingfeng; Zhang, Zhihu; Shan, Yongle; Zou, Wei

    2015-10-01

    To analyze the expression levels of heat shock protein70 (HSPs70) and HSPs70 mRNA in different exposure to manganese, and research the neuroprotective effect on the career exposure to manganese. From 2008 to 2009, with cross-sectional study design, and in a locomotive and rolling stock works, by stratified random sampling method, the exposed sample consisted of 180 welders from different welding shops and 100 unexposed in the last three years, non-welder controls with age-matched workers of similar socioeconomic status from the same industry. The control workers had not been exposed to neurotoxic chemicals. The mRNA expressions of four different metabolic enzyme were detected by SYBR Green I quantitative real-time polymerase chain reaction. The expression levels of the two enzymes mRNA in different exposure to manganese were analyzed. The expressions of HSPs70 were detected by Western blot. The concentration of air manganese was determined by GFAAS. The average concentration of 8 h time (8h-TWA) was used to express the level of individual exposure to manganese, according to the air manganese workplace occupational exposure limit (8h-TWA=0.15 mg/m3), the exposed group is divided into high exposed group (>0.15 mg/m3) and low exposure group (<0.15 mg/m3). The individuals exposed to manganese dose of exposed group ((0.25±0.31) mg/m3) was higher than the control group ((0.06±0.02) mg/m3) (t=6.15, P=0.001); individuals exposed to manganese dose of high exposure group for (0.42±0.34) mg/m3, which was higher than low exposure group (0.09±0.07) mg/m3 (t=9.80, P=0.001). HSPs70 mRNA and protein of exposure group (5.65±0.21, 3.26±0.15) were higher than the reference group (0.41±0.03, 1.32±0.12) (t=18.91, t=8.68, P=0.001). HSP70 mRNA and protein of high exposure group (6.48±0.37, 3.67±0.26) were higher than the low exposure group (5.15±0.23, 3.02±0.19) (t=3.24, t=2.01, P=0.003, P=0.043). The expression of peripheral blood lymphocytes HSPs70 level and HSPs70 mRNA

  8. Fluorescent discharge lamp

    Science.gov (United States)

    Mukai, E.; Otsuka, H.; Nomi, K.; Honmo, I.

    1982-01-01

    A rapidly illuminating fluorescent lamp 1,200 mm long and 32.5 mm in diameter with an interior conducting strip which is compatible with conventional fixtures and ballasts is described. The fluorescent lamp is composed of a linear glass tube, electrodes sealed at both ends, mercury and raregas sealed in the glass tube, a fluorescent substance clad on the inner walls of the glass tube, and a clad conducting strip extending the entire length of the glass tube in the axial direction on the inner surface of the tube.

  9. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  10. Expression of calmodulin mRNA in rat olfactory neuroepithelium.

    Science.gov (United States)

    Biffo, S; Goren, T; Khew-Goodall, Y S; Miara, J; Margolis, F L

    1991-04-01

    A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.

  11. Studies on the role of NonA in mRNA biogenesis

    International Nuclear Information System (INIS)

    Kozlova, Natalia; Braga, Jose; Lundgren, Josefin; Rino, Jose; Young, Patrick; Carmo-Fonseca, Maria; Visa, Neus

    2006-01-01

    The NonA protein of Drosophila melanogaster is an abundant nuclear protein that belongs to the DBHS (Drosophila behavior, human splicing) protein family. The DBHS proteins bind both DNA and RNA in vitro and have been involved in different aspects of gene expression, including pre-mRNA splicing, transcription regulation and nuclear retention of mRNA. We have used double-stranded RNA interference in Drosophila S2 cells to silence the expression of NonA and to investigate its role in mRNA biogenesis. We show that knockdown of NonA does not affect transcription nor splicing. We demonstrate that NonA forms a complex with the essential nuclear export factor NXF1 in an RNA-dependent manner. We have constructed stable S2 cell lines that express full-length and truncated NXF1 fused to GFP in order to perform fluorescence recovery after photobleaching experiments. We show that knockdown of NonA reduces the intranuclear mobility of NXF1-GFP associated with poly(A) + RNA in vivo, while the mobility of the truncated NXF1-GFP that does not bind RNA is not affected. Our data suggest that NonA facilitates the intranuclear mobility of mRNP particles

  12. Site-Specific Covalent Conjugation of Modified mRNA by tRNA Guanine Transglycosylase.

    Science.gov (United States)

    Ehret, Fabian; Zhou, Cun Yu; Alexander, Seth C; Zhang, Dongyang; Devaraj, Neal K

    2018-03-05

    Modified mRNA (mod-mRNA) has recently been widely studied as the form of RNA useful for therapeutic applications due to its high stability and lowered immune response. Herein, we extend the scope of the recently established RNA-TAG (transglycosylation at guanosine) methodology, a novel approach for genetically encoded site-specific labeling of large mRNA transcripts, by employing mod-mRNA as substrate. As a proof of concept, we covalently attached a fluorescent probe to mCherry encoding mod-mRNA transcripts bearing 5-methylcytidine and/or pseudouridine substitutions with high labeling efficiencies. To provide a versatile labeling methodology with a wide range of possible applications, we employed a two-step strategy for functionalization of the mod-mRNA to highlight the therapeutic potential of this new methodology. We envision that this novel and facile labeling methodology of mod-RNA will have great potential in decorating both coding and noncoding therapeutic RNAs with a variety of diagnostic and functional moieties.

  13. HIV-1 matrix dependent membrane targeting is regulated by Gag mRNA trafficking.

    Directory of Open Access Journals (Sweden)

    Jing Jin

    Full Text Available Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV post-transcriptional regulatory element (PRE mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.

  14. A mRNA and cognate microRNAs localize in the nucleolus.

    Science.gov (United States)

    Reyes-Gutierrez, Pablo; Ritland Politz, Joan C; Pederson, Thoru

    2014-01-01

    We previously discovered that a set of 5 microRNAs are concentrated in the nucleolus of rat myoblasts. We now report that several mRNAs are also localized in the nucleoli of these cells as determined by microarray analysis of RNA from purified nucleoli. Among the most abundant of these nucleolus-localized mRNAs is that encoding insulin-like growth factor 2 (IGF2), a regulator of myoblast proliferation and differentiation. The presence of IGF2 mRNA in nucleoli was confirmed by fluorescence in situ hybridization, and RT-PCR experiments demonstrated that these nucleolar transcripts are spliced, thus arriving from the nucleoplasm. Bioinformatics analysis predicted canonically structured, highly thermodynamically stable interactions between IGF2 mRNA and all 5 of the nucleolus-localized microRNAs. These results raise the possibility that the nucleolus is a staging site for setting up particular mRNA-microRNA interactions prior to export to the cytoplasm.

  15. Reviews in fluorescence 2007

    CERN Document Server

    Lakowicz, Joseph R; Geddes, Chris D

    2009-01-01

    This fourth volume in the Springer series summarizes the year's progress in fluorescence, with authoritative analytical reviews specialized enough for professional researchers, yet also appealing to a wider audience of scientists in related fields.

  16. Introduction to fluorescence

    CERN Document Server

    Jameson, David M

    2014-01-01

    "An essential contribution to educating scientists in the principles of fluorescence. It will also be an important addition to the libraries of practitioners applying the principles of molecular fluorescence."-Ken Jacobson, Kenan Distinguished Professor of Cell Biology and Physiology, University of North Carolina at Chapel Hill"An exquisite compendium of fluorescence and its applications in biochemistry enriched by a very exciting historical perspective. This book will become a standard text for graduate students and other scientists."-Drs. Zygmunt (Karol) Gryczynski and Ignacy Gryczynski, University of North Texas Health Science Center"… truly a masterwork, combining clarity, precision, and good humor. The reader, novice or expert, will be pleased with the text and will not stop reading. It is a formidable account of the fluorescence field, which has impacted the life sciences so considerably in the last 60 years."-Jerson L. Silva, M.D., Ph.D., Professor and Director, National Institute of Science and Tech...

  17. Fluorescence (Multiwave) Confocal Microscopy.

    Science.gov (United States)

    Welzel, J; Kästle, Raphaela; Sattler, Elke C

    2016-10-01

    In addition to reflectance confocal microscopy, multiwave confocal microscopes with different laser wavelengths in combination with exogenous fluorophores allow fluorescence mode confocal microscopy in vivo and ex vivo. Fluorescence mode confocal microscopy improves the contrast between the epithelium and the surrounding soft tissue and allows the depiction of certain structures, like epithelial tumors, nerves, and glands. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Blot hybridization analysis of TCR genes of T cells for five people exposed in a radiation accident

    International Nuclear Information System (INIS)

    Min Rui; Liu Benti; Cheng Tianmin; Yang Rujun; Meng Xiangshun; Xiao Jinsong

    1996-01-01

    Human lymphocyte total DNA was prepared in agarose plug by mixing cells with low melting agarose, and two restriction endonucleases were used for digestion of the total DNA with human α and β TCR cDNA probes. The total digested DNA from five people who were whole body exposed to 2.0-2.5 Gy ionizing radiation in an accident 4.5 years ago was hybridized by Southern blot method. The results showed that no obvious difference in hybridization bands was found between controls and the five victims when hybridizations were fulfilled in the total DNA which was digested by Hind III restriction endonuclease with both α and β probes. However, when the total DNA was digested with restriction endonuclease EcoR I and was hybridized with TCR α probe, four of the five exposed people showed a different hybridizing band pattern compared with the controls. The results are also discussed

  19. Detection of anti-HIV-1 IgG antibodies in whole saliva by GACELISA and Western blot assays.

    Science.gov (United States)

    Matee, M I; Lyamuya, E F; Simon, E; Mbena, E C; Kagoma, C; Samaranayake, L P; Scheutz, F

    1996-05-01

    The present study, based on 158 HIV seropositives and 167 HIV seronegatives, demonstrates that saliva collected with the Omni-SAL device and tested with GACELISA (an IgG antibody capture ELISA) is an effective non-invasive alternative to serum for anti-HIV IgG antibody screening. The study also shows that a conventional serum Western blot kit can be used, with slight modifications, for confirmatory testing of saliva specimens. Collecting saliva with the Omni-SAL device had a very good acceptance rate among Tanzanian subjects, and although this diagnostic method is not yet known by the general public, 65% of the study participants preferred to give saliva instead of blood for HIV testing.

  20. Evaluation of a new eastern blotting technique for the analysis of ginsenoside Re in American ginseng berry pulp extracts.

    Science.gov (United States)

    Morinaga, Osamu; Uto, Takuhiro; Yuan, Chun-Su; Tanaka, Hiroyuki; Shoyama, Yukihiro

    2010-06-01

    A new eastern blotting technique has been established for ginsenoside Re (G-Re) contained in American ginseng berry pulp extracts. G-Re in American ginseng berry pulp was extracted using 100% methanol, 100% ethanol, 50% aqueous methanol, and 50% aqueous ethanol. The combined crude extracts were applied onto a polyethersulfone membrane and developed using the methanol-water-acetic acid solvent system (45:55:1 v/v). Separated components were immunostained using anti-G-Re monoclonal antibody. G-Re was first specifically detected and then quantitatively analyzed using NIH Imaging software. We also confirmed that the most suitable solvent was 50% aqueous methanol for extracting G-Re from American ginseng berry pulp. (c) 2009 Elsevier B.V. All rights reserved.

  1. mRNA and protein dataset of autophagy markers (LC3 and p62) in several cell lines

    Science.gov (United States)

    Gómez-Sánchez, Rubén; Yakhine-Diop, Sokhna M.S.; Rodríguez-Arribas, Mario; Bravo-San Pedro, José M.; Martínez-Chacón, Guadalupe; Uribe-Carretero, Elisabet; Pinheiro de Castro, Diana C.J.; Pizarro-Estrella, Elisa; Fuentes, José M.; González-Polo, Rosa A.

    2016-01-01

    We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I) to the autophagosomal membrane where it is lipidated (LC3-II)) and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates), (Klionsky et al., 2016) [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR). To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1) to block fusion of autophagosomes with lysosomes. PMID:27054171

  2. Standardisation of Western blotting to detect HTLV-1 antibodies synthesised in the central nervous system of HAM/TSP patients

    Directory of Open Access Journals (Sweden)

    Luiz Claudio Pereira Ribeiro

    2013-09-01

    Full Text Available Intrathecal synthesis of human T-lymphotropic virus type 1 (HTLV-1 antibodies (Abs represents conclusive evidence of a specific immune response in the central nervous system of HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP patients. Western blotting (WB for HTLV Abs in serum is a confirmatory test for HTLV-1 infection. The aim of this study was to standardise the Western blot to demonstrate the intrathecal pattern of Abs against HTLV-1 proteins in HAM/TSP patients. Paired cerebrospinal fluid (CSF and serum samples were selected from 20 patients with definite HAM/TSP, 19 HTLV-1 seronegative patients and two HTLV-1 patients without definite HAM/TSP. The presence of reactive bands of greater intensity in the CSF compared to serum (or bands in only the CSF indicated the intrathecal synthesis of anti-HTLV-1 Abs. All definite HAM/TSP patients presented with an intrathecal synthesis of anti-HTLV-1 Abs; these Abs were not detected in the control patients. The most frequent intrathecal targets of anti-HTLV-1 Abs were GD21, rgp46-I and p24 and, to a lesser extent, p19, p26, p28, p32, p36, p53 gp21 and gp46. The intrathecal immune response against env (GD21 and rgp46-I and gag (p24 proteins represents the most important humoral pattern in HAM/TSP. This response may be used as a diagnostic marker, considering the frequent association of intrathecal anti-HTLV-1 Ab synthesis with HAM/TSP and the pathogenesis of this neurological disease.

  3. Reverse line blot probe design and polymerase chain reaction optimization for bloodmeal analysis of ticks from the eastern United States.

    Science.gov (United States)

    Scott, M C; Harmon, J R; Tsao, J I; Jones, C J; Hickling, G J

    2012-05-01

    Determining the host preference of vector ticks is vital to elucidating the eco-epidemiology of the diseases they spread. Detachment of ticks from captured hosts can provide evidence of feeding on those host species, but only for those species that are feasible to capture. Recently developed, highly sensitive molecular assays show great promise in allowing host selection to be determined from minute traces of host DNA that persist in recently molted ticks. Using methods developed in Europe as a starting-point, we designed 12S rDNA mitochondrial gene probes suitable for use in a reverse line blot (RLB) assay of ticks feeding on common host species in the eastern United States. This is the first study to use the 12S mitochondrial gene in a RLB bloodmeal assay in North America. The assay combines conventional PCR with a biotin-labeled primer and reverse line blots that can be stripped and rehybridized up to 20 times, making the method less expensive and more straightforward to interpret than previous methods of tick bloodmeal identification. Probes were designed that target the species, genus, genus group, family, order, or class of eight reptile, 13 birds, and 32 mammal hosts. After optimization, the RLB assay correctly identified the current hostspecies for 99% of ticks [Amblyomma americanum (L.) and eight other ixodid tick species] collected directly from known hosts. The method identified previous-host DNA for approximately half of all questing ticks assayed. Multiple bloodmeal determinations were obtained in some instances from feeding and questing ticks; this pattern is consistent with previous RLB studies but requires further investigation. Development of this probe library, suitable for eastern U.S. ecosystems, opens new avenues for eco-epidemiological investigations of this region's tick-host systems.

  4. Southern blot analysis of skin biopsies for human papillomavirus DNA: renal allograft recipients in south-eastern Queensland.

    Science.gov (United States)

    Trenfield, K; Salmond, C A; Pope, J H; Hardie, I R

    1993-01-01

    The 104 skin biopsies from 34 patients who attended a Renal Transplant Unit in Brisbane over 12 months included 40 squamous cell carcinoma (SCC), 22 solar keratoses, 4 hyperkeratoses, 18 warts and 11 basal cell carcinoma (BCC). Human papillomavirus (HPV) DNA was identified by Southern blot hybridisation using, as individual probes, purified insert DNA from recombinant HPV 1, 2, 3 or 3/10, 4, 5 or 5/8, 7, 11, 16, 18 and 41 under relaxed conditions and characterised by restriction enzyme analysis and Southern blot hybridisation under more stringent conditions. Genomic HPV DNA was characterised in 7 skin biopsies from 4 renal allograft recipients (RARs): HPV 1A in a SCC (20 copies/cell) and a BCC (10 copies/cell) from the one patient, HPV 36 (20 copies/cell) in a SCC, HPV 1A [symbol: see text] 1000 copies/cell) in a wart and HPV 2B (200-800 copies/cell) in 3 warts from the one patient. Only HPV 1A in the SCC exhibited a significant degree of subtype variation. HPV DNA was identified in another 5 skin biopsies from another 4 RARs: HPV 3A in a wart and a hyperkeratosis, HPV 3/10-related DNA in 2 solar keratoses and HPV 5/8-related DNA in another (20-50 copies/cell). The incidence of HPV 5 (or 5-related HPVs) in RAR SCC was very low and that of HPV DNA in RAR warts was lower than that recorded elsewhere but this was not due to insensitivity of the assays. There was no evidence for a role for HPV in the aetiology of skin cancer in RARs in south-eastern Queensland but the possibility remains that as yet unidentified HPV types are involved.

  5. [Western Blot diagnostic yield for simultaneous antibody-detection in patients with human cysticercosis, hydatidosis, and human fascioliasis].

    Science.gov (United States)

    Davelois, Kelly; Escalante, Hermes; Jara, César

    2016-01-01

    . To determine the diagnostic yield using western blotting to simultaneously detect antibodies in patients with human cysticercosis, hydatidosis, and human fascioliasis. Materials and methods . Cross-sectional study of diagnostic yield assessment. Excretory/secretory antigens were obtained from Taenia solium larvae, Echinococcus granulosus cysts, and the adult flukes of Fasciola hepática, which were then separated using the polyacrylamide gel electrophoresis technique, transferred, and attached to a nitrocellulose membrane to be probed with sera from the patient infected with the three parasites. The sensitivity of the technique was assessed using 300 individual serum samples, 60 pools of two parasites, and 20 pools of three parasites with 75 sera from patients with other parasites, 10 from patients with other diseases, and 15 from patients without parasites. Results . The technique revealed 13 glycoproteins (GP): GP 35, 31, 24, 23, 18, 17, 14, and 13 kDa for cysticercosis; GP 8, 16, and 21 kDa for hydatidosis; and GP 17 and 23 kDa for fascioliasis. The test detected the presence of antibodies with a sensitivity of 96% (95% confidence interval [CI] = 94.62-98.54%) in the detection of one or the thirteen bands, a specificity of 100% (95% CI = 99.50-100.00%); individually, there was a sensitivity for cysticercosis of 97% (95% CI = 93.16-100.00%), for hydatidosis of 94% (95% CI = 88.85-99.15%) and for fascioliasis of 96% (95% CI = 91.66-100.00%). Conclusions . Western blotting is effective in the simultaneous detection of antibodies in patients with human cysticercosis, hydatidosis, and fascioliasis, and it can be used as a diagnostic test to either rule out or confirm the presence of antibodies in endemic areas.

  6. Diagnostic potential of Western blot analysis of sera from dogs with leishmaniasis in endemic areas and significance of the pattern.

    Science.gov (United States)

    Aisa, M J; Castillejo, S; Gallego, M; Fisa, R; Riera, M C; de Colmenares, M; Torras, S; Roura, X; Sentis, J; Portus, M

    1998-02-01

    Serum samples collected from 237 dogs in Catalonia (northeastern Spain) were screened by Western blot analysis to detect the presence of antibodies specific to different Leishmania infantum polypeptide fractions. Leishmaniasis was confirmed in 72 of these dogs by direct examination and/or culture. Another 165 animals from the Priorat region were studied periodically for 2-8 years between 1987 and 1995, giving a total of 565 determinations. A control group of 93 dogs from nonendemic areas was also studied. Sera from dogs with leishmaniasis recognized antigens with molecular weights ranging from 12 to 85 kD. The most sensitive antigens were those of 70, 65, 46, 30, 28, 14, and 12 kD, which were recognized by 75%, 75%, 78%, 75%, 81%, 79%, and 75%, respectively, of the sera from dogs with positive parasitologic examination results. Antigens of 70 and 65 kD were also recognized by two dogs from nonendemic areas. Antigens of 14 and 12 kD were the first to be recognized by sera of asymptomatic dogs with titers less than the cut-off value of the dot-ELISA that increased during the longitudinal study, and the presence of antibodies specific for these fractions was observed for up to six years before seroconversion observed by dot-ELISA. These antibodies were also the first to disappear in dogs in which the disease was self-limited. The study corroborates the high sensitivity and specificity of Western blots in the diagnosis of canine leishmaniasis when the bands of low molecular weight (less than 46 kD) are considered, and indicates that fractions of 14 and 12 kD are useful in detecting early forms of the disease.

  7. Murine interleukin 1 receptor. Direct identification by ligand blotting and purification to homogeneity of an interleukin 1-binding glycoprotein

    International Nuclear Information System (INIS)

    Bird, T.A.; Gearing, A.J.; Saklatvala, J.

    1988-01-01

    Functional receptors (IL1-R) for the proinflammatory cytokine interleukin 1 (IL1) were solubilized from plasma membranes of the NOB-1 subclone of murine EL4 6.1 thymoma cells using the zwitterionic detergent 3[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Membrane extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and ligand blotted with 125 I-labeled recombinant human IL1 alpha in order to reveal proteins capable of specifically binding IL1. A single polydisperse polypeptide of Mr approximately equal to 80,000 was identified in this way, which bound IL1 alpha and IL1 beta with the same affinity as the IL1-R on intact NOB-1 cells (approximately equal to 10(-10) M). The IL1-binding polypeptide was only seen in membranes from IL1-R-bearing cells and did not react with interleukin 2, tumor necrosis factor alpha, or interferon. IL1-R was purified to apparent homogeneity from solubilized NOB-1 membranes by affinity chromatography on wheat germ agglutinin-Sepharose and IL1 alpha-Sepharose. Gel electrophoresis and silver staining of purified preparations revealed a single protein of Mr approximately equal to 80,000 which reacted positively in the ligand-blotting procedure and which we identify as the ligand-binding moiety of the murine IL1-R. Purified IL1-R exhibited the same affinity and specificity as the receptor on intact cells. The relationship of this protein to proteins identified by covalent cross-linking studies is discussed

  8. Fluorescence Image Segmentation by using Digitally Reconstructed Fluorescence Images

    OpenAIRE

    Blumer, Clemens; Vivien, Cyprien; Oertner, Thomas G; Vetter, Thomas

    2011-01-01

    In biological experiments fluorescence imaging is used to image living and stimulated neurons. But the analysis of fluorescence images is a difficult task. It is not possible to conclude the shape of an object from fluorescence images alone. Therefore, it is not feasible to get good manual segmented nor ground truth data from fluorescence images. Supervised learning approaches are not possible without training data. To overcome this issues we propose to synthesize fluorescence images and call...

  9. mRNA localization mechanisms in Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Lysangela R Alves

    Full Text Available Asymmetric mRNA localization is a sophisticated tool for regulating and optimizing protein synthesis and maintaining cell polarity. Molecular mechanisms involved in the regulated localization of transcripts are widespread in higher eukaryotes and fungi, but not in protozoa. Trypanosomes are ancient eukaryotes that branched off early in eukaryote evolution. We hypothesized that these organisms would have basic mechanisms of mRNA localization. FISH assays with probes against transcripts coding for proteins with restricted distributions showed a discrete localization of the mRNAs in the cytoplasm. Moreover, cruzipain mRNA was found inside reservosomes suggesting new unexpected functions for this vacuolar organelle. Individual mRNAs were also mobilized to RNA granules in response to nutritional stress. The cytoplasmic distribution of these transcripts changed with cell differentiation, suggesting that localization mechanisms might be involved in the regulation of stage-specific protein expression. Transfection assays with reporter genes showed that, as in higher eukaryotes, 3'UTRs were responsible for guiding mRNAs to their final location. Our results strongly suggest that Trypanosoma cruzi have a core, basic mechanism of mRNA localization. This kind of controlled mRNA transport is ancient, dating back to early eukaryote evolution.

  10. Nine New Fluorescent Probes

    Science.gov (United States)

    Lin, Tsung-I.; Jovanovic, Misa V.; Dowben, Robert M.

    1989-06-01

    Absorption and fluorescence spectroscopic studies are reported here for nine new fluorescent probes recently synthesized in our laboratories: four pyrene derivatives with substituents of (i) 1,3-diacetoxy-6,8-dichlorosulfonyl, (ii) 1,3-dihydroxy-6,8-disodiumsulfonate, (iii) 1,3-disodiumsulfonate, and (iv) l-ethoxy-3,6,8-trisodiumsulfonate groups, and five [7-julolidino] coumarin derivatives with substituents of (v) 3-carboxylate-4-methyl, (vi) 3- methylcarboxylate, (vii) 3-acetate-4-methyl, (viii) 3-propionate-4-methyl, and (ix) 3-sulfonate-4-methyl groups. Pyrene compounds i and ii and coumarin compounds v and vi exhibit interesting absorbance and fluorescence properties: their absorption maxima are red shifted compared to the parent compound to the blue-green region, and the band width broadens considerably. All four blue-absorbing dyes fluoresce intensely in the green region, and the two pyrene compounds emit at such long wavelengths without formation of excimers. The fluorescence properties of these compounds are quite environment-sensitive: considerable spectral shifts and fluorescence intensity changes have been observed in the pH range from 3 to 10 and in a wide variety of polar and hydrophobic solvents with vastly different dielectric constants. The high extinction and fluorescence quantum yield of these probes make them ideal fluorescent labeling reagents for proteins, antibodies, nucleic acids, and cellular organelles. The pH and hydrophobicity-dependent fluorescence changes can be utilized as optical pH and/or hydrophobicity indicators for mapping environmental difference in various cellular components in a single cell. Since all nine probes absorb in the UV, but emit at different wavelengths in the visible, these two groups of compounds offer an advantage of utilizing a single monochromatic light source (e.g., a nitrogen laser) to achieve multi-wavelength detection for flow cytometry application. As a first step to explore potential application in

  11. Modification of T-cell antigenic properties of tetanus toxoid by SDS-PAGE separation. Implications for T-cell blotting

    DEFF Research Database (Denmark)

    Christensen, C B; Theander, T G

    1997-01-01

    Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20% of the ......Using Tetanus Toxoid (TT) as a model antigen the T-cell Blotting method was evaluated. Peripheral blood mononuclear cell (PBMC) cultures were stimulated by blotted nitrocellulose-bound TT or soluble TT. SDS-Poly-Acrylamide-Gel-Electrophoresis separated TT only induced proliferation in 20......% of the PBMC cultures whereas proliferation was induced in 79% of the same cultures offered similar treated TT (except for the PAGE separation). When T-cell blotting was performed with TT separated in a SDS-agarose matrix, proliferation was induced in 80% of donors responding to soluble TT. The results show...... that SDS-PAGE alters the ability of TT to induce T-cell proliferation, possibly due to unpolymerized acrylamide binding to proteins during SDS-PAGE. The use of SDS-PAGE T-cell blotting in the screening for T-cell antigens must therefore be reconsidered. We suggest the use of SDS-Agarose Gel Electrophoresis...

  12. Dot Blot para determinar la identidad antigénica en vacunas conjugadas contra Streptococcus pneumoniae serotipo 19F

    Directory of Open Access Journals (Sweden)

    Osmir Cabrera-Blanco

    2017-04-01

    Full Text Available Las autoridades regulatorias recomiendan el uso de técnicas de Resonancia Magnética Nuclear o técnicas serológicas para la determinación de la identidad de los antígenos presentes en las vacunas conjugadas. Con la aparición de las vacunas conjugadas multivalentes, se ha hecho necesario recurrir a técnicas inmunoquímicas con la utilización de anticuerpos monoclonales para aumentar la sensibilidad en la determinación de la identidad de los antígenos en dichas vacunas conjugadas. El objetivo del presente trabajo fue establecer las condiciones óptimas de trabajo que permitieran utilizar la técnica del Dot Blot para determinar la identidad de los antígenos en vacunas conjugadas de Streptococcus pneumoniae serotipo 19F. Para ello se estudiaron los tiempos de incubación, la influencia del reactivo en la solución de bloqueo; también las concentraciones óptimas del anticuerpo monoclonal y de los ingredientes farmacéuticos activos, así como los volúmenes de aplicación óptimos para estos y vacunas. Se utilizó un anticuerpo monoclonal contra el polisacárido capsular del serotipo 19F de neumococo. Las muestras empleadas en este trabajo fueron lotes de ingredientes farmacéuticos activos de conjugados de polisacárido capsular 19F y lotes de un candidato vacunal cubano conjugado heptavalente contra neumococos. Los resultados mostraron que para la determinación de la identidad antigénica fueron suficientes 10 µL de muestras de los principios activos a una concentración de 125 µg/mL e igual volumen para las vacunas heptavalentes. Quedó demostrado que una concentración de 1 µg/mL para el anticuerpo monoclonal y tiempos de incubación de 30 min a 37 °C fueron suficientes para la determinación. Estos resultados permiten concluir que quedaron establecidas las condiciones óptimas de trabajo para determinar la identidad antigénica por Dot Blot del polisacárido capsular de S. pneumoniae serotipo 19F presente en las vacunas

  13. Protein Structure and the Sequential Structure of mRNA

    DEFF Research Database (Denmark)

    Brunak, Søren; Engelbrecht, Jacob

    1996-01-01

    entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment, By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets, These signals do not originate from......A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed, We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting...... protein, The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain, A complete search for GenBank nucleotide sequences coding for structural...

  14. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis: interferon-beta treatment increases IL-10 mRNA expression while reducing IL-23 mRNA expression

    DEFF Research Database (Denmark)

    Krakauer, M.; Sorensen, P.; Khademi, M.

    2008-01-01

    volunteers served to confirm initial findings. mRNA was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR). RESULTS: We found elevated expression of interleukin (IL)-23 and IL-10 in untreated MS patients. IFN-beta therapy increased IL-10 and decreased IL-23 expression independently...... of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  15. Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

    Directory of Open Access Journals (Sweden)

    Carlo Cattani

    2011-01-01

    Full Text Available This paper deals with the sequence analysis of acute myeloid leukemia mRNA. Six transcript variants of mlf1 mRNA, with more than 2000 bps, are analyzed by focusing on the autocorrelation of each distribution. Through the correlation matrix, some patches and similarities are singled out and commented, with respect to similar distributions. The comparison of Kolmogorov fractal dimension will be also given in order to classify the six variants. The existence of a fractal shape, patterns, and symmetries are discussed as well.

  16. Detection of melatonin receptor mRNA in human muscle

    International Nuclear Information System (INIS)

    Li Lei

    2004-01-01

    To verify the expression of melatonin receptor mRNA in human, muscle, muscle beside vertebrae was collected to obtain total RNA and the mRNA of melatonin receptor was detected by RT-PCR method. The electrophoretic results of RT-PCR products by mt 1 and MT 2 primer were all positive and the sequence is corresponding with human melatonin receptor cDNA. It suggests that melatonin may act on the muscle beside vertebrae directly and regulate its growth and development. (authors)

  17. Nanosecond fluorescence spectroscopy

    International Nuclear Information System (INIS)

    Leskovar, B.

    1985-03-01

    This article is a summary of a short course lecture given in conjunction with the 1984 Nuclear Science Symposium. Measuring systems for nanosecond fluorescence spectroscopy using single-photon counting techniques are presented. These involve systems based on relaxation-type spark gap light pulser and synchronously pumped mode-locked dye lasers. Furthermore, typical characteristics and optimization of operating conditions of the critical components responsible for the system time resolution are discussed. A short comparison of the most important deconvolution methods for numerical analysis of experimental data is given particularly with respect to the signal-to-noise ratio of the fluorescence signal. 22 refs., 8 figs

  18. Fluorescence uranium determination

    International Nuclear Information System (INIS)

    Fernandez Cellini, R.; Crus Castillo, F. de la; Barrera Pinero, R.

    1960-01-01

    An equipment for analysis of uranium by fluorescence was developed in order to determine it at such a low concentration that it can not be determined by the most sensible analytical methods. this new fluorimeter was adapted to measure the fluorescence emitted by the phosphorus sodium fluoride-sodium carbonate-potasium carbonate-uranyl, being excited by ultraviolet light of 3,650 A the intensity of the light emitted was measure with a photomultiplicator RCA 5819 and the adequate electronic equipment. (Author) 19 refs

  19. Detection of Zika virus using reverse-transcription LAMP coupled with reverse dot blot analysis in saliva.

    Directory of Open Access Journals (Sweden)

    Maite Sabalza

    Full Text Available In recent years, there have been increasing numbers of infectious disease outbreaks that spread rapidly to population centers resulting from global travel, population vulnerabilities, environmental factors, and ecological disasters such as floods and earthquakes. Some examples of the recent outbreaks are the Ebola epidemic in West Africa, Middle East respiratory syndrome coronavirus (MERS-Co in the Middle East, and the Zika outbreak through the Americas. We have created a generic protocol for detection of pathogen RNA and/or DNA using loop-mediated isothermal amplification (LAMP and reverse dot-blot for detection (RDB and processed automatically in a microfluidic device. In particular, we describe how a microfluidic assay to detect HIV viral RNA was converted to detect Zika virus (ZIKV RNA. We first optimized the RT-LAMP assay to detect ZIKV RNA using a benchtop isothermal amplification device. Then we implemented the assay in a microfluidic device that will allow analyzing 24 samples simultaneously and automatically from sample introduction to detection by RDB technique. Preliminary data using saliva samples spiked with ZIKV showed that our diagnostic system detects ZIKV RNA in saliva. These results will be validated in further experiments with well-characterized ZIKV human specimens of saliva. The described strategy and methodology to convert the HIV diagnostic assay and platform to a ZIKV RNA detection assay provides a model that can be readily utilized for detection of the next emerging or re-emerging infectious disease.

  20. New Method for Simultaneous Species-Specific Identification of Equine Strongyles (Nematoda, Strongylida) by Reverse Line Blot Hybridization▿

    Science.gov (United States)

    Traversa, Donato; Iorio, Raffaella; Klei, Thomas R.; Kharchenko, Vitaliy A.; Gawor, Jakub; Otranto, Domenico; Sparagano, Olivier A. E.

    2007-01-01

    The ability of a reverse line blot (RLB) assay to identify 13 common species of equine small strongyles (cyathostomins) and to discriminate them from three Strongylus spp. (large strongyles) was demonstrated. The assay relied on the specific hybridization of PCR-amplified intergenic spacer DNA fragments of the nuclear ribosomal DNA to membrane-bound species-specific probes. All cyathostomins examined were unequivocally identified and simultaneously discriminated from each other and from three large strongyles (Strongylus edentatus, Strongylus equinus, and Strongylus vulgaris). This assay will enable the accurate and rapid identification of equine cyathostomins irrespective of their life cycle stage, opening important avenues for a better understanding of their biology and epidemiology and of the pathogenesis of cyathostomin-associated disease. In particular, this RLB method promises to be a powerful diagnostic tool to determine the roles of individual species in the pathogenesis of mixed infections and to elucidate some aspects of cyathostominosis. Also, it could represent a basic step toward the development of a rapid and simple molecular test for the early detection of drug-resistant genotypes of horse strongyle species. PMID:17626168

  1. Demonstration of monoclonal anti-carcinoembryonic antigen (CEA) antibody internalization by electron microscopy, western blotting and radioimmunoassay.

    Science.gov (United States)

    Tsaltas, G; Ford, C H; Gallant, M

    1992-01-01

    One of the important factors affecting the action of monoclonal antibodies (Mabs) or immunoconjugates on tumour sites depends on whether the Mab is internalized by the cancer cells in question. The underexplored subject of internalization is discussed in this paper, and a number of in vitro techniques for investigating internalization are evaluated, using a model which consists of a well characterized anti-carcinoembryonic antigen (anti-CEA) Mab and a number of CEA expressing human cancer cell lines. Employing two alternative radiolabeling assays, evidence for internalization of the anti-CEA Mab by a CEA-positive colorectal cancer cell line (LS174T) was obtained throughout the time intervals examined (5 min to 150 min). Electronmicroscopy employing horseradish-peroxidase labeled anti-CEA Mab and control antibody permitted direct visualization of anti-CEA Mab-related staining in intracellular compartments of a high CEA-expressor human colorectal cell line (SKCO1). Finally Western blots of samples derived from cytosolic and membrane components of solubilized cells from lung and colonic cancer cell lines provided evidence for internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 minutes. Internalized anti-CEA was detected in all CEA expressing cell lines (LS174T, SKCO1, BENN) but not in the case of a very low CEA expressor line (COLO 320).

  2. Exposure to Sarcocystis spp. in horses from Spain determined by Western blot analysis using Sarcocystis neurona merozoites as heterologous antigen.

    Science.gov (United States)

    Arias, M; Yeargan, M; Francisco, I; Dangoudoubiyam, S; Becerra, P; Francisco, R; Sánchez-Andrade, R; Paz-Silva, A; Howe, D K

    2012-04-30

    Horses serve as an intermediate host for several species of Sarcocystis, all of which utilize canids as the definitive host. Sarcocystis spp. infection and formation of latent sarcocysts in horses often appears to be subclinical, but morbidity can occur, especially when the parasite burden is large. A serological survey was conducted to determine the presence of antibodies against Sarcocystis spp. in seemingly healthy horses from the Galicia region of Spain. Western blot analyses using Sarcocystis neurona merozoites as heterologous antigen suggested greater than 80% seroprevalance of Sarcocystis spp. in a sample set of 138 horses. The serum samples were further tested with enzyme-linked immunosorbent assays (ELISAs) based on recombinant S. neurona-specific surface antigens (rSnSAGs). As expected for horses from the Eastern Hemisphere, less than 4% of the serum samples were positive when analyzed with either the rSnSAG2 or the rSnSAG4/3 ELISAs. An additional 246 horses were tested using the rSnSAG2 ELISA, which revealed that less than 3% of the 384 samples were seropositive. Collectively, the results of this serologic study suggested that a large proportion of horses from this region of Spain are exposed to Sarcocystis spp. Furthermore, the anti-Sarcocystis seroreactivity in these European horses could be clearly distinguished from anti-S. neurona antibodies using the rSnSAG2 and rSnSAG4/3 ELISAs. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

    Directory of Open Access Journals (Sweden)

    NUNES Cáris Maroni

    1997-01-01

    Full Text Available Visceral larva migrans (VLM is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA using the larval excretory-secretory antigen of T. canis (TES, the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa. Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

  4. Methylmethane-sulphonate and X-ray-induced mutations in the Chinese hamster hprt gene: mRNA phenotyping using polymerase chain reactions

    International Nuclear Information System (INIS)

    Chaudhry, M.A.; Fox, Margaret

    1990-01-01

    Alterations in the hprt gene of Chinese hamster cells were determined in 71 spontaneous, methylmethane sulphonate (MMS)-and X-ray induced mutants, using the Southern blot hybridization technique. To investigate the possibility of small deletions, MMS-induced mutants were studied with proves derived from exons 3 and 9 but no evidence of specific deletion of these two exons was found. The polymerase chain reaction (PCR) was used to phenotype hprt transcripts in 48 MMS, X-ray and spontaneous Chinese hamster mutants by amplifying the coding region of their cDNA. Among 22 MMS-induced mutants the message was present in 16 instances. An analysis of 20 X-ray-induced mutants showed the presence of hprt mRNA in 11 of them with five having low levels of transcription. Among six spontaneous mutants, four were negative for mRNA on standard Northern blots and in one the message was only detected after PCR amplification. Direct DNA sequencing of 10 mutants revealed the presence of base substitutions in five of them while a 7 bp deletion was found in another. No mutations were found in another four mutants, suggesting the presence of mutation outside the coding region. (author)

  5. A nucleic acid strand displacement system for the multiplexed detection of tuberculosis-specific mRNA using quantum dots

    Science.gov (United States)

    Gliddon, H. D.; Howes, P. D.; Kaforou, M.; Levin, M.; Stevens, M. M.

    2016-05-01

    The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar range using only a standard laboratory plate reader. We demonstrate the utility of our QD-based system for the detection of two genes selected from a microarray-derived tuberculosis-specific gene expression signature. Levels of up- and downregulated gene transcripts comprising this signature can be combined to give a disease risk score, making the signature more amenable for use as a diagnostic marker. Our QD-based approach to detect these transcripts could pave the way for novel diagnostic assays for tuberculosis.The development of rapid, robust and high performance point-of-care diagnostics relies on the advancement and combination of various areas of research. We have developed an assay for the detection of multiple mRNA molecules that combines DNA nanotechnology with fluorescent nanomaterials. The core switching mechanism is toehold-mediated strand displacement. We have used fluorescent quantum dots (QDs) as signal transducers in this assay, as they bring many benefits including bright fluorescence and multiplexing abilities. The resulting assay is capable of multiplexed detection of long RNA targets against a high concentration of background non-target RNA, with high sensitivity and specificity and limits of detection in the nanomolar

  6. [Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes of the insect Tenebrio molitor].

    Science.gov (United States)

    Bogoliubov, D S; Kiselev, A M; Shabel'nikov, S V; Parfenov, V N

    2012-01-01

    The nucleus ofvitellogenic oocytes of the yellow mealworm, Tenebrio molitor, contains a karyosphere that consists of the condensed chromatin embedded in an extrachromosomal fibrogranular material. Numerous nuclear bodies located freely in the nucleoplasm are also observed. Amongst these bodies, counterparts of nuclear speckles (= interchromatin granule clusters, IGCs) can be identified by the presence of the marker protein SC35. Microinjections of fluorescently tagged methyloligoribonucleotide probes 2'-O-Me(U)22, complementary to poly(A) tails of RNAs, revealed poly(A)+ RNA in the vast majority of IGCs. We found that all T. molitor oocyte IGCs contain heterogeneous ribonucleoprotein (hnRNP) core protein Al that localizes to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and a part of nucleoplasmic IGCs also contain the adapter protein Aly that is known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was observed to colocalize with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner whereas it is RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data suggest on a role of the nucleoplasmic IGCs in mRNA biogenesis and retention in a road to nuclear export.

  7. Associations of ACE Gene Insertion/Deletion Polymorphism, ACE Activity, and ACE mRNA Expression with Hypertension in a Chinese Population

    Science.gov (United States)

    He, Qingfang; Fan, Chunhong; Yu, Min; Wallar, Gina; Zhang, Zuo-Feng; Wang, Lixin; Zhang, Xinwei; Hu, Ruying

    2013-01-01

    Background The present study was designed to explore the association of angiotensin converting enzyme (ACE) gene insertion/deletion (I/D, rs4646994) polymorphism, plasma ACE activity, and circulating ACE mRNA expression with essential hypertension (EH) in a Chinese population. In addition, a new detection method for circulating ACE mRNA expression was explored. Methods The research was approved by the ethics committee of Zhejiang Provincial Center for Disease Prevention and Control. Written informed consent was obtained prior to the investigation. 221 hypertensives (cases) and 221 normotensives (controls) were interviewed, subjected to a physical examination, and provided blood for biochemical and genetic tests. The ACE mRNA expression was analyzed by real time fluorescent quantitative Reverse Transcription PCR (FQ-RT-PCR). We performed logistic regression to assess associations of ACE I/D genotypes, ACE activity, and ACE mRNA expression levels with hypertension. Results The results of the multivariate logistic regression analysis showed that the additive model (ID, DD versus II) of the ACE genotype revealed an association with hypertension with adjusted OR of 1.43(95% CI: 1.04-1.97), and ACE ID genotype with adjusted OR of 1.72(95% CI: 1.01-2.92), DD genotype with adjusted OR of 1.94(95% CI: 1.01-3.73), respectively. In addition, our data also indicate that plasma ACE activity (adjusted OR was 1.13(95% CI: 1.08-1.18)) was significantly related to hypertension. However, the plasma ACE mRNA expressions were not different between the cases and controls. Conclusion ACE I/D polymorphism and ACE activity revealed significant influence on hypertension, while circulating ACE mRNA expression was not important factors associated with hypertension in this Chinese population. The detection of circulating ACE mRNA expression by FQ-RT-PCR might be a useful method for early screening and monitoring of EH. PMID:24098401

  8. Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

    International Nuclear Information System (INIS)

    Li Li; Li Xincang; Li Lu; Wang Jinxing; Jin Wenrui

    2011-01-01

    An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10 -14 mol L -1 . Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

  9. Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA

    Energy Technology Data Exchange (ETDEWEB)

    Li Li [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Li Xincang [School of Life Sciences, Shandong University, Jinan 250100 (China); Li Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2011-01-24

    An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1 x 10{sup -14} mol L{sup -1}. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

  10. Monitoring by fluorescence measurements

    International Nuclear Information System (INIS)

    Malcolme-Lawes, D.J.; Gifford, L.A.

    1981-01-01

    A fluorimetric detector is described in which the fluorescence excitation source may be 3 H, 14 C, 35 S, 147 Pm or 63 Ni. Such a detector can be adapted for use with flowing liquid systems especially liquid chromatography systems. (U.K.)

  11. Fluorescence lifetime based bioassays

    Science.gov (United States)

    Meyer-Almes, Franz-Josef

    2017-12-01

    Fluorescence lifetime (FLT) is a robust intrinsic property and material constant of fluorescent matter. Measuring this important physical indicator has evolved from a laboratory curiosity to a powerful and established technique for a variety of applications in drug discovery, medical diagnostics and basic biological research. This distinct trend was mainly driven by improved and meanwhile affordable laser and detection instrumentation on the one hand, and the development of suitable FLT probes and biological assays on the other. In this process two essential working approaches emerged. The first one is primarily focused on high throughput applications employing biochemical in vitro assays with no requirement for high spatial resolution. The second even more dynamic trend is the significant expansion of assay methods combining highly time and spatially resolved fluorescence data by fluorescence lifetime imaging. The latter approach is currently pursued to enable not only the investigation of immortal tumor cell lines, but also specific tissues or even organs in living animals. This review tries to give an actual overview about the current status of FLT based bioassays and the wide range of application opportunities in biomedical and life science areas. In addition, future trends of FLT technologies will be discussed.

  12. Fluorescent Lamp Replacement Study

    Science.gov (United States)

    2017-07-01

    not be cited for purposes of advertisement. DISPOSITION INSTRUCTIONS: Destroy this document when no longer needed. Do not return to the... recycling , and can be disposed safely in a landfill. (2) LEDs offer reduced maintenance costs and fewer bulb replacements, significantly reducing... recycling . Several fixtures, ballasts and energy efficient fluorescent bulbs that were determined to be in pristine condition were returned to ATC

  13. Cloning and mRNA expression pattern analysis under low ...

    African Journals Online (AJOL)

    This research cloned endochitinase-antifreeze protein precursor (EAPP) gene of Dong-mu 70 rye (Secale cereale) by designing special primers according to Genbank's EAPP gene sequence, and analyzing the influence of low temperature stress on the expression of mRNA with RT-PCR. The results indicated that the ...

  14. Statistical filtering in fluorescence microscopy and fluorescence correlation spectroscopy

    Czech Academy of Sciences Publication Activity Database

    Macháň, Radek; Kapusta, Peter; Hof, Martin

    Roč. 406 , č. 20 (2014), s. 4797-4813 ISSN 1618-2642 R&D Projects: GA ČR GBP208/12/G016 Institutional support: RVO:61388955 Keywords : Filtered fluorescence correlation spectroscopy * Fluorescence lifetime correlation spectroscopy * Fluorescence spectral correlation spectroscopy Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.436, year: 2014

  15. Who's who in fluorescence 2008

    CERN Document Server

    Geddes, Chris D

    2008-01-01

    The Journal of Fluorescence's sixth Who's Who directory publishes the names, contact details, specialty keywords, and a brief description of scientists employing fluorescence methodology and instrumentation in their working lives. This is a unique reference.

  16. Antibody Banding Patterns of the Enzyme-Linked Immunoelectrotransfer Blot and Brain Imaging Findings in Patients With Neurocysticercosis.

    Science.gov (United States)

    Arroyo, Gianfranco; Rodriguez, Silvia; Lescano, Andres G; Alroy, Karen A; Bustos, Javier A; Santivañez, Saul; Gonzales, Isidro; Saavedra, Herbert; Pretell, E Javier; Gonzalez, Armando E; Gilman, Robert H; Tsang, Victor C W; Garcia, Hector H

    2018-01-06

    The enzyme-linked immunoelectrotransfer blot (EITB) assay is the reference serological test for neurocysticercosis (NCC). A positive result on EITB does not always correlate with the presence of active infections in the central nervous system (CNS), and patients with a single viable brain cyst may be EITB negative. Nonetheless, EITB antibody banding patterns appears to be related with the expression of 3 protein families of Taenia solium, and in turn with the characteristics of NCC in the CNS (type, stage, and burden of viable cysts). We evaluated EITB antibody banding patterns and brain imaging findings of 548 NCC cases. Similar banding patterns were grouped into homogeneous classes using latent class analysis. The association between classes and brain imaging findings was assessed. Four classes were identified. Class 1 (patients negative or only positive to the GP50 band, related to the protein family of the same name) was associated with nonviable or single viable parenchymal cysticerci; class 2 (patients positive to bands GP42-39 and GP24, related to the T24-42 protein family, with or without anti-GP50 antibodies) was associated with intraparenchymal viable and nonviable infections; classes 3 and 4 (positive to GP50, GP42-39, and GP24 but also responding to low molecular weight bands GP21, GP18, GP14, and GP13, related to the 8 kDa protein family) were associated with extraparenchymal and intraparenchymal multiple viable cysticerci. EITB antibody banding patterns correlate with brain imaging findings and complement imaging information for the diagnosis of NCC and for staging NCC patients. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  17. Detection of Potentially Diagnostic Leishmania Antigens with Western Blot Analysis of Sera from Patients with Cutaneous and Visceral Leishmaniases

    Directory of Open Access Journals (Sweden)

    Seyyed Javad SEYYEDTABAEI

    2017-06-01

    Full Text Available Background: Visceral leishmaniasis (VL and cutaneous leishmaniasis (CL are important public health problems in Iran. We aimed to evaluate the diagnostic potential of Western blot (WB compared with indirect immunofluorescence test (IFAT to serodiagnosis of leishmaniasis.Methods: This study was performed from 2010-2014 and participants were different parts of Iran. Serum samples were obtained from 43 patients with proven CL, 33 patients with proven VL, 39 patients with other parasitic diseases and 23 healthy individuals. Results: WB sensitivity for CL and VL was 100% and 91%, compared to IFA 4.6% and 87.8%, respectively. Sera from patients with CL and VL recognized numerous antigens with molecular weights ranging from 14 to 68 kDa and 12 to 94 kDa, respectively. The most sensitive antigens were 14 and 16 kDa for CL recognized by 100% of the sera from patients with proven CL and 12, 14 and 16 kDa for VL, recognized by 63.6%, 100% and 63.6% of the sera from patients with proven VL respectively. WB analysis is more sensitive than IFAT for the diagnosis of leishmaniasis particularly in cases of cutaneous leishmaniasis. The 12, 14 and 16 kDa can be valuable diagnostic molecules for serodiagnosis of leishmaniasis because at least two immunogenic molecules were simultaneously detected by all patient sera, as well as produced antibodies against these antigens have no cross-reactivity with other control groups.Conclusion: WB could be useful for screening and serodiagnosis of CL and VL in epidemiologic studies in endemic areas.

  18. Detection of Babesia and Theileria species infection in cattle from Portugal using a reverse line blotting method.

    Science.gov (United States)

    Silva, M G; Marques, P X; Oliva, A

    2010-12-15

    Babesiosis and Theileriosis are tick-borne diseases widespread in tropical and sub-tropical regions with high economic impact worldwide. In Portugal there are at least 4 tick vectors known to be competent for the transmission of Babesia and Theileria sp. identified: Rhipicephalus bursa, Rhipicephalus (Boophilus) annulatus, Ixodes ricinus and Haemaphysalis punctata. All these potential Babesia and Theileria tick vectors are widely distributed in Portugal, although they are predominant in the Southern region. In this study, 1104 cattle blood samples were randomly collected from Central and Southern regions of Portugal and analyzed by PCR-reverse line blotting (RLB) for the detection of Babesia and Theileria sp. Testing indicated that 74.7% of the bovines tested were positive for either Babesia and/or Theileria sp. In addition, five different apicomplexan species, namely, Theileria buffeli, Theileria annulata, Babesia divergens, Babesia bovis, and Babesia bigemina were detected by RLB among the bovines tested. T. buffeli was the most frequently found species, being present in 69.9% of the positive samples either as single infections (52.4%), or as mixed infections (17.5%). The Babesia specie most frequently found was B. divergens, detected in 4.2% of the infected bovines. Overall, infected bovines were found in all regions tested; however the highest number of infected bovines was observed in Évora district (96.2%) and in cattle from Limousin breeds (81.7%). The results indicate widespread Babesia and Theileria infections in Portuguese bovines, suggesting the need for improved control of ticks and tick-borne diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

  19. Development of a quantitative analysis method for mRNA from Mycobacterium leprae and slow-growing acid-fast bacteria

    International Nuclear Information System (INIS)

    Nakanaga, Kazue; Maeda Shinji; Matsuoka, Masanori; Kashiwabara, Yoshiko

    1999-01-01

    This study aimed to develop a specific method for detection and quantitative determination of mRNA that allows estimation of viable counts of M. leprae and other mycobacteria. Of heart-shock protein of 65 kDa (hsp65), mRNA was used as an indicator to discriminate the living cells and died ones. To compare mRNA detections by RNase protection assay (RPA) and Northern blot hybridization (NBH), labelled anti-sense RNA for hsp65 gene of M. leprae was synthesized using plasmid pUC8/N5. The anti-sense RNA synthesized from the template DNA containing about 580 bp (194 to 762) of hsp65 gene. When compared with NBH method, the amount of probe required for the detection by RPA method was 1/30 or less and the detection sensitivity of RPA was also 10 times higher. In addition, complicated procedures were needed to eliminate non-specific reactions in NBH method. These results indicated that RPA method is more convenient and superior for the mRNA detection. However, isotope degradation in the probe used for RPA method might affect the results. Therefore, 33 P of 35 P, of which degradation energy is less that 32 P should be used for labelling. Total RNA was effectively extracted from M. chelonae, M. marinum by AGPC method, but not from M. leprae. In conclusion, RPA is a very effective detection method for these mRNA, but it seems necessary to further improve the sensitivity of detection for a small amount of test materials. (M.N.)

  20. Fas ligand expression in human and mouse cancer cell lines; a caveat on over-reliance on mRNA data

    Directory of Open Access Journals (Sweden)

    Ryan Aideen E

    2006-02-01

    Full Text Available Abstract Background During carcinogenesis, tumors develop multiple mechanisms for evading the immune response, including upregulation of Fas ligand (FasL/CD95L expression. Expression of FasL may help to maintain tumor cells in a state of immune privilege by inducing apoptosis of anti-tumor immune effector cells. Recently this idea has been challenged by studies reporting that tumor cells of varying origin do not express FasL. In the present study, we aimed to comprehensively characterize FasL expression in tumors of both murine and human origin over a 72 hour time period. Methods RNA and protein was extracted from six human (SW620, HT29, SW480, KM12SM, HCT116, Jurkat and three mouse (CMT93, CT26, B16F10 cancer cell lines at regular time intervals over a 72 hour time period. FasL expression was detected at the mRNA level by RT-PCR, using intron spanning primers, and at the protein level by Western Blotting and immunofluorescence, using a polyclonal FasL- specific antibody. Results Expression of FasL mRNA and protein was observed in all cell lines analysed. However, expression of FasL mRNA varied dramatically over time, with cells negative for FasL mRNA at many time points. In contrast, 8 of the 9 cell lines constitutively expressed FasL protein. Thus, cells can abundantly express FasL protein at times when FasL mRNA is absent. Conclusion These findings demonstrate the importance of complete analysis of FasL expression by tumor cells in order to fully characterize its biological function and may help to resolve the discrepancies present in the literature regarding FasL expression and tumor immune privilege.

  1. Development of a quantitative analysis method for mRNA from Mycobacterium leprae and slow-growing acid-fast bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Nakanaga, Kazue; Maeda Shinji; Matsuoka, Masanori; Kashiwabara, Yoshiko [National Inst. of Infectious Diseases, Tokyo (Japan)

    1999-02-01

    This study aimed to develop a specific method for detection and quantitative determination of mRNA that allows estimation of viable counts of M. leprae and other mycobacteria. Of heart-shock protein of 65 kDa (hsp65), mRNA was used as an indicator to discriminate the living cells and died ones. To compare mRNA detections by RNase protection assay (RPA) and Northern blot hybridization (NBH), labelled anti-sense RNA for hsp65 gene of M. leprae was synthesized using plasmid pUC8/N5. The anti-sense RNA synthesized from the template DNA containing about 580 bp (194 to 762) of hsp65 gene. When compared with NBH method, the amount of probe required for the detection by RPA method was 1/30 or less and the detection sensitivity of RPA was also 10 times higher. In addition, complicated procedures were needed to eliminate non-specific reactions in NBH method. These results indicated that RPA method is more convenient and superior for the mRNA detection. However, isotope degradation in the probe used for RPA method might affect the results. Therefore, {sup 33}P of {sup 35}P, of which degradation energy is less that {sup 32}P should be used for labelling. Total RNA was effectively extracted from M. chelonae, M. marinum by AGPC method, but not from M. leprae. In conclusion, RPA is a very effective detection method for these mRNA, but it seems necessary to further improve the sensitivity of detection for a small amount of test materials. (M.N.)

  2. Gad1 mRNA as a reliable indicator of altered GABA release from orexigenic neurons in the hypothalamus.

    Science.gov (United States)

    Dicken, Matthew S; Hughes, Alexander R; Hentges, Shane T

    2015-11-01

    The strength of γ-aminobutyric acid (GABA)-mediated inhibitory synaptic input is a principle determinant of neuronal activity. However, because of differences in the number of GABA afferent inputs and the sites of synapses, it is difficult to directly assay for altered GABA transmission between specific cells. The present study tested the hypothesis that the level of mRNA for the GABA synthetic enzyme glutamate decarboxylase (GAD) can provide a reliable proxy for GABA release. This was tested in a mouse hypothalamic circuit important in the regulation of energy balance. Fluorescent in situ hybridization results show that the expression of Gad1 mRNA (encoding the GAD67 enzyme) was increased in hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons after an overnight fast, consistent with the ability of GABA from these neurons to stimulate food intake. Optogenetic studies confirmed that the observed increase in Gad1 mRNA correlated with an increase in the probability of GABA release from NPY/AgRP neurons onto downstream proopiomelanocortin neurons. Likewise, there was an increase in the readily releasable pool of GABA in NPY/AgRP neurons. Selective inhibition of GAD activity in NPY/AgRP neurons decreased GABA release, indicating that GAD67 activity, which is largely dictated by expression level, is a key determinant of GABA release. Altogether, it appears that Gad expression may be a reliable proxy of altered GABAergic transmission. Examining changes in Gad mRNA as a proxy for GABA release may be particularly helpful when the downstream targets are not known or when limited tools exist for detecting GABA release at a particular synapse. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  3. Synthesis and Fluorescence Spectra of Triazolylcoumarin Fluorescent Dyes

    Institute of Scientific and Technical Information of China (English)

    PENG Xian-fu; LI Hong-qi

    2009-01-01

    Much attention is devoted to fluorescent dyes especially those with potential in versatile applications. Reactions under "click" conditions between nonfluorescent 3 - azidocoumarins and terminal alkynes produced 3 -(1, 2, 3- triazol- 1 - yl)cournarins, a novel type of fluorescent dyes with intense fluorescence. The structures of the new coumarins were characterized by 1H NMR, MS, and IR spectra. Fluorescence spectra measurement demonstrated excellent fluorescence performance of the triazolylcoumarins and this click reaction is a promising candidate for bioconjugation and bioimaging applications since both azide and alkynes are quite inert to biological systems.

  4. Fluorescence spectroscopy of dental calculus

    International Nuclear Information System (INIS)

    Bakhmutov, D; Gonchukov, S; Sukhinina, A

    2010-01-01

    The aim of the present study was to investigate the fluorescence properties of dental calculus in comparison with the properties of adjacent unaffected tooth structure using both lasers and LEDs in the UV-visible range for fluorescence excitation. The influence of calculus color on the informative signal is demonstrated. The optimal spectral bands of excitation and registration of the fluorescence are determined

  5. Fluorescence spectroscopy of dental calculus

    Science.gov (United States)

    Bakhmutov, D.; Gonchukov, S.; Sukhinina, A.

    2010-05-01

    The aim of the present study was to investigate the fluorescence properties of dental calculus in comparison with the properties of adjacent unaffected tooth structure using both lasers and LEDs in the UV-visible range for fluorescence excitation. The influence of calculus color on the informative signal is demonstrated. The optimal spectral bands of excitation and registration of the fluorescence are determined.

  6. Fluorescence Imaging Reveals Surface Contamination

    Science.gov (United States)

    Schirato, Richard; Polichar, Raulf

    1992-01-01

    In technique to detect surface contamination, object inspected illuminated by ultraviolet light to make contaminants fluoresce; low-light-level video camera views fluorescence. Image-processing techniques quantify distribution of contaminants. If fluorescence of material expected to contaminate surface is not intense, tagged with low concentration of dye.

  7. Who's who in fluorescence 2005

    CERN Document Server

    Geddes, Chris D

    2006-01-01

    The Journal of Fluorescence's third Who's Who directory publishes the names, contact details, specialty keywords, photographs, and a brief description of scientists employing fluorescence methodology and instrumentation in their working livesThe directory provides company contact details with a brief list of fluorescence-related products.

  8. Impact of target mRNA structure on siRNA silencing efficiency: A large-scale study.

    Science.gov (United States)

    Gredell, Joseph A; Berger, Angela K; Walton, S Patrick

    2008-07-01

    The selection of active siRNAs is generally based on identifying siRNAs with certain sequence and structural properties. However, the efficiency of RNA interference has also been shown to depend on the structure of the target mRNA, primarily through studies using exogenous transcripts with well-defined secondary structures in the vicinity of the target sequence. While these studies provide a means for examining the impact of target sequence and structure independently, the predicted secondary structures for these transcripts are often not reflective of structures that form in full-length, native mRNAs where interactions can occur between relatively remote segments of the mRNAs. Here, using a combination of experimental results and analysis of a large dataset, we demonstrate that the accessibility of certain local target structures on the mRNA is an important determinant in the gene silencing ability of siRNAs. siRNAs targeting the enhanced green fluorescent protein were chosen using a minimal siRNA selection algorithm followed by classification based on the predicted minimum free energy structures of the target transcripts. Transfection into HeLa and HepG2 cells revealed that siRNAs targeting regions of the mRNA predicted to have unpaired 5'- and 3'-ends resulted in greater gene silencing than regions predicted to have other types of secondary structure. These results were confirmed by analysis of gene silencing data from previously published siRNAs, which showed that mRNA target regions unpaired at either the 5'-end or 3'-end were silenced, on average, approximately 10% more strongly than target regions unpaired in the center or primarily paired throughout. We found this effect to be independent of the structure of the siRNA guide strand. Taken together, these results suggest minimal requirements for nucleation of hybridization between the siRNA guide strand and mRNA and that both mRNA and guide strand structure should be considered when choosing candidate si

  9. Isolation and characterization of human glycophorin A cDNA clones by a synthetic oligonucleotide approach: nucleotide sequence and mRNA structure

    International Nuclear Information System (INIS)

    Siebert, P.D.; Fukuda, M.

    1986-01-01

    In an effort to understand the relationships among and the regulation of human glycophorins, the authors have isolated and characterized several glycophorin A-specific cDNA clones obtained from a human erythroleukemic K562 cell cDNA library. This was accomplished by using mixed synthetic oligonucleotides, corresponding to various regions of the known amino acid sequence, to prime the synthesis of the cDNA as well as to screen the cDNA library. They also used synthetic oligonucleotides to sequence the largest of the glycophorin cDNAs. The nucleotide sequence obtained suggests the presence of a potential leader peptide, consistent with the membrane localization of this glycoprotein. Examination of the structure of glycophorin mRNA by blot hybridization revealed the existence of several electrophoretically distinct mRNAs numbering three or four, depending on the size of the glycophorin cDNA used as a hybridization probe. The smaller cDNA hybridized to three mRNAs of approximately 2.8, 1.7, and 1.0 kilobases. In contrast, the larger cDNA hybridized to an additional mRNA of approximately 0.6 kilobases. Further examination of the relationships between these multiple mRNAs by blot hybridization was conducted with the use of exact-sequence oligonucleotide probes constructed from various regions of the cDNA representing portions of the amino acid sequence of glycophorin A with or without known homology with glycophorin B. In total, the results obtained are consistent with the hypothesis that the three larger mRNAs represent glycophorin A gene transcripts and that the smallest (0.6 kilobase) mRNA may be specific for glycophorin B

  10. 1,25 dihydroxyvitamin D3 (1,25) regulation of c-myc mRNA in HL-60 leukemia cells

    International Nuclear Information System (INIS)

    Simpson, R.U.; Bresnick, E.H.; Begley, D.A.

    1986-01-01

    Recently, 1,25 was shown to induce differentiation and decrease c-myc levels in HL-60 cells. The authors have confirmed these observations by RNA dot blot analysis. Cells treated with 50 nM 1,25 for 4, 24 and 48 hr showed c-myc mRNA levels of 26, 17 and 15% of control respectively. β-Actin mRNA levels were not altered. To ascertain whether 1, 25 regulated c-myc transcriptionally, an HL-60 nuclear RNA runoff assay was developed. Assay of total nuclei transcriptional activity revealed that 50% of RNA elongation was α-amanitin (0.8 μg/ml) sensitive and was linear with nuclei concentration (0.1-1 x 10 7 nuclei). 1,25 (50 nM) treated (45-96 hr) cells had decreased (approx.40%) total transcription rate relative to control. Decreased total RNA synthesis occurred concomitant with NBT reducing activity. 32 P-RNA runoff transcripts from HL-60 nuclei were hybridized to excess (5 μg DNA was excess) Pst I linearized c-myc and β-actin clones (in pBR322) immobilized on nitrocellulose filter. 32 P-RNA input from 2 x 10 6 to 2 x 10 7 cpm yielded linear hybridization signal. Analysis of blot dot intensity revealed no difference in transcription of c-myc in nuclei from 1,25 dosed or control cells. (myc/actin ratios: 1,25 (50 nM, 72 hr) =1.1 +/- 0.3 and control (72 hr) = 1.0, N=3 or 2 or 3 dots ea). These preliminary data suggest 1,25 does not affect c-myc transcription in HL-60 nuclei and may regulate c-myc mRNA post-transcriptionally

  11. Evaluation of the radioimmunoassay, indirect enzyme linked immunosorbent assay, and dot blot assay for the identification of Xanthomonas campestris pv. phaseoli

    Energy Technology Data Exchange (ETDEWEB)

    Malin, E; Belden, E L; Roth, D

    1985-09-01

    A radioimmunoassay (RIA), an indirect competitive enzyme-linked immunosorbent assay (ELISA), and a dot-blot modification of the ELISA were evaluated for detection and identification of Xanthomonas campestris pv. phaseoli (X. c. pv. phaseoli). RIA and the dot blot tests were specific for X. c. pv. phaseoli; however, significant cross reactions occurred in the indirect competitive ELISA when using anti-X. c. pv. phaseoli antiserum against other closely related bacteria. The sensitivity level of all procedures for X. c. pv. phaseoli was approximately l0/sup 5/ colony forming unitsmL. All procedures were unsatisfactory in reliably detecting low levels of X. c. pv. phaseoli directly from extracts of bean seed. However when used in conjunction with ilution plating the dot blot assay and the RIA would be useful in specifically identifying X. c. pv. phaseoli. The relative merits of these tests for identification of X. c. pv. phaseoli are discussed.

  12. Fluorescence fluctuation spectroscopy (FFS)

    CERN Document Server

    Tetin, Sergey

    2012-01-01

    This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers fluorescence fluctuation spectroscopy and includes chapters on such topics as Förster resonance energy transfer (fret) with fluctuation algorithms, protein corona on nanoparticles by FCS, and FFS approaches to the study of receptors in live cells. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers fluorescence fluctuation spectroscopy Contains chapters on such topics as Förster resonance energy transfer (fret) with fluctuation algorithms, protein corona on nanoparticles by FCS, and FFS approaches to the study of receptors in live cells.

  13. Fluorescent quantification of melanin.

    Science.gov (United States)

    Fernandes, Bruno; Matamá, Teresa; Guimarães, Diana; Gomes, Andreia; Cavaco-Paulo, Artur

    2016-11-01

    Melanin quantification is reportedly performed by absorption spectroscopy, commonly at 405 nm. Here, we propose the implementation of fluorescence spectroscopy for melanin assessment. In a typical in vitro assay to assess melanin production in response to an external stimulus, absorption spectroscopy clearly overvalues melanin content. This method is also incapable of distinguishing non-melanotic/amelanotic control cells from those that are actually capable of performing melanogenesis. Therefore, fluorescence spectroscopy is the best method for melanin quantification as it proved to be highly specific and accurate, detecting even small variations in the synthesis of melanin. This method can also be applied to the quantification of melanin in more complex biological matrices like zebrafish embryos and human hair. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Fluorescent nanodiamond for biomedicine

    International Nuclear Information System (INIS)

    Milos Nesladek

    2014-01-01

    NV centers in diamond have gained strong interest as a novel tool for quantum information processing, quantum computing and quantum photonics. These applications are based on fluorescent and spin properties of NV-centres. However, in some conditions NV- can lose an electron and turn to NV0. The occupation of NV0 and NV- charge states depend on the position of their ground states with respect to the Fermi level and the mechanism of the charge transfer. Interestingly, that the charge switch has important implications on applications of fluorescent nanodiamond (fND) to nano-biology and nano-medicine. fND can be used for bio-marking and bio-tracking but also for the monitoring of targeted delivery to the cells. In this presentation we review the current state-of-the art for using fND particles for fluorescent bio imaging in cells and discuss the charge transfer and its luminescence stability by using ultra high sensitive spectroscopy methods to study the NV0 and NV- state occupation. (author)

  15. Increased mRNA expression of a laminin-binding protein in human colon carcinoma: Complete sequence of a full-length cDNA encoding the protein

    International Nuclear Information System (INIS)

    Yow, Hsiukang; Wong, Jau Min; Chen, Hai Shiene; Lee, C.; Steele, G.D. Jr.; Chen, Lanbo

    1988-01-01

    Reliable markers to distinguish human colon carcinoma from normal colonic epithelium are needed particularly for poorly differentiated tumors where no useful marker is currently available. To search for markers the authors constructed cDNA libraries from human colon carcinoma cell lines and screened for clones that hybridize to a greater degree with mRNAs of colon carcinomas than with their normal counterparts. Here they report one such cDNA clone that hybridizes with a 1.2-kilobase (kb) mRNA, the level of which is ∼9-fold greater in colon carcinoma than in adjacent normal colonic epithelium. Blot hybridization of total RNA from a variety of human colon carcinoma cell lines shows that the level of this 1.2-kb mRNA in poorly differentiated colon carcinomas is as high as or higher than that in well-differentiated carcinomas. Molecular cloning and complete sequencing of cDNA corresponding to the full-length open reading frame of this 1.2-kb mRNA unexpectedly show it to contain all the partial cDNA sequence encoding 135 amino acid residues previously reported for a human laminin receptor. The deduced amino acid sequence suggests that this putative laminin-binding protein from human colon carcinomas consists of 295 amino acid residues with interesting features. There is an unusual C-terminal 70-amino acid segment, which is trypsin-resistant and highly negatively charged

  16. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, Charlotte Guldborg; Paludan, Søren Riis

    2006-01-01

    and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...

  17. Collagen mRNA levels changes during colorectal cancer carcinogenesis

    DEFF Research Database (Denmark)

    Skovbjerg, Hanne; Anthonsen, Dorit; Lothe, Inger M B

    2009-01-01

    BACKGROUND: Invasive growth of epithelial cancers is a complex multi-step process which involves dissolution of the basement membrane. Type IV collagen is a major component in most basement membranes. Type VII collagen is related to anchoring fibrils and is found primarily in the basement membrane...... zone of stratified epithelia. Immunohistochemical studies have previously reported changes in steady-state levels of different alpha(IV) chains in several epithelial cancer types. In the present study we aimed to quantitatively determine the mRNA levels of type IV collagen (alpha1/alpha 4/alpha 6......) and type VII collagen (alpha1) during colorectal cancer carcinogenesis. METHODS: Using quantitative RT-PCR, we have determined the mRNA levels for alpha1(IV), alpha 4(IV), alpha 6(IV), and alpha1(VII) in colorectal cancer tissue (n = 33), adenomas (n = 29) and in normal tissue from the same individuals...

  18. Peptide inhibitors of botulinum neurotoxin by mRNA display

    International Nuclear Information System (INIS)

    Yiadom, Kwabena P.A.B.; Muhie, Seid; Yang, David C.H.

    2005-01-01

    Botulinum neurotoxins (BoNTs) are extremely toxic. The metalloproteases associated with the toxins cleave proteins essential for neurotransmitter secretion. Inhibitors of the metalloprotease are currently sought to control the toxicity of BoNTs. Toward that goal, we produced a synthetic cDNA for the expression and purification of the metalloprotease of BoNT/A in Escherichia coli as a biotin-ubiquitin fusion protein, and constructed a combinatorial peptide library to screen for BoNT/A light chain inhibitors using mRNA display. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. The results suggest that mRNA display may provide a general approach in developing peptide inhibitors of BoNTs

  19. Regulation of mRNA translation influences hypoxia tolerance

    International Nuclear Information System (INIS)

    Koritzinsky, M.; Wouters, B.G.; Koumenis, C.

    2003-01-01

    Hypoxia is a heterogenous but common characteristic of human tumours and poor oxygenation is associated with poor prognosis. We believe that the presence of viable hypoxic tumor cells reflects in part an adaptation and tolerance of these cells to oxygen deficiency. Since oxidative phosphorylation is compromized during hypoxia, adaptation may involve both the upregulation of glycolysis as well as downregulation of energy consumption. mRNA translation is one of the most energy costly cellular processes, and we and others have shown that global mRNA translation is rapidly inhibited during hypoxia. However, some mRNAs, including those coding for HIF-1 α and VEGF, remain efficiently translated during hypoxia. Clearly, the mechanisms responsible for the overall inhibition of translation during hypoxia does not compromize the translation of certain hypoxia-induced mRNA species. We therefore hypothesize that the inhibition of mRNA translation serves to promote hypoxia tolerance in two ways: i) through conservation of energy and ii) through differential gene expression involved in hypoxia adaptation. We have recently identified two pathways that are responsible for the global inhibition of translation during hypoxia. The phosphorylation of the eukaryotic initiation factor eIF2 α by the ER resident kinase PERK results in down-regulation of protein synthesis shortly after the onset of hypoxia. In addition, the initiation complex eIF4F is disrupted during long lasting hypoxic conditions. The identification of the molecular pathways responsible for the inhibition of overall translation during hypoxia has rendered it possible to investigate their importance for hypoxia tolerance. We have found that mouse embryo fibroblasts that are knockout for PERK and therefore not able to inhibit protein synthesis efficiently during oxygen deficiency are significantly less tolerant to hypoxia than their wildtype counterparts. We are currently also investigating the functional significance

  20. Exercise increases the plasma membrane content of the Na+ -K+ pump and its mRNA in rat skeletal muscles.

    Science.gov (United States)

    Tsakiridis, T; Wong, P P; Liu, Z; Rodgers, C D; Vranic, M; Klip, A

    1996-02-01

    Muscle fibers adapt to ionic challenges of exercise by increasing the plasma membrane Na+-K+ pump activity. Chronic exercise training has been shown to increase the total amount of Na+-K+ pumps present in skeletal muscle. However, the mechanism of adaptation of the Na+-K+ pump to an acute bout of exercise has not been determined, and it is not known whether it involves alterations in the content of plasma membrane pump subunits. Here we examine the effect of 1 h of treadmill running (20 m/min, 10% grade) on the subcellular distribution and expression of Na+-K+ pump subunits in rat skeletal muscles. Red type I and IIa (red-I/IIa) and white type IIa and IIb (white-IIa/IIb) hindlimb muscles from resting and exercised female Sprague-Dawley rats were removed for subcellular fractionation. By homogenization and gradient centrifugation, crude membranes and purified plasma membranes were isolated and subjected to gel electrophoresis and immunoblotting by using pump subunit-specific antibodies. Furthermore, mRNA was isolated from specific red type I (red-I) and white type IIb (white-IIb) muscles and subjected to Northern blotting by using subunit-specific probes. In both red-I/IIa and white-IIa/IIb muscles, exercise significantly raised the plasma membrane content of the alpha1-subunit of the pump by 64 +/- 24 and 55 +/- 22%, respectively (P < 0.05), and elevated the alpha2-polypeptide by 43 +/- 22 and 94 +/- 39%, respectively (P < 0.05). No significant effect of exercise could be detected on the amount of these subunits in an internal membrane fraction or in total membranes. In addition, exercise significantly increased the alpha1-subunit mRNA in red-I muscle (by 50 +/- 7%; P < 0.05) and the beta2-subunit mRNA in white-IIb muscles (by 64 +/- 19%; P < 0.01), but the alpha2- and beta1-mRNA levels were unaffected in this time period. We conclude that increased presence of alpha1- and alpha2-polypeptides at the plasma membrane and subsequent elevation of the alpha1- and beta2

  1. Cup regulates oskar mRNA stability during oogenesis.

    Science.gov (United States)

    Broyer, Risa M; Monfort, Elena; Wilhelm, James E

    2017-01-01

    The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. In this study, we have found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form. Published by Elsevier Inc.

  2. Lymphotoxin β receptor activation promotes mRNA expression of RelA and pro-inflammatory cytokines TNFα and IL-1β in bladder cancer cells.

    Science.gov (United States)

    Shen, Mo; Zhou, Lianlian; Zhou, Ping; Zhou, Wu; Lin, Xiangyang

    2017-07-01

    The role of inflammation in tumorigenesis and development is currently well established. Lymphotoxin β receptor (LTβR) activation induces canonical and noncanonical nuclear factor (NF)‑κB signaling pathways, which are linked to inflammation‑induced carcinogenesis. In the present study, 5,637 bladder cancer cells were cultured and the activation of LTβR was induced by functional ligand, lymphotoxin (LT) α1β2, and silencing with shRNA. Reverse transcription‑quantitative polymerase chain reaction was utilized to detect the mRNA expression levels of NF‑κB family members RelA and RelB, cytokines including LTα, LTβ, tumor necrosis factor (TNF)α, TNF superfamily member 14, interleukin (IL)‑6 and IL‑1β, and proliferation‑related genes including CyclinD1 and Survivin. The expression of phospho‑p65 was determined by western blotting. Activation of LTβR on bladder cancer 5,637 cells was demonstrated to upregulate the mRNA expression levels of the RELA proto‑oncogene, RelA, by 2.5‑fold compared with unstimulated cells, while no significant change was observed in the RELB proto‑oncogene NF‑κB member mRNA levels. Expression of pro‑inflammatory cytokines tumor necrosis factor (TNF)α and interleukin (IL)‑1β mRNA levels were significantly increased nearly 5‑fold and 1.5‑fold, respectively, following LTβR activation compared with unstimulated cells. The LTβR‑induced upregulation of RelA, TNFα and IL‑1β was decreased by ~33, 27, and 26% respectively when LTβR was silenced via short hairpin RNA. Activation of LTβR had no effect on 5,637 cell growth, despite CyclinD1 and Survivin mRNA levels increasing by ~2.7 and 1.3‑fold, respectively, compared with unstimulated cells. In conclusion, activation of LTβR induced the expression of RelA mRNA levels. LTβR activation might be an important mediator in promoting an inflammatory microenvironment in bladder cancer, via the upregulation of TNFα and IL‑1β mRNA levels. LTβR may

  3. ANCA-GBM dot-blot : Evaluation of an assay in the differential diagnosis of patients presenting with rapidly progressive glomerulonephritis

    NARCIS (Netherlands)

    Rutgers, Abraham; Damoiseaux, Jan; Roozendaal, Caroline; Limburg, Pieter C; Stegeman, Coen A; Tervaert, Jan Willem Cohen

    Rapidly progressive glomerulonephritis (RPGN) is characterized by rapid and progressive loss of renal function and the presence of crescentic glomerulonephritis (CGN). Early diagnosis and appropriate treatment is mandatory to prevent death and/or renal failure. We have evaluated an ANCA-GBM dot-blot

  4. Dot-blot immunoassay of Fasciola gigantica infection using 27 kDa and adult worm regurge antigens in Egyptian patients.

    Science.gov (United States)

    Kamel, Hanan H; Saad, Ghada A; Sarhan, Rania M

    2013-04-01

    The purpose of the present study was to evaluate the potential role of the 27-Kilodalton (KDa) antigen versus Fasciola gigantica adult worm regurge antigens in a DOT-Blot assay and to assess this assay as a practical tool for diagnosis fascioliasis in Egyptian patients. Fasciola gigantica antigen of an approximate molecular mass 27-(KDa) was obtained from adult worms by a simple elution SDS-PAGE. A Dot-Blot was developed comparatively to adult worm regurge antigens for the detection of specific antibodies from patients infected with F. gigantica in Egypt. Control sera were obtained from patients with other parasitic infections and healthy volunteers to assess the test and compare between the antigens. The sensitivity, specificity, positive and negative predictive values of Dot-Blot using the adult worm regurge were 80%, 90%, 94.1%, and 69.2% respectively, while those using 27-KDa were 100% which confirms the diagnostic potential of this antigen. All patients infected with Fasciola were positive, with cross reactivity reported with Schistosoma mansoni serum samples. This 27-KDa Dot-Blot assay showed to be a promising test which can be used for serodiagnosis of fascioliasis in Egyptian patients especially, those presenting with hepatic disease. It is specific, sensitive and easy to perform method for the rapid diagnosis particularly when more complex laboratory tests are unavailable.

  5. Natural selection and algorithmic design of mRNA.

    Science.gov (United States)

    Cohen, Barry; Skiena, Steven

    2003-01-01

    Messenger RNA (mRNA) sequences serve as templates for proteins according to the triplet code, in which each of the 4(3) = 64 different codons (sequences of three consecutive nucleotide bases) in RNA either terminate transcription or map to one of the 20 different amino acids (or residues) which build up proteins. Because there are more codons than residues, there is inherent redundancy in the coding. Certain residues (e.g., tryptophan) have only a single corresponding codon, while other residues (e.g., arginine) have as many as six corresponding codons. This freedom implies that the number of possible RNA sequences coding for a given protein grows exponentially in the length of the protein. Thus nature has wide latitude to select among mRNA sequences which are informationally equivalent, but structurally and energetically divergent. In this paper, we explore how nature takes advantage of this freedom and how to algorithmically design structures more energetically favorable than have been built through natural selection. In particular: (1) Natural Selection--we perform the first large-scale computational experiment comparing the stability of mRNA sequences from a variety of organisms to random synonymous sequences which respect the codon preferences of the organism. This experiment was conducted on over 27,000 sequences from 34 microbial species with 36 genomic structures. We provide evidence that in all genomic structures highly stable sequences are disproportionately abundant, and in 19 of 36 cases highly unstable sequences are disproportionately abundant. This suggests that the stability of mRNA sequences is subject to natural selection. (2) Artificial Selection--motivated by these biological results, we examine the algorithmic problem of designing the most stable and unstable mRNA sequences which code for a target protein. We give a polynomial-time dynamic programming solution to the most stable sequence problem (MSSP), which is asymptotically no more complex

  6. [Effects of lipopolysaccharides extracted from Porphyromonas endodontalis on the expression of IL-1beta mRNA and IL-6 mRNA in osteoblasts].

    Science.gov (United States)

    Yang, Di; Li, Ren; Qiu, Li-Hong; Li, Chen

    2009-04-01

    To quantify the IL-1 beta mRNA and IL-6 mRNA expression induced by lipopolysaccharides (LPS)extracted from Porphyromonas endodontalis(P.e) in osteoblasts, and to relate P.e-LPS to bone absorption pathogenesis in lesions of chronical apical periodontitis. MG63 was treated with different concentrations of P.e-LPS(0-50 microg/mL) for different hours(0-24h). The expression of IL-1 beta mRNA and IL-6 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR).Statistical analysis was performed using one- way ANOVA and Dunnett t test with SPSS11.0 software package. The level of IL-1 beta mRNA and IL-6 mRNA increased significantly after treatment with P.e-LPS at more than 5 microg/mL (P<0.01)and for more than 1 hour (P<0.01), which indicated that P.e-LPS induced osteoblasts to express IL-1 beta mRNA and IL-6 mRNA in dose and time dependent manners. P.e-LPS may promote bone resorption in lesions of chronical apical periodontitis by inducing IL-1 beta mRNA and IL-6 mRNA expression in osteoblasts.

  7. Fluorescent microthermographic imaging

    Energy Technology Data Exchange (ETDEWEB)

    Barton, D.L.

    1993-09-01

    In the early days of microelectronics, design rules and feature sizes were large enough that sub-micron spatial resolution was not needed. Infrared or IR thermal techniques were available that calculated the object`s temperature from infrared emission. There is a fundamental spatial resolution limitation dependent on the wavelengths of light being used in the image formation process. As the integrated circuit feature sizes began to shrink toward the one micron level, the limitations imposed on IR thermal systems became more pronounced. Something else was needed to overcome this limitation. Liquid crystals have been used with great success, but they lack the temperature measurement capabilities of other techniques. The fluorescent microthermographic imaging technique (FMI) was developed to meet this need. This technique offers better than 0.01{degrees}C temperature resolution and is diffraction limited to 0.3 {mu}m spatial resolution. While the temperature resolution is comparable to that available on IR systems, the spatial resolution is much better. The FMI technique provides better spatial resolution by using a temperature dependent fluorescent film that emits light at 612 nm instead of the 1.5 {mu}m to 12 {mu}m range used by IR techniques. This tutorial starts with a review of blackbody radiation physics, the process by which all heated objects emit radiation to their surroundings, in order to understand the sources of information that are available to characterize an object`s surface temperature. The processes used in infrared thermal imaging are then detailed to point out the limitations of the technique but also to contrast it with the FMI process. The FMI technique is then described in detail, starting with the fluorescent film physics and ending with a series of examples of past applications of FMI.

  8. Sorting live stem cells based on Sox2 mRNA expression.

    Directory of Open Access Journals (Sweden)

    Hans M Larsson

    Full Text Available While cell sorting usually relies on cell-surface protein markers, molecular beacons (MBs offer the potential to sort cells based on the presence of any expressed mRNA and in principle could be extremely useful to sort rare cell populations from primary isolates. We show here how stem cells can be purified from mixed cell populations by sorting based on MBs. Specifically, we designed molecular beacons targeting Sox2, a well-known stem cell marker for murine embryonic (mES and neural stem cells (NSC. One of our designed molecular beacons displayed an increase in fluorescence compared to a nonspecific molecular beacon both in vitro and in vivo when tested in mES and NSCs. We sorted Sox2-MB(+SSEA1(+ cells from a mixed population of 4-day retinoic acid-treated mES cells and effectively isolated live undifferentiated stem cells. Additionally, Sox2-MB(+ cells isolated from primary mouse brains were sorted and generated neurospheres with higher efficiency than Sox2-MB(- cells. These results demonstrate the utility of MBs for stem cell sorting in an mRNA-specific manner.

  9. Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii

    Directory of Open Access Journals (Sweden)

    Masayuki Onishi

    2016-12-01

    Full Text Available The unicellular green alga Chlamydomonas reinhardtii is a model organism that provides an opportunity to understand the evolution and functional biology of the lineage that includes the land plants, as well as aspects of the fundamental core biology conserved throughout the eukaryotic phylogeny. Although many tools are available to facilitate genetic, molecular biological, biochemical, and cell biological studies in Chlamydomonas, expression of unselected transgenes of interest (GOIs has been challenging. In most methods used previously, the GOI and a selectable marker are expressed from two separate mRNAs, so that their concomitant expression is not guaranteed. In this study, we developed constructs that allow expression of an upstream GOI and downstream selectable marker from a single bicistronic mRNA. Although this approach in other systems has typically required a translation-enhancing element such as an internal ribosome entry site for the downstream marker, we found that a short stretch of unstructured junction sequence was sufficient to obtain adequate expression of the downstream gene, presumably through post-termination reinitiation. With this system, we obtained robust expression of both endogenous and heterologous GOIs, including fluorescent proteins and tagged fusion proteins, in the vast majority of transformants, thus eliminating the need for tedious secondary screening for GOI-expressing transformants. This improved efficiency should greatly facilitate a variety of genetic and cell-biological studies in Chlamydomonas and also enable new applications such as expression-based screens and large-scale production of foreign proteins.

  10. A fluorescence scanning electron microscope

    International Nuclear Information System (INIS)

    Kanemaru, Takaaki; Hirata, Kazuho; Takasu, Shin-ichi; Isobe, Shin-ichiro; Mizuki, Keiji; Mataka, Shuntaro; Nakamura, Kei-ichiro

    2009-01-01

    Fluorescence techniques are widely used in biological research to examine molecular localization, while electron microscopy can provide unique ultrastructural information. To date, correlative images from both fluorescence and electron microscopy have been obtained separately using two different instruments, i.e. a fluorescence microscope (FM) and an electron microscope (EM). In the current study, a scanning electron microscope (SEM) (JEOL JXA8600 M) was combined with a fluorescence digital camera microscope unit and this hybrid instrument was named a fluorescence SEM (FL-SEM). In the labeling of FL-SEM samples, both Fluolid, which is an organic EL dye, and Alexa Fluor, were employed. We successfully demonstrated that the FL-SEM is a simple and practical tool for correlative fluorescence and electron microscopy.

  11. The clinical value of HPV E6/E7 and STAT3 mRNA detection in cervical cancer screening.

    Science.gov (United States)

    Fan, Yibing; Shen, Zongji

    2018-02-11

    To explore the value of human papillomavirus (HPV) E6/E7 and signal transducer and activator of transcription 3 (STAT3) mRNA detection in the screening of cervical lesions. 192 patients with abnormal ThinPrep cytology test (TCT) results and/or high-risk HPV infection were screened to identify possible cervical lesions in cases. Diagnoses were confirmed by histopathology. Fluorescence in situ hybridization (FISH) was performed to detect and qualify the mRNAs of HPV E6/E7, STAT3, and Survivin in cervical exfoliated cells. In addition, the performance of separate and combined mRNA detection methods were compared with TCT, HR-HPV DNA schemes respectively. 1. Compared with HPVE6/E7 and STAT3 mRNA methods, Survivin mRNA assay had poor specificity (Sp), Youden index (YI) and concordance rate. 2. HPV E6/E7, STAT3, and STAT3 + HR-HPV methods had the best Sp, concordance rate and positive predictive value (PPV) for cervical lesions screening and atypical squamous cells of undetermined significance (ASCUS) triage. For screening of high grade squamous intraepithelial lesions or greater (HSILs+), no difference was observed in the Se of mRNA detection methods in comparison with that of TCT, HR-HPV and TCT + HR-HPV, whereas the false positive rate (FPR) decreased by 41.48%/55.99%/17.19% and the colposcopy referral rate reduced by about 20.00%/25.00%/11.17%. For triage of women with ASCUS, no difference was observed in the Se of mRNA detection methods as compared to that of HR-HPV (χ 2  = 1.05, P > 0.75), while the FPR decreased by 45.83%/37.50%/41.66% and the colposcopy referral rate reduced by 32.42%/22.60%/25.28%, respectively. The Se, YI, and PPV of the combined methods increased in comparison to each method alone. 3. Compared with the TCT + HR-HPV method, HPV E6/E7 + STAT3 method had perfect Sp (95.92%) and PPV (95.40%) for screening HSILs+, the FPR and colposcopy referral rate decreased by 31.06% and 22.48% respectively. 1. The expression of HPV E6/E

  12. Development of a fluorescent cryocooler

    International Nuclear Information System (INIS)

    Edwards, B.C.; Buchwald, M.I.; Epstein, R.I.; Gosnell, T.R.; Mungan, C.E.

    1995-01-01

    Recent work at Los Alamos National Laboratory has demonstrated the physical principles for a new type of solid-state cryocooler based on anti-Stokes fluorescence. Design studies indicate that a vibration-free, low-mass ''fluorescent cryocooler'' could operate for years with efficiencies and cooling powers comparable to current commercial systems. This paper presents concepts for a fluorescent cryocooler, design considerations and expected performance

  13. Up-regulation of Slc39A2(Zip2) mRNA in peripheral blood mononuclear cells from patients with pulmonary tuberculosis.

    Science.gov (United States)

    Tao, Yan-ting; Huang, Qing; Jiang, Ya-li; Wang, Xiao-lei; Sun, Ping; Tian, Yuanyuan; Wu, Hai-liang; Zhang, Min; Meng, Si-bo; Wang, Yu-shu; Sun, Qing; Zhang, Lian-ying

    2013-08-01

    Zinc is the most common trace mineral after iron in the human body. In organisms, zinc transporters help zinc influx and efflux from cells. A previous study has reported that Zip2 was up-regulated over 27-fold in human monocytic THP-1 cells, when intracellular zinc was depleted by TPEN. Our study found Zip2 was over-expressed in leukocytes of asthmatic infants, especially those in which the serum zinc level was lower than those in healthy infants. Pulmonary tuberculosis (PTB) patients have significantly low serum zinc levels. Here we investigated whether Zip2 level was changed in the patients with PTB. Zip2 mRNA and protein levels in peripheral blood mononuclear cells (PBMC) from PTB (n1=23) and healthy controls (n2=42) were detected by quantitative real-time PCR and western blot, respectively. mRNA expression levels of another four zinc transporters, Zip1, Zip6, Zip8 and ZnT1, were detected by quantitative real-time PCR. Zip2 mRNA level was significantly up-regulated in PTB patients (P=0.001), and Zip8 mRNA level was significantly down-regulated compared with control individuals (Plevels of Zip1, Zip6 and ZnT1 in either group (P>0.05). Zip2 protein expression levels increased in PTB patients compared with control individuals. Our study found that knockdown of ZIP2 with siRNA caused a decrease in Zip2 levels in PBMC of PTB patients, while reducing the expression of INF-γ (Pinitial infection control of the human body, by promoting and maintaining the immune response of adaptive T cells.

  14. Thyroid hormone deiodinase type 2 mRNA levels in sea lamprey (Petromyzon marinus) are regulated during metamorphosis and in response to a thyroid challenge.

    Science.gov (United States)

    Stilborn, S Salina M; Manzon, Lori A; Schauenberg, Jennifer D; Manzon, Richard G

    2013-03-01

    Thyroid hormones (THs) are crucial for normal vertebrate development and are the one obligate morphogen that drives amphibian metamorphosis. However, contrary to other metamorphosing vertebrates, lampreys exhibit a sharp drop in serum TH early in metamorphosis, and anti-thyroid agents such as potassium perchlorate (KClO(4)) induce metamorphosis. The type 2 deiodinase (D2) enzyme is a key regulator of TH availability during amphibian metamorphosis. We set out to determine how D2 may be involved in the regulation of lamprey metamorphosis and thyroid homeostasis. We cloned a 1.8Kb Petromyzon marinus D2 cDNA that includes the entire protein coding region and a selenocysteine (Sec) codon. Northern blotting indicated that the lamprey D2 mRNA is the longest reported to date (>9Kb). Using real-time PCR, we showed that intestinal and hepatic D2 mRNA levels were elevated prior to and during the early stages of metamorphosis and then declined dramatically to low levels that were sustained for the remainder of metamorphosis. These data are consistent with previously reported changes in serum TH levels and deiodinase activity. Treatment of larvae with either TH or KClO(4) significantly affected D2 mRNA levels in the intestine and liver. These D2 mRNA levels during metamorphosis and in response to thyroid challenges suggest that D2 may function in the regulation of TH levels during lamprey metamorphosis and the maintenance of TH homeostasis. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. The Minor Allele of rs7574865 in the STAT4 Gene Is Associated with Increased mRNA and Protein Expression.

    Science.gov (United States)

    Lamana, Amalia; López-Santalla, Mercedes; Castillo-González, Raquel; Ortiz, Ana María; Martín, Javier; García-Vicuña, Rosario; González-Álvaro, Isidoro

    2015-01-01

    The T allele of rs7574865 in STAT4 confers risk of developing autoimmune disorders. However, its functional significance remains unclear. Here we analyze how rs7574865 affects the transcription of STAT4 and its protein expression. We studied 201 patients (80% female; median age, 54 years; median disease duration, 5.4 months) from PEARL study. Demographic, clinical, laboratory and therapeutic data were collected at each visit. IL-6 serum levels were measured by enzyme immune assay. The rs7574865 was genotyped using TaqMan probes. The expression levels of STAT4 mRNA were determined at 182 visits from 69 patients using quantitative real-time polymerase chain reaction. STAT4 protein was assessed by western blot in 62 samples from 34 patients. To determine the effect of different variables on the expression of STAT4 mRNA and protein, we performed multivariate longitudinal analyses using generalized linear models. After adjustment for age, disease activity and glucocorticoid dose as confounders, the presence of at least one copy of the T allele of rs7574865 was significantly associated with higher levels of STAT4 mRNA. Similarly, TT patients showed significantly higher levels of STAT4 protein than GG patients. IL-6 induced STAT4 and STAT5 phosphorylation in peripheral blood lymphocytes. Patients carrying at least one T allele of rs7574865 displayed lower levels of serum IL-6 compared to GG homozygous; by contrast the production of C-reactive protein was similar in both populations. Our data suggest that the presence of the rs7574865 T allele enhances STAT4 mRNA transcription and protein expression. It may enhance the signaling of molecules depending on the STAT4 pathway.

  16. The Minor Allele of rs7574865 in the STAT4 Gene Is Associated with Increased mRNA and Protein Expression.

    Directory of Open Access Journals (Sweden)

    Amalia Lamana

    Full Text Available The T allele of rs7574865 in STAT4 confers risk of developing autoimmune disorders. However, its functional significance remains unclear. Here we analyze how rs7574865 affects the transcription of STAT4 and its protein expression.We studied 201 patients (80% female; median age, 54 years; median disease duration, 5.4 months from PEARL study. Demographic, clinical, laboratory and therapeutic data were collected at each visit. IL-6 serum levels were measured by enzyme immune assay. The rs7574865 was genotyped using TaqMan probes. The expression levels of STAT4 mRNA were determined at 182 visits from 69 patients using quantitative real-time polymerase chain reaction. STAT4 protein was assessed by western blot in 62 samples from 34 patients. To determine the effect of different variables on the expression of STAT4 mRNA and protein, we performed multivariate longitudinal analyses using generalized linear models.After adjustment for age, disease activity and glucocorticoid dose as confounders, the presence of at least one copy of the T allele of rs7574865 was significantly associated with higher levels of STAT4 mRNA. Similarly, TT patients showed significantly higher levels of STAT4 protein than GG patients. IL-6 induced STAT4 and STAT5 phosphorylation in peripheral blood lymphocytes. Patients carrying at least one T allele of rs7574865 displayed lower levels of serum IL-6 compared to GG homozygous; by contrast the production of C-reactive protein was similar in both populations.Our data suggest that the presence of the rs7574865 T allele enhances STAT4 mRNA transcription and protein expression. It may enhance the signaling of molecules depending on the STAT4 pathway.

  17. Fluorescence of ceramic color standards

    International Nuclear Information System (INIS)

    Koo, Annette; Clare, John F.; Nield, Kathryn M.; Deadman, Andrew; Usadi, Eric

    2010-01-01

    Fluorescence has been found in color standards available for use in calibration and verification of color measuring instruments. The fluorescence is excited at wavelengths below about 600 nm and emitted above 700 nm, within the response range of silicon photodiodes, but at the edge of the response of most photomultipliers and outside the range commonly scanned in commercial colorimeters. The degree of fluorescence on two of a set of 12 glossy ceramic tiles is enough to introduce significant error when those tiles have been calibrated in one mode of measurement and are used in another. We report the nature of the fluorescence and the implications for color measurement.

  18. Intergenic mRNA molecules resulting from trans-splicing.

    Science.gov (United States)

    Finta, Csaba; Zaphiropoulos, Peter G

    2002-02-22

    Accumulated recent evidence is indicating that alternative splicing represents a generalized process that increases the complexity of human gene expression. Here we show that mRNA production may not necessarily be limited to single genes, as human liver also has the potential to produce a variety of hybrid cytochrome P450 3A mRNA molecules. The four known cytochrome P450 3A genes in humans, CYP3A4, CYP3A5, CYP3A7, and CYP3A43, share a high degree of similarity, consist of 13 exons with conserved exon-intron boundaries, and form a cluster on chromosome 7. The chimeric CYP3A mRNA molecules described herein are characterized by CYP3A43 exon 1 joined at canonical splice sites to distinct sets of CYP3A4 or CYP3A5 exons. Because the CYP3A43 gene is in a head-to-head orientation with the CYP3A4 and CYP3A5 genes, bypassing transcriptional termination can not account for the formation of hybrid CYP3A mRNAs. Thus, the mechanism generating these molecules has to be an RNA processing event that joins exons of independent pre-mRNA molecules, i.e. trans-splicing. Using quantitative real-time polymerase chain reaction, the ratio of one CYP3A43/3A4 intergenic combination was estimated to be approximately 0.15% that of the CYP3A43 mRNAs. Moreover, trans-splicing has been found not to interfere with polyadenylation. Heterologous expression of the chimeric species composed of CYP3A43 exon 1 joined to exons 2-13 of CYP3A4 revealed catalytic activity toward testosterone.

  19. mRNA transfection of mouse and human neural stem cell cultures

    OpenAIRE

    McLenachan, Samuel; Zhang, D.; Palomo, A.B.; Edel, Michael John; Chen, F.K.

    2013-01-01

    The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has ...

  20. Gibberellin (GA3) enhances cell wall invertase activity and mRNA levels in elongating dwarf pea (Pisum sativum) shoots

    Science.gov (United States)

    Wu, L. L.; Mitchell, J. P.; Cohn, N. S.; Kaufman, P. B.

    1993-01-01

    The invertase (EC 3.2.1.26) purified from cell walls of dwarf pea stems to homogeneity has a molecular mass of 64 kilodaltons (kD). Poly(A)+RNA was isolated from shoots of dwarf pea plants, and a cDNA library was constructed using lambda gt11 as an expression vector. The expression cDNA library was screened with polyclonal antibodies against pea cell wall invertase. One invertase cDNA clone was characterized as a full-length cDNA with 1,863 base pairs. Compared with other known invertases, one homologous region in the amino acid sequence was found. The conserved motif, Asn-Asp-Pro-Asn-Gly, is located near the N-terminal end of invertase. Northern blot analysis showed that the amount of invertase mRNA (1.86 kb) was rapidly induced to a maximal level 4 h after GA3 treatment, then gradually decreased to the control level. The mRNA level at 4 h in GA3-treated peas was fivefold higher than that of the control group. The maximal increase in activity of pea cell wall invertase elicited by GA3 occcured at 8 h after GA3 treatment. This invertase isoform was shown immunocytochemically to be localized in the cell walls, where a 10-fold higher accumulation occurred in GA3-treated tissue compared with control tissue. This study indicates that the expression of the pea shoot cell-wall invertase gene could be regulated by GA3 at transcriptional and/or translational levels.

  1. Phorbol esters induce interleukin 2 mRNA in sensitive but not in resistant EL4 cells

    International Nuclear Information System (INIS)

    Harrison, J.R.; Lynch, K.R.; Sando, J.J.

    1986-01-01

    Phorbol ester (PE) sensitive EL4 cells are growth-inhibited and produce interleukin 2 (IL2) when treated with PE. Resistant EL4 cells lack both responses. To determine whether the defect in resistant cells occurs pre or post-transcriptionally, an assay for IL2 mRNA was developed using a synthetic oligonucleotide to mouse IL2 as a probe. Total RNA (15 μg) from cells +/- PE was electrophoresed, blotted onto a cationic nylon membrane, and probed with radiolabeled oligomer. This probe hybridized to a 1.1 kb band in RNA from PE-treated sensitive cells. This RNA was detectable within 3h of PE administration, was clearly visible by 6h, and peaked by 9 to 12h. No bands hybridizing with the IL2 probe were detected in RNA isolated from unstimulated cells or from resistant EL4 cells at any time following PE stimulation. Since levels of the protooncogene c-myc have been shown to decrease in a number of cell lines during differentiation and growth inhibition, total RNA from EL4 cells was probed with a nick-translated plasmid containing the protein coding region of the c-myc gene. In PE sensitive cells, levels of c-myc RNA are markedly reduced by 3h. In a pilot experiment with resistant cells, c-myc levels appeared to remain constant. These results demonstrate that PE induced IL2 mRNA in PE sensitive but not resistant EL4 cells. Sensitive and resistant EL4 cell lines provide a useful model for the investigation of the regulation of gene expression by PE

  2. Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA*

    Science.gov (United States)

    Woo, Ho-Hyung; Baker, Terri; Laszlo, Csaba; Chambers, Setsuko K.

    2013-01-01

    CSF-1 mRNA 3′UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3′UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3′UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of

  3. A glimpse at mRNA dynamics reveals cellular domains and rapid trafficking through granules

    NARCIS (Netherlands)

    Gemert, Alice Myriam Christi van

    2011-01-01

    mRNA transport and targeting are essential to gene expression regulation. Specific mRNA sequences can bind several proteins and together form RiboNucleoProtein particles (RNP). The various proteins within the RNP determine mRNA fate: translation, transport or decay. RNP composition varies with

  4. Expression of tomato prosystemin gene in Arabidopsis reveals systemic translocation of its mRNA and confers necrotrophic fungal resistance.

    Science.gov (United States)

    Zhang, Haiyan; Yu, Pengli; Zhao, Jiuhai; Jiang, Hongling; Wang, Haiyang; Zhu, Yingfang; Botella, Miguel A; Šamaj, Jozef; Li, Chuanyou; Lin, Jinxing

    2018-01-01

    Systemin (SYS), an octadecapeptide hormone processed from a 200-amino-acid precursor (prosystemin, PS), plays a central role in the systemic activation of defense genes in tomato in response to herbivore and pathogen attacks. However, whether PS mRNA is transferable and its role in systemic defense responses remain unknown. We created the transgenic tomato PS gene tagged with the green fluorescent protein (PS-GFP) using a shoot- or root-specific promoter, and the constitutive 35S promoter in Arabidopsis. Subcellular localization of PS-/SYS-GFP was observed using confocal laser scanning microscopy and gene transcripts were determined using quantitative real-time PCR. In Arabidopsis, PS protein can be processed and SYS is secreted. Shoot-/root-specific expression of PS-GFP in Arabidopsis, and grafting experiments, revealed that the PS mRNA moves in a bi-directional manner. We also found that ectopic expression of PS improves Arabidopsis resistance to the necrotrophic fungus Botrytis cinerea, consistent with substantial upregulation of the transcript levels of specific pathogen-responsive genes. Our results provide novel insights into the multifaceted mechanism of SYS signaling transport and its potential application in genetic engineering for increasing pathogen resistance across diverse plant families. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  5. Characterization of Sm14 related components in different helminths by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis

    Directory of Open Access Journals (Sweden)

    Nilton Thaumaturgo

    2002-10-01

    Full Text Available Sm14 was the first fatty acid-binding protein homologue identified in helminths. Thereafter, members of the same family were identified in several helminth species, with high aminoacid sequence homology between them. In addition, immune crossprotection was also reported against Fasciola hepatica infection, in animals previously immunized with the Schistosoma mansoni vaccine candidate, r-Sm14. In the present study, data on preliminary sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis of nine different helminth extracts focusing the identification of Sm14 related proteins, is reported. Out of these, three extracts - Ascaris suum (males and females, Echinostoma paraensei, and Taenia saginata - presented components that comigrated with Sm14 in SDS-PAGE, and that were recognized by anti-rSm14 policlonal serum, in Western blotting tests.

  6. Detection of human papilloma virus 16 and 18 DNA sequences by southern blot hybridization in oral leukoplakia and squamous cell carcinoma.

    Science.gov (United States)

    Khanna, Rahul; Rao, G R K; Tiwary, S K; Rai, Ashish; Khanna, Seema; Khanna, A K

    2009-04-01

    The etiopathological role of human papilloma virus (HPV) in the causation of oral cancer is till a subject of speculation. We used the technique of Southern blot hybridization to detect the presence of HPV types 16 & 18 in biopsy specimens from oral cancer and leukoplakia patients as well as normal oral mucosal biopsies. The prevalence of either HPV type 16 or 18 was found in 64.5% (29/45) of oral cancer, 40%(12/30) of leukoplakia and 20%(9/45) of normal oral mucosal biopsies. No association could be demonstrated between tobacco usage habits or a history of genital warts with HPV prevalence. A significant finding was that none of the oral cancer patients were negative for both: a history of tobacco usage as well as presence of HPV infection, on Southern blot hybridization.

  7. MiR-520b suppresses proliferation of hepatoma cells through targeting ten-eleven translocation 1 (TET1) mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Weiying; Lu, Zhanping; Gao, Yuen [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin (China); Song, Tianqiang, E-mail: tjchi@hotmai.com [Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China)

    2015-05-08

    Accumulating evidence indicates that microRNAs are able to act as oncogenes or tumor suppressor genes in human cancer. We previously reported that miR-520b was down-regulated in hepatocellular carcinoma (HCC) and its deregulation was involved in hepatocarcinogenesis. In the present study, we report that miR-520b suppresses cell proliferation in HCC through targeting the ten-eleven translocation 1 (TET1) mRNA. Notably, we identified that miR-520b was able to target 3′-untranslated region (3′UTR) of TET1 mRNA by luciferase reporter gene assays. Then, we revealed that miR-520b was able to reduce the expression of TET1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blotting analysis. In terms of function, 5-ethynyl-2-deoxyuridine (EdU) incorporation and colony formation assays demonstrated that the forced miR-520b expression remarkably inhibited proliferation of hepatoma cells, but TET1 overexpression could rescue the inhibition of cell proliferation mediated by miR-520b. Furthermore, anti-miR-520b enhanced proliferation of hepatoma cells, whereas silencing of TET1 abolished anti-miR-520b-induced acceleration of cell proliferation. Then, we validated that the expression levels of miR-520b were negatively related to those of TET1 mRNA in clinical HCC tissues. Thus, we conclude that miR-520b depresses proliferation of liver cancer cells through targeting 3′UTR of TET1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • TET1 is a novel target gene of miR-520b. • TET1 is upregulated in clinical HCC tissues. • MiR-520b is negatively correlated with TET1 in clinical HCC tissues. • MiR-520b depresses the proliferation of HCC cells through targeting TET1 mRNA.

  8. MiR-30e suppresses proliferation of hepatoma cells via targeting prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA

    Energy Technology Data Exchange (ETDEWEB)

    Feng, Guoxing [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Shi, Hui [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin (China); Li, Jiong; Yang, Zhe [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Fang, Runping; Ye, Lihong [State Key Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin (China); Zhang, Weiying, E-mail: zhwybao@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China); Zhang, Xiaodong, E-mail: zhangxd@nankai.edu.cn [State Key Laboratory of Medicinal Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai University, Tianjin (China)

    2016-04-08

    Aberrant microRNA expression has been shown to be characteristic of many cancers. It has been reported that the expression levels of miR-30e are decreased in liver cancer tissues. However, the role of miR-30e in hepatocellular carcinoma remains poorly understood. In the present study, we investigated the significance of miR-30e in hepatocarcinogenesis. Bioinformatics analysis reveals a putative target site of miR-30e in the 3′-untranslated region (3′UTR) of prolyl 4-hydroxylase subunit alpha-1 (P4HA1) mRNA. Moreover, luciferase reporter gene assays verified that miR-30e directly targeted 3′UTR of P4HA1 mRNA. Then, we demonstrated that miR-30e was able to reduce the expression of P4HA1 at the levels of mRNA and protein using reverse transcription-polymerase chain reaction and Western blot analysis. Enforced expression of miR-30e suppressed proliferation of HepG2 cells by 5-ethynyl-2-deoxyuridine (EdU) assay and reduced colony formation of these cells by colony formation analysis. Conversely, anti-miR-30e enhanced the proliferation of hepatoma cells in vitro. Interestingly, the ectopic expression of P4HA1 could efficiently rescue the inhibition of cell proliferation mediated by miR-30e in HepG2 cells. Meanwhile, silencing of P4HA1 abolished the anti-miR-30e-induced proliferation of cells. Clinically, quantitative real-time PCR showed that miR-30e was down-regulated in liver tumor tissues relative to their peritumor tissues. The expression levels of miR-30e were negatively correlated to those of P4HA1 mRNA in clinical liver tumor tissues. Thus, we conclude that miR-30e suppresses proliferation of hepatoma cells through targeting P4HA1 mRNA. Our finding provides new insights into the mechanism of hepatocarcinogenesis. - Highlights: • P4HA1 is a novel target gene of miR-30e. • P4HA1 is increased in clinical HCC tissues. • MiR-30e is negatively correlated with P4HA1 in clinical HCC tissues. • MiR-30e suppresses the proliferation of HCC cells through

  9. Matrin 3 binds and stabilizes mRNA.

    Directory of Open Access Journals (Sweden)

    Maayan Salton

    Full Text Available Matrin 3 (MATR3 is a highly conserved, inner nuclear matrix protein with two zinc finger domains and two RNA recognition motifs (RRM, whose function is largely unknown. Recently we found MATR3 to be phosphorylated by the protein kinase ATM, which activates the cellular response to double strand breaks in the DNA. Here, we show that MATR3 interacts in an RNA-dependent manner with several proteins with established roles in RNA processing, and maintains its interaction with RNA via its RRM2 domain. Deep sequencing of the bound RNA (RIP-seq identified several small noncoding RNA species. Using microarray analysis to explore MATR3's role in transcription, we identified 77 transcripts whose amounts depended on the presence of MATR3. We validated this finding with nine transcripts which were also bound to the MATR3 complex. Finally, we demonstrated the importance of MATR3 for maintaining the stability of several of these mRNA species and conclude that it has a role in mRNA stabilization. The data suggest that the cellular level of MATR3, known to be highly regulated, modulates the stability of a group of gene transcripts.

  10. The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2

    Science.gov (United States)

    Kerr, Niall; Holmes, Fiona E.; Hobson, Sally-Ann; Vanderplank, Penny; Leard, Alan; Balthasar, Nina; Wynick, David

    2015-01-01

    The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal1, Gal2 and the less studied Gal3 (GalR1–3 gene products). There is a wealth of data on expression of Gal1–3 at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal1 or Gal2 receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal1-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal1-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal1-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal2-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal1 in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs). PMID:26292267

  11. Establishment and application of a modified membrane-blot assay for Rhizomucor miehei lipases aimed at improving their methanol tolerance and thermostability.

    Science.gov (United States)

    He, Dong; Luo, Wen; Wang, Zhiyuan; Lv, Pengmei; Yuan, Zhenhong; Huang, Shaowei; Xv, Jingliang

    2017-07-01

    Directed evolution has been proved an effective way to improve the stability of proteins, but high throughput screening assays for directed evolution with simultaneous improvement of two or more properties are still rare. In this study, we aimed to establish a membrane-blot assay for use in the high-throughput screening of Rhizomucor miehei lipases (RMLs). With the assistance of the membrane-blot screening assay, a mutant E47K named G10 that showed improved thermal stability was detected in the first round of error-prone PCR. Using G10 as the parent, two variants G10-11 and G10-20 that showed improved thermal stability and methanol tolerance without loss of activity compared to the wild type RML were obtained. The T 50 60 -value of G10-11 and G10-20 increased by 12°C and 6.5°C, respectively. After incubation for 1h, the remaining residual activity of G10-11 and G10-20 was 63.45% and 74.33%, respectively, in 50% methanol, and 15.98% and 30.22%, respectively, in 80% methanol. Thus, we successfully developed a membrane-blot assay that could be used for the high-throughput screening of RMLs with improved thermostability and methanol tolerance. Based on our findings, we believe that our newly developed membrane-blot assay will have potential applications in directed evolution in the future. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Fluorescing macerals from wood precursors

    Energy Technology Data Exchange (ETDEWEB)

    Stout, S A; Bensley, D F

    1987-01-01

    A preliminary investigation into the origin of wood-derived macerals has established the existence of autofluorescent maceral precursors in the secondary xylem of swamp-inhabiting plant species. The optical character and fluorescent properties of microtomed thin-sections of modern woods from the Florida Everglades and Okefenokee Swamp, Georgia are compared to the character and properties of their peatified equivalents from various Everglades and Okefenokee peat horizons and their lignitic equivalents from the Brandon lignite of Vermont and the Trail Ridge lignitic peat from northern Florida. The inherent fluorescence of woody cell walls is believed to be caused by lignin though other cell wall components may contribute. The fluorescence spectra for several wood and cell types had a ..gamma../sub m//sub a//sub x/ of 452 nm and Q value of 0.00. The color as observed in blue light and the spectral geometry as measured in UV light of peatified and lignitic woody cell walls (potential textinites) may change progressively during early coalification. Cell wall-derived maceral material is shown to maintain its fluorescing properties after being converted to a structureless material, perhaps a corpohuminite or humodetrinite precursor. Fluorescing xylem cell contents, such as condensed tannins or essential oils, can maintain the fluorescent character through early coalification. Xylem cell walls and xylem cell contents are shown to provide fluorescing progenitor materials which would not require subsequent infusion with 'lipid' materials to account for their fluorescence as phytoclast material or as macerals in coal. 35 references.

  13. Assessing Photosynthesis by Fluorescence Imaging

    Science.gov (United States)

    Saura, Pedro; Quiles, Maria Jose

    2011-01-01

    This practical paper describes a novel fluorescence imaging experiment to study the three processes of photochemistry, fluorescence and thermal energy dissipation, which compete during the dissipation of excitation energy in photosynthesis. The technique represents a non-invasive tool for revealing and understanding the spatial heterogeneity in…

  14. Development of EMab-51, a Sensitive and Specific Anti-Epidermal Growth Factor Receptor Monoclonal Antibody in Flow Cytometry, Western Blot, and Immunohistochemistry.

    Science.gov (United States)

    Itai, Shunsuke; Kaneko, Mika K; Fujii, Yuki; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Handa, Saori; Chang, Yao-Wen; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

    2017-10-01

    The epidermal growth factor receptor (EGFR) is a member of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and is involved in cell growth and differentiation. EGFR homodimers or heterodimers with other HER members, such as HER2 and HER3, activate downstream signaling cascades in many cancers. In this study, we developed novel anti-EGFR monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. First, we expressed the full-length or ectodomain of EGFR in LN229 glioblastoma cells and then immunized mice with LN229/EGFR or ectodomain of EGFR, and performed the first screening using enzyme-linked immunosorbent assays. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical (fourth screening) analyses. Among 100 mAbs, only one clone EMab-51 (IgG 1 , kappa) reacted with EGFR in Western blot analysis. Finally, immunohistochemical analyses with EMab-51 showed sensitive and specific reactions against oral cancer cells, warranting the use of EMab-51 to detect EGFR in pathological analyses of EGFR-expressing cancers.

  15. Time-resolved fluorescence spectroscopy

    International Nuclear Information System (INIS)

    Gustavsson, Thomas; Mialocq, Jean-Claude

    2007-01-01

    This article addresses the evolution in time of light emitted by a molecular system after a brief photo-excitation. The authors first describe fluorescence from a photo-physical point of view and discuss the characterization of the excited state. Then, they explain some basic notions related to fluorescence characterization (lifetime and decays, quantum efficiency, so on). They present the different experimental methods and techniques currently used to study time-resolved fluorescence. They discuss basic notions of time resolution and spectral reconstruction. They briefly present some conventional methods: intensified Ccd cameras, photo-multipliers and photodiodes associated with a fast oscilloscope, and phase modulation. Other methods and techniques are more precisely presented: time-correlated single photon counting (principle, examples, and fluorescence lifetime imagery), streak camera (principle, examples), and optical methods like the Kerr optical effect (principle and examples) and fluorescence up-conversion (principle and theoretical considerations, examples of application)

  16. Tumor protein D52 expression is post-transcriptionally regulated by T-cell intercellular antigen (TIA) 1 and TIA-related protein via mRNA stability.

    Science.gov (United States)

    Motohashi, Hiromi; Mukudai, Yoshiki; Ito, Chihiro; Kato, Kosuke; Shimane, Toshikazu; Kondo, Seiji; Shirota, Tatsuo

    2017-05-04

    Although tumor protein D52 (TPD52) family proteins were first identified nearly 20 years ago, their molecular regulatory mechanisms remain unclear. Therefore, we investigated the post-transcriptional regulation of TPD52 family genes. An RNA immunoprecipitation (RIP) assay showed the potential binding ability of TPD52 family mRNAs to several RNA-binding proteins, and an RNA degradation assay revealed that TPD52 is subject to more prominent post-transcriptional regulation than are TPD53 and TPD54. We subsequently focused on the 3'-untranslated region (3'-UTR) of TPD52 as a cis -acting element in post-transcriptional gene regulation. Several deletion mutants of the 3'-UTR of TPD52 mRNA were constructed and ligated to the 3'-end of a reporter green fluorescence protein gene. An RNA degradation assay revealed that a minimal cis -acting region, located in the 78-280 region of the 5'-proximal region of the 3'-UTR, stabilized the reporter mRNA. Biotin pull-down and RIP assays revealed specific binding of the region to T-cell intracellular antigen 1 (TIA-1) and TIA-1-related protein (TIAR). Knockdown of TIA-1/TIAR decreased not only the expression, but also the stability of TPD52 mRNA; it also decreased the expression and stability of the reporter gene ligated to the 3'-end of the 78-280 fragment. Stimulation of transforming growth factor-β and epidermal growth factor decreased the binding ability of these factors, resulting in decreased mRNA stability. These results indicate that the 78-280 fragment and TIA-1/TIAR concordantly contribute to mRNA stability as a cis -acting element and trans -acting factor(s), respectively. Thus, we here report the specific interactions between these elements in the post-transcriptional regulation of the TPD52 gene. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  17. The combination of quantitative PCR and western blot detecting CP4-EPSPS component in Roundup Ready soy plant tissues and commercial soy-related foodstuffs.

    Science.gov (United States)

    Xiao, Xiao; Wu, Honghong; Zhou, Xinghu; Xu, Sheng; He, Jian; Shen, Wenbiao; Zhou, Guanghong; Huang, Ming

    2012-06-01

    With the widespread use of Roundup Ready soy (event 40-3-2) (RRS), the comprehensive detection of genetically modified component in foodstuffs is of significant interest, but few protein-based approaches have been found useful in processed foods. In this report, the combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different RRS plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing both meat and plant protein concentrates. The validity of the 2 methods was confirmed first. We also showed that the CP4-EPSPS protein existed in different RRS plant tissues. In certain cases, the results from the western blot and the qPCR were not consistent. To be specific, at least 2 degraded fragments of CP4-EPSPS protein (35.5 and 24.6 kDa) were observed. For dried bean curd crust and deep-fried bean curd, a degraded protein fragment with the size of 24.6 kDa appeared, while cp4-epsps gene could not be traced by qPCR. In contrast, we found a signal of cp4-epsps DNA in 3 foodstuffs, including soy-containing ham cutlet product, meat ball, and sausage by qPCR, while CP4-EPSPS protein could not be detected by western blot in such samples. Our study therefore concluded that the combination of DNA- and protein-based methods would compensate each other, thus resulting in a more comprehensive detection from nucleic acid and protein levels. The combination of quantitative PCR (qPCR) and western blot was used to detect cp4-epsps gene and its protein product in different Roundup Ready soy (event 40-3-2) plant tissues and commercial soy-containing foodstuffs. The foods included those of plant origin produced by different processing procedures and also some products containing a combination of both meat and plant protein concentrates. This study indicated that the combination of DNA- and protein-based methods

  18. Detection of KatG Gen Mutation on Mycobacterium Tuberculosis by Means of PCR-Dot Blot Hybridization with 32P Labeled Oligonucleotide Probe Methods

    International Nuclear Information System (INIS)

    Maria Lina R; Budiman Bela; Andi Yasmon

    2009-01-01

    Handling and controlling of tuberculosis, a disease caused by Mycobacterium tuberculosis (MTB), is now complicated since there are many MTBs that are resistant against anti-tuberculosis drugs such as isoniazid. The drug resistance could occurred due to the inadequate and un-regular drug utilization that cause gene mutation of the drug target such as katG gene for isoniazid. The molecular biology techniques such as the PCR- dot blot hybridization with radioisotope ( 32 P) labeled oligonucleotide probe, has been reported as a technique that is more sensitive and rapid for detection of gene mutations related with drug resistances. Hence, the aim of this study was to apply the PCR- dot blot hybridization technique using 32 P labeled oligonucleotide probe for detection of single mutation at codon 315 of katG gene of MTBs that rise the isoniazid resistance. In this study, we used 89 sputum specimens and a standard MTB (MTB H 37 RV) as a control. DNA extractions were performed by the BOOM method and the phenol chloroform for sputum samples and standard MTB, respectively. Primers used for PCR technique were Pt8 and Pt9 and RTB59 and RTB36 for detecting tuberculosis causing Mycobacterium and the existence of katG gene, respectively. Both of the primers are specific for IS6110 region and katG gene, respectively. PCR products were detected by an agarose gel electrophoresis technique. Dot blot hybridization with 32 P-oligonucleotide probe 315mu was performed to detect mutation at codon 315 of tested samples. Results of the PCR using primer Pt8 and Pt9 showed that all sputum specimens had positive results. Mutation detection by PCR- dot blot hybridization with 32 P-oligonucleotide probe 315mu, revealed that 11 of 89 tested samples had a mutation at their codon 315 of katG gene. Based upon these results, it is concluded that PCR-dot blot hybridization with 32 P-oligonucleotide probe is a technique that is rapid and highly specific and sensitive for detection of mutation at codon

  19. Single Molecule Spectroscopy of Fluorescent Proteins

    NARCIS (Netherlands)

    Blum, Christian; Subramaniam, Vinod

    2009-01-01

    The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the

  20. Fluorescent multiplex cell flow systems and methods

    KAUST Repository

    Merzaban, Jasmeen; Abuelela, Ayman F.; Mohammad, Amal Jehad

    2017-01-01

    scanning system emits multiple electromagnetic wavelengths simultaneously it cause multiple fluorescent labels having different excitation wavelength maximums to fluoresce. The system can simultaneously capture real-time fluorescence images from at least

  1. Fluorescent standards for photodynamic therapy

    Science.gov (United States)

    Belko, N.; Kavalenka, S.; Samtsov, M.

    2016-08-01

    Photodynamic therapy is an evolving technique for treatment of various oncological diseases. This method employs photosensitizers - species that lead to death of tumor cells after the photoactivation. For further development and novel applications of photodynamic therapy new photosensitizers are required. After synthesis of a new photosensitizer it is important to know its concentration in different biological tissues after its administration and distribution. The concentration is frequently measured by the extraction method, which has some disadvantages, e.g. it requires many biological test subjects that are euthanized during the measurement. We propose to measure the photosensitizer concentration in tissue by its fluorescence. For this purpose fluorescent standards were developed. The standards are robust and simple to produce; their fluorescence signal does not change with time. The fluorescence intensity of fluorescent standards seems to depend linearly on the dye concentration. A set of standards thus allow the calibration of a spectrometer. Finally, the photosensitizer concentration can be determined by the fluorescence intensity after comparing the corresponding spectrum with spectra of the set of fluorescent standards. A biological test subject is not euthanized during this kind of experiment. We hope this more humane technique can be used in future instead of the extraction method.

  2. Relative quantification of mRNA: comparison of methods currently used for real-time PCR data analysis

    Directory of Open Access Journals (Sweden)

    Koppel Juraj

    2007-12-01

    Full Text Available Abstract Background Fluorescent data obtained from real-time PCR must be processed by some method of data analysis to obtain the relative quantity of target mRNA. The method chosen for data analysis can strongly influence results of the quantification. Results To compare the performance of six techniques which are currently used for analysing fluorescent data in real-time PCR relative quantification, we quantified four cytokine transcripts (IL-1β, IL-6 TNF-α, and GM-CSF in an in vivo model of colonic inflammation. Accuracy of the methods was tested by quantification on samples with known relative amounts of target mRNAs. Reproducibility of the methods was estimated by the determination of the intra-assay and inter-assay variability. Cytokine expression normalized to the expression of three reference genes (ACTB, HPRT, SDHA was then determined using the six methods for data analysis. The best results were obtained with the relative standard curve method, comparative Ct method and with DART-PCR, LinRegPCR and Liu & Saint exponential methods when average amplification efficiency was used. The use of individual amplification efficiencies in DART-PCR, LinRegPCR and Liu & Saint exponential methods significantly impaired the results. The sigmoid curve-fitting (SCF method produced medium performance; the results indicate that the use of appropriate type of fluorescence data and in some instances manual selection of the number of amplification cycles included in the analysis is necessary when the SCF method is applied. We also compared amplification efficiencies (E and found that although the E values determined by different methods of analysis were not identical, all the methods were capable to identify two genes whose E values significantly differed from other genes. Conclusion Our results show that all the tested methods can provide quantitative values reflecting the amounts of measured mRNA in samples, but they differ in their accuracy and

  3. Fluorescence molecular tomography in the presence of background fluorescence

    International Nuclear Information System (INIS)

    Soubret, Antoine; Ntziachristos, Vasilis

    2006-01-01

    Fluorescence molecular tomography is an emerging imaging technique that resolves the bio-distribution of engineered fluorescent probes developed for in vivo reporting of specific cellular and sub-cellular targets. The method can detect fluorochromes in picomole amounts or less, imaged through entire animals, but the detection sensitivity and imaging performance drop in the presence of background, non-specific fluorescence. In this study, we carried out a theoretical and an experimental investigation on the effect of background fluorescence on the measured signal and on the tomographic reconstruction. We further examined the performance of three subtraction methods based on physical models of photon propagation, using experimental data on phantoms and small animals. We show that the data pre-processing with subtraction schemes can improve image quality and quantification when non-specific background florescence is present

  4. Identification of a noncanonically transcribed subgenomic mRNA of infectious bronchitis virus and other gammacoronaviruses.

    Science.gov (United States)

    Bentley, Kirsten; Keep, Sarah May; Armesto, Maria; Britton, Paul

    2013-02-01

    Coronavirus subgenomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving transcription regulatory sequences (TRSs) located in the 5' leader sequence (TRS-L) and upstream of each structural and group-specific gene (TRS-B). Several gammacoronaviruses including infectious bronchitis virus (IBV) contain a putative open reading frame (ORF), localized between the M gene and gene 5, which is controversial due to the perceived absence of a TRS. We have studied the transcription of a novel sgmRNA associated with this potential ORF and found it to be transcribed via a previously unidentified noncanonical TRS-B. Using an IBV reverse genetics system, we demonstrated that the template-switching event during intergenic region (IR) sgmRNA synthesis occurs at the 5' end of the noncanonical TRS-B and recombines between nucleotides 5 and 6 of the 8-nucleotide consensus TRS-L. Introduction of a complete TRS-B showed that higher transcription levels are achieved by increasing the number of nucleotide matches between TRS-L and TRS-B. Translation of a protein from the sgmRNA was demonstrated using enhanced green fluorescent protein, suggesting the translation of a fifth, novel, group-specific protein for IBV. This study has resolved an issue concerning the number of ORFs expressed by members of the Gammacoronavirus genus and proposes the existence of a fifth IBV accessory protein. We confirmed previous reports that coronaviruses can produce sgmRNAs from noncanonical TRS-Bs, which may expand their repertoire of proteins. We also demonstrated that noncanonical TRS-Bs may provide a mechanism by which coronaviruses can control protein expression levels by reducing sgmRNA synthesis.

  5. Functions of the nonsense-mediated mRNA decay pathway in Drosophila development.

    Directory of Open Access Journals (Sweden)

    Mark M Metzstein

    2006-12-01

    Full Text Available Nonsense-mediated mRNA decay (NMD is a cellular surveillance mechanism that degrades transcripts containing premature translation termination codons, and it also influences expression of certain wild-type transcripts. Although the biochemical mechanisms of NMD have been studied intensively, its developmental functions and importance are less clear. Here, we describe the isolation and characterization of Drosophila "photoshop" mutations, which increase expression of green fluorescent protein and other transgenes. Mapping and molecular analyses show that photoshop mutations are loss-of-function mutations in the Drosophila homologs of NMD genes Upf1, Upf2, and Smg1. We find that Upf1 and Upf2 are broadly active during development, and they are required for NMD as well as for proper expression of dozens of wild-type genes during development and for larval viability. Genetic mosaic analysis shows that Upf1 and Upf2 are required for growth and/or survival of imaginal cell clones, but this defect can be overcome if surrounding wild-type cells are eliminated. By contrast, we find that the PI3K-related kinase Smg1 potentiates but is not required for NMD or for viability, implying that the Upf1 phosphorylation cycle that is required for mammalian and Caenorhabditis elegans NMD has a more limited role during Drosophila development. Finally, we show that the SV40 3' UTR, present in many Drosophila transgenes, targets the transgenes for regulation by the NMD pathway. The results establish that the Drosophila NMD pathway is broadly active and essential for development, and one critical function of the pathway is to endow proliferating imaginal cells with a competitive growth advantage that prevents them from being overtaken by other proliferating cells.

  6. Cloning the human lysozyme cDNA: Inverted Alu repeat in the mRNA and in situ hybridization for macrophages and Paneth cells

    International Nuclear Information System (INIS)

    Chung, L.P.; Keshav, S.; Gordon, S.

    1988-01-01

    Lysozyme is a major secretory product of human and rodent macrophages and a useful marker for myelomonocytic cells. Based on the known human lysozyme amino acid sequence, oligonucleotides were synthesized and used as probes to screen a phorbol 12-myristate 13-acetate-treated U937 cDNA library. A full-length human lysozyme cDNA clone, pHL-2, was obtained and characterized. Sequence analysis shows that human lysozyme, like chicken lysozyme, has in 18-amino-acid-long signal peptide, but unlike the chicken lysozyme cDNA, the human lysozyme cDNA has a >1-kilobase-long 3' nontranslated sequence. Interestingly, within this 3' region, an inverted repeat of the Alu family of repetitive sequences was discovered. In RNA blot analyses, DNA probes prepared from pHL-2 can be used to detect lysozyme mRNA not only from human but also from mouse and rat. Moreover, by in situ hybridization, complementary RNA transcripts have been used as probes to detect lysozyme mRNA in mouse macrophages and Paneth cells. This human lysozyme cDNA clone is therefore likely to be a useful molecular probe for studying macrophage distribution and gene expression

  7. Decreased A20 mRNA and protein expression in peripheral blood mononuclear cells in patients with type 2 diabetes and latent autoimmune diabetes in adults.

    Science.gov (United States)

    Cheng, Liqing; Zhang, Dongmei; Jiang, Youzhao; Deng, Wuquan; Wu, Qi'nan; Jiang, Xiaoyan; Chen, Bing

    2014-12-01

    A20 is a negative regulator of nuclear factor kappa B activation and the central gatekeeper in inflammation and immunity. While its role in type 1 diabetes has been widely studied, its expression level in immune cells from type 2 diabetes (T2D) and latent autoimmune diabetes in adult (LADA) patients remains unclear. This study aimed to clarify whether the expression of A20 is altered in patients with T2D or LADA. Quantitative real-time polymerase chain reaction and western blotting were utilized to determine the expression of A20 mRNA and protein respectively in peripheral blood mononuclear cells (PBMCs) from patients with T2D (n=36) or LADA (n=17) and sex- and age-matched healthy controls (n=34). The mRNA and protein expression of A20 in PBMCs from T2D and LADA patients was significantly decreased compared with healthy controls (P1 year since diagnosis) (P<0.05). Our results suggest that decreased expression of A20 in PBMCs may be involved in the pathogenesis of diabetes, and targeting A20 may offer a potential therapeutic tool in the treatment of diabetes. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  8. Glycophospholipid Formulation with NADH and CoQ10 Significantly Reduces Intractable Fatigue in Western Blot-Positive ‘Chronic Lyme Disease’ Patients: Preliminary Report

    Directory of Open Access Journals (Sweden)

    Garth L. Nicolson

    2012-03-01

    Full Text Available Background: An open label 8-week preliminary study was conducted in a small number of patients to determine if a combination oral supplement containing a mixture of phosphoglycolipids, coenzyme Q10 and microencapsulated NADH and other nutrients could affect fatigue levels in long-term, Western blot-positive, multi-symptom ‘chronic Lyme disease’ patients (also called ‘post-treatment Lyme disease’ or ‘post Lyme syndrome’ with intractable fatigue. Methods: The subjects in this study were 6 males (mean age = 45.1 ± 12.4 years and 10 females (mean age = 54.6 ± 7.4 years with ‘chronic Lyme disease’ (determined by multiple symptoms and positive Western blot analysis that had been symptomatic with chronic fatigue for an average of 12.7 ± 6.6 years. They had been seen by multiple physicians (13.3 ± 7.6 and had used many other remedies, supplements and drugs (14.4 ± 7.4 without fatigue relief. Fatigue was monitored at 0, 7, 30 and 60 days using a validated instrument, the Piper Fatigue Scale.Results: Patients in this preliminary study responded to the combination test supplement, showing a 26% reduction in overall fatigue by the end of the 8-week trial (p< 0.0003. Analysis of subcategories of fatigue indicated that there were significant improvements in the ability to complete tasks and activities as well as significant improvements in mood and cognitive abilities. Regression analysis of the data indicated that reductions in fatigue were consistent and occurred with a high degree of confidence (R2= 0.998. Functional Foods in Health and Disease 2012, 2(3:35-47 Conclusions: The combination supplement was a safe and effective method to significantly reduce intractable fatigue in long-term patients with Western blot-positive ‘chronic Lyme disease.’

  9. Contribution of dot-blot assay to the diagnosis and management of myositis: a three-year practice at a university hospital centre.

    Science.gov (United States)

    Martel, Clothilde; Vignaud, Guillaume; Liozon, Eric; Magy, Laurent; Gallouedec, Gael; Ly, Kim; Bezanahary, Holly; Cypierre, Anne; Lapébie, François-Xavier; Palat, Sylvain; Gondran, Guillaume; Jauberteau, Marie-Odile; Fauchais, Anne-Laure

    2016-01-01

    Idiopathic inflammatory myopathies (IIM) are heterogeneous autoimmune diseases with wide clinical spectrum that may lead to delayed diagnosis. The aim of this study was to examine the impact of IIM-specific dot-blot assay on diagnostic process of patients presenting with muscular or systemic symptoms evocating of IIM. We collected all the prescriptions of an IIM specific dot-blot assay (8 autoantigens including Jo-1, PL-7, PL-12, SRP, Mi-2, Ku, PM/Scl and Scl-70) over a 38-month period. 316 myositis dot-blot assays (MSD) were performed in 274 patients (156 women, mean age 53±10.6 years) referring for muscular and/or systemic symptoms suggesting IIM. The timing of dot prescription through the diagnostic process was highly variable: without (35%), concomitantly (16%) or after electromyographic studies (35%). Fifty-nine patients (22%) had IIM according to Bohan and Peter's criteria. Among them, 29 (49%) had positive dot (8 Jo-1, 6 PM-Scl, 5 PL-12, 5 SRP, 2 Mi-2, 2 PL-7 and 1 Ku). Various other diagnoses were performed including 35 autoimmune disease or granulomatosis (12%), 19 inflammatory rheumatic disease (7%), 16 non inflammatory muscular disorders (6%), 10 drug-induced myalgia (4%), 11 infectious myositis (4%). Except 11 borderline SRP results and one transient PM-Scl, MSD was positive only in one case of IIM. Dot allowed clinicians to correct diagnosis in 4 cases and improved the diagnosis of IIM subtypes in 4 cases. This study reflects the interest of myositis dot in the rapid diagnosis process of patients with non-specific muscular symptoms leading to various diagnoses including IIM.

  10. Instructive for disposal of fluorescent

    International Nuclear Information System (INIS)

    Salazar Vargas, Gerlin

    2014-01-01

    An instructive is established for the management system of waste fluorescent lamps, ensuring the storage, collection, transportation, and final disposal. The lamp is changed by an official of the Seccion de Matenimiento Construccion of the Oficina de Servicios Generales or is produced with the support of an official of the unit. The fluorescent should be deposited in stock of materials of the building maintenance section or unit specified with the help of a staff and in appropriate conditions. The fluorescent lamp is transported according to the guidelines in the manual. A responsible company is contracted by la Vicerrectoria de Administracion of the Universidad de Costa Rica dedicated to the transport and proper handling of fluorescent lamps [es

  11. ANTAGONISTIC POTENTIAL OF FLUORESCENT Pseudomonas ...

    African Journals Online (AJOL)

    Prof. Adipala Ekwamu

    GROWTH OF TOMATO CHALLENGED WITH PHTOPATHOGENS ... This study focused on the antagonistic potential of fluorescent Pseudomonas in vitro, and its inoculation effect on growth .... the 5 days old culture in starch agar with Lugol's.

  12. Sulphate reduction and vertical distribution of sulphate-reducing bacteria quantified by rRNA slot-blot hybridization in a coastal marine sediment

    DEFF Research Database (Denmark)

    Sahm, K.; MacGregor, BJ; Jørgensen, BB

    1999-01-01

    In the past, enumeration of sulphate-reducing bacteria (SRB) by cultivation-based methods generally contradicted measurements of sulphate reduction, suggesting unrealistically high respiration rates per cell. Here, we report evidence that quantification of SRB rRNA by slot-blot hybridization......, directly above the sulphate reduction maximum. Cell numbers calculated by converting the relative contribution of SRB rRNA to the percentage of DAPI-stained cells indicated a population size for SRB of 2.4-6.1 x 10(8) cells cm(-3) wet sediment. Cellular sulphate reduction rates calculated on the basis...

  13. Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

    Science.gov (United States)

    Yamamoto, Norio; Yamashita, Yoko; Yoshioka, Yasukiyo; Nishiumi, Shin; Ashida, Hitoshi

    2016-08-01

    Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  14. Standardization of micro-enzyme-linked immunosorbent assay (ELISA) and Western blot for detection of Trypanosoma cruzi antibodies using extracts from Mexican strains as antigens.

    Science.gov (United States)

    Sánchez, B; Monteón, V; Reyes, P A; Espinoza, B

    2001-01-01

    This report describes two assays for the detection of anti-Trypanosoma cruzi antibodies using Mexican strains of the parasite and the concordance with two assays previously evaluated at the Instituto Nacional de Cardiología Ignacio Chávez in Mexico City. Micro-enzyme-linked immunosorbent assay (ELISA) and Western blot were used for the detection of T. cruzi antibodies with a total extract of epimastigote from Ninoa and Queretaro, which are Mexican strains of T. cruzi. To standardize these methods, a total of 246 serum samples was used. In addition, sera from six confirmed Mexican chronic individuals in the asymptomatic phase were also used for comparison with the Argentinean antigen. ELISA was 100% specific in that no false positive results were found with sera of both healthy individuals and non-Chagasic cardiopaths. Sera from individuals infected with Leishmania sp. showed approximately 16% of cross-reaction with ELISA. The test showed a positive predictive value of 90% and a negative predictive value of 100%. Western blot was also a highly sensitive test for detecting chronic Chagasic symptomatic patients from Mexico because no false negative results were obtained. Furthermore, it was possible to use Western blot to detect seven immunodominant antigens of approximately 30, 32, 40, 42, 65, 70, and 83 kDa. Concordance with two previous standardized tests at the Instituto Nacional de Cardiología showed a Kappa index of 0.96, indicating high concordance between the results obtained at these two laboratories. Finally, ELISA using Ninoa antigen extract was more sensitive than ELISA with an Argentinean extract, which failed to detect individuals in the chronic asymptomatic phase (undetermined phase) of infection. This study indicates that ELISA and Western blot using Ninoa and/or Queretaro extracts of T. cruzi as antigens are useful tools in the detection of individuals who have been exposed to T. cruzi both in the undetermined/asymptomatic and symptomatic phases

  15. Use of the water-soluble fluor sodium salicylate for fluorographic detection of tritium in thin-layer chromatograms and nitrocellulose blots

    International Nuclear Information System (INIS)

    Lucher, L.A.; Lego, T.

    1989-01-01

    We have determined that sodium salicylate, a water-soluble fluor which we use routinely for fluorography with polyacrylamide gels, is also useful for fluorography with thin-layer media. Detection of 3 H-labeled material applied to thin-layer chromatography plates, or nitrocellulose membranes, can be enhanced up to 150-fold after treatment with an aqueous solution of 2 M sodium salicylate, while detection of 35 S-labeled material is enhanced only about 2-fold. We demonstrate the utility of sodium salicylate fluorography in detecting 3H-labeled palmitic acid following thin-layer chromatography and 3 H-labeled proteins following blotting to nitrocellulose

  16. X-ray fluorescence holography

    CERN Document Server

    Hayashi, K; Takahashi, Y

    2003-01-01

    X-ray fluorescence holography (XFH) is a new structural analysis method of determining a 3D atomic arrangement around fluorescing atoms. We developed an XFH apparatus using advanced X-ray techniques and succeeded in obtaining high-quality hologram data. Furthermore, we introduced applications to the structural analysis of a thin film and the environment around dopants and, discussed the quantitative analysis of local lattice distortion. (author)

  17. Fluorescent Nanodiamonds in Biomedical Applications.

    Science.gov (United States)

    Mitura, Katarzyna Anna; Włodarczyk, Elżbieta

    2018-04-18

    Nanoparticles have an extended surface and a large surface area, which is the ratio of the size of the surfacearea to the volume. A functionalized surface can give rise to more modifications and therefore allows this nanomaterial to have new properties. Fluorescent molecules contain fluorophore, which is capable of being excited via the absorption of light energy at a specific wavelength and subsequently emitting radiation energy of a longer wavelength. A chemically modified surface of nanodiamond (ND; by carboxylation) demonstrated biocompatibility with DNA, cytochrome C, and antigens. In turn, fluorescent nanodiamonds (FNDs) belong to a group of new nanomaterials. Their surface can be modified by joining functional groups such as carboxyl, hydroxyl, or amino, after which they can be employed as a fluorescence agent. Their fluorescent properties result from defects in the crystal lattice. FNDs reach dimensions of 4-100 nm, have attributes such as photostability, long fluorescence lifetimes (10 ns), and fluorescence emission between 600 and 700 nm. They are also nontoxic, chemically inert, biocompatible, and environmentally harmless. The main purpose of this article was to present the medical applications of various types of modified NDs.

  18. Fluorescence detection of esophageal neoplasia

    Science.gov (United States)

    Borisova, E.; Vladimirov, B.; Avramov, L.

    2008-06-01

    White-light endoscopy is well-established and wide used modality. However, despite the many technological advances that have been occurred, conventional endoscopy is suboptimal and usually detects advanced stage lesions. The limitations of standard endoscopy initiate development of spectroscopic techniques, additional to standard endoscopic equipment. One of the most sensitive approaches is fluorescence spectroscopy of gastrointestinal mucosa for neoplasia detection. In the recent study delta-aminolevulinic acid/Protoporphyrin IX (5-ALA/PpIX) is used as fluorescent marker for dysplasia and tumor detection in esophagus. The 5-ALA is administered per os six hours before measurements at dose 20 mg/kg weight. Excitation source has max of emission at 405 nm and light is delivered by the standard light guide of the endoscopic equipment. Through endoscopic instrumental channel a fiber is applied to return information about fluorescence to microspectrometer. Spectral features observed during endoscopic investigations could be distinct as the next regions: 450-630 nm region, where tissue autofluorescence is observed; 630-710 nm region, where fluorescence of PpIX is clearly pronounced; 530-580 nm region, where minima in the autofluorescence signal are observed, related to reabsorption of blood. The lack of fluorescence peaks in the red spectral area for normal mucosa is an indication for selective accumulation of 5-ALA/PpIX only in abnormal sites Very good correlation between fluorescence signals and histology examination of the lesions investigated is achieved.

  19. Effect of conjugated linoleic acids on the activity and mRNA expression of 5- and 15-lipoxygenases in human macrophages.

    Science.gov (United States)

    Stachowska, Ewa; Dziedziejko, Violetta; Safranow, Krzysztof; Jakubowska, Katarzyna; Olszewska, Maria; Machaliñski, Bogusław; Chlubek, Dariusz

    2007-06-27

    Lipoxygenases are a family of non-heme enzyme dioxygenases. The role of lipoxygenases is synthesis of hydroperoxides of fatty acids, which perform signaling functions in the body. Studies on conjugated linoleic acids (CLAs) as fatty acids with a potential anti-atherosclerotic function have recently been initiated. The aim of the study was to test the effect of CLAs and linoleic acid on 5- and 15-lipoxygenase (5-LO, 15-LO-1) enzyme activity, their mRNA expression, and concentration in the cells. It was also desired to determine whether the CLAs are substrates for the enzymes. For the experiments monocytic cell line (THP-1) and monocytes obtained from human venous blood were used. Monocytes were differentiated to macrophages: THP-1 (CD14+) by PMA administration (100 nM for 24 h) and monocytes from blood (CD14+) by 7-day cultivation with the autologous serum (10%). After differentiation, macrophages were cultured with 30 microM CLAs or linoleic acid for 48 h. The 15- and 5-lipoxygenase products were measured by HPLC method. mRNA expression and protein content were analyzed by real-time PCR and Western blot analysis. The in vitro studies proved that both CLA isomers are not substrates for 15-LO-1; in ex vivo studies hydroxydecadienoic acid (HODE) concentration was significantly reduced (p = 0.019). The trans-10,cis-12 CLA isomer reduced HODE concentration by 28% (p = 0.046) and the cis-9,trans-11 CLA isomer by 35% (p = 0.028). In macrophages obtained from THP-1 fatty acids did not change significantly mRNA expression of the majority of the investigated genes. CLAs did not change the content of 5-LO and 15-LO-1 proteins in macrophages obtained from peripheral blood. Linoleic acid induced 15-LO-1 expression (2.6 times, p < 0.05). CLAs may perform the function of an inhibitor of lipoxygenase 15-LO-1 activity in macrophages.

  20. [Expression of Jagged1 mRNA in human epithelial ovarian carcinoma tissues and effect of RNA interference of Jagged1 on growth of xenograft in nude mice].

    Science.gov (United States)

    Liu, G Y; Gao, Z H; Li, L; Song, T T; Sheng, X G

    2016-06-25

    To investigate the expression of Jagged1 in human epithelial ovarian carcinoma tissues and the effect of Jagged1 on growth of xenograft in nude mice. (1) Forty-eight cases of ovarian cancer and 30 cases of patients with benign epithelial ovarian tumor in the Henan Province Xinxiang Central Hospital during Feb. 2011 to Mar. 2014 were enrolled in this study. The mRNA expression of Jagged1, Notch1 and the downstream target genes Hes1, Hey1 were analyzed by using realtime PCR method. (2) The ovarian cancer xenograft models in nude mice were constructed by injecting SKOV3 cells in axillary subcutaneouswere. The nude mice were randomly divided into Jagged1 interference group, blank plasmid group and control group. Each group had 10 mice. They were transfected with pcDNA3.1(+)-siRNA-Jagged1, blank plasmid pDC3.1 and phosphate buffer, respectively. The tumor volumes and tumor masses were measured 14 days after transfection and the inhibition rate was calculated. The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues after transfection in each group was detected by using realtime PCR technique and the relative protein expression of Jagged1, Notch1, Hes1 and Hey1 in xenograft tissues was detected by utilizing western blot method. (1) The relative mRNA expression of Jagged1, Notch1, Hes1 and Hey1 in ovarian cancer tissues were higher than benign ovarian tumor tissues, the differences were statistically significant (Ptissues of nude micein Jagged1 interference group were lower than that in the other two groups, the differences were statistically significant (Ptissues of nude mice among the three groups (P>0.05). Jagged1 is highly expressed in epithelial ovarian carcinoma. Jagged1 gene interference in xenograft tumor can inhibit ovarian cancer cell growth and improve tumor suppressor rate, which probably play roles by inhibiting Notch1 signaling pathway.

  1. FLUORESCENCE DIAGNOSIS FOR RECURRENT BLADDER CANCER

    Directory of Open Access Journals (Sweden)

    R. V. Ulyanov

    2017-01-01

    Full Text Available The clinical case of successful use of local fluorescence spectroscopy combined with fluorescence imaging during cystoscopy for diagnosis of recurrent bladder cancer is represented in the article. Histological study of fluorescent foci confirmed tumor growth (urothelial carcinoma in all areas with high levels of diagnostic parameter. In the fluorescent focus with low diagnostic parameter inflammation was detected.

  2. Physical change in cytoplasmic messenger ribonucleoproteins in cells treated with inhibitors of mRNA transcription

    International Nuclear Information System (INIS)

    Dreyfuss, G.; Adam, S.A.; Choi, Y.D.

    1984-01-01

    Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared

  3. Comparison of RT-PCR-Dot blot hybridization based on radioisotope 32P with conventional RT-PCR and commercial ELISA Assays for blood screening of HIV-1

    International Nuclear Information System (INIS)

    Maria Lina R; Andi Yasmon

    2011-01-01

    There are many commercial ELISA and rapid test kits that have been used for blood screening; however, the kits can give false positive and negative results. Therefore, RT-PCR (Reverse Transcription Polymerase Chain Reaction) - Dot Blot Hybridization based on radioisotope 32 P (RDBR) method was developed in this research, to compare the method with the conventional RT-PCR and commercial ELISA Enzyme-Linked lmmunosorbent Assay) kit. This method is efficient for screening of large blood specimens and surveillance study. Eighty seven samples were used and serum of the samples were tested by ELISA to detect HIV-1. The HIV-l RNA genome was extracted from plasma samples and tested using the RT-PCR and RDBR methods. Of 87 samples that were tested, the rates of positive testing of the RT-PCR, the RDBR, and the ELISA were 71.26%, 74.71%, and 80.46%, respectively. The RDBR (a combination of RTPCR and dot blot hybridization) was more sensitive than conventional RT-PCR by showing 3.45% in increase number of positive specimens. The results showed that of 9 samples (10.34%) were negative RDBR and positive ELISA, while 4 samples (4.60%) were negative ELISA and positive RDBR. The two methods showed slightly difference in the results but further validation is still needed. However, RDBR has high potential as an alternative method for screening of blood in large quantities when compared to method of conventional RT-PCR and ELISA. (author)

  4. A combined RT-PCR and dot-blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius).

    Science.gov (United States)

    Lopez-Jimena, B; Cherif, N; Garcia-Rosado, E; Infante, C; Cano, I; Castro, D; Hammami, S; Borrego, J J; Alonso, M C

    2010-10-01

    To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin-labelled probes resulted in the identification of both genotypes separately. The application of the RT-PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46.87% of viral nervous necrosis virus carriers. The combination of RT-PCR and blot hybridization increases the detection rate up to 90.62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56.25%). This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time. © 2010 The Authors. Journal compilation © 2010 The Society for Applied Microbiology.

  5. Demonstration of functional low-density lipoprotein receptors by protein blotting in fibroblasts from a subject with homozygous receptor-negative familial hypercholesterolemia

    International Nuclear Information System (INIS)

    Semenkovich, C.F.; Ostlund, R.E. Jr.; Yang, J.; Reaban, M.E.

    1985-01-01

    We report the detection of low-density lipoprotein (LDL) receptors by the technique of receptor blotting in fibroblasts from a patient with homozygous familial hypercholesterolemia (FHC) previously classified as ''receptor negative.'' Solubilized receptors were electrophoresed, transferred to nitrocellulose paper, treated with LDL followed by radiolabeled antibody to LDL, and visualized by autoradiography. GM 2000 FHC fibroblasts revealed LDL receptors with an apparent molecular weight of approximately 140,000, the same as in normal cells. LDL receptor activity by blotting in GM 2000 cells was greatly diminished in comparison with normal cells, but was calcium dependent. Receptor activity was also detectable by conventional monolayer binding and degradation assays. Thus, GM 2000 cells have profoundly diminished LDL receptor activity, but retain the genetic capacity to make LDL receptor material of normal molecular weight that is capable of binding LDL. Previous studies have demonstrated the presence of trace amounts of immunoreactive LDL receptor protein in fibroblasts from some receptor-negative FHC homozygotes. These studies are extended by demonstrating the ability of this material to bind LDL

  6. Application of FTA sample collection and DNA purification system on the determination of CTG trinucleotide repeat size by PCR-based Southern blotting.

    Science.gov (United States)

    Hsiao, K M; Lin, H M; Pan, H; Li, T C; Chen, S S; Jou, S B; Chiu, Y L; Wu, M F; Lin, C C; Li, S Y

    1999-01-01

    Myotonic dystrophy (DM) is caused by a CTG trinucleotide expansion mutation at exon 15 of the myotonic dystrophy protein kinase gene. The clinical severity of this disease correlates with the length of the CTG trinucleotide repeats. Determination of the CTG repeat length has been primarily relied on by Southern blot analysis of restriction enzyme-digested genomic DNA. The development of PCR-based Southern blotting methodology provides a much more sensitive and simpler protocol for DM diagnosis. However, the quality of the template and the high (G+C) ratio of the amplified region hamper the use of PCR on the diagnosis of DM. A modified PCR protocol to amplify different lengths of CTG repeat region using various concentrations of 7deaza-dGTP has been reported (1). Here we describe a procedure including sample collection, DNA purification, and PCR analysis of CTG repeat length without using 7-deaza-dGTP. This protocol is very sensitive and convenient because only a small number of nucleate cells are needed for detection of CTG expansion. Therefore, it could be very useful in clinical and prenatal diagnosis as well as in prevalence study of DM.

  7. Effect of in vitro estrogenic pesticides on human oestrogen receptor α and β mRNA levels

    DEFF Research Database (Denmark)

    Theander Grünfeld, Heidi; Bonefeld-Jørgensen, Eva Cecilie

    2004-01-01

    of the ERα mRNA level, but only significantly for prochloraz, dieldrin, and tolchlofos-methyl. Alone no pesticides affected the ERβ mRNA level significantly, but chlorpyrifos increased the mRNA level weakly. Co-exposure with E2 elicited a significant increased ERβ mRNA level by prochloraz, fenarimol...

  8. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    Science.gov (United States)

    Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

    2013-01-01

    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

  9. Cytoplasmic Control of Sense-Antisense mRNA Pairs

    Directory of Open Access Journals (Sweden)

    Flore Sinturel

    2015-09-01

    Full Text Available Transcriptome analyses have revealed that convergent gene transcription can produce many 3′-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3′-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3′-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3′-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5′-3′ cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression.

  10. Cytoplasmic Control of Sense-Antisense mRNA Pairs.

    Science.gov (United States)

    Sinturel, Flore; Navickas, Albertas; Wery, Maxime; Descrimes, Marc; Morillon, Antonin; Torchet, Claire; Benard, Lionel

    2015-09-22

    Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Regulation of mRNA translation during mitosis.

    Science.gov (United States)

    Tanenbaum, Marvin E; Stern-Ginossar, Noam; Weissman, Jonathan S; Vale, Ronald D

    2015-08-25

    Passage through mitosis is driven by precisely-timed changes in transcriptional regulation and protein degradation. However, the importance of translational regulation during mitosis remains poorly understood. Here, using ribosome profiling, we find both a global translational repression and identified ~200 mRNAs that undergo specific translational regulation at mitotic entry. In contrast, few changes in mRNA abundance are observed, indicating that regulation of translation is the primary mechanism of modulating protein expression during mitosis. Interestingly, 91% of the mRNAs that undergo gene-specific regulation in mitosis are translationally repressed, rather than activated. One of the most pronounced translationally-repressed genes is Emi1, an inhibitor of the anaphase promoting complex (APC) which is degraded during mitosis. We show that full APC activation requires translational repression of Emi1 in addition to its degradation. These results identify gene-specific translational repression as a means of controlling the mitotic proteome, which may complement post-translational mechanisms for inactivating protein function.

  12. IER5 gene's mRNA expression after irradiation

    International Nuclear Information System (INIS)

    Ding Kuke; Shen Jingjing; Xu Lili; Li Yanling; Zhou Ping; Ma Binrong; Zhao Zengqiang; Sui Jianli; Zhou Pingkun

    2008-01-01

    Objective: To explore the effect of irradiation on IER5 gene expression. Methods: Two kinds of cells (AHH-1 and HeLa) and the BALB/c-nu mice inoculated with tumor cells were exposed to 60 Co γ- rays and analyzed by real-time PCR. The above-mentioned irradiated objects were firstly divided into groups by different doses and post-radiation time, then mRNA were extracted and reverse-transcripted to DNA before real-time PCR test. Results: Under the same condition, AHH-1 was more sensitive to radiation than HeLa. The dose level corresponding to the expression peak of AHH-1 was less than that of HeLa. For AHH-1 cells, the response to 2 Gy irradiation was earlier than that to 10 Gy. But there was not remarkable difference for HeLa response between 2 and 10 Gy, and the top transcriptional levels for both cells nearly simultaneously appeared at 2 h after irradiation. In addition, the IER5 gene of human liver tumor was more sensitive than that of lung cancer and brain tumor. Conclusions: IER5 might be a candidate biomarker of radiation injury, and had the potential value in radiation-therapy for liver tumor. (authors)

  13. In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

    DEFF Research Database (Denmark)

    Nielsen, M. E.; Rasmussen, I. A.; Kristensen, S. G.

    2011-01-01

    significantly with the expression of AMHRII, but did not correlate with any of the hormones in the follicular fluid. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the follicular......Human small antral follicles (diameter 3-9 mm) were obtained from ovaries surgically removed for fertility preservation. From the individual aspirated follicles, granulosa cells and the corresponding follicular fluid were isolated in 64 follicles, of which 55 were available for mRNA analysis (24...... and to the follicular fluid concentrations of AMH, inhibin-B, progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated...

  14. Kan du blot blive buddreng..

    DEFF Research Database (Denmark)

    Henriksen, Carsten

    2011-01-01

    ADHD? Adfærdsvanskelig? Diagnoserne står i kø, når vi i dag skal forklare, hvorfor Emil ikke kan følge med i skolen. Men startskuddet til den videnskabelige opdeling af de børn, der ikke følger flertallets læringskurve, lød allerede for 100 år siden.......ADHD? Adfærdsvanskelig? Diagnoserne står i kø, når vi i dag skal forklare, hvorfor Emil ikke kan følge med i skolen. Men startskuddet til den videnskabelige opdeling af de børn, der ikke følger flertallets læringskurve, lød allerede for 100 år siden....

  15. Heterologous overexpression of sfCherry fluorescent protein in Nannochloropsis salina

    Directory of Open Access Journals (Sweden)

    Nam Kyu Kang

    2015-12-01

    Full Text Available Oleaginous microalgae of the Nannochloropsis genus are considered excellent candidates for biofuels and value-added products owing to their high biomass productivity and lipid content. Here, we report the first overexpression and detection of a heterologous sfCherry fluorescent protein in Nannochloropsis salina in order to develop a transformation toolbox for future genetic improvements. Particle bombardment was employed for transformation, and expression of Shble under the control of TUB and UEP promoters, cloned from N. salina, was used to confer resistance to Zeocin antibiotics, resulting in 5.9 and 4.7 transformants per 108 cells, respectively. Stable integration of the markers into the genome was confirmed using a restriction enzyme site-directed amplification (RESDA PCR. The expression of sfCherry fluorescent protein was confirmed by Western blot analysis and confocal microscopy. These results suggest new possibilities of efficient genetic engineering of Nannochloropsis for the production of biofuels and other biochemicals.

  16. Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

    Energy Technology Data Exchange (ETDEWEB)

    Bloch, Donald B., E-mail: bloch@helix.mgh.harvard.edu; Nobre, Rita A.; Bernstein, Gillian A.; Yang, Wei-Hong

    2011-09-10

    Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: {yields} A two-hybrid assay was developed to study interactions in macromolecular complexes. {yields} The assay was applied to interactions between components of mRNA P-bodies. {yields} The assay effectively and efficiently identified protein interaction domains. {yields} P-body assembly in mammalian cells differs from that in other species.

  17. Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

    International Nuclear Information System (INIS)

    Bloch, Donald B.; Nobre, Rita A.; Bernstein, Gillian A.; Yang, Wei-Hong

    2011-01-01

    Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5' cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species. -- Research highlights: → A two-hybrid assay was developed to study interactions in macromolecular complexes. → The assay was applied to interactions between components of mRNA P-bodies. → The assay effectively and efficiently identified protein interaction domains. → P-body assembly in mammalian cells differs from that in other species.

  18. Plasmonics Enhanced Smartphone Fluorescence Microscopy

    KAUST Repository

    Wei, Qingshan; Acuna, Guillermo; Kim, Seungkyeum; Vietz, Carolin; Tseng, Derek; Chae, Jongjae; Shir, Daniel; Luo, Wei; Tinnefeld, Philip; Ozcan, Aydogan

    2017-01-01

    Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

  19. Fluorescence and phosphorescence of rutin

    Energy Technology Data Exchange (ETDEWEB)

    Bondarev, Stanislav L., E-mail: bondarev@imaph.bas-net.by [Minsk State Higher Radioengineering College, 220005 Minsk (Belarus); Knyukshto, Valeri N. [B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, 220072 Minsk (Belarus)

    2013-10-15

    Rutin is one of the most promising flavonoid from a pharmacological and biochemical point of view. Here we have explored its spectroscopic and photophysical properties at room temperature and 77 K using steady-state absorption-luminescence methods and pulse spectroscopy equipment. By excitation into the absorption band 1 of rutin in methanol at room temperature the normal Stokes' shifted fluorescence with a maximum at 415 nm and quantum yield of 2×10{sup −4} was revealed. However, by excitation into the bands 2 and 3 any emission wasn’t observed. At 77 K in ethanol glass we have observed fluorescence at 410 nm and phosphorescence at 540 nm for the first time. As a result the adequate energetic scheme including the lowest electronic excited singlet at 26000 cm{sup −1} and triplet at 19600 cm{sup −1} states was proposed. -- Highlights: • Rutin fluorescence and phosphorescence at 77 K were revealed for the first time. • Room temperature fluorescence is determined by maximum at 415 nm and yield of 2×10{sup −4}. • Violation of Vavilov–Kasha rule by excitation into the absorption bands 2 and 3. • Fluorescence and phosphorescence in rutin are caused by the allowed π, π{sup (⁎)} transitions.

  20. Plasmonics Enhanced Smartphone Fluorescence Microscopy

    KAUST Repository

    Wei, Qingshan

    2017-05-12

    Smartphone fluorescence microscopy has various applications in point-of-care (POC) testing and diagnostics, ranging from e.g., quantification of immunoassays, detection of microorganisms, to sensing of viruses. An important need in smartphone-based microscopy and sensing techniques is to improve the detection sensitivity to enable quantification of extremely low concentrations of target molecules. Here, we demonstrate a general strategy to enhance the detection sensitivity of a smartphone-based fluorescence microscope by using surface-enhanced fluorescence (SEF) created by a thin metal-film. In this plasmonic design, the samples are placed on a silver-coated glass slide with a thin spacer, and excited by a laser-diode from the backside through a glass hemisphere, generating surface plasmon polaritons. We optimized this mobile SEF system by tuning the metal-film thickness, spacer distance, excitation angle and polarization, and achieved ~10-fold enhancement in fluorescence intensity compared to a bare glass substrate, which enabled us to image single fluorescent particles as small as 50 nm in diameter and single quantum-dots. Furthermore, we quantified the detection limit of this platform by using DNA origami-based brightness standards, demonstrating that ~80 fluorophores per diffraction-limited spot can be readily detected by our mobile microscope, which opens up new opportunities for POC diagnostics and sensing applications in resource-limited-settings.

  1. Noncoding RNA mediated traffic of foreign mRNA into chloroplasts reveals a novel signaling mechanism in plants.

    Directory of Open Access Journals (Sweden)

    Gustavo Gómez

    Full Text Available Communication between chloroplasts and the nucleus is one of the milestones of the evolution of plants on earth. Proteins encoded by ancestral chloroplast-endogenous genes were transferred to the nucleus during the endosymbiotic evolution and originated this communication, which is mainly dependent on specific transit-peptides. However, the identification of nuclear-encoded proteins targeted to the chloroplast lacking these canonical signals suggests the existence of an alternative cellular pathway tuning this metabolic crosstalk. Non-coding RNAS (NcRNAs are increasingly recognized as regulators of gene expression as they play roles previously believed to correspond to proteins. Avsunviroidae family viroids are the only noncoding functional RNAs that have been reported to traffic inside the chloroplasts. Elucidating mechanisms used by these pathogens to enter this organelle will unearth novel transport pathways in plant cells. Here we show that a viroid-derived NcRNA acting as a 5'UTR-end mediates the functional import of Green Fluorescent Protein (GFP mRNA into chloroplast. This claim is supported by the observation at confocal microscopy of a selective accumulation of GFP in the chloroplast of the leaves expressing the chimeric vd-5'UTR/GFP and by the detection of the GFP mRNA in chloroplasts isolated from cells expressing this construct. These results support the existence of an alternative signaling mechanism in plants between the host cell and chloroplasts, where an ncRNA functions as a key regulatory molecule to control the accumulation of nuclear-encoded proteins in this organelle. In addition, our findings provide a conceptual framework to develop new biotechnological tools in systems using plant chloroplast as bioreactors. Finally, viroids of the family Avsunviroidae have probably evolved to subvert this signaling mechanism to regulate their differential traffic into the chloroplast of infected cells.

  2. Stable transformation of sunflower (Helianthus annuus L.) using a non-meristematic regeneration protocol and green fluorescent protein as a vital marker.

    Science.gov (United States)

    Müller, A; Iser, M; Hess, D

    2001-10-01

    Stable transformation of sunflower was achieved using a non-meristematic hypocotyl explant regeneration protocol of public inbred HA300B. Uniformly transformed shoots were obtained after co-cultivation with Agrobacterium tumefaciens carrying a gfp (green fluorescent protein) gene containing an intron that blocks expression of gfp in Agrobacterium. Easily detectable, bright green fluorescence of transformed tissues was used to establish an optimal regeneration and transformation procedure. By Southern blot analysis, integration of the gfp and nptll genes was confirmed. Stable transformation efficiency was 0.1%. From 68 T1 plants analyzed, 17 showed transmission of transgene DNA and 15 of them contained the intact gfp gene. Expression of gfp was detected in 10 T1 plants carrying the intact gfp gene using a fluorimetric assay or western blot analysis. Expression of the nptll gene was confirmed in 13 T1 plants. The transformation system enables the rapid transfer of agronomically important genes.

  3. Fluorescence confocal microscopy for pathologists.

    Science.gov (United States)

    Ragazzi, Moira; Piana, Simonetta; Longo, Caterina; Castagnetti, Fabio; Foroni, Monica; Ferrari, Guglielmo; Gardini, Giorgio; Pellacani, Giovanni

    2014-03-01

    Confocal microscopy is a non-invasive method of optical imaging that may provide microscopic images of untreated tissue that correspond almost perfectly to hematoxylin- and eosin-stained slides. Nowadays, following two confocal imaging systems are available: (1) reflectance confocal microscopy, based on the natural differences in refractive indices of subcellular structures within the tissues; (2) fluorescence confocal microscopy, based on the use of fluorochromes, such as acridine orange, to increase the contrast epithelium-stroma. In clinical practice to date, confocal microscopy has been used with the goal of obviating the need for excision biopsies, thereby reducing the need for pathological examination. The aim of our study was to test fluorescence confocal microscopy on different types of surgical specimens, specifically breast, lymph node, thyroid, and colon. The confocal images were correlated to the corresponding histological sections in order to provide a morphologic parallel and to highlight current limitations and possible applications of this technology for surgical pathology practice. As a result, neoplastic tissues were easily distinguishable from normal structures and reactive processes such as fibrosis; the use of fluorescence enhanced contrast and image quality in confocal microscopy without compromising final histologic evaluation. Finally, the fluorescence confocal microscopy images of the adipose tissue were as accurate as those of conventional histology and were devoid of the frozen-section-related artefacts that can compromise intraoperative evaluation. Despite some limitations mainly related to black/white images, which require training in imaging interpretation, this study confirms that fluorescence confocal microscopy may represent an alternative to frozen sections in the assessment of margin status in selected settings or when the conservation of the specimen is crucial. This is the first study to employ fluorescent confocal microscopy on

  4. Bioinspired nanocomplex for spatiotemporal imaging of sequential mRNA expression in differentiating neural stem cells.

    Science.gov (United States)

    Wang, Zhe; Zhang, Ruili; Wang, Zhongliang; Wang, He-Fang; Wang, Yu; Zhao, Jun; Wang, Fu; Li, Weitao; Niu, Gang; Kiesewetter, Dale O; Chen, Xiaoyuan

    2014-12-23

    Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions.

  5. Nonparametric testing for DNA copy number induced differential mRNA gene expression

    NARCIS (Netherlands)

    van Wieringen, W.N.; van de Wiel, M.A.

    2009-01-01

    The central dogma of molecular biology relates DNA with mRNA. Array CGH measures DNA copy number and gene expression microarrays measure the amount of mRNA. Methods that integrate data from these two platforms may uncover meaningful biological relationships that further our understanding of cancer.

  6. Coordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

    KAUST Repository

    Arae, Toshihiro

    2017-04-18

    Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.

  7. Primary induction of vitellogenin mRNA in the rooster by 17beta-estradiol.

    Science.gov (United States)

    Burns, A T; Deeley, R G; Gordon, J I; Udell, D S; Mullinix, K P; Goldberger, R F

    1978-01-01

    We have studied the kinetics of vitellogenin mRNA accumulation in rooster liver after a primary injection of 17beta-estradiol. The levels of vitellogenin mRNA have been determined both by hybridization of total cellular RNA to vitellogenin cDNA and by translation of vitellogenin mRNA in a wheat germ cell-free system. The results obtained by both methods of analysis are in good agreement and indicate that vitellogenin mRNA is present in the liver of normal roosters at a level of 0-5 molecules per liver cell and increases in amount during the 3 days following injection of estrogen, reaching a level of almost 6000 molecules per cell at the peak of the response. The level of vitellogenin mRNA declined exponentially during the next 14 days with a half-life of 29 hr, reaching a level of less than 10 molecules per cell at 17 days after injection of the hormone. The levels of vitellogenin mRNA after stimulation with estrogen have been correlated with the in vivo rate of synthesis of the vitellogenin polypeptide. The results indicate that the rate of vitellogenin synthesis is closely correlated with the level of vitellogenin mRNA. On the basis of these findings, we conclude that vitellogenin mRNA does not exist in the liver in an untranslated form after withdrawal from estrogen. PMID:273910

  8. Survivin mRNA antagonists using locked nucleic acid, potential for molecular cancer therapy

    DEFF Research Database (Denmark)

    Fisker, Niels; Westergaard, Majken; Hansen, Henrik Frydenlund

    2007-01-01

    We have investigated the effects of different locked nucleic acid modified antisense mRNA antagonists against Survivin in a prostate cancer model. These mRNA antagonists were found to be potent inhibitors of Survivin expression at low nanomolar concentrations. Additionally there was a pronounced ...

  9. Coordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

    KAUST Repository

    Arae, Toshihiro; Isai, Shiori; Sakai, Akira; Mineta, Katsuhiko; Hirai, Masami Yokota; Suzuki, Yuya; Kanaya, Shigehiko; Yamaguchi, Junji; Naito, Satoshi; Chiba, Yukako

    2017-01-01

    stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were

  10. In situ localization of chalcone synthase mRNA in pea root nodule development.

    NARCIS (Netherlands)

    Yang, W.C.; Canter Cremers, H.C.J.; Hogendijk, P.; Katinakis, P.; Wijffelman, C.A.; Franssen, H.J.; Kammen, van A.; Bisseling, T.

    1992-01-01

    In this paper studies on the role of flavonoids in pea root nodule development are reported. Flavonoid synthesis was followed by localizing chalcone synthase (CHS) mRNA in infected pea roots and in root nodules. In a nodule primordium, CHS mRNA is present in all cells of the primordium. Therefore it

  11. Responses of mRNA expression of PepT1 in small intestine to ...

    African Journals Online (AJOL)

    To study the effect of circulation small peptides concentration on mRNA expression in small intestine, graded amount of soybean small peptides (SSP) were infused into lactating goats through duodenal fistulas. Peptide-bound amino acid (PBAA) concentration in arterial plasma and the mRNA expression of PepT1 was ...

  12. Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons

    NARCIS (Netherlands)

    Mochida, Sumiko; Poulain, Bernard; Eisel, Ulrich; Binz, Thomas; Kurazono, Hisao; Niemann, Heiner; Tauc, Ladislav; Bullock, Theodore H.

    1990-01-01

    Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in

  13. Serum detection of IgG antibodies against Demodex canis by western blot in healthy dogs and dogs with juvenile generalized demodicosis.

    Science.gov (United States)

    Ravera, Ivan; Ferreira, Diana; Gallego, Laia Solano; Bardagí, Mar; Ferrer, Lluís

    2015-08-01

    The aim of this study was to investigate the presence of canine immunoglobulins (Ig) G against Demodex proteins in the sera of healthy dogs and of dogs with juvenile generalized demodicosis (CanJGD) with or without secondary pyoderma. Demodex mites were collected from dogs with CanJGD. Protein concentration was measured and a western blot technique was performed. Pooled sera from healthy dogs reacted mainly with antigen bands ranging from 55 to 72 kDa. Pooled sera from dogs with CanJGD without secondary pyoderma reacted either with 10 kDa antigen band or 55 to 72 kDa bands. Pooled sera from dogs with CanJGD with secondary pyoderma reacted only with a 10 kDa antigen band. The results of this study suggest that both healthy dogs and dogs with CanJGD develop a humoral response against different proteins of Demodex canis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Application of a Reverse Line Blot hybridisation assay for the species-specific identification of cyathostomins (Nematoda, Strongylida) from benzimidazole-treated horses in the Slovak Republic.

    Science.gov (United States)

    Cernanská, Dana; Paoletti, Barbara; Králová-Hromadová, Ivica; Iorio, Raffaella; Cudeková, Patrícia; Milillo, Piermarino; Traversa, Donato

    2009-03-09

    Five horse farms located in eastern Slovakia were investigated for the presence of benzimidazole-resistant strongyles by faecal egg count reduction test and egg hatch assay. Coprocultures were prepared for each farm from faecal samples taken pre- and post-treatment and harvested larvae were molecularly examined with a Reverse Line Blot assay. Faecal egg count reduction values ranged from 0 to 52.5% and all farms were positive for benzimidazole-resistant cyathostomins. Seven benzimidazole-resistant cyathostomin species were molecularly identified on farms before and also after treatment. These data demonstrate that resistance to benzimidazoles is well established in cyathostomin populations from horse farms in the Slovak Republic and that the molecular assay was able to determine the species-specific distribution of resistant cyathostomins under field conditions.

  15. Sulphate reduction and vertical distribution of sulphate-reducing bacteria quantified by rRNA slot-blot hybridization in a coastal marine sediment

    DEFF Research Database (Denmark)

    Sahm, K.; MacGregor, BJ; Jørgensen, BB

    1999-01-01

    In the past, enumeration of sulphate-reducing bacteria (SRB) by cultivation-based methods generally contradicted measurements of sulphate reduction, suggesting unrealistically high respiration rates per cell. Here, we report evidence that quantification of SRB rRNA by slot-blot hybridization...... between 18% and 25% to the prokaryotic rRNA pool. The dominant SRB were related to complete oxidizing genera (Desulphococcus, Desulphosarcina and Desulphobacterium), while Desulpho-bacter could not be detected. The vertical profile and quantity of rRNA from SRB was compared with sulphate reduction rates......, directly above the sulphate reduction maximum. Cell numbers calculated by converting the relative contribution of SRB rRNA to the percentage of DAPI-stained cells indicated a population size for SRB of 2.4-6.1 x 10(8) cells cm(-3) wet sediment. Cellular sulphate reduction rates calculated on the basis...

  16. Study on sensitivity of southern blotting hybridization using a 32P-labeled probe of PCR products in detecting human cytomegalovirus

    International Nuclear Information System (INIS)

    Bu Hengfu; Chen Juan; Shen Rongsen; Ma Liren; Xu Yongqiang

    1996-01-01

    Southern blotting hybridization (SBH) using a 32 P-labeled probe is one of the most practical methods for genetic diagnosis of pathogen. On the basis of establishing PCR and nested PCR for detecting human cytomegalovirus (HCMV), a 32 P-labeled probe was prepared with the amplified products of 613 bp PCR outer primers and hybridized with 300 bp inner primer amplified product, resulting in increase in detecting sensitivity from 17 ng (in 1.2% agarose electrophoresis) before SBH to 500 pg (autoradiographed), in other words, increasing the sensitivity of detecting HCMV by 10 2 dilutions after using SBH. The method of PCR and SBH using a 32 P-labeled probe could detect less than 1 gene copy of HCMV, therefore, it is a rapid and reliable diagnosis method for detecting HCMV latent infection

  17. Hairpin-Hairpin Molecular Beacon Interactions for Detection of Survivin mRNA in Malignant SW480 Cells.

    Science.gov (United States)

    Ratajczak, Katarzyna; Krazinski, Bartlomiej E; Kowalczyk, Anna E; Dworakowska, Beata; Jakiela, Slawomir; Stobiecka, Magdalena

    2018-05-07

    Cancer biomarkers offer unique prospects for the development of cancer diagnostics and therapy. One of such biomarkers, protein survivin (Sur), exhibits strong antiapoptotic and proliferation-enhancing properties and is heavily expressed in multiple cancers. Thus, it can be utilized to provide new modalities for modulating the cell-growth rate, essential for effective cancer treatment. Herein, we have focused on the development of a new survivin-based cancer detection platform for colorectal cancer cells SW480 using a turn-on fluorescence oligonucleotide molecular beacon (MB) probe, encoded to recognize Sur messenger RNA (mRNA). Contrary to the expectations, we have found that both the complementary target oligonucleotide strands as well as the single- and double-mismatch targets, instead of exhibiting the anticipated simple random conformations, preferentially formed secondary structure motifs by folding into small-loop hairpin structures. Such a conformation may interfere with, or even undermine, the biorecognition process. To gain better understanding of the interactions involved, we have replaced the classical Tyagi-Kramer model of interactions between a straight target oligonucleotide strand and a hairpin MB with a new model to account for the hairpin-hairpin interactions as the biorecognition principle. A detailed mechanism of these interactions has been proposed. Furthermore, in experimental work, we have demonstrated an efficient transfection of malignant SW480 cells with SurMB probes containing a fluorophore Joe (SurMB-Joe) using liposomal nanocarriers. The green emission from SurMB-Joe in transfected cancer cells, due to the hybridization of the SurMB-Joe loop with Sur mRNA hairpin target, corroborates Sur overexpression. On the other hand, healthy human-colon epithelial cells CCD 841 CoN show only negligible expression of survivin mRNA. These experiments provide the proof-of-concept for distinguishing between the cancer and normal cells by the proposed

  18. Fluorescence detection of dental calculus

    Science.gov (United States)

    Gonchukov, S.; Biryukova, T.; Sukhinina, A.; Vdovin, Yu

    2010-11-01

    This work is devoted to the optimization of fluorescence dental calculus diagnostics in optical spectrum. The optimal wavelengths for fluorescence excitation and registration are determined. Two spectral ranges 620 - 645 nm and 340 - 370 nm are the most convenient for supra- and subgingival calculus determination. The simple implementation of differential method free from the necessity of spectrometer using was investigated. Calculus detection reliability in the case of simple implementation is higher than in the case of spectra analysis at optimal wavelengths. The use of modulated excitation light and narrowband detection of informative signal allows us to decrease essentially its diagnostic intensity even in comparison with intensity of the low level laser dental therapy.

  19. A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis.

    Science.gov (United States)

    Lin, Wen-Wei; Chen, I-Ju; Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R; Cheng, Tian-Lu

    2016-01-01

    Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15-120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.

  20. The potential role of IGF-I receptor mRNA in rats with diabetic retinopathy

    Institute of Scientific and Technical Information of China (English)

    匡洪宇; 邹伟; 刘丹; 史榕荇; 程丽华; 殷慧清; 刘晓民

    2003-01-01

    Objective To evaluate the potential role of insulin-like growth factor-1 receptor mRNA(IGF-IR mRNA) in the onset and development of retinopathy in diabetic rats.Methods A diabetic model was duplicated in Wistar rats. The early changes in the retina were examined using light and transmission electron microscopy. Expression of IGF-IR mRNA was analyzed using in situ hybridization.Results Weak expression of IGF-IR mRNA(5%) was found in retinas of normal rats, but was significantly increased (15% and 18%) in the retinas of diabetic rats after 3 and 6 months of diabetes (P<0.01). In situ hybridization and morphological study demonstrated that there was a positive correlation between IGF-IR mRNA expression and retinal changes at various stages.Conclusion Increased IGF-IR mRNA might play an important role in the onset and development of diabetic retinopathy.

  1. Simultaneous isolation of mRNA and native protein from minute samples of cells

    DEFF Research Database (Denmark)

    Petersen, Tonny Studsgaard; Andersen, Claus Yding

    2014-01-01

    Precious biological samples often lack a sufficient number of cells for multiple procedures, such as extraction of mRNA while maintaining protein in a non-denatured state suitable for subsequent characterization. Here we present a new method for the simultaneous purification of mRNA and native...... in their native state for traditional protein assays. We validated the procedure using neonatal rat ovaries and small numbers of human granulosa cells, demonstrating the extraction of mRNA suitable for gene expression analysis with simultaneous isolation of native proteins suitable for downstream characterization...... proteins from samples containing small numbers of cells. Our approach utilizes oligodeoxythymidylate [oligo(dT)25]-coated paramagnetic beads in an optimized reaction buffer to isolate mRNA comparable in quantity and quality to mRNA isolated with existing methods, while maintaining the proteins...

  2. Biomolecule-to-fluorescent-color encoder: modulation of fluorescence emission via DNA structural changes

    Science.gov (United States)

    Nishimura, Takahiro; Ogura, Yusuke; Yamada, Kenji; Ohno, Yuko; Tanida, Jun

    2014-01-01

    A biomolecule-to-fluorescent-color (B/F) encoder for optical readout of biomolecular information is proposed. In the B/F encoder, a set of fluorescence wavelengths and their intensity levels are used for coding of a biomolecular signal. A hybridization chain reaction of hairpin DNAs labeled with fluorescent reporters was performed to generate the fluorescence color codes. The fluorescence is modulated via fluorescence resonance energy transfer, which is controlled by DNA structural changes. The results demonstrate that fluorescent color codes can be configured based on two wavelengths and five intensities using the B/F encoder, and the assigned codes can be retrieved via fluorescence measurements. PMID:25071950

  3. Impairment of FOS mRNA stabilization following translation arrest in granulocytes from myelodysplastic syndrome patients.

    Science.gov (United States)

    Feng, Xiaomin; Shikama, Yayoi; Shichishima, Tsutomu; Noji, Hideyoshi; Ikeda, Kazuhiko; Ogawa, Kazuei; Kimura, Hideo; Takeishi, Yasuchika; Kimura, Junko

    2013-01-01

    Although quantitative and qualitative granulocyte defects have been described in myelodysplastic syndromes (MDS), the underlying molecular basis of granulocyte dysfunction in MDS is largely unknown. We recently found that FOS mRNA elevation under translation-inhibiting stimuli was significantly smaller in granulocytes from MDS patients than in healthy individuals. The aim of this study is to clarify the cause of the impaired FOS induction in MDS. We first examined the mechanisms of FOS mRNA elevation using granulocytes from healthy donors cultured with the translation inhibitor emetine. Emetine increased both transcription and mRNA stability of FOS. p38 MAPK inhibition abolished the emetine-induced increase of FOS transcription but did not affect FOS mRNA stabilization. The binding of an AU-rich element (ARE)-binding protein HuR to FOS mRNA containing an ARE in 3'UTR was increased by emetine, and the knockdown of HuR reduced the FOS mRNA stabilizing effect of emetine. We next compared the emetine-induced transcription and mRNA stabilization of FOS between MDS patients and healthy controls. Increased rates of FOS transcription by emetine were similar in MDS and controls. In the absence of emetine, FOS mRNA decayed to nearly 17% of initial levels in 45 min in both groups. In the presence of emetine, however, 76.7±19.8% of FOS mRNA remained after 45 min in healthy controls, versus 37.9±25.5% in MDS (Pknowledge, this is the first report demonstrating attenuation of stress-induced FOS mRNA stabilization in MDS granulocytes.

  4. Temporal profiles of age-dependent changes in cytokine mRNA expression and glial cell activation after status epilepticus in postnatal rat hippocampus.

    Science.gov (United States)

    Järvelä, Juha T; Lopez-Picon, Francisco R; Plysjuk, Anna; Ruohonen, Saku; Holopainen, Irma E

    2011-04-08

    Status epilepticus (SE) is proposed to lead to an age-dependent acute activation of a repertoire of inflammatory processes, which may contribute to neuronal damage in the hippocampus. The extent and temporal profiles of activation of these processes are well known in the adult brain, but less so in the developing brain. We have now further elucidated to what extent inflammation is activated by SE by investigating the acute expression of several cytokines and subacute glial reactivity in the postnatal rat hippocampus. SE was induced by an intraperitoneal (i.p.) injection of kainic acid (KA) in 9- and 21-day-old (P9 and P21) rats. The mRNA expression of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), matrix metalloproteinase-9 (MMP-9), glial-derived neurotrophic factor (GDNF), interferon gamma (IFN-γ), and transforming growth factor-beta 1 (TGF-β1) were measured from 4 h up to 3 days after KA injection with real-time quantitative PCR (qPCR). IL-1β protein expression was studied with ELISA, GFAP expression with western blotting, and microglial and astrocyte morphology with immunohistochemistry 3 days after SE. SE increased mRNA expression of IL-1β, TNF-α and IL-10 mRNA in hippocampus of both P9 and P21 rats, their induction being more rapid and pronounced in P21 than in P9 rats. MMP-9 expression was augmented similarly in both age groups and GDNF expression augmented only in P21 rats, whereas neither IFN-γ nor TGF-β1 expression was induced in either age group. Microglia and astrocytes exhibited activated morphology in the hippocampus of P21 rats, but not in P9 rats 3 d after SE. Microglial activation was most pronounced in the CA1 region and also detected in the basomedial amygdala. Our results suggest that SE provokes an age-specific cytokine expression in the acute phase, and age-specific glial cell activation in the subacute phase as verified now in the postnatal rat hippocampus. In the juvenile hippocampus

  5. X-ray fluorescence spectrometry

    International Nuclear Information System (INIS)

    Vries, J.L. de.

    1976-01-01

    The seventh edition of Philips' Review of Literature on x-ray fluorescence spectrometry starts with a list of conference proceedings on the subject, organised by the Philips organisation at regular intervals in various European countries. It is followed by a list of bulletins. The bibliography is subdivided according to spectra, equipment, applications and absorption analysis

  6. X-ray fluorescence spectrometry

    International Nuclear Information System (INIS)

    Ray, N.B.

    1977-01-01

    The principle, instrument and procedure of X-ray fluorescence spectrometry are described. It is a rapid, simple and sensitive method for the trace analysis of elements from sodium to uranium in powder, liquid or metal samples. (M.G.B.)

  7. A fluorescent probe for ecstasy.

    Science.gov (United States)

    Masseroni, D; Biavardi, E; Genovese, D; Rampazzo, E; Prodi, L; Dalcanale, E

    2015-08-18

    A nanostructure formed by the insertion in silica nanoparticles of a pyrene-derivatized cavitand, which is able to specifically recognize ecstasy in water, is presented. The absence of effects from interferents and an efficient electron transfer process occurring after complexation of ecstasy, makes this system an efficient fluorescent probe for this popular drug.

  8. Erythrocyte fluorescence and lead intoxication.

    Science.gov (United States)

    Clark, K G

    1976-01-01

    Blood samples from people exposed to inorganic lead were examined by fluorescence microscopy for excess erythrocyte porphyrin. With continued lead absorption, fluorescent erythrocytes appeared in the circulation of workers handling this metal or its compounds, and they progressively increased in number and brilliance. These changes ensued if the blood lead concentration was maintained above 2-42 mumol/l (50 mug/100 ml), and preceded any material fall in the haemoglobin value. At one factory, 62-5% of 81 symptomless workers showed erythrocyte fluorescence attributable to the toxic effects of lead. Excess fluorocytes were found in blood samples from a child with pica and three of her eight siblings. These four were subsequently shown to have slightly increased blood lead concentrations (2-03 to 2-32 mumol/l). Fluorescence microscopy for excess erythrocyte porphyrin is a sensitive method for the detection of chronic lead intoxication. A relatively slight increase in the blood lead is associated with demonstrabel changes in erythrocyte porphyrin content. The procedure requires little blood, and may be performed upon stored samples collected for lead estimation. The results are not readily influenced by contamination, and provide good confirmatory evidence for the absorption of biochemically active lead. PMID:963005

  9. Fluorescent sensors based on bacterial fusion proteins

    International Nuclear Information System (INIS)

    Mateu, Batirtze Prats; Pum, Dietmar; Sleytr, Uwe B; Toca-Herrera, José L; Kainz, Birgit

    2014-01-01

    Fluorescence proteins are widely used as markers for biomedical and technological purposes. Therefore, the aim of this project was to create a fluorescent sensor, based in the green and cyan fluorescent protein, using bacterial S-layers proteins as scaffold for the fluorescent tag. We report the cloning, expression and purification of three S-layer fluorescent proteins: SgsE-EGFP, SgsE-ECFP and SgsE-13aa-ECFP, this last containing a 13-amino acid rigid linker. The pH dependence of the fluorescence intensity of the S-layer fusion proteins, monitored by fluorescence spectroscopy, showed that the ECFP tag was more stable than EGFP. Furthermore, the fluorescent fusion proteins were reassembled on silica particles modified with cationic and anionic polyelectrolytes. Zeta potential measurements confirmed the particle coatings and indicated their colloidal stability. Flow cytometry and fluorescence microscopy showed that the fluorescence of the fusion proteins was pH dependent and sensitive to the underlying polyelectrolyte coating. This might suggest that the fluorescent tag is not completely exposed to the bulk media as an independent moiety. Finally, it was found out that viscosity enhanced the fluorescence intensity of the three fluorescent S-layer proteins. (paper)

  10. A single bout of whole-leg, peristaltic pulse external pneumatic compression upregulates PGC-1α mRNA and endothelial nitric oxide sythase protein in human skeletal muscle tissue.

    Science.gov (United States)

    Kephart, Wesley C; Mobley, C Brooks; Fox, Carlton D; Pascoe, David D; Sefton, JoEllen M; Wilson, Trent J; Goodlett, Michael D; Kavazis, Andreas N; Roberts, Michael D; Martin, Jeffrey S

    2015-07-01

    What is the central question of this study? Does 60 min of peristaltic pulse external pneumatic compression (EPC) alter gene and protein expression patterns related to metabolism, vascular biology, redox balance and inflammation in vastus lateralis biopsy samples? What is the main finding and its importance? A single bout of EPC transiently upregulates PGC-1α mRNA, while also upregulating endothelial nitric oxide synthase protein and nitric oxide metabolite concentrations in vastus lateralis biopsy samples. We investigated whether a single 60 min bout of whole-leg, lower pressure external pneumatic compression (EPC) altered select vascular, metabolic, antioxidant and inflammation-related mRNAs. Ten participants (eight male, two female; aged 22.0 ± 0.4 years) reported to the laboratory 4 h postprandial, and vastus lateralis muscle biopsies were obtained before (PRE) and 1 and 4 h after EPC treatment. Messenger RNA expression was analysed using real-time RT-PCR, and significant mRNA findings were investigated further by Western blot analysis of respective protein concentrations. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) mRNA increased by 77% 1 h following EPC compared with PRE levels (P = 0.005), but no change in protein concentration 1 or 4 h post-EPC was observed. Increases in endothelial nitric oxide sythase (eNOS) mRNA (+44%) and superoxide dismutase 2 (SOD2) mRNA (+57%) 1 h post-EPC as well as an increase in interleukin-10 mRNA (+132%) 4 h post-EPC compared with PRE levels were observed, but only approached significance (P = 0.076, 0.077 and 0.074, respectively). Interestingly, eNOS protein (+40%, P = 0.025) and nitrate and nitrite (NOx) concentrations (+69%, P = 0.025) increased 1-4 h post-EPC. Moreover, SOD2 protein tended to increase from PRE to 4 h post-EPC (+43%, P = 0.074), although no changes in tissue 4-hydroxnonenal levels was observed. An acute bout of EPC transiently upregulates PGC-1α mRNA, while also upregulating e

  11. Constitutive mRNA expression and protein activity levels of nine ABC efflux transporters in seven permanent cell lines derived from different tissues of rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    Fischer, Stephan; Loncar, Jovica; Zaja, Roko; Schnell, Sabine; Schirmer, Kristin; Smital, Tvrtko; Luckenbach, Till

    2011-01-25

    Permanent fish cell lines have become common model systems for determining ecotoxicological effects of pollutants. For these cell lines little is known on the cellular active transport mechanisms that control the amount of a compound entering the cell, such as the MXR (multixenobiotic resistance) system mediated by ATP binding cassette (ABC) transport proteins. Therefore, for toxic evaluation of chemicals with those cells information on MXR is important. We here present data on constitutive mRNA expression and protein activity levels of a series of ABC efflux transporters in seven permanent cell lines derived from liver (RTL-W1; R1) and liver hepatoma (RTH-149), gill (RTgill-W1), gonad (RTG-2), gut (RTgutGC) and brain (RTbrain) of rainbow trout (Oncorhynchus mykiss). In addition to known transporters abcb1 (designated here abcb1a), abcb11, abcc1-3, abcc5 and abcg2, we quantified expression levels of a newly identified abcb1 isoform (abcb1b) and abcc4, previously unknown in trout. Quantitative real time PCR (qPCR) indicated that mRNA of the examined ABC transporters was constitutively expressed in all cell lines. Transporter mRNA expression patterns were similar in all cell lines, with expression levels of abcc transporters being 80 to over 1000 fold higher than for abcg2, abcb1a/b and abcb11 (abcc1-5>abcg2>abcb1a/b, 11). Transporter activity in the cell lines was determined by measuring uptake of transporter type specific fluorescent substrates in the presence of activity inhibitors. The combination of the ABCB1 and ABCC transporter substrate calcein-AM with inhibitors cyclosporine A, PSC833 and MK571 resulted in a concentration-dependent fluorescence increase of up to 3-fold, whereas reversin 205 caused a slight, but not concentration-dependent fluorescence increase. Accumulation of the dyes Hoechst 33342 and 2',7'-dichlorodihydrofluorescein diacetate was basically unchanged in the presence of Ko134 and taurocholate, respectively, indicating low Abcg2 and Abcb11

  12. Note sur la présence de lames aménagées par technique de Kostienki dans les couches gravettiennes du Blot (Cerzat,Haute-Loire).

    OpenAIRE

    Klaric , Laurent

    2000-01-01

    International audience; The unprecedented presence of Kostienki-technique prepared blades (also called Kostienki knives) in the Gravettian layers at Le Blot leads to a new analysis of these artefacts. Thorough technological study has pointed to the possible role of these items as cores, in association with or complementary to burin-forms, in particular context of backed-bladelet production. Le Blot is the second French site yielding such artefacts, the other being Corbiac (Dordogne). The aim ...

  13. Fluorescent Protein Approaches in Alpha Herpesvirus Research

    Directory of Open Access Journals (Sweden)

    Ian B. Hogue

    2015-11-01

    Full Text Available In the nearly two decades since the popularization of green fluorescent protein (GFP, fluorescent protein-based methodologies have revolutionized molecular and cell biology, allowing us to literally see biological processes as never before. Naturally, this revolution has extended to virology in general, and to the study of alpha herpesviruses in particular. In this review, we provide a compendium of reported fluorescent protein fusions to herpes simplex virus 1 (HSV-1 and pseudorabies virus (PRV structural proteins, discuss the underappreciated challenges of fluorescent protein-based approaches in the context of a replicating virus, and describe general strategies and best practices for creating new fluorescent fusions. We compare fluorescent protein methods to alternative approaches, and review two instructive examples of the caveats associated with fluorescent protein fusions, including describing several improved fluorescent capsid fusions in PRV. Finally, we present our future perspectives on the types of powerful experiments these tools now offer.

  14. The hypoxic proteome is influenced by gene-specific changes in mRNA translation

    International Nuclear Information System (INIS)

    Koritzinsky, Marianne; Seigneuric, Renaud; Magagnin, Michael G.; Beucken, Twan van den; Lambin, Philippe; Wouters, Bradly G.

    2005-01-01

    Background and purpose: Hypoxia causes a rapid reduction in mRNA translation efficiency. This inhibition does not affect all mRNA species to the same extent and can therefore contribute significantly to hypoxia-induced differential protein expression. Our aim in this study was to characterize changes in gene expression during acute hypoxia and evaluate the contribution of regulation via mRNA translation on these changes. For each gene, the contribution of changes in mRNA abundance versus mRNA translation was determined. Materials and methods: DU145 prostate carcinoma cells were exposed to 4 h of hypoxia ( 2 ). Efficiently translated mRNAs were isolated by sedimentation through a sucrose gradient. Affymetrix microarray technology was used to evaluate both the transcriptional and translational contribution to gene expression. Results were validated by quantitative PCR. Results: One hundred and twenty genes were more than 4-fold upregulated by hypoxia in the efficiently translated fraction of mRNA, in comparison to only 76 genes at the level of transcription. Of the 50 genes demonstrating the largest changes in translation, 11 were found to be more than 2-fold over represented in the translated fraction in comparison to their overall transcriptional level. The gene with the highest translational contribution to its induction was CITED-2, which is a negative regulator of HIF-1 transcriptional activity. Conclusions: Gene-specific regulation of mRNA translation contributes significantly to differential gene expression during hypoxia

  15. Complement mRNA in the mammalian brain: responses to Alzheimer's disease and experimental brain lesioning.

    Science.gov (United States)

    Johnson, S A; Lampert-Etchells, M; Pasinetti, G M; Rozovsky, I; Finch, C E

    1992-01-01

    This study describes evidence in the adult human and rat brain for mRNAs that encode two complement (C) proteins, C1qB and C4. C proteins are important effectors of humoral immunity and inflammation in peripheral tissues but have not been considered as normally present in brain. Previous immunocytochemical studies showed that C proteins are associated with plaques, tangles, and dystrophic neurites in Alzheimer's disease (AD), but their source is unknown. Combined immunocytochemistry and in situ hybridization techniques show C4 mRNA in pyramidal neurons and C1qB mRNA in microglia. Primary rat neuron cultures also show C1qB mRNA. In the cortex from AD brains, there were two- to threefold increases of C1qB mRNA and C4 mRNA, and increased C1qB mRNA prevalence was in part associated with microglia. As a model for AD, we examined entorhinal cortex perforant path transection in the rat brain, which caused rapid increases of C1qB mRNA in the ipsilateral, but not contralateral, hippocampus and entorhinal cortex. The role of brain-derived acute and chronic C induction during AD and experimental lesions can now be considered in relation to functions of C proteins that pertain to cell degeneration and/or cell preservation and synaptic plasticity.

  16. The mRNA expression of XRCC repair genes in mice after γ-ray radiation

    International Nuclear Information System (INIS)

    Wang Qin; Yue Jingyin; Li Jin; Mu Chuanjie; Fan Feiyue

    2006-01-01

    Objective: To investigate the role of XRCC repair genes in radioresistance of IRM-2 inbred mice. Methods: Northern hybridization was used to measure mRNA expression of XRCC1 and XRCC5 genes in IRM-2 inbred mice. ICR/JCL and 615 after exposure to different doses of γ-ray radiation at different postirradiation time. Results: The levels of XRCC1 and XRCC5 mRNA expression in control IRM-2 mice were higher significantly than those in their control parental mice (P<0.01 and P<0.05). The mRNA expression of XRCC genes in ICR/JCL and 615 mice all increased to some extent after exposure 1, 2 and 4 Gy radiation. But the levels were significantly higher at 2h postirradiation (P<0.05) . The levels of XRCC mRNA expression in IRM-2 mice did not increase significnatly compared with the control mice after exposure 1 and 2 Gy radiation. But the levels of XRCC1 and XRCC5 mRNA expression increased markedly at 4Gy 1h postirradiation (P<0.05 and P<0.01). Conclusion: The basal levels of XRCC1 and XRCC5 mRNA expression in IRM-2 mice were high. The high level of XRCC5 mRNA expression was involved in the repair of DNA double strand breaks induced by higher dose radiation, which perhaps was one of radioresistance causes of IRM-2 mice. (authors)

  17. Nonsense mutations in the human β-globin gene affect mRNA metabolism

    International Nuclear Information System (INIS)

    Baserga, S.J.; Benz, E.J. Jr.

    1988-01-01

    A number of premature translation termination mutations (nonsense mutations) have been described in the human α- and β-globin genes. Studies on mRNA isolated from patients with β 0 -thalassemia have shown that for both the β-17 and the β-39 mutations less than normal levels of β-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human β-globin mRNA). In vitro studies using the cloned β-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. The authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human β-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation

  18. NONOates regulate KCl cotransporter-1 and -3 mRNA expression in vascular smooth muscle cells.

    Science.gov (United States)

    Di Fulvio, Mauricio; Lauf, Peter K; Shah, Shalin; Adragna, Norma C

    2003-05-01

    Nitric oxide (NO) donors regulate KCl cotransport (KCC) activity and cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in sheep erythrocytes and in primary cultures of rat vascular smooth muscle cells (VSMCs), respectively. In this study, we used NONOates as rapid and slow NO releasers to provide direct evidence implicating NO as a regulator of KCC3 gene expression at the mRNA level. In addition, we used the expression of KCC3 mRNA to further investigate the mechanism of action of these NO donors at the cellular level. Treatment of VSMCs with rapid NO releasers, like NOC-5 and NOC-9, as well as with the direct NO-independent soluble guanylyl cyclase (sGC) stimulator YC-1, acutely increased KCC3 mRNA expression in a concentration- and time-dependent manner. The slow NO releaser NOC-18 had no effect on KCC3 gene expression. A specific NO scavenger completely prevented the NONOate-induced KCC3 mRNA expression. Inhibition of sGC with LY-83583 blocked the NONOate- and YC-1-induced KCC3 mRNA expression. This study shows that in primary cultures of rat VSMCs, the fast NO releasers NOC-9 and NOC-5, but not the slow NO releaser NOC-18, acutely upregulate KCC3 mRNA expression in a NO/sGC-dependent manner.

  19. Structural design of intrinsically fluorescent oxysterols

    DEFF Research Database (Denmark)

    Nåbo, Lina J; Modzel, Maciej; Krishnan, Kathiresan

    2018-01-01

    Oxysterols are oxidized derivatives of cholesterol with many important biological functions. Trafficking of oxysterols in and between cells is not well studied, largely due to the lack of appropriate oxysterol analogs. Intrinsically fluorescent oxysterols present a new route towards direct...... observation of intracellular oxysterol trafficking by fluorescence microscopy. We characterize the fluorescence properties of the existing fluorescent 25-hydroxycholesterol analog 25-hydroxycholestatrienol, and propose a new probe with an extended conjugated system. The location of both probes inside...

  20. Staphylococcus aureus RNAIII binds to two distant regions of coa mRNA to arrest translation and promote mRNA degradation.

    Directory of Open Access Journals (Sweden)

    Clément Chevalier

    2010-03-01

    Full Text Available Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation.

  1. A novel link between Sus1 and the cytoplasmic mRNA decay machinery suggests a broad role in mRNA metabolism

    Directory of Open Access Journals (Sweden)

    Llopis Ana

    2010-03-01

    Full Text Available Abstract Background Gene expression is achieved by the coordinated action of multiple factors to ensure a perfect synchrony from chromatin epigenetic regulation through to mRNA export. Sus1 is a conserved mRNA export/transcription factor and is a key player in coupling transcription initiation, elongation and mRNA export. In the nucleus, Sus1 is associated to the transcriptional co-activator SAGA and to the NPC associated complex termed TREX2/THSC. Through these associations, Sus1 mediates the nuclear dynamics of different gene loci and facilitate the export of the new transcripts. Results In this study, we have investigated whether the yeast Sus1 protein is linked to factors involved in mRNA degradation pathways. We provide evidence for genetic interactions between SUS1 and genes coding for components of P-bodies such as PAT1, LSM1, LSM6 and DHH1. We demonstrate that SUS1 deletion is synthetic lethal with 5'→3' decay machinery components LSM1 and PAT1 and has a strong genetic interaction with LSM6 and DHH1. Interestingly, Sus1 overexpression led to an accumulation of Sus1 in cytoplasmic granules, which can co-localise with components of P-bodies and stress granules. In addition, we have identified novel physical interactions between Sus1 and factors associated to P-bodies/stress granules. Finally, absence of LSM1 and PAT1 slightly promotes the Sus1-TREX2 association. Conclusions In this study, we found genetic and biochemical association between Sus1 and components responsible for cytoplasmic mRNA metabolism. Moreover, Sus1 accumulates in discrete cytoplasmic granules, which partially co-localise with P-bodies and stress granules under specific conditions. These interactions suggest a role for Sus1 in gene expression during cytoplasmic mRNA metabolism in addition to its nuclear function.

  2. Negative regulation of neuromedin U mRNA expression in the rat pars tuberalis by melatonin.

    Directory of Open Access Journals (Sweden)

    Sayaka Aizawa

    Full Text Available The pars tuberalis (PT is part of the anterior pituitary gland surrounding the median eminence as a thin cell layer. The characteristics of PT differ from those of the pars distalis (PD, such as cell composition and gene expression, suggesting that the PT has a unique physiological function compared to the PD. Because the PT highly expresses melatonin receptor type 1, it is considered a mediator of seasonal and/or circadian signals of melatonin. Expression of neuromedin U (NMU that is known to regulate energy balance has been previously reported in the rat PT; however, the regulatory mechanism of NMU mRNA expression and secretion in the PT are still obscure. In this study, we examined both the diurnal change of NMU mRNA expression in the rat PT and the effects of melatonin on NMU in vivo. In situ hybridization and quantitative PCR analysis of laser microdissected PT samples revealed that NMU mRNA expression in the PT has diurnal variation that is high during the light phase and low during the dark phase. Furthermore, melatonin administration significantly suppressed NMU mRNA expression in the PT in vivo. On the other hand, 48 h fasting did not have an effect on PT-NMU mRNA expression, and the diurnal change of NMU mRNA expression was maintained. We also found the highest expression of neuromedin U receptor type 2 (NMUR2 mRNA in the third ventricle ependymal cell layer, followed by the arcuate nucleus and the spinal cord. These results suggest that NMU mRNA expression in the PT is downregulated by melatonin during the dark phase and shows diurnal change. Considering that NMU mRNA in the PT showed the highest expression level in the brain, PT-NMU may act on NMUR2 in the brain, especially in the third ventricle ependymal cell layer, with a circadian rhythm.

  3. A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

    International Nuclear Information System (INIS)

    Toki, Yasumichi; Sasaki, Katsunori; Tanaka, Hiroki; Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro; Torimoto, Yoshihiro; Ohtake, Takaaki; Kohgo, Yutaka

    2016-01-01

    Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.

  4. A selective splicing variant of hepcidin mRNA in hepatocellular carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Toki, Yasumichi [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Sasaki, Katsunori, E-mail: k-sasaki@asahikawa-med.ac.jp [Department of Gastrointestinal Immunology and Regenerative Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Tanaka, Hiroki [Department of Legal Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Yamamoto, Masayo; Hatayama, Mayumi; Ito, Satoshi; Ikuta, Katsuya; Shindo, Motohiro; Hasebe, Takumu; Nakajima, Shunsuke; Sawada, Koji; Fujiya, Mikihiro [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, Hokkaido 078-8510 (Japan); Torimoto, Yoshihiro [Oncology Center, Asahikawa Medical University Hospital, Hokkaido 078-8510 (Japan); Ohtake, Takaaki; Kohgo, Yutaka [Department of Gastroenterology, International University of Health and Welfare Hospital, Tochigi 329-2763 (Japan)

    2016-08-05

    Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells. - Highlights: • An aberrant splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene. • Absolute quantification of hepcidin mRNA by digital PCR amplification. • Hepatoma-derived cell lines have significant copies of variant-type hepcidin mRNA. • Truncated preprohepcidin is secreted from cells without posttranslational cleavage.

  5. Identification of stress responsive genes by studying specific relationships between mRNA and protein abundance.

    Science.gov (United States)

    Morimoto, Shimpei; Yahara, Koji

    2018-03-01

    Protein expression is regulated by the production and degradation of mRNAs and proteins but the specifics of their relationship are controversial. Although technological advances have enabled genome-wide and time-series surveys of mRNA and protein abundance, recent studies have shown paradoxical results, with most statistical analyses being limited to linear correlation, or analysis of variance applied separately to mRNA and protein datasets. Here, using recently analyzed genome-wide time-series data, we have developed a statistical analysis framework for identifying which types of genes or biological gene groups have significant correlation between mRNA and protein abundance after accounting for potential time delays. Our framework stratifies all genes in terms of the extent of time delay, conducts gene clustering in each stratum, and performs a non-parametric statistical test of the correlation between mRNA and protein abundance in a gene cluster. Consequently, we revealed stronger correlations than previously reported between mRNA and protein abundance in two metabolic pathways. Moreover, we identified a pair of stress responsive genes ( ADC17 and KIN1 ) that showed a highly similar time series of mRNA and protein abundance. Furthermore, we confirmed robustness of the analysis framework by applying it to another genome-wide time-series data and identifying a cytoskeleton-related gene cluster (keratin 18, keratin 17, and mitotic spindle positioning) that shows similar correlation. The significant correlation and highly similar changes of mRNA and protein abundance suggests a concerted role of these genes in cellular stress response, which we consider provides an answer to the question of the specific relationships between mRNA and protein in a cell. In addition, our framework for studying the relationship between mRNAs and proteins in a cell will provide a basis for studying specific relationships between mRNA and protein abundance after accounting for potential

  6. T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

    Science.gov (United States)

    Fauser, S; Kern, P

    1997-04-01

    In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

  7. On the Spatial Organization of mRNA, Plasmids, and Ribosomes in a Bacterial Host Overexpressing Membrane Proteins.

    Directory of Open Access Journals (Sweden)

    Lieke A van Gijtenbeek

    2016-12-01

    Full Text Available By using fluorescence imaging, we provide a time-resolved single-cell view on coupled defects in transcription, translation, and growth during expression of heterologous membrane proteins in Lactococcus lactis. Transcripts encoding poorly produced membrane proteins accumulate in mRNA-dense bodies at the cell poles, whereas transcripts of a well-expressed homologous membrane protein show membrane-proximal localization in a translation-dependent fashion. The presence of the aberrant polar mRNA foci correlates with cessation of cell division, which is restored once these bodies are cleared. In addition, activation of the heat-shock response and a loss of nucleoid-occluded ribosomes are observed. We show that the presence of a native-like N-terminal domain is key to SRP-dependent membrane localization and successful production of membrane proteins. The work presented gives new insights and detailed understanding of aberrant membrane protein biogenesis, which can be used for strategies to optimize membrane protein production.

  8. Transfection of Eimeria mitis with yellow fluorescent protein as reporter and the endogenous development of the transgenic parasite.

    Directory of Open Access Journals (Sweden)

    Mei Qin

    Full Text Available BACKGROUND: Advancements have been made in the genetic manipulation of apicomplexan parasites. Both the in vitro transient and in vivo stable transfection of Eimeria tenella have been developed successfully. Herein, we report the transient and stable transfection of Eimeria mitis. METHODS AND FINDINGS: Sporozoites of E. mitis transfected with enhanced yellow fluorescent protein (EYFP expression plasmid were inoculated into chickens via the cloacal route. The recovered fluorescent oocysts were sorted by fluorescence activated cell sorting (FACS and then passaged 6 generations successively in chickens. The resulting population was analyzed by genome walking and Western blot. The endogenous development of the transgenic E. mitis was observed and its reproduction potential was tested. The stable transfection of E. mitis was developed. Genome walking confirmed the random integration of plasmid DNA into the genome; while Western blot analysis demonstrated the expression of foreign proteins. Constitutive expression of EYFP was observed in all stages of merogony, gametogony and sporogony. The peak of the transgenic oocyst output was delayed by 24 h and the total oocyst reproduction was reduced by 7-fold when compared to the parental strain. CONCLUSION: Stable transfection of E. mitis was successfully developed. The expression of foreign antigens in the transgenic parasites will facilitate the development of transgenic E. mitis as a vaccine vector.

  9. Plasmonic enhancement of ultraviolet fluorescence

    Science.gov (United States)

    Jiao, Xiaojin

    Plasmonics relates to the interaction between electromagnetic radiation and conduction electrons at metallic interfaces or in metallic nanostructures. Surface plasmons are collective electron oscillations at a metal surface, which can be manipulated by shape, texture and material composition. Plasmonic applications cover a broad spectrum from visible to near infrared, including biosensing, nanolithography, spectroscopy, optoelectronics, photovoltaics and so on. However, there remains a gap in this activity in the ultraviolet (UV, research. Motivating factors in the study of UV Plasmonics are the direct access to biomolecular resonances and native fluorescence, resonant Raman scattering interactions, and the potential for exerting control over photochemical reactions. This dissertation aims to fill in the gap of Plasmonics in the UV with efforts of design, fabrication and characterization of aluminium (Al) and magnesium (Mg) nanostructures for the application of label-free bimolecular detection via native UV fluorescence. The first contribution of this dissertation addresses the design of Al nanostructures in the context of UV fluorescence enhancement. A design method that combines analytical analysis with numerical simulation has been developed. Performance of three canonical plasmonic structures---the dipole antenna, bullseye nanoaperture and nanoaperture array---has been compared. The optimal geometrical parameters have been determined. A novel design of a compound bullseye structure has been proposed and numerically analyzed for the purpose of compensating for the large Stokes shift typical of UV fluorescence. Second, UV lifetime modification of diffusing molecules by Al nanoapertures has been experimentally demonstrated for the first time. Lifetime reductions of ~3.5x have been observed for the high quantum yield (QY) laser dye p-terphenyl in a 60 nm diameter aperture with 50 nm undercut. Furthermore, quantum-yield-dependence of lifetime reduction has been

  10. HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels

    DEFF Research Database (Denmark)

    Hviid, Thomas Vauvert F; Hylenius, Sine; Rørbye, Christina

    2003-01-01

    between mother and fetus in several ways. Finally, the expression of membrane-bound HLA-G and soluble HLA-G has been proposed to influence the outcome of pregnancy, and an aberrant HLA-G expression in pre-eclamptic placentas and spontaneous abortions has been reported. Here, an association between certain...... HLA-G polymorphisms and the mRNA levels of the different alternatively spliced HLA-G isoforms in first trimester trophoblast cell populations is reported. Several alternatively spliced HLA-G mRNA isoforms, including a 14-bp polymorphism in the 3'UTR end (exon 8) of the HLA-G gene, are expressed...

  11. Rendimiento diagnóstico del Western Blot para detectar simultáneamente anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana

    Directory of Open Access Journals (Sweden)

    Kelly Davelois

    Full Text Available Objetivo. Determinar el rendimiento diagnóstico de la técnica de Western Blot para detectar simultáneamente anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana. Materiales y métodos. Estudio transversal de evaluación de prueba diagnóstica. Se obtuvieron los antígenos de excreción-secreción de las larvas de Taenia solium, quistes de Echinococcus granulosus; y la forma adulta de Fasciola hepática; que luego fueron separados electroforéticamente en geles de poliacrilamida individuales, transferidos y fijados a una membrana de nitrocelulosa para ser enfrentados con sueros de pacientes con las tres parasitosis. La sensibilidad de la técnica se evaluó empleando 300 sueros individuales, 60 pools de dos parasitosis y 20 pools de tres parasitosis y la especificidad con 75 sueros de pacientes con otras parasitosis, 10 de pacientes con otras enfermedades y 15 sueros de personas no parasitadas. Resultados. La técnica reconoció trece glicoproteínas (GP: GP 35, 31, 24, 23, 18, 17, 14 y 13 kDa para cisticercosis, GP 8,16 y 21 kDa para hidatidosis y GP: 17 y 23 kDa para fascioliasis. La prueba detectó la presencia de anticuerpos alcanzando una sensibilidad de 96% (IC95%: 94,62-98,54% en la detección de una o las trece bandas, una especificidad de 100% (IC95%: 99,50 - 100,00%; individualmente, se tuvo una sensibilidad para cisticercosis de 97% (IC95%: 93,16-100%, para hidatidosis de 94% (IC95%: 88,85-99,15% y para fascioliasis de 96% (IC95%: 91,66-100%. Conclusiones. La prueba de Western blot es eficaz en la detección, simultanea de anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana, y puede ser utilizada como prueba de descarte o confirmatoria en zonas endémicas.

  12. Demonstrating Fluorescence with Neon Paper and Plastic

    Science.gov (United States)

    Birriel, Jennifer J.; Roe, Clarissa

    2015-01-01

    Several papers in this journal have dealt with the fluorescence in orange neon plastic, olive oil, and soda. In each case, the fluorescent emission was excited by either green or violet-blue laser light. In this paper, we examine the fluorescent emission spectra of so-called neon colored papers and plastic clipboards available in department and…

  13. Adipose tissue interleukin-18 mRNA and plasma interleukin-18: effect of obesity and exercise

    DEFF Research Database (Denmark)

    Leick, Lotte; Lindegaard, Birgitte; Stensvold, Dorthe

    2007-01-01

    resistance was tested. Furthermore, we speculated that acute exercise and exercise training would regulate AT IL-18 mRNA expression. RESEARCH METHODS AND PROCEDURES: Non-obese subjects with BMI women: n = 18; men; n = 11) and obese subjects with BMI >30 kg/m(2) (women: n = 6; men: n = 7...... of regular physical activity with improved insulin sensitivity.......OBJECTIVES: Obesity and a physically inactive lifestyle are associated with increased risk of developing insulin resistance. The hypothesis that obesity is associated with increased adipose tissue (AT) interleukin (IL)-18 mRNA expression and that AT IL-18 mRNA expression is related to insulin...

  14. Assessing mRNA nuclear export in mammalian cells by microinjection.

    Science.gov (United States)

    Lee, Eliza S; Palazzo, Alexander F

    2017-08-15

    The nuclear export of mRNAs is an important yet little understood part of eukaryotic gene expression. One of the easiest methods for monitoring mRNA export in mammalian tissue culture cells is through the microinjection of DNA plasmids into the nucleus and monitoring the distribution of the transcribed product over time. Here we describe how to setup a microscope equipped with a micromanipulator used in cell microinjections, and we explain how to perform a nuclear mRNA export assay and obtain the nuclear export rate for any given mRNA. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Total Internal Reflection Fluorescence Microscopy Imaging-Guided Confocal Single-Molecule Fluorescence Spectroscopy

    OpenAIRE

    Zheng, Desheng; Kaldaras, Leonora; Lu, H. Peter

    2013-01-01

    We have developed an integrated spectroscopy system combining total internal reflection fluorescence microscopy imaging with confocal single-molecule fluorescence spectroscopy for two-dimensional interfaces. This spectroscopy approach is capable of both multiple molecules simultaneously sampling and in situ confocal fluorescence dynamics analyses of individual molecules of interest. We have demonstrated the calibration with fluorescent microspheres, and carried out single-molecule spectroscop...

  16. A conformation-induced fluorescence method for microRNA detection

    DEFF Research Database (Denmark)

    Aw, Sherry S; Tang, Melissa Xm; Teo, Yin Nah

    2016-01-01

    and quantify microRNAs may aid research into novel aspects of microRNA biology and contribute to the development of diagnostics. By introducing an additional stem loop into the fluorescent RNA Spinach and altering its 3' and 5' ends, we have generated a new RNA, Pandan, that functions as the basis for a micro......MicroRNAs play important roles in a large variety of biological systems and processes through their regulation of target mRNA expression, and show promise as clinical biomarkers. However, their small size presents challenges for tagging or direct detection. Innovation in techniques to sense......RNA sensor. Pandan contains two sequence-variable stem loops that encode complementary sequence for a target microRNA of interest. In its sensor form, it requires the binding of a target microRNA in order to reconstitute the RNA scaffold for fluorophore binding and fluorescence. Binding of the target micro...

  17. Fluorescent scattering by molecules embedded in small particles

    International Nuclear Information System (INIS)

    1982-01-01

    Studies are reported in these areas: double resonance in fluorescent and Raman scattering; surface enhanced Raman scattering; fluorescence by molecules embedded in small particles; fluorescence by a liquid droplet; and fluorescence by conical pits in surfaces

  18. microRNA-independent recruitment of Argonaute 1 to nanos mRNA through the Smaug RNA-binding protein

    OpenAIRE

    Pinder, Benjamin D; Smibert, Craig A

    2012-01-01

    Argonaute 1 directly interacts with the RNA binding protein Smaug in Drosophila, is thereby recruited to the Smaug target nanos mRNA and is required for Smaug-mediated translational repression of the nanos mRNA.

  19. Western blot seroindeterminate individuals for Human T-lymphotropic Virus 1/2 (HTLV-1/2 in Fortaleza (Brazil: a serological and molecular diagnostic and epidemiological approach

    Directory of Open Access Journals (Sweden)

    Santos Terezinha de Jesus Teixeira

    2003-01-01

    Full Text Available How to handle Western blot (WB seroindeterminate individuals for Human T-lymphotropic Virus 1/2 (HTLV-1/2 constitutes a challenge for blood banks and fam ilies. We made a cross-sectional study of 191 enzyme linked immunoassay (EIA reactive individuals from the hematological center (HEMOCE of Fortaleza (Brazil, examining their serological (WB and molecular (PCR diagnosis, and demographic profiles, as well as a possible association of their condition with other infectious pathologies and risk factors. Ethical institutional approval and personal consent were obtained. Out of 191 EIA reactive individuals, 118 were WB seroindeterminate and 73 were seropositive for HTLV-1/2. In the PCR analysis of 41 WB seroindeterminate individuals, 9 (22% were positive and 32 (78% were negative for HTLV-1/2. The demographic analysis indicated a trend towards a predominance of males among the seroindeterminate individuals and females in the seropositive ones. The seroindeterminate individuals were younger than the seropositive ones. We did not find any association of these conditions with syphilis, Chagas disease or HIV or hepatitis, and with risk factors such as breast-feeding, blood transfusion, STD (syphilis and IDU.

  20. Western blot seroindeterminate individuals for Human T-lymphotropic Virus 1/2 (HTLV-1/2 in Fortaleza (Brazil: a serological and molecular diagnostic and epidemiological approach

    Directory of Open Access Journals (Sweden)

    Terezinha de Jesus Teixeira Santos

    Full Text Available How to handle Western blot (WB seroindeterminate individuals for Human T-lymphotropic Virus 1/2 (HTLV-1/2 constitutes a challenge for blood banks and fam ilies. We made a cross-sectional study of 191 enzyme linked immunoassay (EIA reactive individuals from the hematological center (HEMOCE of Fortaleza (Brazil, examining their serological (WB and molecular (PCR diagnosis, and demographic profiles, as well as a possible association of their condition with other infectious pathologies and risk factors. Ethical institutional approval and personal consent were obtained. Out of 191 EIA reactive individuals, 118 were WB seroindeterminate and 73 were seropositive for HTLV-1/2. In the PCR analysis of 41 WB seroindeterminate individuals, 9 (22% were positive and 32 (78% were negative for HTLV-1/2. The demographic analysis indicated a trend towards a predominance of males among the seroindeterminate individuals and females in the seropositive ones. The seroindeterminate individuals were younger than the seropositive ones. We did not find any association of these conditions with syphilis, Chagas disease or HIV or hepatitis, and with risk factors such as breast-feeding, blood transfusion, STD (syphilis and IDU.

  1. Direct visualization of epidermal growth factor receptors (EGFR) in A431 and placental cell membrane by western blot with 125I-EGF

    International Nuclear Information System (INIS)

    Lin, P.H.; Selinfreund, R.; Wharton, W.

    1986-01-01

    Using the western blot technique, they have devised a new procedure that allowed the direct visualization of both the 150KD and the 170KD forms of EGFR by its natural ligand, 125 I-EGF. A431, and placental plasmalemma were purified and solubilized in either SDS-PAGE buffer (without DTT, EDTA) or Triton X-100 (0.5%), resolved on PAGE and electrophoretically transferred onto nitrocellulose (NC) paper. In the absence of boiling, SDS did not denature the EGFR. Although EGER band can be detected after hybridization with 125 I-EGF, the receptor signal was considerably improved with the addition of 0.1% Tween-20. The binding of 125 I-EGF to the both the 150KD and the 170KD bands of the EGFR was specific, reversible and increased with the amount of membrane protein present. The direct visualization of the EGFR using its natural ligand eliminated the necessity for the time consuming antibody preparation. Presently, they are using this technique to identify specific receptors for other ligands

  2. Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

    Science.gov (United States)

    Methogo, Ruth Menque; Dansette, Patrick M.; Klarskov, Klaus

    2007-12-01

    A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

  3. A Western blot-based investigation of the yeast secretory pathway designed for an intermediate-level undergraduate cell biology laboratory.

    Science.gov (United States)

    Hood-Degrenier, Jennifer K

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in two distinct steps of protein secretion were differentiated using a genetic reporter designed specifically to identify defects in the first step of the pathway, the insertion of proteins into the endoplasmic reticulum (Vallen, 2002). We have developed two versions of a Western blotting assay that serves as a second way of distinguishing the two secretory mutants, which we pair with the genetic assay in a 3-wk laboratory module. A quiz administered before and after students participated in the lab activities revealed significant postlab gains in their understanding of the secretory pathway and experimental techniques used to study it. A second survey administered at the end of the lab module assessed student perceptions of the efficacy of the lab activities; the results of this survey indicated that the experiments were successful in meeting a set of educational goals defined by the instructor.

  4. Porcine Cysticercosis: Possible Cross-Reactivity of Taenia hydatigena to GP50 Antigen in the Enzyme-Linked Immunoelectrotransfer Blot Assay.

    Science.gov (United States)

    Muro, Claudio; Gomez-Puerta, Luis A; Flecker, Robert H; Gamboa, Ricardo; Barreto, Percy Vilchez; Dorny, Pierre; Tsang, Victor C W; Gilman, Robert H; Gonzalez, Armando E; Garcia, Hector H; O'Neal, Seth E; For The Cysticercosis Working Group In Peru

    2017-12-01

    The lentil lectin glycoprotein enzyme-linked immunoelectrotransfer blot (LLGP EITB, reported sensitivity 99% and specificity 100%) is used as a serologic marker of exposure to Taenia solium in pigs. However, only a limited number of parasites have been evaluated for cross reactivity. Pigs may host other related cestode infections, including Taenia hydatigena, which have not been formally evaluated for cross-reactions. We investigated a corral in Tumbes, Peru, a region where a cysticercosis elimination demonstration project was completed in 2012. In this corral, 14/19 (73.7%) 6-8-week-old piglets were reactive to GP50 on LLGP EITB, and all had circulating Taenia sp. antigens. From eight necropsied piglets; four were infected with T. hydatigena metacestodes whereas none had evidence of T. solium infection. Two resident dogs were subsequently confirmed to have T. hydatigena taeniasis. These results suggest GP50 cross-reactivity in T. hydatigena- infected pigs, although controlled experimental infection is needed to confirm this hypothesis.

  5. Differential staining of Western blots of human secreted glycoproteins from serum, milk, saliva, and seminal fluid using lectins displaying diverse sugar specificities.

    Science.gov (United States)

    Gilboa-Garber, Nechama; Lerrer, Batya; Lesman-Movshovich, Efrat; Dgani, Orly

    2005-12-01

    Human milk, serum, saliva, and seminal fluid glycoproteins (gps) nourish and protect newborn and adult tissues. Their saccharides, which resemble cell membrane components, may block pathogen adhesion and infection. In the present study, they were examined by a battery of lectins from plants, animals, and bacteria, using hemagglutination inhibition and Western blot analyses. The lectins included galactophilic ones from Aplysia gonad, Erythrina corallodendron, Maclura pomifera (MPL), peanut, and Pseudomonas aeruginosa (PA-IL); fucose-binding lectins from Pseudomonas aeruginosa (PA-IIL), Ralstonia solanacearum (RSL), and Ulex europaeus (UEA-I), and mannose/glucose-binding Con A. The results demonstrated the chosen lectin efficiency for differential analysis of human secreted gps as compared to CBB staining. They unveiled the diversity of these body fluid gp glycans (those of the milk and seminal fluid being highest): the milk gps interacted most strongly with PA-IIL, followed by RSL; the saliva gps with RSL, followed by PA-IIL and MPL; the serum gps with Con A and MPL, followed by PA-IIL and RSL, and the seminal plasma gps with RSL and MPL, followed by UEA-I and PA-IIL. The potential usage of these lectins as probes for scientific, industrial, and medical purposes, and for quality control of the desired gps is clearly indicated.

  6. Myostatin inhibitors in sports drug testing: Detection of myostatin-neutralizing antibodies in plasma/serum by affinity purification and Western blotting.

    Science.gov (United States)

    Walpurgis, Katja; Thomas, Andreas; Schänzer, Wilhelm; Thevis, Mario

    2016-02-01

    Myostatin is a key regulator of skeletal muscle growth and inhibition of its signaling pathway results in an increased muscle mass and function. The aim of this study was to develop a qualitative detection assay for myostatin-neutralizing antibodies for doping control purposes by using immunological approaches. To detect different types of myostatin-neutralizing antibodies irrespective of their amino acid sequence, an immunological assay specific for antibodies directed against myostatin and having a human Fc domain was established. Affinity purification and Western blotting strategies were combined to allow extracting and identifying relevant analytes from 200 μL of plasma/serum in a non-targeted approach. The assay was characterized regarding specificity, linearity, precision, robustness, and recovery. The assay was found to be highly specific, robust, and linear from 0.1 to 1 μg/mL. The precision was successfully specified at three different concentrations and the recovery of the affinity purification was 58%. Within this study, an immunological detection assay for myostatin-neutralizing antibodies present in plasma/serum specimens was developed and successfully characterized. The presented approach can easily be modified to include other therapeutic antibodies and serves as proof-of-concept for the detection of antibody-based myostatin inhibitors in doping control samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. A comparison of DNA extraction protocols from blood spotted on FTA cards for the detection of tick-borne pathogens by Reverse Line Blot hybridization.

    Science.gov (United States)

    Hailemariam, Zerihun; Ahmed, Jabbar Sabir; Clausen, Peter-Henning; Nijhof, Ard Menzo

    2017-01-01

    An essential step in the molecular detection of tick-borne pathogens (TBPs) in blood is the extraction of DNA. When cooled storage of blood under field conditions prior to DNA extraction in a dedicated laboratory is not possible, the storage of blood on filter paper forms a promising alternative. We evaluated six DNA extraction methods from blood spotted on FTA Classic ® cards (FTA cards), to determine the optimal protocol for the subsequent molecular detection of TBPs by PCR and the Reverse Line Blot hybridization assay (RLB). Ten-fold serial dilutions of bovine blood infected with Babesia bovis, Theileria mutans, Anaplasma marginale or Ehrlichia ruminantium were made by dilution with uninfected blood and spotted on FTA cards. Subsequently, DNA was extracted from FTA cards using six different DNA extraction protocols. DNA was also isolated from whole blood dilutions using a commercial kit. PCR/RLB results showed that washing of 3mm discs punched from FTA cards with FTA purification reagent followed by DNA extraction using Chelex ® resin was the most sensitive procedure. The detection limit could be improved when more discs were used as starting material for the DNA extraction, whereby the use of sixteen 3mm discs proved to be most practical. The presented best practice method for the extraction of DNA from blood spotted on FTA cards will facilitate epidemiological studies on TBPs. It may be particularly useful for field studies where a cold chain is absent. Copyright © 2016 Elsevier GmbH. All rights reserved.

  8. Simultaneous and independent detection of C9ORF72 alleles with low and high number of GGGGCC repeats using an optimised protocol of Southern blot hybridisation.

    Science.gov (United States)

    Buchman, Vladimir L; Cooper-Knock, Johnathan; Connor-Robson, Natalie; Higginbottom, Adrian; Kirby, Janine; Razinskaya, Olga D; Ninkina, Natalia; Shaw, Pamela J

    2013-04-08

    Sizing of GGGGCC hexanucleotide repeat expansions within the C9ORF72 locus, which account for approximately 10% of all amyotrophic lateral sclerosis (ALS) cases, is urgently required to answer fundamental questions about mechanisms of pathogenesis in this important genetic variant. Currently employed PCR protocols are limited to discrimination between the presence and absence of a modified allele with more than 30 copies of the repeat, while Southern hybridisation-based methods are confounded by the somatic heterogeneity commonly present in blood samples, which might cause false-negative or ambiguous results. We describe an optimised Southern hybridisation-based protocol that allows confident detection of the presence of a C9ORF72 repeat expansion alongside independent assessment of its heterogeneity and the number of repeat units. The protocol can be used with either a radiolabeled or non-radiolabeled probe. Using this method we have successfully sized the C9ORF72 repeat expansion in lymphoblastoid cells, peripheral blood, and post-mortem central nervous system (CNS) tissue from ALS patients. It was also possible to confidently demonstrate the presence of repeat expansion, although of different magnitude, in both C9ORF72 alleles of the genome of one patient. The suggested protocol has sufficient advantages to warrant adoption as a standard for Southern blot hybridisation analysis of GGGGCC repeat expansions in the C9ORF72 locus.

  9. Application of the Reverse Line Blot Assay for the Molecular Detection of Theileria and Babesia sp. in Sheep and Goat Blood Samples from Pakistan

    Directory of Open Access Journals (Sweden)

    A Rasul

    2013-06-01

    Full Text Available Background: The present study was designed to detect the presence of tick-borne parasites (Theileria and Babesia spp. in 196 blood samples collected from apparently healthy sheep and goats from two provinces, Punjab and Khyber Pukhtoon Khwa, in Pakistan.Methods: Reverse line blot (RLB assay was applied for the parasitic detection by the amplification of hypervariable V4 region of the 18S ribosomal RNA (rRNA gene. A membrane with covalently linked generic and species specific oligonucleotide probes was used for the hybridization of amplified PCR products.Results: Parasites were detected in 16% of the ruminant blood samples under study. Two Theileria species, T. lestoquardi and T. ovis, were identified in samples. 25, of the total 32, infected animals were from Khyber Pukhtoon Khwa.Conclusion: Sheep were more prone to tick borne haemoprotozans as 81% infected samples were sheep as compared to 19% goats (P > 0.001. Risk factor analysis revealed that male (P = 0.03, ani­mals infested by ticks (P = 0.03 and herd composed of sheep only (P = 0.001 were more infected by blood parasites.

  10. A 15-year-long Southern blotting analysis of FMR1 to detect female carriers and for prenatal diagnosis of fragile X syndrome in Taiwan.

    Science.gov (United States)

    Tzeng, C-C; Tsai, L-P; Chang, Y-K; Hung, Y-J; Chang, Y-Y; Su, Y-P; Jiang, J-J; Liang, H-M

    2017-08-01

    Here, we review the results of Southern blotting analyses of the FMR1 gene performed in our reference laboratory in Taiwan over a 15-year period. In total, 725 high-risk women with a family history of fragile X syndrome (FXS) or idiopathic intellectual disability, 3911 low-risk pregnant women without such family history, and prenatal diagnosis data for 32 foetuses from 24 carrier mothers were included. Only 2 carriers were in the low-risk group, which indicated a prevalence of 1 of 1955 women (95% confidence interval: 1/7156-1/539). A total of 100 carriers were found to be in the high-risk group, thus revealing a significantly higher frequency than the low-risk group (100/725 vs 2/3911, P<0.0001). Eight of the 14 foetuses that inherited the maternal mutant allele were verified to have a full mutation, with the smallest maternal pre-mutation allele carrying 56 CGG repeats. The overall findings confirmed that the carrier prevalence among low-risk women in Taiwan is significantly lower than that reported in western countries. Therefore, the most important step for preventing FXS in Taiwan would be to focus on high-risk women by promoting general awareness of this disease and spreading knowledge regarding the benefits of carrier screening and prenatal testing. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  11. Fluorescence detection of dental calculus

    International Nuclear Information System (INIS)

    Gonchukov, S; Sukhinina, A; Vdovin, Yu; Biryukova, T

    2010-01-01

    This work is devoted to the optimization of fluorescence dental calculus diagnostics in optical spectrum. The optimal wavelengths for fluorescence excitation and registration are determined. Two spectral ranges 620 – 645 nm and 340 – 370 nm are the most convenient for supra- and subgingival calculus determination. The simple implementation of differential method free from the necessity of spectrometer using was investigated. Calculus detection reliability in the case of simple implementation is higher than in the case of spectra analysis at optimal wavelengths. The use of modulated excitation light and narrowband detection of informative signal allows us to decrease essentially its diagnostic intensity even in comparison with intensity of the low level laser dental therapy

  12. Sorting fluorescent nanocrystals with DNA

    Energy Technology Data Exchange (ETDEWEB)

    Gerion, Daniele; Parak, Wolfgang J.; Williams, Shara C.; Zanchet, Daniela; Micheel, Christine M.; Alivisatos, A. Paul

    2001-12-10

    Semiconductor nanocrystals with narrow and tunable fluorescence are covalently linked to oligonucleotides. These biocompounds retain the properties of both nanocrystals and DNA. Therefore, different sequences of DNA can be coded with nanocrystals and still preserve their ability to hybridize to their complements. We report the case where four different sequences of DNA are linked to four nanocrystal samples having different colors of emission in the range of 530-640 nm. When the DNA-nanocrystal conjugates are mixed together, it is possible to sort each type of nanoparticle using hybridization on a defined micrometer -size surface containing the complementary oligonucleotide. Detection of sorting requires only a single excitation source and an epifluorescence microscope. The possibility of directing fluorescent nanocrystals towards specific biological targets and detecting them, combined with their superior photo-stability compared to organic dyes, opens the way to improved biolabeling experiments, such as gene mapping on a nanometer scale or multicolor microarray analysis.

  13. Fluorescence spectroscopy for neoplasms control

    Science.gov (United States)

    Bratchenko, I. A.; Kristoforova, Yu. A.; Myakinin, O. O.; Artemyev, D. N.; Kozlov, S. V.; Moryatov, A. A.; Zakharov, V. P.

    2016-04-01

    Investigation of malignant skin tumors diagnosis was performed involving two setups for native tissues fluorescence control in visible and near infrared regions. Combined fluorescence analysis for skin malignant melanomas and basal cell carcinomas was performed. Autofluorescence spectra of normal skin and oncological pathologies stimulated by 457 nm and 785 nm lasers were registered for 74 skin tissue samples. Spectra of 10 melanomas and 27 basal cell carcinomas were registered ex vivo. Skin tumors analysis was made on the basis of autofluorescence spectra intensity and curvature for analysis of porphyrins, lipo-pigments, flavins and melanin. Separation of melanomas and basal cell carcinomas was performed on the basis of discriminant analysis. Overall accuracy of basal cell carcinomas and malignant melanomas separation in current study reached 86.5% with 70% sensitivity and 92.6% specificity.

  14. New Fluorescence Probes for Biomolecules

    Directory of Open Access Journals (Sweden)

    Katarzyna Jurek

    2015-07-01

    Full Text Available Steady state fluorescence measurements have been used for the investigation of interaction between the bovine serum albumin (BSA and fluorescence probes: 3-hydroxy-2,4- bis[(3-methyl-1,3-benzoxazol-2(3H-ylidenemethyl]cyclobut-2-en-1-one (SQ6, 3-hydroxy- 2,4-bis[(3-methyl-1,3-benzothiazol-2(3H-ylidenemethyl]cyclobut-2-en-1-one (SQ7 and 3-hydroxy-2,4-bis[(1,3,3-trimethyl-1,3-dihydro-2H-indol-2-ylidenemethyl]cyclobut-2-en-1-one (SQ8. The binding constant between bovine serum albumin and squarine dyes has been determined by using both the Benesi-Hildebrand and Stern-Volmer equations. The negative value of free energy change indicates the existence of a spontaneous complexation process of BSA with squarine dyes.

  15. Multi Spectral Fluorescence Imager (MSFI)

    Science.gov (United States)

    Caron, Allison

    2016-01-01

    Genetic transformation with in vivo reporter genes for fluorescent proteins can be performed on a variety of organisms to address fundamental biological questions. Model organisms that may utilize an ISS imager include unicellular organisms (Saccharomyces cerevisiae), plants (Arabidopsis thaliana), and invertebrates (Caenorhabditis elegans). The multispectral fluorescence imager (MSFI) will have the capability to accommodate 10 cm x 10 cm Petri plates, various sized multi-well culture plates, and other custom culture containers. Features will include programmable temperature and light cycles, ethylene scrubbing (less than 25 ppb), CO2 control (between 400 ppm and ISS-ambient levels in units of 100 ppm) and sufficient airflow to prevent condensation that would interfere with imaging.

  16. X-ray fluorescence holography.

    Science.gov (United States)

    Hayashi, Kouichi; Happo, Naohisa; Hosokawa, Shinya; Hu, Wen; Matsushita, Tomohiro

    2012-03-07

    X-ray fluorescence holography (XFH) is a method of atomic resolution holography which utilizes fluorescing atoms as a wave source or a monitor of the interference field within a crystal sample. It provides three-dimensional atomic images around a specified element and has a range of up to a few nm in real space. Because of this feature, XFH is expected to be used for medium-range local structural analysis, which cannot be performed by x-ray diffraction or x-ray absorption fine structure analysis. In this article, we explain the theory of XFH including solutions to the twin-image problem, an advanced measuring system, and data processing for the reconstruction of atomic images. Then, we briefly introduce our recent applications of this technique to the analysis of local lattice distortions in mixed crystals and nanometer-size clusters appearing in the low-temperature phase of a shape-memory alloy.

  17. X-ray fluorescence holography

    International Nuclear Information System (INIS)

    Hayashi, Kouichi; Happo, Naohisa; Hosokawa, Shinya; Hu Wen; Matsushita, Tomohiro

    2012-01-01

    X-ray fluorescence holography (XFH) is a method of atomic resolution holography which utilizes fluorescing atoms as a wave source or a monitor of the interference field within a crystal sample. It provides three-dimensional atomic images around a specified element and has a range of up to a few nm in real space. Because of this feature, XFH is expected to be used for medium-range local structural analysis, which cannot be performed by x-ray diffraction or x-ray absorption fine structure analysis. In this article, we explain the theory of XFH including solutions to the twin-image problem, an advanced measuring system, and data processing for the reconstruction of atomic images. Then, we briefly introduce our recent applications of this technique to the analysis of local lattice distortions in mixed crystals and nanometer-size clusters appearing in the low-temperature phase of a shape-memory alloy. (topical review)

  18. Fluorescence lifetime imaging of skin cancer

    Science.gov (United States)

    Patalay, Rakesh; Talbot, Clifford; Munro, Ian; Breunig, Hans Georg; König, Karsten; Alexandrov, Yuri; Warren, Sean; Neil, Mark A. A.; French, Paul M. W.; Chu, Anthony; Stamp, Gordon W.; Dunsby, Chris

    2011-03-01

    Fluorescence intensity imaging and fluorescence lifetime imaging microscopy (FLIM) using two photon microscopy (TPM) have been used to study tissue autofluorescence in ex vivo skin cancer samples. A commercially available system (DermaInspect®) was modified to collect fluorescence intensity and lifetimes in two spectral channels using time correlated single photon counting and depth-resolved steady state measurements of the fluorescence emission spectrum. Uniquely, image segmentation has been used to allow fluorescence lifetimes to be calculated for each cell. An analysis of lifetime values obtained from a range of pigmented and non-pigmented lesions will be presented.

  19. Localization of insulin receptor mRNA in rat brain by in situ hybridization

    International Nuclear Information System (INIS)

    Marks, J.L.; Porte, D. Jr.; Stahl, W.L.; Baskin, D.G.

    1990-01-01

    Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35 S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus

  20. FATHEAD MINNOW VITELLOGENIN: CDNA SEQUENCE AND MRNA AND PROTEIN EXPRESSION AFTER 17 BETA-ESTRADIOL TREATMENT

    Science.gov (United States)

    In the present study, a sensitive ribonuclease protection assay (RPA) for VTG mRNA was developed for the fathead minnow (Pimephales promelas), a species proposed for routine endocrine-disrupting chemical (EDC) screening.

  1. 60Co γ-irradiation enhances expression of GAP-43 mRNA in rat brain

    International Nuclear Information System (INIS)

    Su Bingyin; Cai Wenqin; Zhang Chenggang

    2001-01-01

    Objective: To study the relationship between the expression of GAP-43 mRNA and nerve regeneration in rat brain after 60 Co γ-irradiation. Methods: Wistar rats were subjected to whole-body irradiation with 8 Gy 60 Co γ-rays. The expression of GAP-43 was detected by in situ hybridization histochemistry using Dig-cRNA probe. Results: It was found that the expression of GAP-43 mRNA increased in the cerebral cortex, caudate, putamen, globus pallidum, thalamus and hypothalamus one week after 8 Gy 60 Co γ-irradiation. The peak of GAP-43 mRNA expression was observed in the fourth week and then began to decrease but still remained at a higher than normal level. However, it decreased to a low level after 7 weeks. Conclusion: Enhanced expression of GAP-43 mRNA after 60 Co γ-irradiation in rat brain is associated with nerve regeneration and reconstruction of synapse

  2. Differential expression of PARP1 mRNA in leucocytes of patients ...

    Indian Academy of Sciences (India)

    P. 2011 Differential expression of PARP1 mRNA in leucocytes of patients with Down's syndrome. J. Genet. ... of Alzheimer disease at an earlier age than subjects with- ... family and personal informed consent. .... In effect, they report that.

  3. Nerve growth factor mRNA in brain: localization by in situ hybridization

    International Nuclear Information System (INIS)

    Rennert, P.D.; Heinrich, G.

    1986-01-01

    Nerve Growth Factor is a 118 amino acid polypeptide that plays an important role in the differentiation and survival of neurons. The recent discovery that a mRNA that encodes beta Nerve Growth Factor is present in brain suggests that the Nerve Growth Factor gene may not only regulate gene expression of peripheral but also of central neurons. To identify the site(s) of Nerve Growth Factor mRNA production in the brain and to determine which cells express the Nerve Growth Factor gene, the technique of in situ hybridization was employed. A 32P-labeled RNA probe complementary to Nerve Growth Factor mRNA hybridized to cells in the stratum granulosum of the dentate gyrus and the stratum pyramidale of the hippocampus. These observations identify for the first time cellular sites of Nerve Growth Factor gene expression in the central nervous system, and suggest that Nerve Growth Factor mRNA is produced by neurons

  4. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    International Nuclear Information System (INIS)

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M.

    1990-01-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures

  5. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    Directory of Open Access Journals (Sweden)

    Beatriz M. A. Fontoura

    2013-07-01

    Full Text Available Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses.

  6. Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA

    OpenAIRE

    Dibrov, Sergey; McLean, Jaime; Hermann, Thomas

    2011-01-01

    An oligonucleotide representing a regulatory element of human thymidylate synthase mRNA has been crystallized as a dimer. The structure of the asymmetric dimer has been determined at 1.97 Å resolution.

  7. Naturally occurring BRCA2 alternative mRNA splicing events in clinically relevant samples

    DEFF Research Database (Denmark)

    Fackenthal, James D; Yoshimatsu, Toshio; Zhang, Bifeng

    2016-01-01

    patterns and thereby disrupt gene function. mRNA analyses are therefore among the tests used to interpret the clinical significance of some genetic variants. However, these could be confounded by the appearance of naturally occurring alternative transcripts unrelated to germline sequence variation...... to characterise the spectrum of naturally occurring BRCA2 mRNA alternate-splicing events. METHODS: mRNA was prepared from several blood and breast tissue-derived cells and cell lines by contributing ENIGMA laboratories. cDNA representing BRCA2 alternate splice sites was amplified and visualised using capillary...... or agarose gel electrophoresis, followed by sequencing. RESULTS: We demonstrate the existence of 24 different BRCA2 mRNA alternate-splicing events in lymphoblastoid cell lines and both breast cancer and non-cancerous breast cell lines. CONCLUSIONS: These naturally occurring alternate-splicing events...

  8. Mechanical Stimulation and IGF-1 Enhance mRNA Translation Rate in Osteoblasts Via Activation of the AKT-mTOR Pathway.

    Science.gov (United States)

    Bakker, Astrid D; Gakes, Tom; Hogervorst, Jolanda M A; de Wit, Gerard M J; Klein-Nulend, Jenneke; Jaspers, Richard T

    2016-06-01

    Insulin-like growth factor-1 (IGF-1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF-1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF-1 stimulate protein synthesis in osteoblasts via activation of the AKT-mTOR pathway. MC3T3-E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF-1 (1-100 ng/ml) for 0.5-6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by Western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF-1 (100 ng/ml) reduced IGF-1 gene expression, although PFF enhanced IGF-1 expression. IGF-1 did not affect collagen-I gene expression. IGF-1 dose-dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF-1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF-1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF-1 on bone may thus be partly explained by mTOR-mediated enhanced protein synthesis in osteoblasts. © 2015 Wiley Periodicals, Inc.

  9. Expression of insulin-like growth factor I receptors at mRNA and protein levels during metamorphosis of Japanese flounder (Paralichthys olivaceus).

    Science.gov (United States)

    Zhang, Junling; Shi, Zhiyi; Cheng, Qi; Chen, Xiaowu

    2011-08-01

    Insulin-like growth factor I (IGF-I) is an important regulator of fish growth and development, and its biological actions are initiated by binding to IGF-I receptor (IGF-IR). Our previous study has revealed that IGF-I could play an important role during metamorphosis of Japanese flounder, Paralichthys olivaceus. The analysis of IGF-IR expression thus helps further elucidate the IGF-I regulation of metamorphic processes. In this study, the spatial-temporal expression of two distinct IGF-IR mRNAs was investigated by real-time RT-PCR. The spatial distribution of two IGF-IR mRNAs in adult tissues is largely overlapped, but they exhibit distinct temporal expression patterns during larval development. A remarkable decrease in IGF-IR-2 mRNA was detected during metamorphosis. In contrast, a significant increase in IGF-IR-1 mRNA was determined from pre-metamorphosis to metamorphic completion. These indicate that they may play different function roles during the flounder metamorphosis. The levels and localization of IGF-IR proteins during larval development were further studied by Western blotting and immunohistochemistry. Immunoreactive IGF-IRs were detected throughout larval development, and the IGF-IR proteins displayed a relatively abundant expression during metamorphosis. Moreover, the IGF-IR proteins appeared in key tissues, such as thickened skin beneath the migrating eye, developing intestine, gills and kidney during metamorphosis. These results further suggest that the IGF-I system may be involved in metamorphic development of Japanese flounder. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. The RDE-10/RDE-11 complex triggers RNAi-induced mRNA degradation by association with target mRNA in C. elegans.

    Science.gov (United States)

    Yang, Huan; Zhang, Ying; Vallandingham, Jim; Li, Hua; Li, Hau; Florens, Laurence; Mak, Ho Yi

    2012-04-15

    The molecular mechanisms for target mRNA degradation in Caenorhabditis elegans undergoing RNAi are not fully understood. Using a combination of genetic, proteomic, and biochemical approaches, we report a divergent RDE-10/RDE-11 complex that is required for RNAi in C. elegans. Genetic analysis indicates that the RDE-10/RDE-11 complex acts in parallel to nuclear RNAi. Association of the complex with target mRNA is dependent on RDE-1 but not RRF-1, suggesting that target mRNA recognition depends on primary but not secondary siRNA. Furthermore, RDE-11 is required for mRNA degradation subsequent to target engagement. Deep sequencing reveals a fivefold decrease in secondary siRNA abundance in rde-10 and rde-11 mutant animals, while primary siRNA and microRNA biogenesis is normal. Therefore, the RDE-10/RDE-11 complex is critical for amplifying the exogenous RNAi response. Our work uncovers an essential output of the RNAi pathway in C. elegans.

  11. An operational fluorescence system for crop assessment

    Science.gov (United States)

    Belzile, Charles; Belanger, Marie-Christine; Viau, Alain A.; Chamberland, Martin; Roy, Simon

    2004-03-01

    The development of precision farming requires new tools for plant nutritional stress monitoring. An operational fluorescence system has been designed for vegetation status mapping and stress detection at plant and field scale. The instrument gives relative values of fluorescence at different wavelengths induced by the two-excitation sources. Lightinduced fluorescence has demonstrated successful crop health monitoring and plant nutritional stress detection capabilities. The spectral response of the plants has first been measured with an hyperspectral imager using laser-induced fluorescence. A tabletop imaging fluorometer based on flash lamp technology has also been designed to study the spatial distribution of fluorescence on plant leaves. For field based non-imaging system, LED technology is used as light source to induce fluorescence of the plant. The operational fluorescence system is based on ultraviolet and blue LED to induce fluorescence. Four narrow fluorescence bands centered on 440, 520, 690 and 740nm are detected. The instrument design includes a modular approach for light source and detector. It can accommodate as many as four different light sources and six bands of fluorescence detection. As part of the design for field application, the instrument is compatible with a mobile platform equipped with a GPS and data acquisition system. The current system developed by Telops/GAAP is configured for potato crops fluorescence measurement but can easily be adapted for other crops. This new instrument offers an effective and affordable solution for precision farming.

  12. Amelioration of ultraviolet-B-induced down-regulation of mRNA levels for chloroplast proteins, by high irradiance, is mediated by photosynthesis

    International Nuclear Information System (INIS)

    Mackerness, S.A. H.; Butt, P.J.; Jordan, B.R.; Thomas, B.

    1996-01-01

    The mechanism by which increasing photosynthetically active radiation (PAR) reduces the sensitivity of RNA transcripts to UV-B radiation was studied in pea (Pisum sativum L.). mRNA transcript levels for rbc S, rbc L, cab and psb A were measured over an 8 d experimental period in pea, plants supplemented with UV-B radiation under a range of conditions. Under low light (150 mu-mol m -2 s -1 ), UV-B resulted in a significant decline in the levels of transcripts for all four genes which was prevented by increasing the background irradiance to 350 mu-mol m -2 s -1 (high light) with white light from fluorescent lamps. Increasing CO 2 levels to give photosynthesis rates equivalent to the high light treatment partially protected rbc S and cab transcripts and fully protected rbc L transcripts but did not prevent visible injury. Increasing light with low pressure sodium lamps, which increase photosynthesis but are not effective for activation of the DNA repair enzyme, photolyase, gave results which were not significantly different from white fluorescent high light treatments. Protection by high light was lost in the presence of the photosynthesis inhibitors CCCP and DCMU. The UV-B induced increase in the expression of chalcone synthase (chs) genes was delayed by the treatments which increased photosynthesis rates and conferred protection. The results indicate that photosynthesis plays a key role in the amelioration of UV-B induced decline in mRNA levels for proteins. The minimal role of DNA repair by photolyase indicates that reduction in photosynthesis gene transcripts in response to UV-B represents a specific regulation rather than being a consequence of DNA damage. (author)

  13. Regulation of mRNA Translation Is a Novel Mechanism for Phthalate Toxicity.

    Directory of Open Access Journals (Sweden)

    Jun Ling

    Full Text Available Phthalates are a group of plasticizers that are widely used in many consumer products and medical devices, thus generating a huge burden to human health. Phthalates have been known to cause a number of developmental and reproductive disorders functioning as endocrine modulators. They are also involved in carcinogenesis with mechanisms less understood. To further understand the molecular mechanisms of phthalate toxicity, in this study we reported a new effect of phthalates on mRNA translation/protein synthesis, a key regulatory step of gene expression. Butyl benzyl phthalate (BBP was found to directly inhibit mRNA translation in vitro but showed a complicated pattern of affecting mRNA translation in cells. In human kidney embryonic cell (HEK-293T, BBP increased cap-dependent mRNA translation at lower concentrations but showed inhibitory effect at higher concentrations. Cap-independent translation was not affected. On the other hand, mono (2-ethylhexyl phthalate (MEHP as a major metabolite of another important phthalate di (2-ethylhexyl phthalate (DEHP inhibited both can-dependent and -independent mRNA translation in vivo. In contrast, BBP and MEHP exhibited an overall promoting effect on mRNA translation in cancer cells. Mechanistic studies identified that the level and phosphorylation of eIF4E-BP (eIF4E binding protein and the amount of eIF4GI in eIF4F complex were altered in accordance with the effect of BBP on translation. BBP was also identified to directly bind to eIF4E, providing a further mechanism underlying the regulation of mRNA by phthalate. At the cellular level BBP inhibited normal cell growth but slightly promoted cancer cells (HT29 growth. Overall, this study provides the first evidence that phthalates can directly regulate mRNA translation as a novel mechanism to mediate their biological toxicities.

  14. Lower FOXO3 mRNA expression in granulosa cells is involved in unexplained infertility.

    Science.gov (United States)

    Yamamoto, Hikaru; Yamashita, Yoshiki; Saito, Natsuho; Hayashi, Atsushi; Hayashi, Masami; Terai, Yoshito; Ohmichi, Masahide

    2017-06-01

    The aim of this study was to investigate whether FOXO1 and FOXO3 mRNA expression in granulosa cells is the cause of unexplained infertility. Thirty-one patients aged infertility and 18 with male partner infertility as a control group) whose serum anti-Müllerian hormone level was >0.5 ng/μL were enrolled in the study. All patients underwent oocyte retrieval under a short protocol from June 2012 to October 2013. Real-time PCR was carried out using mRNA extracted from granulosa cells retrieved from mature follicles. We compared FOXO1 and FOXO3 mRNA expression ratios in granulosa cells between the unexplained infertility group and the male infertility group. The relation between FOXO1 and FOXO3 mRNA expression ratios in granulosa cells and assisted reproduction technology clinical outcome was also examined. FOXO3 mRNA expression ratio was significantly lower in the unexplained infertility group than in the male infertility group. Moreover, FOXO3 mRNA expression ratio showed a positive correlation with both the number of retrieved oocytes and serum anti-Müllerian hormone level. A positive correlation was also identified between FOXO1 mRNA expression and total dose of hMG. As well, the number of retrieved oocytes in the unexplained infertility group was statistically lower than that in the male infertility group. A lower FOXO3 mRNA expression in granulosa cells leads to poor oocyte development in patients with unexplained infertility undergoing controlled ovarian stimulation for in vitro fertilization-embryo transfer. © 2017 Japan Society of Obstetrics and Gynecology.

  15. Estandarización de la técnica de Western blot para el diagnóstico de la fasciolosis humana utilizando antígenos de excreción-secreción de Fasciola hepática Western blot technique standardization of the diagnosis of human fasciolosis using Fasciola hepatica excreted-secreted antigens

    Directory of Open Access Journals (Sweden)

    Hermes Escalante

    2011-09-01

    Full Text Available Objetivos. Evaluar la eficacia de la técnica de electroinmunotransferencia (EITB o Western blot utilizando antígenos de excreción-secreción de las formas adultas de Fasciola hepatica (Fh E/S Ag para el diagnóstico de la fasciolosis humana. Materiales y métodos. Los antígenos fueron obtenidos a las 18 horas de incubación en medio Minimum Essential Eagle y preparados a la concentración proteica de 0,15 ug/uL; los cuales, al ser enfrentados con un pool de sueros de pacientes con fasciolosis confirmada por el hallazgo de huevos del parásito en las heces, se detectaron los antígenos de 10, 12, 17, 23, 27, 30, 36, 43, 66 y 136 KDa, con los cuales se desarrolló la técnica de Western blot. La sensibilidad se evaluó empleando sueros de 67 pacientes con fasciolosis, y la especificidad con sueros de 57 pacientes con otras parasitosis y diez sueros de personas no parasitadas. Resultados. De los 67 sueros, 64 reaccionaron con la banda de 23 KDa y 61 con la banda de 17KDa. Estas dos bandas no fueron detectadas por ninguno de los sueros de pacientes con otras parasitosis, ni de personas no parasitadas, siendo por ello consideradas como específicas y diagnósticas. Conclusiones. La sensibilidad de la prueba, utilizando las bandas de 17 y 23 KDa, fue de 95,5 % cuando se presenta reacción positiva en una o en las dos bandas, siendo la especificidad para estos dos antígenos de 100 % con un valor predictivo positivo de 100 % y un valor predictivo negativo de 95,71 %.Objectives. To evaluate the performance of the enzyme-linked immunoelectrotransfer blot assay (EITB, Western blot using excretory/secretory antigens from adult forms of Fasciola hepatica (Fh E/S Ag for the diagnosis of human fasciolosis. Materials and methods. Antigens were obtained after 18 hours of incubation in culture medium Minimum Essential Eagle, prepared at a protein concentration of 0.15 ug/uL and run against a pool of sera of patients with proven fasciolosis (confirmed by the

  16. The cellular uptake of antisense oligonucleotid of E6 mRNA into cervical cancer cells by DOPE-modified hydroxyapatite nanoparticles

    Directory of Open Access Journals (Sweden)

    Negin Saffarzadeh

    2014-10-01

    Full Text Available Objective(s: Although several chemical and physical methods for gene delivery have been introduced, their cytotoxicity, non-specific immune responses and the lack of biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles (NPs and 1,2-dioleoyl-sn-glycero-3-phosphoethanol​amine (DOPE-modified hydroxyapatite NPs was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical cancer cell line were evaluated. Materials and Methods: Calcium nitrate and diammonium phosphate were used for the synthesis of the hydroxyapatite nanoparticle. Thus, they were coated with polyethylene glycol (PEG, DOPE and antisense oligonucleotide of E6 mRNA using a cross-linker. Then, hydroxyapatite NPs and DOPE-modified hydroxyapatite NPs were incubated 48 hours with cervical cancer cells and their uptakes were evaluated by fluorescent microscopy. Results: The hydroxyapatite NPs had different shapes and some agglomeration with average size of 100 nm. The results showed DOPE-modified hydroxyapatite NPs had higher uptake than hydroxyapatite NPs (P

  17. DNA Topoisomerase I Gene Copy Number and mRNA Expression Assessed as Predictive Biomarkers for Adjuvant Irinotecan in Stage II/III Colon Cancer

    DEFF Research Database (Denmark)

    Nygård, Sune Boris; Vainer, Ben; Nielsen, Signe L

    2016-01-01

    FISH and follow-up data were obtained from 534 patients. TOP1 gain was identified in 27 % using a single-probe enumeration strategy (≥ 4 TOP1 signals per cell), and in 31 % when defined by a TOP1/CEN20 ratio ≥ 1.5. The effect of additional irinotecan was not dependent on TOP1 FISH status. TOP1 m......PURPOSE: Prospective-retrospective assessment of the TOP1 gene copy number and TOP1 mRNA expression as predictive biomarkers for adjuvant irinotecan in stage II/III colon cancer (CC). EXPERIMENTAL DESIGN: Formalin-fixed, paraffin-embedded tissue microarrays were obtained from an adjuvant CC trial...... (PETACC3) where patients were randomized to 5-fluorouracil/folinic acid with or without additional irinotecan. TOP1 copy number status was analyzed by fluorescence in situ hybridization (FISH) using a TOP1/CEN20 dual-probe combination. TOP1 mRNA data were available from previous analyses. RESULTS: TOP1...

  18. mRNA transfection of mouse and human neural stem cell cultures.

    Directory of Open Access Journals (Sweden)

    Samuel McLenachan

    Full Text Available The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

  19. mRNA Transfection of Mouse and Human Neural Stem Cell Cultures

    Science.gov (United States)

    McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J.; Chen, Fred K.

    2013-01-01

    The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages. PMID:24386231

  20. mRNA transfection of mouse and human neural stem cell cultures.

    Science.gov (United States)

    McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J; Chen, Fred K

    2013-01-01

    The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

  1. Myeloperoxidase mRNA detection for lineage determination of leukemic blasts: retrospective analysis.

    Science.gov (United States)

    Crisan, D; Anstett, M J

    1995-07-01

    Myeloperoxidase (MPO) mRNA is an early myeloid marker; its detection in the morphologically and immunophenotypically primitive blasts of acute undifferentiated leukemia (AUL) establishes myeloid lineage and allows reclassification as acute myelogenous leukemia with minimal differentiation (AML-MO). We have previously reported a procedure for MPO mRNA detection by RT-PCR (reverse transcription-polymerase chain reaction) and an adaptation for use of routine hematology smears. This variant procedure allows retrospective analysis of mRNA and is used in the present study to evaluate the lineage of leukemic blasts in seven cases with morphology and cytochemistry consistent with AUL. All hematology smears used in this study were air-dried, unstained or Wright-stained and stored at room temperature for periods varying between 3 days and 2 years. MPO mRNA was detected in six cases, establishing the myeloid lineage of the blasts and the diagnosis of AML-MO. In the remaining case, the blasts were MPO mRNA negative, confirming the diagnosis of AUL. The RT-PCR procedure for retrospective mRNA analysis is useful in the clinical setting, due to its high specificity and sensitivity, speed (less than 24 h), safety (no radioactivity) and convenient use of routine hematology smears; it is particularly attractive in clinical situations when fresh or frozen specimens are no longer available at the time when the need for molecular diagnostics becomes apparent.

  2. Comparison of the performance in detection of HPV infections between the high-risk HPV genotyping real time PCR and the PCR-reverse dot blot assays.

    Science.gov (United States)

    Zhang, Lahong; Dai, Yibei; Chen, Jiahuan; Hong, Liquan; Liu, Yuhua; Ke, Qiang; Chen, Yiwen; Cai, Chengsong; Liu, Xia; Chen, Zhaojun

    2018-01-01

    A new multiplex real-time PCR assay, the high-risk HPV genotyping real time PCR assay (HR HPV RT-PCR), has been developed to detect 15 high-risk HPV types with respective viral loads. In this report, a total of 684 cervical specimens from women diagnosed with vaginitis were assessed by the HR HPV RT-PCR and the PCR reaction and reverse dot blot (PCR-RDB) assays, using a PCR-sequencing method as a reference standard. A total coincidence of 97.7% between the HR HPV RT PCR and the PCR-RDB assays was determined with a Kappa value of 0.953. The HR HPV RT PCR assay had sensitivity, specificity, and concordance rates (accuracy) of 99.7%, 99.7%, and 99.7%, respectively, as confirmed by PCR-sequencing, while the PCR-RDB assay had respective rates of 98.8%, 97.1%, and 98.0%. The overall rate of HPV infection, determined by PCR-sequencing, in women diagnosed with vaginitis was 49.85%, including 36.26% of single infection and 13.6% of multiple infections. The most common infections among the 15 high-risk HPV types in women diagnosed with vaginitis were HPV-52, HPV-16, and HPV-58, with a total detection rate of 10.23%, 7.75%, and 5.85%, respectively. We conclude that the HR HPV RT PCR assay exhibits better clinical performance than the PCR-RDB assay, and is an ideal alternative method for HPV genotyping. In addition, the HR HPV RT PCR assay provides HPV DNA viral loads, and could serve as a quantitative marker in the diagnosis and treatment of single and multiple HPV infections. © 2017 Wiley Periodicals, Inc.

  3. Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment

    Directory of Open Access Journals (Sweden)

    Tetsuya Okuda

    2017-02-01

    Full Text Available Protein modification by O-linked N-acetylglucosamine (O-GlcNAcylation is one of the post transcriptional modifications occurring on cellular proteins. This paper provides a data set relating to the O-GlcNAcylation of cellular proteins detected by RL2 and CTD110.6 antibodies, which are commonly used for detection of protein O-GlcNAcylation, in 2-deoxy-d-glucose (2DG-treated human teratocarcinoma NCCIT cells in support of the research article entitled “A novel, promoter-based, target-specific assay identifies 2-deoxy-d-glucose as an inhibitor of globotriaosylceramide biosynthesis” (Okuda et al., 2009 [1]. The main article described a suppressive effect of 2DG on an Sp1 target gene in NCCIT cells and discussed the relationship between the effect of 2DG and O-GlcNAcylation status of Sp1. The data in this paper complements this relationship by Western blotting and clearly showed that the 2DG treatment increased O-GlcNAcylation of cellular proteins in NCCIT cells, whereas the RL2 and CTD110.6 epitopes were detected in a different manner. The RL2 epitope was detected on Sp1 during 2DG treatment, and the level was transiently increased at 24 h. In contrast, the CTD110.6 epitope became detectable on Sp1 over 72 h after 2DG treatment, and then the other proteins containing CTD110.6 epitopes also appeared in the cell lysates and the anti-Sp1 antibody precipitates.

  4. Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

    International Nuclear Information System (INIS)

    Okita, Naoyuki; Ashizawa, Daisuke; Ohta, Ryo; Abe, Hideaki; Tanuma, Sei-ichi

    2010-01-01

    Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V max ) and the michaelis constant (k m ) of PARG reaction were 4.46 μM and 128.33 μmol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 μM. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.

  5. HIV-1/2 indeterminate Western blot results: follow-up of asymptomatic blood donors in Belo Horizonte, Minas Gerais, Brazil

    Directory of Open Access Journals (Sweden)

    CARNEIRO-PROIETTI A.B.F.

    1999-01-01

    Full Text Available The clinical and public health importance of indeterminate results in HIV-1/2 testing is still difficult to evaluate in volunteer blood donors. At Fundação Hemominas, HIV-1/2 ELISA is used as the screening test and, if reactive, is followed by Western blot (WB. We have evaluated 84 blood donors who had repeatedly reactive ELISA tests for HIV-1/2, but indeterminate WB results. Sixteen of the 84 donors (19.0% had history of sexually transmitted diseases; 18/84 (21.4% informed receiving or paying for sex; 3/84 (3.6% had homosexual contact; 2/26 women (7.6% had past history of multiple illegal abortions and 3/84 (3.6% had been previously transfused. Four out of 62 donors (6.5% had positive anti-nuclear factor (Hep2, with titles up to 1:640. Parasitological examination of the stool revealed eggs of S. mansoni in 4/62 (6.4% donors and other parasites in 8/62 (12.9%. Five (5.9% of the subjects presented overt seroconversion for HIV-1/2, 43/84 (51.2% had negative results on the last visit, while 36/84 (42.9% remained WB indeterminate. Although some conditions could be found associated with the HIV-1/2 indeterminate WB results and many donors had past of risky behavior, the significance of the majority of the results remains to be determined.

  6. Accurate and Practical Identification of 20 Fusarium Species by Seven-Locus Sequence Analysis and Reverse Line Blot Hybridization, and an In Vitro Antifungal Susceptibility Study▿†

    Science.gov (United States)

    Wang, He; Xiao, Meng; Kong, Fanrong; Chen, Sharon; Dou, Hong-Tao; Sorrell, Tania; Li, Ruo-Yu; Xu, Ying-Chun

    2011-01-01

    Eleven reference and 25 clinical isolates of Fusarium were subject to multilocus DNA sequence analysis to determine the species and haplotypes of the fusarial isolates from Beijing and Shandong, China. Seven loci were analyzed: the translation elongation factor 1 alpha gene (EF-1α); the nuclear rRNA internal transcribed spacer (ITS), large subunit (LSU), and intergenic spacer (IGS) regions; the second largest subunit of the RNA polymerase gene (RPB2); the calmodulin gene (CAM); and the mitochondrial small subunit (mtSSU) rRNA gene. We also evaluated an IGS-targeted PCR/reverse line blot (RLB) assay for species/haplotype identification of Fusarium. Twenty Fusarium species and seven species complexes were identified. Of 25 clinical isolates (10 species), the Gibberella (Fusarium) fujikuroi species complex was the commonest (40%) and was followed by the Fusarium solani species complex (FSSC) (36%) and the F. incarnatum-F. equiseti species complex (12%). Six FSSC isolates were identified to the species level as FSSC-3+4, and three as FSSC-5. Twenty-nine IGS, 27 EF-1α, 26 RPB2, 24 CAM, 18 ITS, 19 LSU, and 18 mtSSU haplotypes were identified; 29 were unique, and haplotypes for 24 clinical strains were novel. By parsimony informative character analysis, the IGS locus was the most phylogenetically informative, and the rRNA gene regions were the least. Results by RLB were concordant with multilocus sequence analysis for all isolates. Amphotericin B was the most active drug against all species. Voriconazole MICs were high (>8 μg/ml) for 15 (42%) isolates, including FSSC. Analysis of larger numbers of isolates is required to determine the clinical utility of the seven-locus sequence analysis and RLB assay in species classification of fusaria. PMID:21389150

  7. Discordant human T-lymphotropic virus screening with Western blot confirmation: evaluation of the dual-test algorithm for US blood donations.

    Science.gov (United States)

    Stramer, Susan L; Townsend, Rebecca L; Foster, Gregory A; Johnson, Ramona; Weixlmann, Barbara; Dodd, Roger Y

    2018-03-01

    Human T-lymphotropic virus (HTLV) blood donation screening has used a dual-testing algorithm beginning with either a chemiluminescent immunoassay or enzyme-linked immunosorbent screening assay (ELISA). Before the availability of a licensed HTLV supplemental assay, repeat-reactive (RR) samples on a first assay (Assay 1) were retested with a second screening assay (Assay 2). Donors with RR results by Assay 2 were deferred from blood donation and further tested using an unlicensed supplemental test to confirm reactivity while nonreactive (NR) donors remained eligible for donation until RR on a subsequent donation. This "dual-test" algorithm was replaced in May 2016 with the requirement that all RRs by Assay 1 be further tested by a licensed HTLV supplemental test (Western blot [WB]). In this study, we have requalified the dual-test algorithm using the available licensed HTLV WB. We tested 100 randomly selected HTLV RRs on screening Assay 1 (Abbott PRISM chemiluminescent immunoassay) but NR on screening Assay 2 (Avioq ELISA) by a Food and Drug Administration-licensed WB (MP Biomedicals) to ensure that no confirmed positives were among those that were RR by Assay 1 but NR by Assay 2. Of the 100 samples evaluated, 79 of 100 were WB seronegative, 21 of 100 indeterminate, and 0 of 100 seropositive. Of the 79 of 100 seronegative specimens, 73 of 79 did not express any bands on WB. We demonstrated that none of the 100 samples RR on Assay 1 but NR on Assay 2 were confirmed positive. This algorithm prevents such donors from requiring further testing and from being deferred. © 2018 AABB.

  8. Low Proviral Load is Associated with Indeterminate Western Blot Patterns in Human T-Cell Lymphotropic Virus Type 1 Infected Individuals: Could Punctual Mutations be Related?

    Directory of Open Access Journals (Sweden)

    Camila Cánepa

    2015-10-01

    Full Text Available Background: indeterminate Western blot (WB patterns are a major concern for diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1 infection, even in non-endemic areas. Objectives: (a to define the prevalence of indeterminate WB among different populations from Argentina; (b to evaluate if low proviral load (PVL is associated with indeterminate WB profiles; and (c to describe mutations in LTR and tax sequence of these cases. Results: Among 2031 samples, 294 were reactive by screening. Of them, 48 (16.3% were WB indeterminate and of those 15 (31.3% were PCR+. Quantitative real-time PCR (qPCR was performed to 52 HTLV-1+ samples, classified as Group 1 (G1: 25 WB+ samples from individuals with pathologies; Group 2 (G2: 18 WB+ samples from asymptomatic carriers (AC; and Group 3 (G3: 9 seroindeterminate samples from AC. Median PVL was 4.78, 2.38, and 0.15 HTLV-1 copies/100 PBMCs, respectively; a significant difference (p=0.003 was observed. Age and sex were associated with PVL in G1 and G2, respectively. Mutations in the distal and central regions of Tax Responsive Elements (TRE 1 and 2 of G3 were observed, though not associated with PVL.The 8403A>G mutation of the distal region, previously related to high PVL, was absent in G3 but present in 50% of WB+ samples (p = 0.03. Conclusions: indeterminateWBresults confirmed later as HTLV-1 positive may be associated with low PVL levels. Mutations in LTR and tax are described; their functional relevance remains to be determined.

  9. [Analytic study of dot blotting for the detection of anti-Jo-1, anti-M2, anti-ribosomes and anti-LKM].

    Science.gov (United States)

    Huguet, S; Sghiri, R; Ballot, E; Johanet, C

    2004-01-01

    The Cyto-Dot 4 HM043 kit commercialised by BMD, has replaced the Cyto-Dot HM010 kit that allowed three auto-antibodies detection (anti-Jo-1, anti-M2 and anti-ribosomal protein). Detection of anti-LKM1 auto-antibody was added. These four auto-antibodies have in common only the intracytoplasmic localisation of their respective antigen. The aim of our study was to evaluate this new kit using 104 sera and to compare our results with reference techniques (indirect immunofluorescence IF for anti-M2, anti-ribosomal protein and anti-LKM1, double immunodiffusion ID for anti-Jo-1 and anti-LKM1, western blotting WB for anti-M2) and with Cyto-Dot HM010. The one hundred and four sera were divided into five groups: Group I (n = 12) with anti-Jo-1 detected by ID; Group II (n = 28) with 26 anti-M2 positive by IF and WB, 2 anti-M2 positive only by WB; Group III (n = 10) with anti-ribosomal protein detected by IF 5 of which precipitated by ID; Group IV (n = 32) with anti-LKM1 by IF and ID divided into 18 AIH2 and 14 HCV; Group V (n = 22) consisting of 14 healthy individuals and 8 patients with hypergammaglobulinemia. Results of this study are similar to those of Cyto-Dot HM010 for the three auto-antibodies already in use. Cyto-Dot 4 is a very good anti-LKM1 confirmation method as it is ID. Copyright John Libbey Eurotext 2003.

  10. Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

    International Nuclear Information System (INIS)

    Lazarus, Kyren A.; Zhao, Zhe; Knower, Kevin C.; To, Sarah Q.; Chand, Ashwini L.; Clyne, Colin D.

    2013-01-01

    Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E 2 ), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E 2 , showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E 2 treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer

  11. Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Lazarus, Kyren A. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122 (Australia); Zhao, Zhe; Knower, Kevin C. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); To, Sarah Q. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia); Chand, Ashwini L. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Clyne, Colin D., E-mail: Colin.clyne@princehenrys.org [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia)

    2013-08-30

    Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.

  12. Fluorescent nanoparticles for intracellular sensing: A review

    International Nuclear Information System (INIS)

    Ruedas-Rama, Maria J.; Walters, Jamie D.; Orte, Angel; Hall, Elizabeth A.H.

    2012-01-01

    Highlights: ► Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. ► Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. ► Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

  13. Fluorescent nanoparticles for intracellular sensing: A review

    Energy Technology Data Exchange (ETDEWEB)

    Ruedas-Rama, Maria J., E-mail: mjruedas@ugr.esmailto [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Walters, Jamie D. [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, UK CB2 1QT (United Kingdom); Orte, Angel [Department of Physical Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071, Granada (Spain); Hall, Elizabeth A.H., E-mail: lisa.hall@biotech.cam.ac.uk [Department of Chemical Engineering and Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT (United Kingdom)

    2012-11-02

    Highlights: Black-Right-Pointing-Pointer Analytical applications of fluorescent nanoparticles (NPs) in intracellular sensing. Black-Right-Pointing-Pointer Critical review on performance of QDots, metal NPs, silica NPs, and polymer NPs. Black-Right-Pointing-Pointer Highlighted potential of fluorescence lifetime imaging microscopy (FLIM). - Abstract: Fluorescent nanoparticles (NPs), including semiconductor NPs (Quantum Dots), metal NPs, silica NPs, polymer NPs, etc., have been a major focus of research and development during the past decade. The fluorescent nanoparticles show unique chemical and optical properties, such as brighter fluorescence, higher photostability and higher biocompatibility, compared to classical fluorescent organic dyes. Moreover, the nanoparticles can also act as multivalent scaffolds for the realization of supramolecular assemblies, since their high surface to volume ratio allow distinct spatial domains to be functionalized, which can provide a versatile synthetic platform for the implementation of different sensing schemes. Their excellent properties make them one of the most useful tools that chemistry has supplied to biomedical research, enabling the intracellular monitoring of many different species for medical and biological purposes. In this review, we focus on the developments and analytical applications of fluorescent nanoparticles in chemical and biological sensing within the intracellular environment. The review also points out the great potential of fluorescent NPs for fluorescence lifetime imaging microscopy (FLIM). Finally, we also give an overview of the current methods for delivering of fluorescent NPs into cells, where critically examine the benefits and liabilities of each strategy.

  14. Three-dimensional fluorescence lifetime tomography

    International Nuclear Information System (INIS)

    Godavarty, Anuradha; Sevick-Muraca, Eva M.; Eppstein, Margaret J.

    2005-01-01

    Near-infrared fluorescence tomography using molecularly targeted lifetime-sensitive, fluorescent contrast agents have applications for early-stage cancer diagnostics. Yet, although the measurement of fluorescent lifetime imaging microscopy (FLIM) is extensively used in microscopy and spectroscopy applications, demonstration of fluorescence lifetime tomography for medical imaging is limited to two-dimensional studies. Herein, the feasibility of three-dimensional fluorescence-lifetime tomography on clinically relevant phantom volumes is established, using (i) a gain-modulated intensified charge coupled device (CCD) and modulated laser diode imaging system, (ii) two fluorescent contrast agents, e.g., Indocyanine green and 3-3'-Diethylthiatricarbocyanine iodide differing in their fluorescence lifetime by 0.62 ns, and (iii) a two stage approximate extended Kalman filter reconstruction algorithm. Fluorescence measurements of phase and amplitude were acquired on the phantom surface under different target to background fluorescence absorption (70:1, 100:1) and fluorescence lifetime (1:1, 2.1:1) contrasts at target depths of 1.4-2 cm. The Bayesian tomography algorithm was employed to obtain three-dimensional images of lifetime and absorption owing to the fluorophores

  15. Multispectral open-air intraoperative fluorescence imaging.

    Science.gov (United States)

    Behrooz, Ali; Waterman, Peter; Vasquez, Kristine O; Meganck, Jeff; Peterson, Jeffrey D; Faqir, Ilias; Kempner, Joshua

    2017-08-01

    Intraoperative fluorescence imaging informs decisions regarding surgical margins by detecting and localizing signals from fluorescent reporters, labeling targets such as malignant tissues. This guidance reduces the likelihood of undetected malignant tissue remaining after resection, eliminating the need for additional treatment or surgery. The primary challenges in performing open-air intraoperative fluorescence imaging come from the weak intensity of the fluorescence signal in the presence of strong surgical and ambient illumination, and the auto-fluorescence of non-target components, such as tissue, especially in the visible spectral window (400-650 nm). In this work, a multispectral open-air fluorescence imaging system is presented for translational image-guided intraoperative applications, which overcomes these challenges. The system is capable of imaging weak fluorescence signals with nanomolar sensitivity in the presence of surgical illumination. This is done using synchronized fluorescence excitation and image acquisition with real-time background subtraction. Additionally, the system uses a liquid crystal tunable filter for acquisition of multispectral images that are used to spectrally unmix target fluorescence from non-target auto-fluorescence. Results are validated by preclinical studies on murine models and translational canine oncology models.

  16. Red and Green Fluorescence from Oral Biofilms.

    Science.gov (United States)

    Volgenant, Catherine M C; Hoogenkamp, Michel A; Krom, Bastiaan P; Janus, Marleen M; Ten Cate, Jacob M; de Soet, Johannes J; Crielaard, Wim; van der Veen, Monique H

    2016-01-01

    Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation) as compared to the sucrose grown biofilms (cariogenic simulation). Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red) were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries.

  17. Red and Green Fluorescence from Oral Biofilms.

    Directory of Open Access Journals (Sweden)

    Catherine M C Volgenant

    Full Text Available Red and green autofluorescence have been observed from dental plaque after excitation by blue light. It has been suggested that this red fluorescence is related to caries and the cariogenic potential of dental plaque. Recently, it was suggested that red fluorescence may be related to gingivitis. Little is known about green fluorescence from biofilms. Therefore, we assessed the dynamics of red and green fluorescence in real-time during biofilm formation. In addition, the fluorescence patterns of biofilm formed from saliva of eight different donors are described under simulated gingivitis and caries conditions. Biofilm formation was analysed for 12 hours under flow conditions in a microfluidic BioFlux flow system with high performance microscopy using a camera to allow live cell imaging. For fluorescence images dedicated excitation and emission filters were used. Both green and red fluorescence were linearly related with the total biomass of the biofilms. All biofilms displayed to some extent green and red fluorescence, with higher red and green fluorescence intensities from biofilms grown in the presence of serum (gingivitis simulation as compared to the sucrose grown biofilms (cariogenic simulation. Remarkably, cocci with long chain lengths, presumably streptococci, were observed in the biofilms. Green and red fluorescence were not found homogeneously distributed within the biofilms: highly fluorescent spots (both green and red were visible throughout the biomass. An increase in red fluorescence from the in vitro biofilms appeared to be related to the clinical inflammatory response of the respective saliva donors, which was previously assessed during an in vivo period of performing no-oral hygiene. The BioFlux model proved to be a reliable model to assess biofilm fluorescence. With this model, a prediction can be made whether a patient will be prone to the development of gingivitis or caries.

  18. Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency

    International Nuclear Information System (INIS)

    Ruijter, Jan M.; Hoff, Maurice J. B. van den; Lorenz, Peter; Tuomi, Jari M.; Hecker, Michael

    2014-01-01

    The analysis of quantitative PCR data usually does not take into account the fact that the increase in fluorescence depends on the monitoring chemistry, the input of ds-DNA or ss-cDNA, and the directionality of the targeting of probes or primers. The monitoring chemistries currently available can be categorized into six groups: (A) DNA-binding dyes; (B) hybridization probes; (C) hydrolysis probes; (D) LUX primers; (E) hairpin primers; and (F) the QZyme system. We have determined the kinetics of the increase in fluorescence for each of these groups with respect to the input of both ds-DNA and ss-cDNA. For the latter, we also evaluated mRNA and cDNA targeting probes or primers. This analysis revealed three situations. Hydrolysis probes and LUX primers, compared to DNA-binding dyes, do not require a correction of the observed quantification cycle. Hybridization probes and hairpin primers require a correction of −1 cycle (dubbed C-lag), while the QZyme system requires the C-lag correction and an efficiency-dependent C-shift correction. A PCR efficiency value can be derived from the relative increase in fluorescence in the exponential phase of the amplification curve for all monitoring chemistries. In case of hydrolysis probes, LUX primers and hairpin primers, however, this should be performed after cycle 12, and for the QZyme system after cycle 19, to keep the overestimation of the PCR efficiency below 0.5 %. (author)

  19. Fluorescence imaging spectrometer optical design

    Science.gov (United States)

    Taiti, A.; Coppo, P.; Battistelli, E.

    2015-09-01

    The optical design of the FLuORescence Imaging Spectrometer (FLORIS) studied for the Fluorescence Explorer (FLEX) mission is discussed. FLEX is a candidate for the ESA's 8th Earth Explorer opportunity mission. FLORIS is a pushbroom hyperspectral imager foreseen to be embarked on board of a medium size satellite, flying in tandem with Sentinel-3 in a Sun synchronous orbit at a height of about 815 km. FLORIS will observe the vegetation fluorescence and reflectance within a spectral range between 500 and 780 nm. Multi-frames acquisitions on matrix detectors during the satellite movement will allow the production of 2D Earth scene images in two different spectral channels, called HR and LR with spectral resolution of 0.3 and 2 nm respectively. A common fore optics is foreseen to enhance by design the spatial co-registration between the two spectral channels, which have the same ground spatial sampling (300 m) and swath (150 km). An overlapped spectral range between the two channels is also introduced to simplify the spectral coregistration. A compact opto-mechanical solution with all spherical and plane optical elements is proposed, and the most significant design rationales are described. The instrument optical architecture foresees a dual Babinet scrambler, a dioptric telescope and two grating spectrometers (HR and LR), each consisting of a modified Offner configuration. The developed design is robust, stable vs temperature, easy to align, showing very high optical quality along the whole field of view. The system gives also excellent correction for transverse chromatic aberration and distortions (keystone and smile).

  20. UCP2 mRNA expression is dependent on glucose metabolism in pancreatic islets

    International Nuclear Information System (INIS)

    Dalgaard, Louise T.

    2012-01-01

    Highlights: ► UCP2 mRNA levels are decreased in islets of Langerhans from glucokinase deficient mice. ► UCP2 mRNA up-regulation by glucose is dependent on glucokinase. ► Absence of UCP2 increases GSIS of glucokinase heterozygous pancreatic islets. ► This may protect glucokinase deficient mice from hyperglycemic damages. -- Abstract: Uncoupling Protein 2 (UCP2) is expressed in the pancreatic β-cell, where it partially uncouples the mitochondrial proton gradient, decreasing both ATP-production and glucose-stimulated insulin secretion (GSIS). Increased glucose levels up-regulate UCP2 mRNA and protein levels, but the mechanism for UCP2 up-regulation in response to increased glucose is unknown. The aim was to examine the effects of glucokinase (GK) deficiency on UCP2 mRNA levels and to characterize the interaction between UCP2 and GK with regard to glucose-stimulated insulin secretion in pancreatic islets. UCP2 mRNA expression was reduced in GK+/− islets and GK heterozygosity prevented glucose-induced up-regulation of islet UCP2 mRNA. In contrast to UCP2 protein function UCP2 mRNA regulation was not dependent on superoxide generation, but rather on products of glucose metabolism, because MnTBAP, a superoxide dismutase mimetic, did not prevent the glucose-induced up-regulation of UCP2. Glucose-stimulated insulin secretion was increased in UCP2−/− and GK+/− islets compared with GK+/− islets and UCP2 deficiency improved glucose tolerance of GK+/− mice. Accordingly, UCP2 deficiency increased ATP-levels of GK+/− mice. Thus, the compensatory down-regulation of UCP2 is involved in preserving the insulin secretory capacity of GK mutant mice and might also be implicated in limiting disease progression in MODY2 patients.

  1. Protein functional features are reflected in the patterns of mRNA translation speed.

    Science.gov (United States)

    López, Daniel; Pazos, Florencio

    2015-07-09

    The degeneracy of the genetic code makes it possible for the same amino acid string to be coded by different messenger RNA (mRNA) sequences. These "synonymous mRNAs" may differ largely in a number of aspects related to their overall translational efficiency, such as secondary structure content and availability of the encoded transfer RNAs (tRNAs). Consequently, they may render different yields of the translated polypeptides. These mRNA features related to translation efficiency are also playing a role locally, resulting in a non-uniform translation speed along the mRNA, which has been previously related to some protein structural features and also used to explain some dramatic effects of "silent" single-nucleotide-polymorphisms (SNPs). In this work we perform the first large scale analysis of the relationship between three experimental proxies of mRNA local translation efficiency and the local features of the corresponding encoded proteins. We found that a number of protein functional and structural features are reflected in the patterns of ribosome occupancy, secondary structure and tRNA availability along the mRNA. One or more of these proxies of translation speed have distinctive patterns around the mRNA regions coding for certain protein local features. In some cases the three patterns follow a similar trend. We also show specific examples where these patterns of translation speed point to the protein's important structural and functional features. This support the idea that the genome not only codes the protein functional features as sequences of amino acids, but also as subtle patterns of mRNA properties which, probably through local effects on the translation speed, have some consequence on the final polypeptide. These results open the possibility of predicting a protein's functional regions based on a single genomic sequence, and have implications for heterologous protein expression and fine-tuning protein function.

  2. ZMS regulation of M2 muscarinic receptor mRNA stability requires protein factor

    International Nuclear Information System (INIS)

    Zhang Yongfang; Xia Zongqin; Hu Ya'er

    2010-01-01

    Aim The aim of this work is to study the elevation mechanism of ZMS on muscarinic M2 receptor mRNA expression. Methods Actinomycin D was added to cultured CHOm2 cells to stop the de novo synthesis of M2 receptor mRNA and samples were taken at various times to determine the time course of mRNA of M2 receptor with real-time quantitative RT-PCR. Half-life of M2 receptor mRNA and the effect of ZMS on the half-life was obtained from the slope of the exponential curves. Cycloheximide was added at 4 h prior to and 24 h after the addition of ZMS to examine the effect of de novo protein synthesis on the action of ZMS. Results The half-life of m2 mRNA was prolonged by ZMS treatment without cycloheximide (4.75±0.54 h and 2.13 h±0.23 h for ZMS and vehicle treated groups, respectively, P<0.05). When cycloheximide was added to the culture medium 4h prior to the addition of ZMS, the effect of ZMS in prolonging the half-life of m2 mRNA disappeared (3.06 h±0.23 h and 3.00 h±l.20 h for cells with and without ZMS, respectively). However, when the ZMS was added to the medium 24h prior to the addition of cycloheximide, the action of ZMS was not abolished by cycloheximide (half-life was 5.43 h±1.13 h and 2.46 h±0.09 h for cells with and without ZMS, respectively). Conclusion These data suggest that de novo protein synthesis was required for the increase in M2 mRNA stability induced by ZMS. (authors)

  3. The ribosome structure controls and directs mRNA entry, translocation and exit dynamics

    International Nuclear Information System (INIS)

    Kurkcuoglu, Ozge; Doruker, Pemra; Jernigan, Robert L; Sen, Taner Z; Kloczkowski, Andrzej

    2008-01-01

    The protein-synthesizing ribosome undergoes large motions to effect the translocation of tRNAs and mRNA; here, the domain motions of this system are explored with a coarse-grained elastic network model using normal mode analysis. Crystal structures are used to construct various model systems of the 70S complex with/without tRNA, elongation factor Tu and the ribosomal proteins. Computed motions reveal the well-known ratchet-like rotational motion of the large subunits, as well as the head rotation of the small subunit and the high flexibility of the L1 and L7/L12 stalks, even in the absence of ribosomal proteins. This result indicates that these experimentally observed motions during translocation are inherently controlled by the ribosomal shape and only partially dependent upon GTP hydrolysis. Normal mode analysis further reveals the mobility of A- and P-tRNAs to increase in the absence of the E-tRNA. In addition, the dynamics of the E-tRNA is affected by the absence of the ribosomal protein L1. The mRNA in the entrance tunnel interacts directly with helicase proteins S3 and S4, which constrain the mRNA in a clamp-like fashion, as well as with protein S5, which likely orients the mRNA to ensure correct translation. The ribosomal proteins S7, S11 and S18 may also be involved in assuring translation fidelity by constraining the mRNA at the exit site of the channel. The mRNA also interacts with the 16S 3' end forming the Shine–Dalgarno complex at the initiation step; the 3' end may act as a 'hook' to reel in the mRNA to facilitate its exit

  4. Expression and clinicopathological significance of Mel-18 and Bmi-1 mRNA in gastric carcinoma.

    Science.gov (United States)

    Lu, You-Wei; Li, Jin; Guo, Wei-Jian

    2010-11-08

    The Polycomb group (PcG) genes are a class of regulators responsible for maintaining homeotic gene expression throughout cell division. PcG expression is deregulated in some types of human cancer. Both Bmi-1 and Mel-18 are of the key PcG proteins. We investigate the expression and clinicopathological roles of Mel-18 and Bmi-1 mRNA in gastric cancer. The expression of Mel-18 and Bmi-1 in a series of 71 gastric cancer tissues and paired normal mucosal tissues distant from the tumorous lesion was assayed by quantitative real time RT-PCR. The correlation between Mel-18 and Bmi-1 mRNA expression, and between Mel-18 or Bmi-1 mRNA level and clinicopathological characteristics were analyzed. Expression of Mel-18 and Bmi-1 genes was variably detected, but overexpression of Bmi-1 mRNA and decreased expression of Mel-18 mRNA were the most frequent alteration. In addition, the expression of Bmi-1 and Mel-18 mRNA inversely correlates in gastric tumors. Moreover, a significant positive correlation between Bmi-1 overexpression and tumor size, depth of invasion, or lymph node metastasis, and a significant negative correlation between Mel-18 low-expression with lymph node metastasis or the clinical stage were observed. Our data suggest that Mel-18 and Bmi-1 may play crucial but opposite roles in gastric cancer. Decreased Mel-18 and increased Bmi-1 mRNA expression was associated with the carcinogenesis and progression of gastric cancer. It is possible to list Bmi-1 and Mel-18 as biomarkers for predicting the prognosis of gastric cancer.

  5. Association of chemerin mRNA expression in human epicardial adipose tissue with coronary atherosclerosis

    Directory of Open Access Journals (Sweden)

    Wang Linjie

    2011-10-01

    Full Text Available Abstract Background Growing evidence suggests that epicardial adipose tissue (EAT may play a key role in the pathogenesis and development of coronary artery disease (CAD by producing several inflammatory adipokines. Chemerin, a novel adipokine, has been reported to be involved in regulating immune responses and glucolipid metabolism. Given these properties, chemerin may provide an interesting link between obesity, inflammation and atherosclerosis. In this study, we sought to determine the relationship of chemerin expression in EAT and the severity of coronary atherosclerosis in Han Chinese patients. Methods Serums and adipose tissue biopsies (epicardial and thoracic subcutaneous were obtained from CAD (n = 37 and NCAD (n = 16 patients undergoing elective cardiac surgery. Gensini score was used to assess the severity of CAD. Serum levels of chemerin, adiponectin and insulin were measured by ELISA. Chemerin protein expression in adipose tissue was detected by immunohistochemistry. The mRNA levels of chemerin, chemR23, adiponectin and TNF-alpha in adipose tissue were detected by RT-PCR. Results We found that EAT of CAD group showed significantly higher levels of chemerin and TNF-alpha mRNA, and significantly lower level of adiponectin mRNA than that of NCAD patients. In CAD group, significantly higher levels of chemerin mRNA and protein were observed in EAT than in paired subcutaneous adipose tissue (SAT, whereas such significant difference was not found in NCAD group. Chemerin mRNA expression in EAT was positively correlated with Gensini score (r = 0.365, P P P P P P P > 0.05. Conclusions The expressions of chemerin mRNA and protein are significantly higher in EAT from patients with CAD in Han Chinese patients. Furthermore, the severity of coronary atherosclerosis is positive correlated with the level of chemerin mRNA in EAT rather than its circulating level.

  6. mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

    Science.gov (United States)

    Kropp, Jenna; Khatib, Hasan

    2015-01-01

    In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

  7. Consequences of metaphase II oocyte cryopreservation on mRNA content.

    Science.gov (United States)

    Chamayou, S; Bonaventura, G; Alecci, C; Tibullo, D; Di Raimondo, F; Guglielmino, A; Barcellona, M L

    2011-04-01

    We studied the consequences of freezing/thawing processes on mRNA contents in MII oocytes after slow-freezing/rapid thawing (SF/RT) and vitrification/warming (V/W) protocols, and compared the results to fresh MII oocytes. We quantified the nuclear transcript mRNA responsible for the translation of proteins belonging either to trans-regulatory protein family or to functional structural proteins such as proteins involved in DNA structural organization (NAP1L1, TOP1, H1F0H1), chromosomal structure maintenance (SMC, SCC3, RAD21, SMC1A, SMC1B, STAG3, REC8), mitochondrial energetic pathways (ATP5GJ, SDHC), cell cycle regulation and processes (CLTA, MAPK6, CKS2) and staminal cell potency-development competence stage (DPPA3, OCT4, FOXJ2). Surplus MII oocytes were donated from patients in IVF cycles and divided in three groups of 15 oocytes. Group 1 was comprised of non-cryopreserved oocytes and Groups 2 and 3 underwent SF/RT and V/W procedures, respectively. There was an overall decrease of mRNA extracted from cryopreserved oocytes compared to control group. Only 39.4% of mRNA content were preserved after SF/RT while 63.3% of mRNA content were maintained after V/W. Oocyte cryopreservation is associated with molecular injury associated with the decrease of stored mRNA. However the V/W protocol is more conservative than SF/RT resulting in a level of mRNA sufficient to maintain biologic functions in the subsequent fertilized oocyte. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. DNA nanotechnology and fluorescence applications.

    Science.gov (United States)

    Schlichthaerle, Thomas; Strauss, Maximilian T; Schueder, Florian; Woehrstein, Johannes B; Jungmann, Ralf

    2016-06-01

    Structural DNA nanotechnology allow researchers to use the unique molecular recognition properties of DNA strands to construct nanoscale objects with almost arbitrary complexity in two and three dimensions. Abstracted as molecular breadboards, DNA nanostructures enable nanometer-precise placement of guest molecules such as proteins, fluorophores, or nanoparticles. These assemblies can be used to study biological phenomena with unprecedented control over number, spacing, and molecular identity. Here, we give a general introduction to structural DNA nanotechnology and more specifically discuss applications of DNA nanostructures in the field of fluorescence and plasmonics. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer.

    Science.gov (United States)

    Blackwell, Daniel J; Zak, Taylor J; Robia, Seth L

    2016-09-20

    The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. A set of enhanced green fluorescent protein concatemers for quantitative determination of nuclear localization signal strength.

    Science.gov (United States)

    Böhm, Jennifer; Thavaraja, Ramya; Giehler, Susanne; Nalaskowski, Marcus M

    2017-09-15

    Regulated transport of proteins between nucleus and cytoplasm is an important process in the eukaryotic cell. In most cases, active nucleo-cytoplasmic protein transport is mediated by nuclear localization signal (NLS) and/or nuclear export signal (NES) motifs. In this study, we developed a set of vectors expressing enhanced GFP (EGFP) concatemers ranging from 2 to 12 subunits (2xEGFP to 12xEGFP) for analysis of NLS strength. As shown by in gel GFP fluorescence analysis and αGFP Western blotting, EGFP concatemers are expressed as fluorescent full-length proteins in eukaryotic cells. As expected, nuclear localization of concatemeric EGFPs decreases with increasing molecular weight. By oligonucleotide ligation this set of EGFP concatemers can be easily fused to NLS motifs. After determination of intracellular localization of EGFP concatemers alone and fused to different NLS motifs we calculated the size of a hypothetic EGFP concatemer showing a defined distribution of EGFP fluorescence between nucleus and cytoplasm (n/c ratio = 2). Clear differences of the size of the hypothetic EGFP concatemer depending on the fused NLS motif were observed. Therefore, we propose to use the size of this hypothetic concatemer as quantitative indicator for comparing strength of different NLS motifs. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Als2 mRNA splicing variants detected in KO mice rescue severe motor dysfunction phenotype in Als2 knock-down zebrafish.

    Science.gov (United States)

    Gros-Louis, Francois; Kriz, Jasna; Kabashi, Edor; McDearmid, Jonathan; Millecamps, Stéphanie; Urushitani, Makoto; Lin, Li; Dion, Patrick; Zhu, Qinzhang; Drapeau, Pierre; Julien, Jean-Pierre; Rouleau, Guy A

    2008-09-01

    Recessive ALS2 mutations are linked to three related but slightly different neurodegenerative disorders: amyotrophic lateral sclerosis, hereditary spastic paraplegia and primary lateral sclerosis. To investigate the function of the ALS2 encoded protein, we generated Als2 knock-out (KO) mice and zAls2 knock-down zebrafish. The Als2(-/-) mice lacking exon 2 and part of exon 3 developed mild signs of neurodegeneration compatible with axonal transport deficiency. In contrast, zAls2 knock-down zebrafish had severe developmental abnormalities, swimming deficits and motor neuron perturbation. We identified, by RT-PCR, northern and western blotting novel Als2 transcripts in mouse central nervous system. These Als2 transcripts were present in Als2 null mice as well as in wild-type littermates and some rescued the zebrafish phenotype. Thus, we speculate that the newly identified Als2 mRNA species prevent the Als2 KO mice from developing severe neurodegenerative disease and might also regulate the severity of the motor neurons phenotype observed in ALS2 patients.

  12. Enhanced accumulation of Kir4.1 protein, but not mRNA, in a murine model of cuprizone-induced demyelination.

    Science.gov (United States)

    Nakajima, Mitsunari; Kawamura, Takuya; Tokui, Ryuji; Furuta, Kohei; Sugino, Mami; Nakanishi, Masayuki; Okuyama, Satoshi; Furukawa, Yoshiko

    2013-11-06

    Two channel proteins, inwardly rectifying potassium channel 4.1 (Kir4.1) and water channel aquaporin-4 (AQP4), were recently identified as targets of an autoantibody response in patients with multiple sclerosis and neuromyelitis optica, respectively. In the present study, we examined the expression patterns of Kir4.1 and AQP4 in a mouse model of demyelination induced by cuprizone, a copper chelator. Demyelination was confirmed by immunohistochemistry using an anti-proteolipid protein antibody in various brain regions, including the corpus callosum, of cuprizone-fed mice. Activation of microglial and astroglial cells was also confirmed by immunohistochemistry, using an anti-ionized calcium binding adapter molecule and a glial fibrillary acidic protein antibody. Western blot analysis revealed the induction of Kir4.1 protein, but not AQP4, in the cortex of cuprizone-fed mice. Immunohistochemical analysis confirmed the Kir4.1 protein induction in microvessels of the cerebral cortex. Real-time polymerase chain reaction analysis revealed that mRNA levels of Kir4.1 and AQP4 in the cortex did not change during cuprizone administration. These findings suggest that enhanced accumulation of Kir4.1 protein in the brain with an inflammatory condition facilitates the autoantibody formation against Kir4.1 in patients with multiple sclerosis. © 2013 Published by Elsevier B.V.

  13. Metallochaperone for Cu,Zn-superoxide dismutase (CCS) protein but not mRNA is higher in organs from copper-deficient mice and rats.

    Science.gov (United States)

    Prohaska, Joseph R; Broderius, Margaret; Brokate, Bruce

    2003-09-15

    Cu,Zn-superoxide dismutase (SOD1) is an abundant metalloenzyme important in scavenging superoxide ions. Cu-deficient rats and mice have lower SOD1 activity and protein, possibly because apo-SOD1 is degraded faster than holo-SOD1. SOD1 interacts with and requires its metallochaperone CCS for donating copper. We produced dietary Cu deficiency in rodents to determine if the reduction in SOD1 was related to the level of its specific metallochaperone CCS. CCS levels determined by immunoblot were 2- to 3-fold higher in liver, heart, kidney, and brain from male Cu-deficient rats and mice under a variety of conditions. CCS was also higher in livers of Cu-deficient dams. Interestingly, CCS levels in brain of Cu-deficient mice were also higher even though SOD1 activity and protein were not altered, suggesting that the rise in CCS is correlated with altered Cu status rather than a direct result of lower SOD1. A DNA probe specific for rat CCS detected a single transcript by Northern blot hybridization with liver RNA. CCS mRNA levels in mouse and rat liver were not altered by dietary treatment. These results suggest a posttranscriptional mechanism for higher CCS protein when Cu is limiting in the cell, perhaps due to slower protein turnover. Elevation in CCS level is one of the most dramatic alterations in Cu binding proteins accompanying Cu deficiency and may be useful to assess Cu status.

  14. Course of c-myc mRNA expression in the regenerating mouse testis determined by competitive reverse transcriptase polymerase chain reaction.

    Science.gov (United States)

    Amendola, R

    1994-11-01

    The c-myc proto-oncogene is a reliable marker of the "G0-early G1" transition, and its down-regulation is believed to be necessary to obtain cellular differentiation. In murine spermatogenesis, the level of c-myc transcripts does not correlate with the rate of cellular division. Proliferation of supposed staminal spermatogonia to reproduce themselves is induced with a local 5 Gy X-ray dose in 90-day-old C57Bl/6 mice. c-myc quantification by a newly developed competitive reverse transcriptase polymerase chain reaction (RT-PCR) was carried out to follow the expression course of this proto-oncogene. Damage and restoration of spermatogenesis were analyzed at days 3, 6, 9, 10, 13, 30, and 60 after injury by relative testes/body weight determination and histological examination. Proliferative status was determined by histone H3 Northern blot analysis. c-myc mRNA level was 10 times higher after 3 days in the irradiated animals compared to the controls. An increasing number of copies were noted up to 10 days, but promptly decreased to the base level found for irradiated mice from 13 to 60 days. Interestingly, the expression of histone H3 detected S phase only in testes at 60 days from damage.

  15. Fluorescence Imaging of Posterior Spiracles from Second and Third Instars of Forensically Important Chrysomya rufifacies (Diptera: Calliphoridae).

    Science.gov (United States)

    Flores, Danielle; Miller, Amy L; Showman, Angelique; Tobita, Caitlyn; Shimoda, Lori M N; Sung, Carl; Stokes, Alexander J; Tomberlin, Jeffrey K; Carter, David O; Turner, Helen

    2016-11-01

    Entomological protocols for aging blowfly (Diptera: Calliphoridae) larvae to estimate the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analyzing fly larvae differ, most rely on light microscopy, genetic analysis, or, more rarely, electron microscopy. This pilot study sought to improve resolution of larval stage in the forensically important blowfly Chrysomya rufifacies using high-content fluorescence microscopy and biochemical measures of developmental marker proteins. We established fixation and mounting protocols, defined a set of measurable morphometric criteria and captured developmental transitions of 2nd instar to 3rd instar using both fluorescence microscopy and anti-ecdysone receptor Western blot analysis. The data show that these instars can be distinguished on the basis of robust, nonbleaching, autofluorescence of larval posterior spiracles. High-content imaging techniques using confocal microscopy, combined with morphometric and biochemical techniques, may therefore aid forensic entomologists in estimating TOC. © 2016 American Academy of Forensic Sciences.

  16. Fluorescence Imaging of Posterior Spiracles from Second and Third Instars of Forensically-important Chrysomya rufifacies (Diptera: Calliphoridae)*

    Science.gov (United States)

    Flores, Danielle; Miller, Amy L.; Showman, Angelique; Tobita, Caitlyn; Shimoda, Lori M.N.; Sung, Carl; Stokes, Alexander J.; Tomberlin, Jeffrey K.; Carter, David O.; Turner, Helen

    2016-01-01

    Entomological protocols for aging blow fly (Diptera: Calliphoridae) larvae to estimate the time of colonization (TOC) are commonly used to assist in death investigations. While the methodologies for analysing fly larvae differ, most rely on light microscopy, genetic analysis or, more rarely, electron microscopy. This pilot study sought to improve resolution of larval stage in the forensically-important blow fly Chrysomya rufifacies using high-content fluorescence microscopy and biochemical measures of developmental marker proteins. We established fixation and mounting protocols, defined a set of measurable morphometric criteria and captured developmental transitions of 2nd instar to 3rd instar using both fluorescence microscopy and anti-ecdysone receptor Western blot analysis. The data show that these instars can be distinguished on the basis of robust, non-bleaching, autofluorescence of larval posterior spiracles. High content imaging techniques using confocal microscopy, combined with morphometric and biochemical techniques, may therefore aid forensic entomologists in estimating TOC. PMID:27706817

  17. Detection of Counterfeit Tequila by Fluorescence Spectroscopy

    Directory of Open Access Journals (Sweden)

    José Manuel de la Rosa Vázquez

    2015-01-01

    Full Text Available An ultraviolet (UV light induced fluorescence study to discriminate fake tequila from genuine ones is presented. A portable homemade system based on four light emitting diodes (LEDs from 255 to 405 nm and a miniature spectrometer was used. It has been shown that unlike fake and silver tequila, which produce weak fluorescence signal, genuine mixed, rested, and aged tequilas show high fluorescence emission in the range from 400 to 750 nm. The fluorescence intensity grows with aging in 100% agave tequila. Such fluorescence differences can even be observed with naked eyes. The presented results demonstrate that the fluorescence measurement could be a good method to detect counterfeit tequila.

  18. Confocal fluorescence techniques in industrial application

    Science.gov (United States)

    Eggeling, Christian; Gall, Karsten; Palo, Kaupo; Kask, Peet; Brand, Leif

    2003-06-01

    The FCS+plus family of evaluation tools for confocal fluorescence spectroscopy, which was developed during recent years, offers a comprehensive view to a series of fluorescence properties. Originating in fluorescence correlation spectroscopy (FCS) and using similar experimental equipment, a system of signal processing methods such as fluorescence intensity distribution analysis (FIDA) was created to analyze in detail the fluctuation behavior of fluorescent particles within a small area of detection. Giving simultaneous access to molecular parameters like concentration, translational and rotational diffusion, molecular brightness, and multicolor coincidence, this portfolio was enhanced by more traditional techniques of fluorescence lifetime as well as time-resolved anisotropy determination. The cornerstones of the FCS+plus methodology will be shortly described. The inhibition of a phosphatase enzyme activity gives a comprehensive industrial application that demonstrates FCS+plus' versatility and its potential for pharmaceutical drug discovery.

  19. A comparison of the immune parameters of dogs infected with visceral leishmaniasis using Western blot and neutralization techniques Comparação dos parâmetros imunológicos de cães infectados com leishmaniose visceral usando as técnicas de Western blot e neutralização

    Directory of Open Access Journals (Sweden)

    Yeda L. Nogueira

    2007-12-01

    Full Text Available The Western blot technique was used to demonstrate the presence of antibodies in the blood of dogs that presented canine visceral leishmaniasis. This technique was used against some specific molecules present in the lysate of the promastigote form of Leshmania chagasi.Through the association of the results of the Western blot technique with the morphological alterations seen as a result of the serum neutralization technique performed in McCoy cells (which mimetizes the macrophage it was possible to observe the role of some molecules of great relevance in determining the disease in symptomatic dogs as well as that of some other molecules associated with asymptomatic infected dogs that may become transmitters as well as differentiating them as asymptomatic resistant dogs. In the sera analyses carried out during the immunobloting a variation of 9 to 27 immunoreacting bands was observed, which were then compared using Dice's similarity coefficient. In the dendrogram constructed on the basis of the coefficient, 50% similarity was observed among the total number of reagent bands with the promastigote lysate, thus creating five groups. The main difference observed related to the clinical condition of the dogs: symptomatic and asymptomatic dogs were found in separate groups. The asymptomatic group of dogs was distributed in two different places in the dendrogram because they presented two different behavior patterns regarding the cellular morphology in the serum neutralization reaction: the presence or absence of cellular lysis. According to this analysis it is possible to evaluate the immune status and associate it with specific markers observed in the reaction found in the Western blot strips.A técnica de Western blot foi utilizada para demonstrar a presença de anticorpos do soro de cães, que apresentavam leishmaniose visceral canina, contra algumas moléculas específicas no lisado da forma promastigota de Leshmania chagasi.Através da associa

  20. Fluorescence fluctuation spectroscopy (FFS), part A

    CERN Document Server

    Tetin, Sergey

    2013-01-01

    This new volume of Methods in Enzymology continues the legacy of this premier serial by containing quality chapters authored by leaders in the field. This volume covers Fluorescence Fluctuation SpectroscopyContains chapters on such topics as Time-integrated fluorescence cumulant analysis, Pulsed Interleaved Excitation, and raster image correlation spectroscopy and number and brightness analysis.Continues the legacy of this premier serial with quality chapters authored by leaders in the fieldCovers fluorescence fluctuation spectroscopyContains chapte