Sample records for activation western blot

  1. Western blotting using capillary electrophoresis.

    PubMed

    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.

  2. Western blotting.

    PubMed

    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.

  3. The western blot

    USDA-ARS?s Scientific Manuscript database

    Western blotting is a technique that involves the separation of proteins by gel electrophoresis, their blotting or transfer to a membrane, and selective immunodetection of an immobilized antigen. This is an important and routine method for protein analysis that depends on the specificity of antibod...

  4. Protein purification and analysis: next generation Western blotting techniques.

    PubMed

    Mishra, Manish; Tiwari, Shuchita; Gomes, Aldrin V

    2017-11-01

    Western blotting is one of the most commonly used techniques in molecular biology and proteomics. Since western blotting is a multistep protocol, variations and errors can occur at any step reducing the reliability and reproducibility of this technique. Recent reports suggest that a few key steps, such as the sample preparation method, the amount and source of primary antibody used, as well as the normalization method utilized, are critical for reproducible western blot results. Areas covered: In this review, improvements in different areas of western blotting, including protein transfer and antibody validation, are summarized. The review discusses the most advanced western blotting techniques available and highlights the relationship between next generation western blotting techniques and its clinical relevance. Expert commentary: Over the last decade significant improvements have been made in creating more sensitive, automated, and advanced techniques by optimizing various aspects of the western blot protocol. New methods such as single cell-resolution western blot, capillary electrophoresis, DigiWest, automated microfluid western blotting and microchip electrophoresis have all been developed to reduce potential problems associated with the western blotting technique. Innovative developments in instrumentation and increased sensitivity for western blots offer novel possibilities for increasing the clinical implications of western blot.

  5. Western Blotting of the Endocannabinoid System.

    PubMed

    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. The Design of a Quantitative Western Blot Experiment

    PubMed Central

    Taylor, Sean C.; Posch, Anton

    2014-01-01

    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. PMID:24738055

  7. Western blotting: an introduction.

    PubMed

    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.

  8. Single cell–resolution western blotting

    PubMed Central

    Kang, Chi-Chih; Yamauchi, Kevin A; Vlassakis, Julea; Sinkala, Elly; Duncombe, Todd A; Herr, Amy E

    2017-01-01

    This protocol describes how to perform western blotting on individual cells to measure cell-to-cell variation in protein expression levels and protein state. like conventional western blotting, single-cell western blotting (scWB) is particularly useful for protein targets that lack selective antibodies (e.g., isoforms) and in cases in which background signal from intact cells is confounding. scWB is performed on a microdevice that comprises an array of microwells molded in a thin layer of a polyacrylamide gel (PAG). the gel layer functions as both a molecular sieving matrix during PAGE and a blotting scaffold during immunoprobing. scWB involves five main stages: (i) gravity settling of cells into microwells; (ii) chemical lysis of cells in each microwell; (iii) PAGE of each single-cell lysate; (iv) exposure of the gel to UV light to blot (immobilize) proteins to the gel matrix; and (v) in-gel immunoprobing of immobilized proteins. Multiplexing can be achieved by probing with antibody cocktails and using antibody stripping/reprobing techniques, enabling detection of 10+ proteins in each cell. We also describe microdevice fabrication for both uniform and pore-gradient microgels. to extend in-gel immunoprobing to gels of small pore size, we describe an optional gel de-cross-linking protocol for more effective introduction of antibodies into the gel layer. once the microdevice has been fabricated, the assay can be completed in 4–6 h by microfluidic novices and it generates high-selectivity, multiplexed data from single cells. the technique is relevant when direct measurement of proteins in single cells is needed, with applications spanning the fundamental biosciences to applied biomedicine. PMID:27466711

  9. Serological investigation of enterococcal infections using western blot.

    PubMed

    Sulaiman, A; Rakita, R M; Arduino, R C; Patterson, J E; Steckelberg, J M; Singh, K V; Murray, B E

    1996-10-01

    To assess the usefulness of Western blot in the diagnosis of enterococcal infections, a pilot study was conducted with a newly developed Western blot using sera from patients with confirmed enterococcal infections. Sera from 17 of 19 patients with enterococcal endocarditis reacted strongly to enterococcal antigens on the Western blot, and most produced specific bands at molecular weights 98 kDa and 54 kDa. Sera from patients with bacteremic cholangitis and pyelonephritis reacted frequently as well, but the pattern of bands was different from that observed with endocarditis. Eighty-five percent of 26 sera tested from patients with bacteremia and associated deep-seated infections (endocarditis, cholangitis, and pyelonephritis) were positive on Western blot, compared to 30% of sera from bacteremic patients with no clinically determined deep focus of infection (p < 0.001).

  10. Multiplexed Western Blotting Using Microchip Electrophoresis.

    PubMed

    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.

  11. Multistrip western blotting to increase quantitative data output.

    PubMed

    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. Western blotting using chemiluminescent substrates.

    PubMed

    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.

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

    PubMed

    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.

  14. Prolonged incubation and stacked film exposure improve sensitivity in western blotting.

    PubMed

    Luo, Haitao; Rankin, Gary O; Straley, Shannon; Chen, Yi Charlie

    2011-01-01

    Western blotting is a basic technique for protein detection. For proteins of less abundance or antibodies of poorer quality, an increased sensitivity is often desired. Although it is commonly known that higher concentrations of antibodies and prolonged film exposure times will help improve sensitivity in western blots, both measures come with their own risks, and it is often unclear to which extent these measures should be applied. We conducted time-course studies to investigate protein-antibody interactions and primary antibody-secondary antibody interactions in western blotting. We also propose a protocol of stacked film exposure and have tested it in standard curves and cancer cell samples. Our study found that protein-primary antibody interactions and primary antibody-secondary antibody interactions could take a longer time than commonly used "one hour" or "overnight", and in some cases longer than 48h, to reach its maximum binding. We also show that the modified protocol of stacked film exposure works well for both standard curves and biological samples, reaching a maximum sensitivity in western blots without blurring target signals or increasing backgrounds. In addition to regular optimization of antibody concentrations and film exposure time, a prolonged incubation with antibodies and stacked film exposure will also help improve sensitivity and reduce background in western blotting. Copyright © 2011 Elsevier Inc. All rights reserved.

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

    PubMed

    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.

  16. Western Blot of Stained Proteins from Dried Polyacrylamide Gels

    NASA Technical Reports Server (NTRS)

    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.

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

    ERIC Educational Resources Information Center

    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…

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

    PubMed

    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.

  19. Treponema pallidum western blot: comparison with the FTA-ABS test as a confirmatory test for syphilis.

    PubMed

    Backhouse, J L; Nesteroff, S I

    2001-01-01

    We developed a Treponema pallidum Western blot and compared the results with Treponema pallidum particle agglutination (TPPA) and fluorescent treponemal antibody absorption (FTA-ABS) tests. The Western blot was deemed reactive if the serum reacted with at least three major antigenic bands (TpN47, TpN44.5, TpN17, TpN15). The sensitivities of the Western blot, TPPA and FTA-ABS, were all 100% and the specificities of the Western blot, TPPA and FTA-ABS were 100%, 100% and 94.5% respectively. In 52 problem sera, reactive in only one treponemal test, the agreement between the Western blot and TPPA (61.5%) was significantly better than between Western blot and FTA-ABS (38.5%). The individual sensitivities and specificities of TpN47, TpN44.5, TpN17, TpN15 were 100%, 100%, 96%, 100% and 20%, 96%, 100%, 100% respectively. We conclude that the Western blot is a useful additional confirmatory test or alternative to the FTA-ABS and that a more sensitive and specific criterion for the Western blot would be reactivity with TpN15 and two of the three other major antigens.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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

  4. Clinical application of a rapid method using agarose gel electrophoresis and Western blotting to evaluate von Willebrand factor protease activity.

    PubMed

    Kirzek, D M; Rick, M E

    2001-03-01

    A method for evaluating the activity of the von Willebrand factor (vWF) protease is described, and a clinical application is illustrated. The procedure utilizes gel electrophoresis, Western blotting, and luminographic detection methods to evaluate the distribution of vWF multimers before and after incubation of clinical samples under conditions that favor proteolysis by this enzyme. Physiologically, the high-molecular-weight multimers of vWF are cleaved by the vWF protease under conditions of high shear stress in parts of the arterial circulation; cleavage of vWF multimers is also observed after exposure of vWF to denaturing agents in vitro and thus can serve as a laboratory test for the activity of the protease. vWF protease activity is decreased or absent in patients with thrombotic thrombocytopenic purpura due to an inhibiting autoantibody, and this leads to high levels of noncleaved vWF and to life-threatening thrombosis, thrombocytopenia and anemia. The assay evaluates the activity of the protease by assessing the cleavage of vWF multimers after patient plasmas are incubated in vitro under denaturing conditions. With the use of these electrophoresis and Western blotting techniques, patient plasmas can be rapidly assessed for the activity of the vWF protease which may aid in the treatment strategy for these patients.

  5. Human thyrotropin receptor subunits characterized by thyrotropin affinity purification and western blotting.

    PubMed

    Leedman, P J; Newman, J D; Harrison, L C

    1989-07-01

    We studied the subunit structure of the human TSH receptor in thyroid tissue from patients with Graves' disease and multinodular goiter by TSH affinity chromatography, immunoprecipitation with Graves' immunoglobulins (Igs), and a modified technique of Western blotting. Human TSH receptor-binding activity was purified about 1,270-fold by sequential affinity chromatography on wheat germ lectin-agarose and TSH-agarose. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of nonreduced affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed three noncovalently linked subunits of 70,000, 50,000, and 35,000 mol wt. When reduced, a major subunit of 25,000 mol wt was identified. When 3 mol/L NaCl was used to elute affinity-purified receptors only the 50,000 mol wt nonreduced subunit was detected. This subunit bound [125I]bovine TSH and was precipitated by Graves' Igs. Modifications to the conventional Western blotting technique enabled thyroglobulin components (approximately 220,000 mol wt), thyroid microsomal antigen (a doublet of approximately 110,000 mol wt), and putative TSH receptor subunits of 70,000 and 50,000 mol wt to be identified in thyroid particulate membranes by Graves' Igs. Blotting of affinity-purified receptors eluted in sodium dodecyl sulfate sample buffer revealed subunits of either 70,000 or 50,000 mol wt, with a minority of Graves' serum samples. We conclude that the nonreduced human TSH receptor is an oligomeric complex comprising three different subunits of 70,000, 50,000, and 35,000 mol wt. The reduced receptor exists as a single subunit of 25,000 mol wt, which may be disulfide linked to form the higher mol wt forms. The 70,000 and 50,000 mol wt subunits contain epitopes that bind Graves' Igs in modified Western blots, thus directly confirming that the human TSH receptor is a target for Graves' Igs.

  6. Automated capillary Western dot blot method for the identity of a 15-valent pneumococcal conjugate vaccine.

    PubMed

    Hamm, Melissa; Ha, Sha; Rustandi, Richard R

    2015-06-01

    Simple Western is a new technology that allows for the separation, blotting, and detection of proteins similar to a traditional Western except in a capillary format. Traditionally, identity assays for biological products are performed using either an enzyme-linked immunosorbent assay (ELISA) or a manual dot blot Western. Both techniques are usually very tedious, labor-intensive, and complicated for multivalent vaccines, and they can be difficult to transfer to other laboratories. An advantage this capillary Western technique has over the traditional manual dot blot Western method is the speed and the automation of electrophoresis separation, blotting, and detection steps performed in 96 capillaries. This article describes details of the development of an automated identity assay for a 15-valent pneumococcal conjugate vaccine, PCV15-CRM197, using capillary Western technology. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Protein analysis through Western blot of cells excised individually from human brain and muscle tissue

    PubMed Central

    Koob, A.O.; Bruns, L.; Prassler, C.; Masliah, E.; Klopstock, T.; Bender, A.

    2016-01-01

    Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. PMID:22402104

  8. Protein analysis through Western blot of cells excised individually from human brain and muscle tissue.

    PubMed

    Koob, A O; Bruns, L; Prassler, C; Masliah, E; Klopstock, T; Bender, A

    2012-06-15

    Comparing protein levels from single cells in tissue has not been achieved through Western blot. Laser capture microdissection allows for the ability to excise single cells from sectioned tissue and compile an aggregate of cells in lysis buffer. In this study we analyzed proteins from cells excised individually from brain and muscle tissue through Western blot. After we excised individual neurons from the substantia nigra of the brain, the accumulated surface area of the individual cells was 120,000, 24,000, 360,000, 480,000, 600,000 μm2. We used an optimized Western blot protocol to probe for tyrosine hydroxylase in this cell pool. We also took 360,000 μm2 of astrocytes (1700 cells) and analyzed the specificity of the method. In muscle we were able to analyze the proteins of the five complexes of the electron transport chain through Western blot from 200 human cells. With this method, we demonstrate the ability to compare cell-specific protein levels in the brain and muscle and describe for the first time how to visualize proteins through Western blot from cells captured individually. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. A new background subtraction method for Western blot densitometry band quantification through image analysis software.

    PubMed

    Gallo-Oller, Gabriel; Ordoñez, Raquel; Dotor, Javier

    2018-06-01

    Since its first description, Western blot has been widely used in molecular labs. It constitutes a multistep method that allows the detection and/or quantification of proteins from simple to complex protein mixtures. Western blot quantification method constitutes a critical step in order to obtain accurate and reproducible results. Due to the technical knowledge required for densitometry analysis together with the resources availability, standard office scanners are often used for the imaging acquisition of developed Western blot films. Furthermore, the use of semi-quantitative software as ImageJ (Java-based image-processing and analysis software) is clearly increasing in different scientific fields. In this work, we describe the use of office scanner coupled with the ImageJ software together with a new image background subtraction method for accurate Western blot quantification. The proposed method represents an affordable, accurate and reproducible approximation that could be used in the presence of limited resources availability. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    PubMed

    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.

  11. Single-Cell Western Blotting after Whole-Cell Imaging to Assess Cancer Chemotherapeutic Response

    PubMed Central

    2015-01-01

    Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors. PMID:25226230

  12. β-actin as a loading control for plasma-based Western blot analysis of major depressive disorder patients.

    PubMed

    Zhang, Rufang; Yang, Deyu; Zhou, Chanjuan; Cheng, Ke; Liu, Zhao; Chen, Liang; Fang, Liang; Xie, Peng

    2012-08-15

    Western blot analysis is a commonly used technique for determining specific protein levels in clinical samples. For normalization of protein levels in Western blot, a suitable loading control is required. On account of its relatively high and constant expression, β-actin has been widely employed in Western blot of cell cultures and tissue extracts. However, β-actin's presence in human plasma and this protein's putative role as a plasma-based loading control for Western blot analysis remain unknown. In this study, an enzyme-linked immunosorbent assay was used to determine the concentration of β-actin in human plasma, which is 6.29±0.54 ng/ml. In addition, the linearity of β-actin immunostaining and loaded protein amount was evaluated by Western blot, and a fine linearity (R²=0.974±0.012) was observed. Furthermore, the expression of plasma β-actin in major depressive disorder subjects and healthy controls was compared. The data revealed no statistically significant difference between these two groups. Moreover, the total coefficient of variation for β-actin expression in the two groups was 9.2±1.2%. These findings demonstrate that β-actin is present in human plasma and may possibly be used as a suitable loading control for plasma-based Western blot analysis in major depressive disorder. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Quantum dot-based western blot for sensitive detection of pig serum antibody to actinobacillus pleuropneumoniae

    NASA Astrophysics Data System (ADS)

    Cişmileanu, Ana; Sima, Cornelia; Grigoriu, Constantin

    2007-08-01

    A quantum dot - immunoglobulin conjugate specific for pig IgG, was obtained by carbodiimide chemistry. We used a Western blot technique for detecting specific antibodies against Actinobacillus pleuropneumoniae (A. pp), which cause porcine pleuropneumonia. The antigen used in this technique was Apx haemolysin which is an important virulence factor of A. pp and it induces protective immunity in vaccined pigs. The detection on Western blot membrane was possible at 1/50 dilution of quantum dot conjugate at a dilution of pig serum till 1/6400. The results for pig serum demonstrated a higher sensitivity of QD-based Western blot technique for the presence of antibodies specific for Apx haemolysin in comparison with similar classical techniques (with coloured substrate for enzyme present in secondary antibody conjugate).

  14. Western blotting revisited: critical perusal of underappreciated technical issues.

    PubMed

    Gorr, Thomas A; Vogel, Johannes

    2015-04-01

    The most commonly used semiquantitative analysis of protein expression still employs protein separation by denaturing SDS-PAGE with subsequent Western blotting and quantification of the resulting ODs of bands visualized with specific antibodies. However, many questions regarding this procedure are usually ignored, although still in need of answering: Does isolation or separation procedure harm the integrity or affect modifications (e.g., phosphorylation) of the protein of interest? Does denaturation reduce binding of antibodies used for detection? Should denaturation be performed or should a native gel be run? How can artificial degradations or aggregations be distinguished from biological relevant ones? If the antibody detects multiple bands (which is not uncommon), which one(s) should be taken into account for quantification and why? Which loading control protein should be chosen and is it really "housekeeping" and how can this be verified? Is the image acquisition system linear and does it come with a sufficient dynamic range? How to account and control for background staining? This article is intended to address these questions and raise the readers awareness to possible Western blot alternatives in the attempt of minimizing possible pitfalls that might loom anywhere from protein isolation to acquisition of final quantitative data. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. A Study of Rubisco through Western Blotting and Tissue Printing Techniques

    ERIC Educational Resources Information Center

    Ma, Zhong; Cooper, Cynthia; Kim, Hyun-Joo; Janick-Buckner, Diane

    2009-01-01

    We describe a laboratory exercise developed for a cell biology course for second-year undergraduate biology majors. It was designed to introduce undergraduates to the basic molecular biology techniques of Western blotting and immunodetection coupled with the technique of tissue printing in detecting the presence, relative abundance, and…

  16. Improved high-throughput quantification of luminescent microplate assays using a common Western-blot imaging system.

    PubMed

    Hawkins, Liam J; Storey, Kenneth B

    2017-01-01

    Common Western-blot imaging systems have previously been adapted to measure signals from luminescent microplate assays. This can be a cost saving measure as Western-blot imaging systems are common laboratory equipment and could substitute a dedicated luminometer if one is not otherwise available. One previously unrecognized limitation is that the signals captured by the cameras in these systems are not equal for all wells. Signals are dependent on the angle of incidence to the camera, and thus the location of the well on the microplate. Here we show that: •The position of a well on a microplate significantly affects the signal captured by a common Western-blot imaging system from a luminescent assay.•The effect of well position can easily be corrected for.•This method can be applied to commercially available luminescent assays, allowing for high-throughput quantification of a wide range of biological processes and biochemical reactions.

  17. Ferulic acid enhances IgE binding to peanut allergens in western blots.

    USDA-ARS?s Scientific Manuscript database

    Phenolic compounds at high concentrations are known to form insoluble complexes with proteins. We hypothesized that this complex formation could interfere with Western blot and ELISA assays for peanut allergens. To verify this, three simple phenolic compounds (ferulic, caffeic, and chlorogenic acids...

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

    EPA Science Inventory

    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...

  19. Ferulic acid enhances IgE binding to peanut allergens in western blots.

    USDA-ARS?s Scientific Manuscript database

    Because phenolic compounds can precipitate or complex with proteins, we postulated that interactions of phenolics with IgE antibodies help enhance IgE binding to peanut allergens in Western blots. Three different phenolics, such as, ferulic, caffeic and chlorogenic acids were examined. Each was mixe...

  20. Serological differentiation of murine typhus and epidemic typhus using cross-adsorption and Western blotting.

    PubMed

    La Scola, B; Rydkina, L; Ndihokubwayo, J B; Vene, S; Raoult, D

    2000-07-01

    Differentiation of murine typhus due to Rickettsia typhi and epidemic typhus due to Rickettsia prowazekii is critical epidemiologically but difficult serologically. Using serological, epidemiological, and clinical criteria, we selected sera from 264 patients with epidemic typhus and from 44 patients with murine typhus among the 29,188 tested sera in our bank. These sera cross-reacted extensively in indirect fluorescent antibody assays (IFAs) against R. typhi and R. prowazekii, as 42% of the sera from patients with epidemic typhus and 34% of the sera from patients with murine typhus exhibited immunoglobulin M (IgM) and/or IgG titers against the homologous antigen (R. prowazekii and R. typhi, respectively) that were more than one dilution higher than those against the heterologous antigen. Serum cross-adsorption studies and Western blotting were performed on sera from 12 selected patients, 5 with murine typhus, 5 with epidemic typhus, and 2 suffering from typhus of undetermined etiology. Differences in IFA titers against R. typhi and R. prowazekii allowed the identification of the etiological agent in 8 of 12 patients. Western blot studies enabled the identification of the etiological agent in six patients. When the results of IFA and Western blot studies were considered in combination, identification of the etiological agent was possible for 10 of 12 patients. Serum cross-adsorption studies enabled the differentiation of the etiological agent in all patients. Our study indicates that when used together, Western blotting and IFA are useful serological tools to differentiate between R. prowazekii and R. typhi exposures. While a cross-adsorption study is the definitive technique to differentiate between infections with these agents, it was necessary in only 2 of 12 cases (16.7%), and the high costs of such a study limit its use.

  1. Serological Differentiation of Murine Typhus and Epidemic Typhus Using Cross-Adsorption and Western Blotting

    PubMed Central

    La Scola, Bernard; Rydkina, Lena; Ndihokubwayo, Jean-Bosco; Vene, Sirkka; Raoult, Didier

    2000-01-01

    Differentiation of murine typhus due to Rickettsia typhi and epidemic typhus due to Rickettsia prowazekii is critical epidemiologically but difficult serologically. Using serological, epidemiological, and clinical criteria, we selected sera from 264 patients with epidemic typhus and from 44 patients with murine typhus among the 29,188 tested sera in our bank. These sera cross-reacted extensively in indirect fluorescent antibody assays (IFAs) against R. typhi and R. prowazekii, as 42% of the sera from patients with epidemic typhus and 34% of the sera from patients with murine typhus exhibited immunoglobulin M (IgM) and/or IgG titers against the homologous antigen (R. prowazekii and R. typhi, respectively) that were more than one dilution higher than those against the heterologous antigen. Serum cross-adsorption studies and Western blotting were performed on sera from 12 selected patients, 5 with murine typhus, 5 with epidemic typhus, and 2 suffering from typhus of undetermined etiology. Differences in IFA titers against R. typhi and R. prowazekii allowed the identification of the etiological agent in 8 of 12 patients. Western blot studies enabled the identification of the etiological agent in six patients. When the results of IFA and Western blot studies were considered in combination, identification of the etiological agent was possible for 10 of 12 patients. Serum cross-adsorption studies enabled the differentiation of the etiological agent in all patients. Our study indicates that when used together, Western blotting and IFA are useful serological tools to differentiate between R. prowazekii and R. typhi exposures. While a cross-adsorption study is the definitive technique to differentiate between infections with these agents, it was necessary in only 2 of 12 cases (16.7%), and the high costs of such a study limit its use. PMID:10882661

  2. Protein blotting protocol for beginners.

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

    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

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

    PubMed

    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.

  6. Normalized Quantitative Western Blotting Based on Standardized Fluorescent Labeling.

    PubMed

    Faden, Frederik; Eschen-Lippold, Lennart; Dissmeyer, Nico

    2016-01-01

    Western blot (WB) analysis is the most widely used method to monitor expression of proteins of interest in protein extracts of high complexity derived from diverse experimental setups. WB allows the rapid and specific detection of a target protein, such as non-tagged endogenous proteins as well as protein-epitope tag fusions depending on the availability of specific antibodies. To generate quantitative data from independent samples within one experiment and to allow accurate inter-experimental quantification, a reliable and reproducible method to standardize and normalize WB data is indispensable. To date, it is a standard procedure to normalize individual bands of immunodetected proteins of interest from a WB lane to other individual bands of so-called housekeeping proteins of the same sample lane. These are usually detected by an independent antibody or colorimetric detection and do not reflect the real total protein of a sample. Housekeeping proteins-assumed to be constitutively expressed mostly independent of developmental and environmental states-can greatly differ in their expression under these various conditions. Therefore, they actually do not represent a reliable reference to normalize the target protein's abundance to the total amount of protein contained in each lane of a blot.Here, we demonstrate the Smart Protein Layers (SPL) technology, a combination of fluorescent standards and a stain-free fluorescence-based visualization of total protein in gels and after transfer via WB. SPL allows a rapid and highly sensitive protein visualization and quantification with a sensitivity comparable to conventional silver staining with a 1000-fold higher dynamic range. For normalization, standardization and quantification of protein gels and WBs, a sample-dependent bi-fluorescent standard reagent is applied and, for accurate quantification of data derived from different experiments, a second calibration standard is used. Together, the precise quantification of

  7. The early days of blotting.

    PubMed

    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.

  8. Comparative evaluation of western blotting in hepatic and pulmonary cystic echinococcosis.

    PubMed

    Akisu, C; Delibas, S B; Bicmen, C; Ozkoc, S; Aksoy, U; Turgay, N

    2006-12-01

    Many serological tests are widely used in the diagnosis of cystic echinococcosis (CE), caused by the larval stages of Echinococcus granulosus. The present study was carried for differentiation between hepatic and pulmonary cystic echinococcosis by Western Blotting (WB). A total of 121 sera from patients with hepatic CE (37), pulmonary CE (31) and controls (53; consisting of six healthy, seven Hymenolepis nana infection, 20 hepatic and 20 pulmonary diseases other than CE) were examined. In all of the CE patients, E. gronulosus infection was confirmed by surgical intervention. Sera were previously tested using IHA and ELISA to detect the E. gronulosus specific antibodies. Sera from hepatic cases of CE reacted with 16 polypeptides of 6-116 kDa and sera from pulmonary cases of CE reacted with 14 polypeptides of 4-130 kDa by Western Blotting. The WB test enabled the detection of antibodies in the hepatic CE samples for proteins of 24, 32 34, 44-46 and 52-54 kDa in molecular weight in 78.4%, 75.7%, 78.4% and 89.2% of the patients, respectively. In the pulmonary CE samples sera WB test enabled the detection of antibodies 24, 44-46, 100, 110, 116 and 120 124 kDa in molecular weight in 81.3%, 75.0%, 87.5%, 71.9%, 84.4% and 65.6% of the patients, respectively. We indicated that the antigenic components of high molecular weight can be good candidates for differentiation of hepatic CE from pulmonary CE.

  9. A Streamlined Western Blot Exercise: An Efficient and Greener Approach in the Laboratory Classroom

    ERIC Educational Resources Information Center

    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…

  10. Fluorescent Labeling of Proteins and Its Application to SDS-PAGE and Western Blotting.

    PubMed

    Alba, F Javier; Bartolomé, Salvador; Bermúdez, Antonio; Daban, Joan-Ramon

    2015-01-01

    This chapter describes very simple fluorescent methods developed in our laboratory allowing the rapid monitoring of total protein patterns on both sodium dodecyl sulfate (SDS) polyacrylamide gels and western blots. The noncovalent dye Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one) is used for the sensitive staining of proteins in SDS gels. This method is compatible with the electroblotting of protein bands and with the staining of the resulting blot with the covalent dye MDPF (2-methoxy-2,4-diphenyl-3(2H)-furanone). These staining procedures are applied sequentially; there is no need to run a duplicate unstained gel for protein blotting. Furthermore, since only the adduct formed by the reaction of MDPF with proteins is fluorescent, there is no need to destain the membrane after protein labeling. In addition, MDPF staining is compatible with further immunodetection of specific bands with polyclonal antibodies. Finally, using the adequate conditions described below, MDPF staining does not preclude the N-terminal sequence analysis of proteins in selected bands.

  11. A Proteomics Approach to the Protein Normalization Problem: Selection of Unvarying Proteins for MS-Based Proteomics and Western Blotting.

    PubMed

    Wiśniewski, Jacek R; Mann, Matthias

    2016-07-01

    Proteomics and other protein-based analysis methods such as Western blotting all face the challenge of discriminating changes in the levels of proteins of interest from inadvertent changes in the amount loaded for analysis. Mass-spectrometry-based proteomics can now estimate the relative and absolute amounts of thousands of proteins across diverse biological systems. We reasoned that this new technology could prove useful for selection of very stably expressed proteins that could serve as better loading controls than those traditionally employed. Large-scale proteomic analyses of SDS lysates of cultured cells and tissues revealed deglycase DJ-1 as the protein with the lowest variability in abundance among different cell types in human, mouse, and amphibian cells. The protein constitutes 0.069 ± 0.017% of total cellular protein and occurs at a specific concentration of 34.6 ± 8.7 pmol/mg of total protein. Since DJ-1 is ubiquitous and therefore easily detectable with several peptides, it can be helpful in normalization of proteomic data sets. In addition, DJ-1 appears to be an advantageous loading control for Western blot that is superior to those used commonly used, allowing comparisons between tissues and cells originating from evolutionarily distant vertebrate species. Notably, this is not possible by the detection and quantitation of housekeeping proteins, which are often used in the Western blot technique. The approach introduced here can be applied to select the most appropriate loading controls for MS-based proteomics or Western blotting in any biological system.

  12. Detection of Diverse and High Molecular Weight Nesprin-1 and Nesprin-2 Isoforms Using Western Blotting.

    PubMed

    Carthew, James; Karakesisoglou, Iakowos

    2016-01-01

    Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (>800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins.

  13. Problem-Solving Test: Southwestern Blotting

    ERIC Educational Resources Information Center

    Szeberényi, József

    2014-01-01

    Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA,…

  14. Use of a sensitive EnVision +-based detection system for Western blotting: avoidance of streptavidin binding to endogenous biotin and biotin-containing proteins in kidney and other tissues.

    PubMed

    Banks, Rosamonde E; Craven, Rachel A; Harnden, Patricia A; Selby, Peter J

    2003-04-01

    Western blotting remains a central technique in confirming identities of proteins, their quantitation and analysis of various isoforms. The biotin-avidin/streptavidin system is often used as an amplification step to increase sensitivity but in some tissues such as kidney, "nonspecific" interactions may be a problem due to high levels of endogenous biotin-containing proteins. The EnVision system, developed for immunohistochemical applications, relies on binding of a polymeric conjugate consisting of up to 100 peroxidase molecules and 20 secondary antibody molecules linked directly to an activated dextran backbone, to the primary antibody. This study demonstrates that it is also a viable and sensitive alternative detection system in Western blotting applications.

  15. Problem-solving test: Southwestern blotting.

    PubMed

    Szeberényi, József

    2014-01-01

    Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA, deletion mutants, expression plasmid, transfection, RNA polymerase II, promoter, Shine-Dalgarno sequence, polyadenylation element, affinity chromatography, Northern blotting, immunoprecipitation, sodium dodecylsulfate, autoradiography, tandem repeats. Copyright © 2014 The International Union of Biochemistry and Molecular Biology.

  16. Western blot (immunoblot) assay of small, round-structured virus associated with an acute gastroenteritis outbreak in Tokyo.

    PubMed

    Hayashi, Y; Ando, T; Utagawa, E; Sekine, S; Okada, S; Yabuuchi, K; Miki, T; Ohashi, M

    1989-08-01

    Small, round-structured virus (SRSV) was detected in a stool specimen of a patient during an acute gastroenteritis outbreak in Tokyo and was tentatively named SRSV-9. SRSV-9 was purified by sucrose velocity gradient centrifugation after CsCl density gradient centrifugation. The buoyant density of SRSV-9 appeared to be 1.36 g/ml in CsCl. A Western blot (immunoblot) assay using the biotin-avidin system revealed that SRSV-9 was antigenically related to the Hawaii agent but distinct from the Norwalk agent and contained a single major structural protein with a molecular size of 63.0 +/- 0.6 kilodaltons. The prevalence of SRSV-9 infection in Tokyo was surveyed by the Western blot antibody assay by using a crude virus preparation as the antigen. Seroconversion was observed in 56.5% of the patients involved in the outbreaks from which SRSV was detected by electron microscopy.

  17. Interaction of murine intestinal mast cell proteinase with inhibitors (serpins) in blood; analysis by SDS-PAGE and western blotting.

    PubMed Central

    Irvine, J; Newlands, G F; Huntley, J F; Miller, H R

    1990-01-01

    The interaction of mouse intestinal mast cell proteinase (IMCP) with serine proteinase inhibitors (serpins) in blood was analysed: (i) by examining the capacity of the inhibitors in blood to block the binding of the irreversible serine esterase inhibitor [3H]diisopropyl fluorophosphate (DFP); (ii) by Western blotting. The binding of [3H]DFP to IMCP was blocked very rapidly by inhibitors in mouse serum and, by Western blotting, this inhibition was associated with the appearance of a 73,000 MW proteinase/inhibitor complex together with a series of higher (greater than 100,000) MW complexes. IMCP was not dissociated from these complexes when electrophoresed under reducing conditions, although prior heat treatment of mouse serum (60 for 30-160 min) abolished the formation of all proteinase/inhibitor complexes. Similarly, the activity of a 48,000 MW inhibitor of chymotrypsin was abolished by heat treatment. A titration experiment established that between 0.5 and 5 mg IMCP were inhibited per ml of serum. The properties and MW of the IMCP inhibitor complexes are typical of serpins and suggest that IMCP secreted during intestinal immunological reactions would be rapidly and irreversibly inactivated by plasma-derived inhibitors. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:2312150

  18. Identification of recombinant human EPO variants in greyhound plasma and urine by ELISA, LC-MS/MS and western blotting: a comparative study.

    PubMed

    Timms, Mark; Steel, Rohan; Vine, John

    2016-02-01

    The recombinant human erythropoietins epoetin alfa (Eprex®), darbepoetin (Aranesp®) and methoxy polyethylene glycol-epoetin beta (Mircera®) were administered to greyhounds for 7, 10 and 14 days respectively. Blood and urine samples were collected and analysed for erythropoietin by ELISA, LC-MS/MS and western blotting. Limits of confirmation in plasma for western blotting and LC-MS/MS methods ranged from a low of 2.5mIU/mL, and closely matched the sensitivity of ELISA screening. Copyright © 2015 John Wiley & Sons, Ltd.

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

    PubMed

    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.

  20. Validation of endothelin B receptor antibodies reveals two distinct receptor-related bands on Western blot.

    PubMed

    Barr, Travis P; Kornberg, Daniel; Montmayeur, Jean-Pierre; Long, Melinda; Reichheld, Stephen; Strichartz, Gary R

    2015-01-01

    Antibodies are important tools for the study of protein expression but are often used without full validation. In this study, we used Western blots to characterize antibodies targeted to the N or C terminal (NT or CT, respectively) and the second or third intracellular loop (IL2 or IL3, respectively) of the endothelin B receptor (ETB). The IL2-targeted antibody accurately detected endogenous ETB expression in rat brain and cultured rat astrocytes by labeling a 50-kDa band, the expected weight of full-length ETB. However, this antibody failed to detect transfected ETB in HEK293 cultures. In contrast, the NT-targeted antibody accurately detected endogenous ETB in rat astrocyte cultures and transfected ETB in HEK293 cultures by labeling a 37-kDa band but failed to detect endogenous ETB in rat brain. Bands detected by the CT- or IL3-targeted antibody were found to be unrelated to ETB. Our findings show that functional ETB can be detected at 50 or 37kDa on Western blot, with drastic differences in antibody affinity for these bands. The 37-kDa band likely reflects ETB processing, which appears to be dependent on cell type and/or culture condition. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Outbreak of larval Echinococcus multilocularis infection in Japanese monkey (Macaca fuscata) in a zoo, Hokkaido: western blotting patterns in the infected monkeys.

    PubMed

    Sato, Chiaki; Kawase, Shiro; Yano, Shoki; Nagano, Hideki; Fujimoto, Satoshi; Kobayashi, Nobuyuki; Miyahara, Kazuro; Yamada, Kazutaka; Sato, Motoyoshi; Kobayashi, Yoshiyasu

    2005-01-01

    A high prevalence of larval Echinococcus multilocularis (Em) infection was found in zoo primates in Hokkaido, Japan. In October 1997, a Japanese monkey (Macaca fuscata) died and histopathologically diagnosed as alveolar hydatidosis. Serum samples were collected from the remaining Japanese monkeys and examined for antibodies against Em by enzyme-linked immunosorbent assay and western blotting. Serological tests showed 12 more animals of the remaining 57 monkeys were possibly infected. Ultrasonography revealed that nine of these 12 animals had a cystic lesion in the liver. The band patterns of western blotting in the monkeys were very similar to those in human.

  2. The Dot Blot ELISA.

    ERIC Educational Resources Information Center

    Gerbig, Donald G., Jr.; Fenk, Christopher J.; Goodhart, Amy S.

    2000-01-01

    Uses two laboratory techniques, Enzyme Linked Immunosorbent Assay (ELISA) and Western Blot, to demonstrate antibody-antigen binding concepts. Includes a list of required materials and directions for the procedure, and makes suggestions for classroom applications. (Contains 13 references.) (YDS)

  3. A research design for the quantification of the neuropeptides substance p and calcitonin gene-related Peptide in rat skin using Western blot analysis.

    PubMed

    Lapin, Guilherme Abbud Franco; Hochman, Bernardo; Nishioka, Michele Akemi; Maximino, Jessica Ruivo; Chadi, Gerson; Ferreira, Lydia Masako

    2015-06-01

    To describe and standardize a protocol that overcomes the technical limitations of Western blot (WB) analysis in the quantification of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) following nociceptive stimuli in rat skin. Male Wistar rats (Rattus norvegicus albinus) weighing 250 to 350 g were used in this study. Elements of WB analysis were adapted by using specific manipulation of samples, repeated cycles of freezing and thawing, more thorough maceration, and a more potent homogenizer; increasing lytic reagents; promoting greater inhibition of protease activity; and using polyvinylidene fluoride membranes as transfer means for skin-specific protein. Other changes were also made to adapt the WB analysis to a rat model. University research center. Western blot analysis adapted to a rat model. This research design has proven effective in collecting and preparing skin samples to quantify SP and CGRP using WB analysis in rat skin. This study described a research design that uses WB analysis as a reproducible, technically accessible, and cost-effective method for the quantification of SP and CGRP in rat skin that overcomes technical biases.

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

    PubMed

    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.

  5. Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis.

    PubMed

    Schüchner, Stefan; Andorfer, Peter; Mudrak, Ingrid; Ogris, Egon

    2016-08-17

    Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film.

  6. Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis

    PubMed Central

    Schüchner, Stefan; Andorfer, Peter; Mudrak, Ingrid; Ogris, Egon

    2016-01-01

    Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film. PMID:27531616

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

    PubMed

    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.

  8. Avoiding pitfalls of internal controls: validation of reference genes for analysis by qRT-PCR and Western blot throughout rat retinal development.

    PubMed

    Rocha-Martins, Maurício; Njaine, Brian; Silveira, Mariana S

    2012-01-01

    Housekeeping genes have been commonly used as reference to normalize gene expression and protein content data because of its presumed constitutive expression. In this paper, we challenge the consensual idea that housekeeping genes are reliable controls for expression studies in the retina through the investigation of a panel of reference genes potentially suitable for analysis of different stages of retinal development. We applied statistical tools on combinations of retinal developmental stages to assess the most stable internal controls for quantitative RT-PCR (qRT-PCR). The stability of expression of seven putative reference genes (Actb, B2m, Gapdh, Hprt1, Mapk1, Ppia and Rn18s) was analyzed using geNorm, BestKeeper and Normfinder software. In addition, several housekeeping genes were tested as loading controls for Western blot in the same sample panel, using Image J. Overall, for qRT-PCR the combination of Gapdh and Mapk1 showed the highest stability for most experimental sets. Actb was downregulated in more mature stages, while Rn18s and Hprt1 showed the highest variability. We normalized the expression of cyclin D1 using various reference genes and demonstrated that spurious results may result from blind selection of internal controls. For Western blot significant variation could be seen among four putative internal controls (β-actin, cyclophilin b, α-tubulin and lamin A/C), while MAPK1 was stably expressed. Putative housekeeping genes exhibit significant variation in both mRNA and protein content during retinal development. Our results showed that distinct combinations of internal controls fit for each experimental set in the case of qRT-PCR and that MAPK1 is a reliable loading control for Western blot. The results indicate that biased study outcomes may follow the use of reference genes without prior validation for qRT-PCR and Western blot.

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

    PubMed

    Eaton, Samantha L; Roche, Sarah L; Llavero Hurtado, Maica; Oldknow, Karla J; Farquharson, Colin; Gillingwater, Thomas H; Wishart, Thomas M

    2013-01-01

    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 that normalisation

  10. Confirmation of the immunoreactivity of monoclonal anti-human C-terminal EGFR antibodies in bronze Corydoras Corydoras aeneus (Callichthyidae Teleostei) by Western Blot method.

    PubMed

    Mytych, Jennifer; Satora, Leszek; Kozioł, Katarzyna

    2018-02-01

    Bronze corydoras (Corydoras aeneus) uses the distal part of the intestine as accessory respiratory organ. Our previous study showed the presence of epidermal growth factor receptor (EGFR) cytoplasmic domain in the digestive tract of the bronze corydoras. In this study, using Western Blot method, we validated the results presented in the previous research. In detail, results of Western Blot analysis on digestive and respiratory part of bronze corydoras intestine homogenates confirmed the immunoreactivity of anti-cytoplasmic domain (C-terminal) human EGFR antibodies with protein band of approximately 180kDa (EGFR molecular weight). This indicates a high homology of EGFR domain between these species and the possibility of such antibody use in bronze corydoras. Statistically significantly higher EGFR expression was observed in the respiratory part of intestine when compared to the digestive part. This implies higher proliferation activity and angiogenesis of epithelium in this part of intestine, creating conditions for air respiration. Therefore, Corydoras aeneus may be considered as a model organism for the molecular studies of the mechanisms of epithelial proliferation initiation and inhibition depending on hypoxia and normoxia. Copyright © 2017. Published by Elsevier GmbH.

  11. Detection of proteins on blot transfer membranes.

    PubMed

    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.

  12. Clinical usefulness of Western blotting and ELISA avidity for the diagnosis of human toxocariasis.

    PubMed

    Rudzińska, M; Kowalewska, B; Sikorska, K

    2017-01-01

    The serodiagnosis of human toxocariasis is difficult. Specific IgGs detected routinely with ELISA based on Toxocara excretory-secretory (TES) antigens often persist for years at an elevated level, which does not allow either the differentiation between an active and persistent infection or monitoring of the effect of treatment. Additionally, false-positive results may occur in co-infections with other helminths due to cross-reactions. We evaluated the usefulness of an IgG avidity index (AI) and a Western blotting (WB) IgG in the diagnosis of patients suspected of Toxocara infection. We studied 138 subjects who were submitted to serological testing two or more times. Confirmation of an infection by WB was achieved in 73.2% of patients. A high AI was obtained in 89.1% of patients, and low AI and borderline AI were found in only 10.9%. Low and borderline values of AI remained at similar levels in subsequent studies over 2-3 years. The results showed the necessity of obligatory verification of all ELISA IgG positive and questionable results by WB. The index of IgG avidity may be helpful in excluding recent infection, but its usefulness in detecting an active phase of invasion requires further research. © 2016 John Wiley & Sons Ltd.

  13. An assessment of a Western blot method for the investigation of canine cutaneous adverse food reactions.

    PubMed

    Maina, Elisa; Matricoti, Irina; Noli, Chiara

    2018-06-01

    Adverse food reaction (AFR) is diagnosed with a two month elimination diet (ED), followed by challenge with the original food. To evaluate reactivity of selected EDs and performance of a Western blot serological test for the diagnosis of AFR. Twenty five food reactive (FR) and 13 non food reactive (NFR) privately owned dogs. Sera were tested for antibodies against hydrolyzed poultry feather (RCA), hydrolyzed soy (PHA), hydrolyzed fish (FUH), limited antigen horse and potato (THP), fresh horse meat and the offending food for each FR dog as documented by provocative challenge. Fourteen sera were negative and two positive to all foods. Sera from five of 13 NFR and three of 25 FR dogs were reactive to hydrolyzed foods. The RCA diet was recognized by four of 38, FUH by six of 38 and PHA by one of 28 samples. THP was recognized by 14 of 33 and fresh horse by one of ten dogs that had never eaten horse meat. The test correctly identified one of 15 dogs allergic to FUH. Twenty of 25 FR sera were negative for the dogs' respective offending foods (20% sensitivity), whereas four of 13 NFR sera were positive to the dogs' usual diets (69% specificity). Western blot analysis cannot be considered as a valid tool for the diagnosis of AFR; it may serve as an aid in selecting an ED. © 2018 ESVD and ACVD.

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

    PubMed

    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.

  15. Cy5 total protein normalization in Western blot analysis.

    PubMed

    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.

  16. Analysis of immunostaining and western blotting of endothelin 1 and its receptors in mitral stenosis

    PubMed Central

    Leão, Sydney Correia; Dashwood, Michael R.; de Andrade, Mateus Santana; Santos, Nicolas Nascimento; Teles, Olivia Regina Lins Leal; de Souza, Williasmin Batista; Rodrigues, Tania Maria de Andrade

    2015-01-01

    Introduction Rheumatic Fever represents a serious public health problem in developing countries, with thousands of new cases each year. It is an autoimmune disease, which occurs in response to infection by streptococcus A. Objective The aim of this study was to evaluate the immunolabeling and protein expression for endothelin-1 and 3 (ET-1, ET-3) and its receptors (ETA, ETB) in rheumatic mitral valves. Methods Immunohistochemistry was used to identify ET-1/ET-3 and ETA/ETB receptors in rheumatic and control mitral valves. Quantitative analysis of immunostaining for ET-1/ET-3 and ETA/ETB receptors was performed. In addition, western blot analysis was carried out to assess protein levels in tissue samples. Results ET-1 and ETA receptor immunostaining predominated in stenotic valves, mainly associated with fibrotic regions, inflammatory areas and neovascularization. Quantitative analysis showed that the average area with positive expression of ET-1 was 18.21±14.96%. For ETA and ETB, the mean expressed areas were respectively 15.06±13.13% and 9.20±11.09%. ET-3 did not have a significant expression. The correlation between the expression of both endothelin receptors were strongly positive (R=0.74, P=0.02), but the correlation between ET-1 and its receptor were negative for both ETA (R=-0.37, P=0.25), and ETB (R=-0.14, P=0.39). This data was supported by western blot analysis. Conclusion The strong correlation between ET-1 and its receptors suggests that both play a role in the pathophysiology of rheumatic mitral valve stenosis and may potentially act as biomarkers of this disease. PMID:26107453

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

    PubMed

    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. Nonenzymatic chemiluminescent detection and quantitation of total protein on Western and slot blots allowing subsequent immunodetection and sequencing.

    PubMed

    Alba, F J; Daban, J R

    1997-10-01

    We have studied the light emission efficiency of proteins labeled with different fluorescent dyes chemically excited by the bis(2,4,6-trichlorophenyl)oxalate (TCPO)-H2O2 reaction. Using this peroxyoxalate chemiluminescence system, the best results were obtained with proteins covalently labeled with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF). Blotted proteins on polyvinylidene difluoride (PVDF) membranes can be labeled rapidly with MDPF. Our results demonstrate that energy from the excited intermediate produced in the TCPO-H2O2 reaction can be efficiently transferred to MDPF-labeled proteins in solution and on PVDF membranes. Although this nonenzymatic chemiluminescent system produces a background emission that reduces the sensitivity, the method developed in this work allows detection of 5 ng of protein in blots after 5 min exposure to X-ray film. Chemiluminescence of MDPF-labeled proteins on Western and slot blots may also be detected and quantified using a charge-coupled device (CCD) camera or a storage phosphor imaging system. This chemiluminescent method allows the staining of the total electrophoretic pattern but does not preclude further N-terminal sequencing and immunodetection of specific bands.

  19. Evaluation of the Western blotting method for the diagnosis of congenital toxoplasmosis.

    PubMed

    Capobiango, Jaqueline Dario; Monica, Thaís Cabral; Ferreira, Fernanda Pinto; Mitsuka-Breganó, Regina; Navarro, Italmar Teodorico; Garcia, João Luis; Reiche, Edna Maria Vissoci

    To evaluate the Western blotting method for the detection of IgG anti-Toxoplasma gondii (T. gondii) (IgG-WB) in the serum of children with suspected congenital toxoplasmosis. We accompanied 47 mothers with acquired toxoplasmosis in pregnancy and their children, between June of 2011 and June of 2014. The IgG-WB was done in house and the test was considered positive if the child had antibodies that recognized at least one band on IgG blots different from the mother's or with greater intensity than the corresponding maternal band, during the first three months of life. 15 children (15.1%) met the criteria for congenital toxoplasmosis and 32 (32.3%) had the diagnosis excluded. The symptoms were observed in 12 (80.0%) children and the most frequent were cerebral calcification in 9 (60.0%), chorioretinitis in 8 (53.3%), and hydrocephalus in 4 (26.6%). IgM antibodies anti-T. gondii detected by chemiluminescence (CL) were found in 6 (40.0%) children and the polymerase chain reaction (PCR) for detection of T. gondii DNA was positive in 5 of 7 performed (71.4%). The sensitivity of IgG-WB was of 60.0% [95% confidence interval (CI) 32.3-83.7%] and specificity 43.7% (95% CI 26.7-62.3%). The sensitivity of IgG-WB increased to 76.0 and 89.1% when associated to the research of IgM anti-T. gondii or PCR, respectively. The IgG-WB showed greater sensitivity than the detection of IgM anti-T. gondii; therefore, it can be used for the diagnosis of congenital toxoplasmosis in association with other congenital infection markers. Copyright © 2016 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  20. Comparison of a serum indirect fluorescent antibody test with two Western blot tests for the diagnosis of equine protozoal myeloencephalitis.

    PubMed

    Duarte, Paulo C; Daft, Barbara M; Conrad, Patricia A; Packham, Andrea E; Gardner, Ian A

    2003-01-01

    A serum indirect fluorescent antibody test (IFAT) was compared with a Western blot (WB) and a modified Western blot (mWB) for diagnosis of equine protozoal myeloencephalitis (EPM). Using receiver-operating characteristic (ROC) analysis, the area under the curve of the IFAT was greater than the areaunder the curves of the WB and the mWB (P = 0.025 and P = 0.044, respectively). There was no statistically significant difference between the areas under the curves of the WBs (P > 0.05). On the basis of an arbitrarily chosen cut-off titer for a positive test result of 1:80 for the IFAT and interpreting weak positive WB results as positive test results, the sensitivities and 95% confidence intervals (CI) of all 3 tests were identical and equal to 88.9% (51.8-99.7%). The specificities and 95% CIs of the IFAT, WB, and mWB test were 100% (91-100%), 87.2% (72.6-95.7%), and 69.2% (52.4-83%), respectively. The overall accuracy of the IFAT was shown to be better than that of the WBs and, therefore, the test has potential for use in the diagnosis of EPM caused by Sarcocystis neurona.

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

    PubMed

    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

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

    PubMed

    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.

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

    PubMed Central

    Ericson, Brad L.; Carlson, Darby J.

    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. PMID:27298753

  4. A Western blot-based investigation of the yeast secretory pathway designed for an intermediate-level undergraduate cell biology laboratory.

    PubMed

    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.

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

    PubMed

    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. Contribution of a Comparative Western Blot Method to Early Postnatal Diagnosis of Congenital Syphilis.

    PubMed

    Marangoni, Antonella; Foschi, Claudio; Capretti, Maria Grazia; Nardini, Paola; Compri, Monica; Corvaglia, Luigi Tommaso; Faldella, Giacomo; Cevenini, Roberto

    2016-05-01

    Serology has a pivotal role in the diagnosis of congenital syphilis (CS), but problems arise because of the passive transfer of IgG antibodies across the placenta. The aim of this study was to assess the diagnostic value of a comparative Western blot (WB) method finalized to match the IgG immunological profiles of mothers and their own babies at birth in order to differentiate between passively transmitted maternal antibodies and antibodies synthesized by the infants against Treponema pallidum Thirty infants born to mothers with unknown or inadequate treatment for syphilis were entered in a retrospective study, conducted at St. Orsola-Malpighi Hospital, Bologna, Italy. All of the infants underwent clinical, instrumental, and laboratory examinations, including IgM WB testing. For the retrospective study, an IgG WB assay was performed by blotting T. pallidum antigens onto nitrocellulose sheets and incubating the strips with serum specimens from mother-child pairs. CS was diagnosed in 11 out of the 30 enrolled infants; 9/11 cases received the definitive diagnosis within the first week of life, whereas the remaining two were diagnosed later because of increasing serological test titers. The use of the comparative IgG WB testing performed with serum samples from mother-child pairs allowed a correct CS diagnosis in 10/11 cases. The CS diagnosis was improved by a strategy combining comparative IgG WB results with IgM WB results, leading to a sensitivity of 100%. The comparative IgG WB test is thus a welcome addition to the conventional laboratory methods used for CS diagnosis, allowing identification and adequate treatment of infected infants and avoiding unnecessary therapy of uninfected newborns. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  7. Glyceraldehyde-3-phosphate dehydrogenase: a universal internal control for Western blots in prokaryotic and eukaryotic cells.

    PubMed

    Wu, Yonghong; Wu, Min; He, Guowei; Zhang, Xiao; Li, Weiguang; Gao, Yan; Li, Zhihui; Wang, Zhaoyan; Zhang, Chenggang

    2012-04-01

    In the current study, we examined the expression level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein in a number of organisms and the stability of GAPDH under various conditions. Our results revealed that GAPDH is present in multiple Escherichia coli strains, the yeast strain GS115, Caenorhabditis elegans, rat PC12 cells, and both mouse and rat brain. Furthermore, GAPDH was stably expressed under different concentrations of inducer and at different times of induction in E. coli (BL21) cells and yeast GS115 cells. Stable expression of GAPDH protein was also observed in C.elegans and PC12 cells that were treated with different concentrations of paraquat or sodium sulfite, respectively. In addition, we were able to detect and identify the endogenous gapA protein in E.coli via immunoprecipitation and MALDI-TOF-MS analysis. Endogenous gapA protein and exogenously expressed (subcloned) GAPDH proteins were detected in E. coli BL21 but not for gapC. With the exception of gapC in E. coli, the various isoforms of GAPDH possessed enzymatic activity. Finally, sequence analysis revealed that the GAPDH proteins were 76% identical, with the exception of E. coli gapC. Taken together, our results indicate that GAPDH could be universally used as an internal control for the Western blot analysis of prokaryotic and eukaryotic samples. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  8. Western blot analysis of sera from dogs with suspected food allergy.

    PubMed

    Favrot, Claude; Linek, Monika; Fontaine, Jacques; Beco, Luc; Rostaher, Ana; Fischer, Nina; Couturier, Nicolas; Jacquenet, Sandrine; Bihain, Bernard E

    2017-04-01

    Food allergy is often suspected in dogs with clinical signs of atopic dermatitis. This diagnosis is confirmed with an elimination diet and a subsequent challenge with regular food. Laboratory tests for the diagnosis of food allergy in dogs are unreliable and/or technically difficult. Cyno-DIAL ® is a Western blot method that might assist with the selection of an appropriate elimination diet. To evaluate the performance of Cyno-DIAL ® for the selection of an elimination diet and diagnosis of food allergy. Thirty eight dogs with atopic dermatitis completed an elimination diet. Combining the results of the diet trials and the challenges, 14 dogs were classified as food allergic (FA), 22 as nonfood-allergic and two as ambiguous cases. Amongst all dogs and amongst dogs with a clinical diagnosis of FA, 3% and 7% (respectively) were positive to Royal Canin Anallergenic ® , Vet-Concept Kanguru ® or Vet-Concept Dog Sana ® ; 8% and 7% to Hill's d/d Duck and Rice ® ; 8% and 21% to Hill's z/d Ultra Allergen Free ® ; 53% and 64% to Eukanuba Dermatosis FP ® ; and 32% and 43% to a home-cooked diet of horse meat, potatoes and zucchini. The specificity and sensitivity of Cyno-DIAL ® for diagnosing food allergy were 73% and 71%, respectively. Although Cyno-DIAL ® was considered potentially useful for identifying appropriate foods for elimination diet trials, it cannot be recommended for the diagnosis of food allergy. The Cyno-DIAL ® test performed better than some previously evaluated ELISA-based tests. © 2017 ESVD and ACVD.

  9. A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis.

    PubMed

    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.

  10. Identification of Yeast V-ATPase Mutants by Western Blots Analysis of Whole Cell Lysates

    NASA Astrophysics Data System (ADS)

    Parra-Belky, Karlett

    2002-11-01

    A biochemistry laboratory was designed for an undergraduate course to help students better understand the link between molecular engineering and biochemistry. Students identified unknown yeast strains with high specificity using SDS-PAGE and Western blot analysis of whole cell lysates. This problem-solving exercise is a common application of biochemistry in biotechnology research. Three different strains were used: a wild-type and two mutants for the proton pump vacuolar ATPase (V-ATPase). V-ATPases are multisubunit enzymes and the mutants used were deletion mutants; each lacked one structural gene of the complex. After three, three-hour labs, mutant strains were easily identified by the students and distinguished from wild-type cells analyzing the pattern of SDS-PAGE distribution of proteins. Identifying different subunits of one multimeric protein allowed for discussion of the structure and function of this metabolic enzyme, which captured the interest of the students. The experiment can be adapted to other multimeric protein complexes and shows improvement of the described methodology over previous reports, perhaps because the problem and its solution are representative of the type of techniques currently used in research labs.

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

    PubMed

    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.

  12. A Secondary Antibody-Detecting Molecular Weight Marker with Mouse and Rabbit IgG Fc Linear Epitopes for Western Blot Analysis

    PubMed Central

    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. PMID:27494183

  13. Interrogation of Multidrug Resistance (MDR1) P-glycoprotein (Pgp, ABCB1) Expression in Human Pancreatic Carcinoma Cells: Correlation of 99mTc-Sestamibi Uptake with Western Blot Analysis

    PubMed Central

    Harpstrite, Scott E.; Gu, Hannah; Natarajan, Radhika; Sharma, Vijay

    2014-01-01

    Objective Histopathological studies indicate approximately 63% of pancreatic tumors express MDR1 Pgp and its polymorphic variants. However, Pgp expression detected at the messenger RNA or protein level does not always correlate with functional transport activity. Because Pgp transport activity is affected by specific mutations as well as the phosphorylation state of the protein, altered or less active forms of Pgp may also be detected by PCR or immunohistochemistry, which do not accurately reflect the status of tumor cell resistance. To interrogate status of functional expression of MDR1 Pgp in MiaPaCa-2 and PANC-1 cells, cellular transport studies using 99mTc-Sestamibi were performed and correlated with western blot analysis. Methods Biochemical transport assays in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells, human epidermal carcinoma drug sensitive KB-3-1 cells and human breast carcinoma MCF-7 cells (negative controls), and human epidermal carcinoma drug resistant KB-8-5 cells, human breast carcinoma stably transfected with Pgp MCF-7/MDR1Pgp cells, and liver carcinoma HepG2 cells (positive controls) were performed. Protein levels were determined using a monoclonal antibody C219. Results 99mTc-Sestamibi demonstrates accumulation in human pancreatic carcinoma MiaPaCa-2 and PANC-1 cells. Uptake profiles are not affected by treatment with LY335979, a Pgp-inhibitor, and correlate to Western blot analysis. Conclusions These cellular transport studies indicate an absence of Pgp at a functional level in MiaPaCa-2 and PANC-1 cells. Because major pancreatic tumors originate from pancreatic duct and 99mTc-Sestamibi undergoes a dominant hepatobiliary mode of excretion, it would not be a sensitive probe for imaging pancreatic adenocarcinomas. Following interrogation of the functional status of Pgp in other pancreatic carcinoma cells, chemotherapeutic drugs that are also MDR1 substrates could offer alternative therapeutics for treating pancreatic adenocarcinomas

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

    PubMed

    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).

  15. Detection of autoimmunity in early primary Epstein-Barr virus infection by Western blot analysis.

    PubMed

    Mascia, M T; Sandri, G; Guerzoni, C; Roncaglia, R; Mantovani, G; Ferri, C

    2008-01-01

    The Epstein-Barr virus (EBV) represents a potentially important factor in the pathogenesis of certain autoimmune disorders such as systemic lupus erythematosus (SLE), and Sjögren's syndrome, probably through a molecular mimicry mechanism. Several studies have focused on the relationship between previous EBV infection and clinically overt connective tissue diseases (CTDs), while the aim of this study was to investigate the immunological alterations during the early phase of primary acute EBV infection by means of ENA Western blotting (WB) analysis. This technique is able to detect a wide spectrum of anti-ENA autoantibodies, potentially directed against diverse epitopes of the same antigen. Sera from 54 subjects (F/M=24/30, mean age 17+/-6 SD years) with primary acute EBV infection were analysed using indirect immunofluorescence (IF) on Hep-2 cells for ANA, and both ELISA and WB for ENA. Only 8 ANA+ and no ENA+ were found by means of IF and ELISA techniques, respectively; however, one or more ENA autoantibodies were detected in 24/54 (44%) sera using WB. The autoantibodies were no longer present at the second evaluation. Subjects with immunological alterations had not developed any significant clinical manifestations at a 5-year follow-up. This study demonstrated the appearance of autoantibody production in a high proportion of individuals with primary acute EBV infection; interestingly, the observed serological subsets are quite similar to clinical SLE clusters. Moreover, the absence of immunological disorders during the follow-up reinforces the role of multiple genetic and/or environmental co-factors in the pathogenesis of CTDs.

  16. Evaluation of the Aspergillus Western Blot IgG Kit for Diagnosis of Chronic Aspergillosis

    PubMed Central

    Oliva, A.; Flori, P.; Hennequin, C.; Dubus, J.-C.; Reynaud-Gaubert, M.; Charpin, D.; Vergnon, J. M.; Gay, P.; Colly, A.; Piarroux, R.; Pelloux, H.

    2014-01-01

    Immunoprecipitin detection (IPD) is the current reference confirmatory technique for anti-Aspergillus antibody detection; however, the lack of standardization is a critical drawback of this assay. In this study, we evaluated the performance of the Aspergillus Western blot (Asp-WB) IgG kit (LDBio Diagnostics, Lyon, France), a recently commercialized immunoblot assay for the diagnosis of various clinical presentations of chronic aspergillosis. Three hundred eight serum samples from 158 patients with aspergillosis sensu lato (s.l.) were analyzed. More specifically, 267 serum samples were derived from patients with Aspergillus disease, including 89 cases of chronic pulmonary aspergillosis, 10 of aspergilloma, and 32 of allergic bronchopulmonary aspergillosis, while 41 samples were from patients with Aspergillus colonization, including 15 cystic fibrosis (CF) and 12 non-CF patients. For blood donor controls, the Asp-WB specificity was 94%, while the kit displayed a sensitivity for the aspergillosis s.l. diagnosis of 88.6%, with a diagnostic odds ratio (DOR) of 119 (95% confidence interval [CI], 57 to 251). The DOR values were 185.22 (95% CI,78.79 to 435.45) and 43.74 (95% CI, 15.65 to 122.20) for the diagnosis of Aspergillus disease and Aspergillus colonization, respectively. Among the patients, the sensitivities of the Asp-WB in the diagnosis of Aspergillus colonization were 100% and 41.7% in CF and non-CF patients, respectively. The Asp-WB yielded fewer false-negative results than did IPD. In conclusion, the Asp-WB kit performed well for the diagnosis of various clinical presentations of aspergillosis in nonimmunocompromised patients, with an enhanced standardization and a higher sensitivity than with IPD, which is the current reference method. PMID:25392351

  17. Diffusion blotting: a rapid and simple method for production of multiple blots from a single gel.

    PubMed

    Olsen, Ingrid; Wiker, Harald G

    2015-01-01

    A very simple and fast method for diffusion blotting of proteins from precast SDS-PAGE gels on a solid plastic support was developed. Diffusion blotting for 3 min gives a quantitative transfer of 10 % compared to 1-h electroblotting. For each subsequent blot from the same gel a doubling of transfer time is necessary to obtain the same amount of protein onto each blot. High- and low-molecular-weight components are transferred equally efficiently when compared to electroblotting. However, both methods do give a higher total transfer of the low-molecular-weight proteins compared to the large proteins. The greatest advantage of diffusion blotting is that several blots can be made from each lane, thus enabling testing of multiple antisera on virtually identical blots. The gel remains on the plastic support, which prevents it from stretching or shrinking. This ensures identical blots and facilitates more reliable molecular weight determination. Furthermore the proteins remaining in the gel can be stained with Coomassie Brilliant Blue or other methods for exact and easy comparison with the developed blots. These advantages make diffusion blotting the method of choice when quantitative protein transfer is not required.

  18. Far-western blotting as a solution to the non-specificity of the anti-erythropoietin receptor antibody

    PubMed Central

    Fecková, Barbora; Kimáková, Patrícia; Ilkovičová, Lenka; Szentpéteriová, Erika; Debeljak, Nataša; Solárová, Zuzana; Sačková, Veronika; Šemeláková, Martina; Bhide, Mangesh; Solár, Peter

    2016-01-01

    The erythropoietin receptor (EpoR) is a member of the cytokine receptor family. The interaction between erythropoietin (Epo) and EpoR is important for the production and maturation of erythroid cells, resulting in the stimulation of hematopoiesis. The fact that EpoR was also detected in neoplastic cells has opened the question about the relevance of anemia treatment with recombinant Epo in cancer patients. Numerous studies have reported pro-stimulating and anti-apoptotic effects of Epo in cancer cells, thus demonstrating EpoR functionality in these cells. By contrast, a previous study claims the absence of EpoR in tumor cells. This apparent discrepancy is based, according to certain authors, on the use of non-specific anti-EpoR antibodies. With the aim of bypassing the direct detection of EpoR with an anti-EpoR antibody, the present authors propose a far-western blot methodology, which in addition, confirms the interaction of Epo with EpoR. Applying this technique, the presence of EpoR and its interaction with Epo in human ovarian adenocarcinoma A2780 and normal human umbilical vein endothelial cells was confirmed. Furthermore, modified immunoprecipitation of EpoR followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis confirmed a 57 kDa protein as a human Epo-interacting protein in both cell lines. PMID:27446474

  19. 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.

    PubMed

    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.

  20. Evaluation of the Aspergillus Western blot IgG kit for diagnosis of chronic aspergillosis.

    PubMed

    Oliva, A; Flori, P; Hennequin, C; Dubus, J-C; Reynaud-Gaubert, M; Charpin, D; Vergnon, J M; Gay, P; Colly, A; Piarroux, R; Pelloux, H; Ranque, S

    2015-01-01

    Immunoprecipitin detection (IPD) is the current reference confirmatory technique for anti-Aspergillus antibody detection; however, the lack of standardization is a critical drawback of this assay. In this study, we evaluated the performance of the Aspergillus Western blot (Asp-WB) IgG kit (LDBio Diagnostics, Lyon, France), a recently commercialized immunoblot assay for the diagnosis of various clinical presentations of chronic aspergillosis. Three hundred eight serum samples from 158 patients with aspergillosis sensu lato (s.l.) were analyzed. More specifically, 267 serum samples were derived from patients with Aspergillus disease, including 89 cases of chronic pulmonary aspergillosis, 10 of aspergilloma, and 32 of allergic bronchopulmonary aspergillosis, while 41 samples were from patients with Aspergillus colonization, including 15 cystic fibrosis (CF) and 12 non-CF patients. For blood donor controls, the Asp-WB specificity was 94%, while the kit displayed a sensitivity for the aspergillosis s.l. diagnosis of 88.6%, with a diagnostic odds ratio (DOR) of 119 (95% confidence interval [CI], 57 to 251). The DOR values were 185.22 (95% CI,78.79 to 435.45) and 43.74 (95% CI, 15.65 to 122.20) for the diagnosis of Aspergillus disease and Aspergillus colonization, respectively. Among the patients, the sensitivities of the Asp-WB in the diagnosis of Aspergillus colonization were 100% and 41.7% in CF and non-CF patients, respectively. The Asp-WB yielded fewer false-negative results than did IPD. In conclusion, the Asp-WB kit performed well for the diagnosis of various clinical presentations of aspergillosis in nonimmunocompromised patients, with an enhanced standardization and a higher sensitivity than with IPD, which is the current reference method. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. Anti-Peptide Monoclonal Antibodies Generated for Immuno-Multiple Reaction Monitoring-Mass Spectrometry Assays Have a High Probability of Supporting Western blot and ELISA*

    PubMed Central

    Schoenherr, Regine M.; Saul, Richard G.; Whiteaker, Jeffrey R.; Yan, Ping; Whiteley, Gordon R.; Paulovich, Amanda G.

    2015-01-01

    Immunoaffinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) has recently been developed for quantitative analysis of peptide and protein expression. As part of this technology, antibodies are generated to short, linear, tryptic peptides that are well-suited for detection by mass spectrometry. Despite its favorable analytical performance, a major obstacle to widespread adoption of immuno-MRM is a lack of validated affinity reagents because commercial antibody suppliers are reluctant to commit resources to producing anti-peptide antibodies for immuno-MRM while the market is much larger for conventional technologies, especially Western blotting and ELISA. Part of this reluctance has been the concern that affinity reagents generated to short, linear, tryptic peptide sequences may not perform well in traditional assays that detect full-length proteins. In this study, we test the feasibility and success rates of generating immuno-MRM monoclonal antibodies (mAbs) (targeting tryptic peptide antigens) that are also compatible with conventional, protein-based immuno-affinity technologies. We generated 40 novel, peptide immuno-MRM assays and determined that the cross-over success rates for using immuno-MRM monoclonals for Western blotting is 58% and for ELISA is 43%, which compare favorably to cross-over success rates amongst conventional immunoassay technologies. These success rates could most likely be increased if conventional and immuno-MRM antigen design strategies were combined, and we suggest a workflow for such a comprehensive approach. Additionally, the 40 novel immuno-MRM assays underwent fit-for-purpose analytical validation, and all mAbs and assays have been made available as a resource to the community via the Clinical Proteomic Tumor Analysis Consortium's (CPTAC) Antibody (http://antibodies.cancer.gov) and Assay Portals (http://assays.cancer.gov), respectively. This study also represents the first

  2. Southern blotting.

    PubMed

    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

  3. Far Western: probing membranes.

    PubMed

    Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

    2007-08-01

    INTRODUCTIONThe far-Western technique described in this protocol is fundamentally similar to Western blotting. In Western blots, an antibody is used to detect a query protein on a membrane. In contrast, in a far-Western blot (also known as an overlay assay) the antibody is replaced by a recombinant GST fusion protein (produced and purified from bacteria), and the assay detects the interaction of this protein with target proteins on a membrane. The membranes are washed and blocked, incubated with probe protein, washed again, and subjected to autoradiography. The GST fusion (probe) proteins are often labeled with (32)P; alternatively, the membrane can be probed with unlabeled GST fusion protein, followed by detection using commercially available GST antibodies. The nonradioactive approach is substantially more expensive (due to the purchase of antibody and detection reagents) than using radioactively labeled proteins. In addition, care must be taken to control for nonspecific interactions with GST alone and a signal resulting from antibody cross-reactivity. In some instances, proteins on the membrane are not able to interact after transfer. This may be due to improper folding, particularly in the case of proteins expressed from a phage expression library. This protocol describes a way to overcome this by washing the membrane in denaturation buffer, which is then serially diluted to permit slow renaturation of the proteins.

  4. Serum detection of IgG antibodies against Demodex canis by western blot in healthy dogs and dogs with juvenile generalized demodicosis.

    PubMed

    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.

  5. Southern blotting.

    PubMed

    Brown, T

    2001-05-01

    Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support, 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 unit describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, and subsequent immobilization 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. 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 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 the second alternate protocol is therefore more rapid and can result in more complete transfer.

  6. A Simple Method to Avoid Nonspecific Signal When Using Monoclonal Anti-Tau Antibodies in Western Blotting of Mouse Brain Proteins.

    PubMed

    Petry, Franck R; Nicholls, Samantha B; Hébert, Sébastien S; Planel, Emmanuel

    2017-01-01

    In Alzheimer's disease and other tauopathies, tau displays several abnormal post-translation modifications such as hyperphosphorylation, truncation, conformation, and oligomerization. Mouse monoclonal antibodies have been raised against such tau modifications for research, diagnostic, and therapeutic purposes. However, many of these primary antibodies are at risk of giving nonspecific signals in common Western blotting procedures. Not because they are unspecific, but because the secondary antibodies used to detect them will also detect the heavy chain of endogenous mouse immunoglobulins (Igs), and give a nonspecific signal at the same molecular weight than tau protein (around 50 kDa). Here, we propose the use of anti-light chain secondary antibodies as a simple and efficient technique to prevent nonspecific Igs signals at around 50 kDa. We demonstrate the efficacy of this method by removing artifactual signals when using monoclonal antibodies directed at tau phosphorylation (AT100, 12E8, AT270), tau truncation (TauC3), tau oligomerization (TOMA), or tau abnormal conformation (Alz50), in wild-type, 3×Tg-AD, and tau knockout mice.

  7. Evaluation of ELISA coupled with Western blot as a surveillance tool for Trichinella infection in wild boar (Sus scrofa).

    PubMed

    Cuttell, Leigh; Gómez-Morales, Maria Angeles; Cookson, Beth; Adams, Peter J; Reid, Simon A; Vanderlinde, Paul B; Jackson, Louise A; Gray, C; Traub, Rebecca J

    2014-01-31

    Trichinella surveillance in wildlife relies on muscle digestion of large samples which are logistically difficult to store and transport in remote and tropical regions as well as labour-intensive to process. Serological methods such as enzyme-linked immunosorbent assays (ELISAs) offer rapid, cost-effective alternatives for surveillance but should be paired with additional tests because of the high false-positive rates encountered in wildlife. We investigated the utility of ELISAs coupled with Western blot (WB) in providing evidence of Trichinella exposure or infection in wild boar. Serum samples were collected from 673 wild boar from a high- and low-risk region for Trichinella introduction within mainland Australia, which is considered Trichinella-free. Sera were examined using both an 'in-house' and a commercially available indirect-ELISA that used excretory-secretory (E/S) antigens. Cut-off values for positive results were determined using sera from the low-risk population. All wild boar from the high-risk region (352) and 139/321 (43.3%) of the wild boar from the low-risk region were tested by artificial digestion. Testing by Western blot using E/S antigens, and a Trichinella-specific real-time PCR was also carried out on all ELISA-positive samples. The two ELISAs correctly classified all positive controls as well as one naturally infected wild boar from Gabba Island in the Torres Strait. In both the high- and low-risk populations, the ELISA results showed substantial agreement (k-value=0.66) that increased to very good (k-value=0.82) when WB-positive only samples were compared. The results of testing sera collected from the Australian mainland showed the Trichinella seroprevalence was 3.5% (95% C.I. 0.0-8.0) and 2.3% (95% C.I. 0.0-5.6) using the in-house and commercial ELISA coupled with WB respectively. These estimates were significantly higher (P<0.05) than the artificial digestion estimate of 0.0% (95% C.I. 0.0-1.1). Real-time PCR testing of muscle from

  8. Introduction to protein blotting.

    PubMed

    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.

  9. Detection of Proteins on Blot Membranes

    PubMed Central

    Goldman, Aaron; Harper, Sandra; Speicher, David W.

    2017-01-01

    Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. PMID:27801518

  10. Detection of Proteins on Blot Membranes.

    PubMed

    Goldman, Aaron; Harper, Sandra; Speicher, David W

    2016-11-01

    Staining of blot membranes enables the visualization of bound proteins. Proteins are usually transferred to blot membranes by electroblotting, by direct spotting of protein solutions, or by contact blots. Staining allows the efficiency of transfer to the membrane to be monitored. This unit describes protocols for staining proteins after electroblotting from polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. The same methods can be used if proteins are directly spotted, either manually or using robotics. Protocols are included for seven general protein stains (amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, India ink, and MemCode) and three fluorescent protein stains (fluorescamine, IAEDANS, and SYPRO Ruby). Also included is an in-depth discussion of the different blot membrane types and the compatibility of different protein stains with downstream applications, such as immunoblotting or N-terminal Edman sequencing. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  11. [Optimization of the immunoelectrophoresis technic (western blot) for the confirmation of human immunodeficiency virus infection (HIV) in Panama].

    PubMed

    Pascale, J M; de Austin, E; de Moreno, N O; Ledezma, C; de Márquez, E; Blanco, R; Quiroz, E; Calzada, J; Vincensini, A R; de Martin, M C

    1995-01-01

    The purpose of this study is to report the results of the authors' investigation to apply the western blot technique (WB UP-LCS) in the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection. To do this, the authors separated the proteins of the HIV-1 virus by electrophoresis, based on their molecular weight, in poliacilamide gel with SDS (SDS-PAGE) during 3 hours at 200 volts. Then they electrotransferred these proteins to nitrocellulose paper during four hours at 200 milliamperes, with the aid of external cooling. The nitrocellulose strips were evaluated considering the incubation time (1 and 16 hours), two conjugates (human anti IgG with Peroxidase and human anti IgG Biotin plus Streptatividine with Peroxidase) and two dilutions of the patients' sera (1/50 and 1/100). Based on their results the Authors conclude that, in the first place, the optimal conditions for the test include a dilution of 1/100 of the patients serum, incubation of the serum for 16 hours and the use of the conjugate of anti human IgG with Biotin and Streptavidine with Peroxidase; secondary, that the immunologic reactivity against proteins p24 and gp 160/120 is the most important diagnostic criterion for the confirmation of infection with HIV-1 and that they obtained a diagnostic correlation of 100% at a cost which was 5 to 7 times less than that of the commercial system.

  12. Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results

    PubMed Central

    Sekizuka, Tsuyoshi; Yamochi, Tadanori; Firouzi, Sanaz; Sato, Tomoo; Umeki, Kazumi; Sasaki, Daisuke; Hasegawa, Hiroo; Kubota, Ryuji; Sobata, Rieko; Matsumoto, Chieko; Kaneko, Noriaki; Momose, Haruka; Araki, Kumiko; Saito, Masumichi; Nosaka, Kisato; Utsunomiya, Atae; Koh, Ki-Ryang; Ogata, Masao; Uchimaru, Kaoru; Iwanaga, Masako; Sagara, Yasuko; Yamano, Yoshihisa; Okayama, Akihiko; Miura, Kiyonori; Satake, Masahiro; Saito, Shigeru; Itabashi, Kazuo; Yamaguchi, Kazunari; Kuroda, Makoto; Watanabe, Toshiki; Okuma, Kazu

    2017-01-01

    ABSTRACT Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens. PMID:28701419

  13. The epidemiology of tick-borne haemoparasites as determined by the reverse line blot hybridization assay in an intensively studied cohort of calves in western Kenya

    PubMed Central

    Njiiri, Nyawira E.; Bronsvoort, B. Mark deC.; Collins, Nicola E.; Steyn, Helena C.; Troskie, Milana; Vorster, Ilse; Thumbi, S.M.; Sibeko, Kgomotso P.; Jennings, Amy; van Wyk, Ilana Conradie; Mbole-Kariuki, Mary; Kiara, Henry; Poole, E. Jane; Hanotte, Olivier; Coetzer, Koos; Oosthuizen, Marinda C.; Woolhouse, Mark; Toye, Philip

    2015-01-01

    The development of sensitive surveillance technologies using PCR-based detection of microbial DNA, such as the reverse line blot assay, can facilitate the gathering of epidemiological information on tick-borne diseases, which continue to hamper the productivity of livestock in many parts of Africa and elsewhere. We have employed a reverse line blot assay to detect the prevalence of tick-borne parasites in an intensively studied cohort of indigenous calves in western Kenya. The calves were recruited close to birth and monitored for the presence of infectious disease for up to 51 weeks. The final visit samples from 453 calves which survived for the study period were analyzed by RLB. The results indicated high prevalences of Theileria mutans (71.6%), T. velifera (62.8%), Anaplasma sp. Omatjenne (42.7%), A. bovis (39.9%), Theileria sp. (sable) (32.7%), T. parva (12.9%) and T. taurotragi (8.5%), with minor occurrences of eight other haemoparasites. The unexpectedly low prevalence of the pathogenic species Ehrlichia ruminantium was confirmed by a species-specific PCR targeting the pCS20 gene region. Coinfection analyses of the seven most prevalent haemoparasites indicated that they were present as coinfections in over 90% of the cases. The analyses revealed significant associations between several of the Theileria parasites, in particular T. velifera with Theileria sp. sable and T. mutans, and T. parva with T. taurotragi. There was very little coinfection of the two most common Anaplasma species, although they were commonly detected as coinfections with the Theileria parasites. The comparison of reverse line blot and serological results for four haemoparasites (T. parva, T. mutans, A. marginale and B. bigemina) indicated that, except for the mostly benign T. mutans, indigenous cattle seem capable of clearing infections of the three other, pathogenic parasites to below detectable levels. Although the study site was located across four agroecological zones, there was

  14. Deep proteomic profiling of vasopressin-sensitive collecting duct cells. I. Virtual Western blots and molecular weight distributions.

    PubMed

    Yang, Chin-Rang; Tongyoo, Pumipat; Emamian, Milad; Sandoval, Pablo C; Raghuram, Viswanathan; Knepper, Mark A

    2015-12-15

    The mouse mpkCCD cell line is a continuous cultured epithelial cell line with characteristics of renal collecting duct principal cells. This line is widely used to study epithelial transport and its regulation. To provide a data resource useful for experimental design and interpretation in studies using mpkCCD cells, we have carried out "deep" proteomic profiling of these cells using three levels of fractionation (differential centrifugation, SDS-PAGE, and HPLC) followed by tandem mass spectrometry to identify and quantify proteins. The analysis of all resulting samples generated 34.6 gigabytes of spectral data. As a result, we identified 6,766 proteins in mpkCCD cells at a high level of stringency. These proteins are expressed over eight orders of magnitude of protein abundance. The data are provided to users as a public data base (https://helixweb.nih.gov/ESBL/Database/mpkFractions/). The mass spectrometry data were mapped back to their gel slices to generate "virtual Western blots" for each protein. For most of the 6,766 proteins, the apparent molecular weight from SDS-PAGE agreed closely with the calculated molecular weight. However, a substantial fraction (>15%) of proteins was found to run aberrantly, with much higher or much lower mobilities than predicted. These proteins were analyzed to identify mechanisms responsible for altered mobility on SDS-PAGE, including high or low isoelectric point, high or low hydrophobicity, physiological cleavage, residence in the lysosome, posttranslational modifications, and expression of alternative isoforms due to alternative exon usage. Additionally, this analysis identified a previously unrecognized isoform of aquaporin-2 with apparent molecular mass <20 kDa. Copyright © 2015 the American Physiological Society.

  15. Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results.

    PubMed

    Kuramitsu, Madoka; Sekizuka, Tsuyoshi; Yamochi, Tadanori; Firouzi, Sanaz; Sato, Tomoo; Umeki, Kazumi; Sasaki, Daisuke; Hasegawa, Hiroo; Kubota, Ryuji; Sobata, Rieko; Matsumoto, Chieko; Kaneko, Noriaki; Momose, Haruka; Araki, Kumiko; Saito, Masumichi; Nosaka, Kisato; Utsunomiya, Atae; Koh, Ki-Ryang; Ogata, Masao; Uchimaru, Kaoru; Iwanaga, Masako; Sagara, Yasuko; Yamano, Yoshihisa; Okayama, Akihiko; Miura, Kiyonori; Satake, Masahiro; Saito, Shigeru; Itabashi, Kazuo; Yamaguchi, Kazunari; Kuroda, Makoto; Watanabe, Toshiki; Okuma, Kazu; Hamaguchi, Isao

    2017-09-01

    Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens. Copyright © 2017 American Society for Microbiology.

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

    PubMed

    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.

  17. Myostatin inhibitors in sports drug testing: Detection of myostatin-neutralizing antibodies in plasma/serum by affinity purification and Western blotting.

    PubMed

    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.

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

    PubMed

    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.

  19. Differential staining of Western blots of human secreted glycoproteins from serum, milk, saliva, and seminal fluid using lectins displaying diverse sugar specificities.

    PubMed

    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.

  20. Lipid-binding analysis using a fat blot assay.

    PubMed

    Munnik, Teun; Wierzchowiecka, Magdalena

    2013-01-01

    Protein-lipid interactions play an important role in lipid metabolism, membrane trafficking and cell -signaling by regulating protein localization, activation, and function. The Fat Blot assay is a relatively simple and inexpensive method to examine these interactions using nitrocellulose membrane-immobilized lipids. The assay is adapted from the method by Dowler et al. (Sci STKE 129:pl6, 2002) and provides qualitative and quantitative information on the relative affinity with which a protein binds to a particular lipid. To perform a Fat Blot assay, serial dilutions of different phospholipids are spotted onto a nitrocellulose membrane. These membranes are then incubated with a lipid-binding protein possessing a GST (or other epitope) tag. The membranes are washed and the protein, which is bound to the membrane by virtue of its interaction with the lipid's head group, is detected by immunoblotting with an antibody against GST (or other epitope). The procedure only requires a few micrograms of protein and is quick, simple and cheap to perform.

  1. Seroprevalence of toxocariasis in hypereosinophilic individuals in Ahwaz, south-western Iran.

    PubMed

    Maraghi, S; Rafiei, A; Hajihossein, R; Sadjjadi, S M

    2012-06-01

    Eosinophilia in human peripheral blood is caused by different agents, including toxocariasis. The present study aimed to evaluate the prevalence of toxocariasis in hypereosinophilic individuals in the city of Ahwaz, located in south-western Iran, using enzyme-linked immunosorbent assay and Western blot techniques. Serum samples were examined from 100 individuals with peripheral blood eosinophilia and also from another 100 individuals without eosinophilia as the control group. In hypereosinophilic individuals seroprevalence antibodies against Toxocara were found in 19 (19%), of whom 12 (63.15%) were female and 7 (36.85%) were male. Positive sera were subsequently confirmed by Western blot. All of the observed bands ranged from 24 to 100 kDa. Antibodies against Toxocara were found in 1% of the control group, but were not confirmed by Western blot. The results showed significant differences between the frequency of infection within age and gender (P < 0.05); the highest prevalence of infection was observed in adults. Differences between the hypereosinophilic and healthy individuals, in terms of Toxocara infection frequency, also proved significant (P < 0.05).The present study thus confirmed the significant prevalence of toxocariasis as a hygienic problem among hypereosinophilic individuals in this area. It is, therefore, necessary to examine these individuals for toxocariasis.

  2. The impact of the Western Ghats on lightning activity on the western coast of India

    NASA Astrophysics Data System (ADS)

    Kamra, A. K.; Nair, A. A.

    2015-06-01

    The effect of the Western Ghats on the lightning activity across the west coast of India around the coastal metropolitan city of Mumbai during the 1998-2012 period is investigated using data from the Lightning Imaging Sensor (LIS) onboard the Tropical Rainfall Measuring Mission (TRMM) satellite. A land-sea contrast of an order of magnitude in the lightning activity is observed even in a small area across the western coast of India. The shape of a zone of high lightning activity formed almost parallel to the Western Ghats during the onset and withdrawal phases of monsoon, strongly suggests the effect of the Western Ghats in its formation. Seasonal variation of the lightning activity in this area and also in each of its four equal sectors (two each over the Arabian Sea and over land) is bi-annual with one peak each in the onset (May/June) and withdrawal months (September/October) of monsoon and a sharp dip to very low values during the monsoon months (July/August) of maximum seasonal rainfall. The lightning activity in each sector is found to increase over the 1998-2012 period. However, the increase in lightning activity over the sector containing Mumbai is found to be greater during the pre- and post-monsoon periods and smaller during the monsoon period as compared to an identical sector immediately south of it.

  3. A Comparison of Antibacterial Activity of Selected Thyme (Thymus) Species by Means of the Dot Blot Test with Direct Bioautographic Detection.

    PubMed

    Orłowska, Marta; Kowalska, Teresa; Sajewicz, Mieczysław; Jesionek, Wioleta; Choma, Irena M; Majer-Dziedzic, Barbara; Szymczak, Grażyna; Waksmundzka-Hajnos, Monika

    2015-01-01

    Bioautography carried out with the aid of thin-layer chromatographic adsorbents can be used to assess antibacterial activity in samples of different origin. It can either be used as a simple and cost-effective detection method applied to a developed chromatogram, or to the dot blot test performed on a chromatographic plate, where total antibacterial activity of a sample is scrutinized. It was an aim of this study to compare antibacterial activity of 18 thyme (Thymus) specimens and species (originating from the same gardening plot and harvested in the same period of time) by means of a dot blot test with direct bioautography. A two-step extraction of herbal material was applied, and at step two the polar fraction of secondary metabolites was obtained under the earlier optimized extraction conditions [methanol-water (27+73, v/v), 130°C]. This fraction was then tested for its antibacterial activity against Bacillus subtilis bacteria. It was established that all investigated extracts exhibited antibacterial activity, yet distinct differences were perceived in the size of the bacterial growth inhibition zones among the compared thyme species. Based on the results obtained, T. citriodorus "golden dwarf" (sample No. 5) and T. marschallianus (sample No. 6) were selected as promising targets for further investigations and possible inclusion in a herbal pharmacopeia, which is an essential scientific novelty of this study.

  4. Estimation of hepatitis E virus (HEV) pig seroprevalence using ELISA and Western blot and comparison between human and pig HEV sequences in Belgium.

    PubMed

    Thiry, Damien; Mauroy, Axel; Saegerman, Claude; Thomas, Isabelle; Wautier, Magali; Miry, Cora; Czaplicki, Guy; Berkvens, Dirk; Praet, Nicolas; van der Poel, Wim; Cariolet, Roland; Brochier, Bernard; Thiry, Etienne

    2014-08-27

    Zoonotic transmission of hepatitis E virus (HEV) is of special concern, particularly in high income countries were waterborne infections are less frequent than in developing countries. High HEV seroprevalences can be found in European pig populations. The aims of this study were to obtain prevalence data on HEV infection in swine in Belgium and to phylogenetically compare Belgian human HEV sequences with those obtained from swine. An ELISA screening prevalence of 73% (95% CI 68.8-77.5) was determined in Belgian pigs and a part of the results were re-evaluated by Western blot (WB). A receiver operating characteristic curve analysis was performed and scenarios varying the ELISA specificity relative to WB were analysed. The seroprevalences estimated by the different scenarios ranged between 69 and 81% and are in agreement with the high exposure of the European pig population to HEV. Pig HEV sequences were genetically compared to those detected in humans in Belgium and a predominance of genotype 3 subtype f was shown in both swine and humans. The high HEV seroprevalence in swine and the close phylogenetic relationships between pig and human HEV sequences further support the risk for zoonotic transmission of HEV between humans and pigs. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Da0324, an inhibitor of nuclear factor-κB activation, demonstrates selective antitumor activity on human gastric cancer cells

    PubMed Central

    Jin, Rong; Xia, Yiqun; Chen, Qiuxiang; Li, Wulan; Chen, Dahui; Ye, Hui; Zhao, Chengguang; Du, Xiaojing; Shi, Dengjian; Wu, Jianzhang; Liang, Guang

    2016-01-01

    Background The transcription factor nuclear factor-κB (NF-κB) is constitutively activated in a variety of human cancers, including gastric cancer. NF-κB inhibitors that selectively kill cancer cells are urgently needed for cancer treatment. Curcumin is a potent inhibitor of NF-κB activation. Unfortunately, the therapeutic potential of curcumin is limited by its relatively low potency and poor cellular bioavailability. In this study, we presented a novel NF-κB inhibitor named Da0324, a synthetic asymmetric mono-carbonyl analog of curcumin. The purpose of this study is to research the expression of NF-κB in gastric cancer and the antitumor activity and mechanism of Da0324 on human gastric cancer cells. Methods The expressions between gastric cancer tissues/cells and normal gastric tissues/cells of NF-κB were evaluated by Western blot. The inhibition viability of compounds on human gastric cancer cell lines SGC-7901, BGC-823, MGC-803, and normal gastric mucosa epithelial cell line GES-1 was assessed with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Absorption spectrum method and high-performance liquid chromatography method detected the stability of the compound in vitro. The compound-induced changes of inducible NF-κB activation in the SGC-7901 and BGC-823 cells were examined by Western blot analysis and immunofluorescence methods. The antitumor activity of compound was performed by clonogenic assay, matrigel invasion assay, flow cytometric analysis, Western blot analysis, and Hoechst 33258 staining assay. Results High levels of p65 were found in gastric cancer tissues and cells. Da0324 displayed higher growth inhibition against several types of gastric cancer cell lines and showed relatively low toxicity to GES-1. Moreover, Da0324 was more stable than curcumin in vitro. Western blot analysis and immunofluorescence methods showed that Da0324 blocked NF-κB activation. In addition, Da0324 significantly inhibited tumor proliferation

  6. Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay.

    PubMed

    Sørensen, M S; Duch, M; Paludan, K; Jørgensen, P; Pedersen, F S

    1992-03-15

    Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.

  7. Discordant human T-lymphotropic virus screening with Western blot confirmation: evaluation of the dual-test algorithm for US blood donations.

    PubMed

    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. Sequential detection of different antigens induced by Epstein-Barr virus and herpes simplex virus in the same Western blot by using dual antibody probes.

    PubMed

    Lin, J C; Pagano, J S

    1986-08-01

    A dual antibody probing technique that permitted a color-coded identification of polypeptides representing different classes of Epstein-Barr virus (EBV) antigens as well as differentiation of the polypeptides induced by different herpesviruses in the same Western blot was developed. When the nitrocellulose sheet was probed first with monoclonal antibody against EBV early antigen diffuse component (EA-D) and then stained with 4-chloro-1-naphthol, four polypeptides specific for EA-D were identified by purple bands. Subsequently, the same nitrocellulose sheet was reprobed with human serum containing antibodies against EBV early antigen, viral capsid antigen, and nuclear antigen and stained with 3,3'-diaminobenzidine. Several brown bands corresponding to early, viral capsid, and nuclear antigen polypeptides were detected. The dual antibody probing technique was used in an analysis to differentiate polypeptides resulting from either EBV or herpes simplex virus infection, either in cells infected by individual virus or in a cell line dually infected by both viruses. On the basis of different colored bands in different lanes of the same gel, 20 polypeptides with molecular weights ranging from 31,000 to 165,000 were identified as herpes simplex virus-specific proteins. These results suggested that the dual antibody probing technique may be applicable in clinical diagnosis for detecting antigens and antibodies derived from different pathogens.

  9. ASBESTOS-INDUCED ACTIVATION OF CELL SIGNALING PATHWAYS IN HUMAN BRONCHIAL EPITHELIAL CELLS

    EPA Science Inventory

    Using respiratory epithelial cells transfected with either superoxide dismutase (SOD) or catalase, the authors tested the hypothesis that the activation of the epidermal growth factor (EGF) receptor signal pathway after asbestos exposure involves an oxidative stress. Western blot...

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

    PubMed

    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.

  11. Activation of Antitumorigenic Stat3beta in Breast Cancer by Splicing Redirection

    DTIC Science & Technology

    2013-07-01

    putative mapped ESEs (shown in green). (B) (Top) RT-PCR and (Bottom) Western Blot analysis of STAT3 a/b levels in MDA-MB-435s cells treated with...codon (PTC), ultimately causing RNA degradation following nonsense mediated decay (NMD). (B) RT-PCR and Western Blot analysis of STAT3 α/β levels in MDA...MB-435s cells treated with 16µM of ST6, ST7 or INV for 4 days. α-tubulin was used as loading control. (C) RT-PCR and Western Blot analysis of STAT3

  12. Automated design of genomic Southern blot probes

    PubMed Central

    2010-01-01

    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 experimentally validate a number

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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'/sup 32/P)-pCp labeled genomic RNA from a related strain. Hybridization was performed at 52/sup 0/C, 50% formamide, 5X SSC. Under these conditions heterologous RNA species must share greater than or equal to 74% sequence homology in order to form stable dsRNA hybrids. Cognate genes of nine members of the Palyam serogroup of orbiviruses were identified andmore » 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.« less

  14. Skin blotting: a noninvasive technique for evaluating physiological skin status.

    PubMed

    Minematsu, Takeo; Horii, Motoko; Oe, Makoto; Sugama, Junko; Mugita, Yuko; Huang, Lijuan; Nakagami, Gojiro; Sanada, Hiromi

    2014-06-01

    The skin performs important structural and physiological functions, and skin assessment represents an important step in identifying skin problems. Although noninvasive techniques for assessing skin status exist, no such techniques for monitoring its physiological status are available. This study aimed to develop a novel skin-assessment technique known as skin blotting, based on the leakage of secreted proteins from inside the skin following overhydration in mice. The applicability of this technique was further investigated in a clinical setting. Skin blotting involves 2 steps: collecting proteins by attaching a damp nitrocellulose membrane to the surface of the skin, and immunostaining the collected proteins. The authors implanted fluorescein-conjugated dextran (F-DEX)-containing agarose gels into mice and detected the tissue distribution of F-DEX under different blotting conditions. They also analyzed the correlations between inflammatory cytokine secretion and leakage following ultraviolet irradiation in mice and in relation to body mass index in humans. The F-DEX in mice was distributed in the deeper and shallower layers of skin and leaked through the transfollicular and transepidermal routes, respectively. Ultraviolet irradiation induced tumor necrosis factor secretion in the epidermis in mice, which was detected by skin blotting, whereas follicular tumor necrosis factor was associated with body mass index in obese human subjects. These results support the applicability of skin blotting for skin assessment. Skin blotting represents a noninvasive technique for assessing skin physiology and has potential as a predictive and diagnostic tool for skin disorders.

  15. The nuclear-factor kappaB pathway is activated in pterygium.

    PubMed

    Siak, Jay Jyh Kuen; Ng, See Liang; Seet, Li-Fong; Beuerman, Roger W; Tong, Louis

    2011-01-05

    Pterygium is a prevalent ocular surface disease with unknown pathogenesis. The authors investigated the role of nuclear factor kappa B (NF-κB) transcription factors in pterygium. Surgically excised primary pterygia were studied compared with uninvolved conjunctiva tissues. NF-κB activation was evaluated using Western blot analysis, ELISA, and DNA-binding assays. Primary pterygium fibroblasts were treated with TNF-α (20 ng/mL), and NF-κB activation was evaluated using immunocytochemistry, Western blot analysis, phospho-IκBα ELISA, and DNA-binding assays. TNF-α stimulation of NF-κB target genes RelB, NFKB2, RANTES, MCP-1, ENA-78, MMP-1, MMP-2, and MMP-3 in pterygium fibroblasts was compared with that in primary tenon fibroblasts by real-time PCR. Phosphorylation of IκBα (Ser32) was increased in pterygia tissues compared with uninvolved conjunctiva tissues, as determined by Western blot analysis and ELISA. IκBα expression was decreased, whereas nuclear RelA and p50 DNA-binding capacities were increased. Within 30 minutes of treatment with TNF-α, pterygium fibroblasts showed increased IκBα phosphorylation and nuclear translocation of RelA and p50. Treatment with TNF-α beyond 12 hours resulted in increased nuclear expression of RelB, p100, and p52. Furthermore, the upregulation of RANTES, MCP-1, ENA-78, MMP-1, MMP-2, and MMP-3 expression was more pronounced in TNF-α-treated pterygium fibroblasts than in tenon fibroblasts. The NF-κB pathway is shown for the first time to be activated in pterygia tissues compared with normal conjunctiva tissues. Stimulation by the inflammatory cytokine TNF-α can activate both canonical and noncanonical NF-κB pathways in pterygium fibroblasts with concomitant upregulation of NF-κB target genes.

  16. Docosahexaenoic Acid (DHA) Provides Neuroprotection in Traumatic Brain Injury Models via Activating Nrf2-ARE Signaling.

    PubMed

    Zhu, Wei; Ding, Yuexia; Kong, Wei; Li, Tuo; Chen, Hongguang

    2018-04-16

    In this study, we explored the neuroprotective effects of docosahexaenoic acid (DHA) in traumatic brain injury (TBI) models. In this study, we first confirmed that DHA was neuroprotective against TBI via the NSS test and Morris water maze experiment. Western blot was conducted to identify the expression of Bax, caspase-3, and Bcl-2. And the cell apoptosis of the TBI models was validated by TUNEL staining. Relationships between nuclear factor erythroid 2-related factor 2-antioxidant response element (Nrf2-ARE) pathway-related genes and DHA were explored by RT-PCR and Western blot. Rats of the DHA group performed remarkably better than those of the TBI group in both NSS test and water maze experiment. DHA conspicuously promoted the expression of Bcl-2 and diminished that of cleaved caspase-3 and Bax, indicating the anti-apoptotic role of DHA. Superoxide dismutase (SOD) activity and cortical malondialdehyde content, glutathione peroxidase (GPx) activity were renovated in rats receiving DHA treatment, implying that the neuroprotective influence of DHA was derived from lightening the oxidative stress caused by TBI. Moreover, immunofluorescence and Western blot experiments revealed that DHA facilitated the translocation of Nrf2 to the nucleus. DHA administration also notably increased the expression of the downstream factors NAD(P)H:quinone oxidoreductase (NQO-1) and heme oxygenase 1(HO-1). DHA exerted neuroprotective influence on the TBI models, potentially through activating the Nrf2- ARE pathway.

  17. Western blotting method (TESAcruzi) as a supplemental test for confirming the presence of anti-Trypanosoma cruzi antibodies in finger prick blood samples from children aged 0-5 years in Brazil.

    PubMed

    Frade, Amanda Farage; Luquetti, Alejandro O; Prata, Aluísio; Ferreira, Antonio Walter

    2011-01-01

    Some Latin American countries have plans for total control and/or eradication of Chagas disease by the main vector (Triatoma infestans) and by blood transfusion. To achieve this, patients with Chagas disease must be identified. A Western blotting test, TESAcruzi, is described as a supplemental test for diagnosis of Chagas disease using samples collected from children <5 years living in different states of Brazil. Blood samples collected by finger prick on filter paper were sent to the test laboratory by a central laboratory to confirm results obtained previously. Ten percent of negative samples, all doubtful and all positive samples were received. Commercial reagents, IgG indirect immunofluorescence, enzyme immunoassay, and a recently introduced TESAcruzi test were used. From 8788 samples, 163 (1.85%) were reactive by IgG-ELISA and 312 (3.55%) by IgG IIF. From these, 77 (0.87%) were reactive in the TESAcruzi test. The results had high clinical value to identify those truly infected. Copyright © 2010 Elsevier B.V. All rights reserved.

  18. Regulation of pancreatic stellate cell activation by Notch3.

    PubMed

    Song, Haiyan; Zhang, Yuxiang

    2018-01-05

    Activated pancreatic stellate cells (PaSCs) are the key cellular source of cancer-associated fibroblasts in the pancreatic stroma of patients with pancreatic ductal adenocarcinoma (PDAC), however, the activation mechanism of PaSCs is not yet known. The Notch signaling pathway, components of which are expressed in stromal cells, is involved in the fibrosis of several organs, including the lung and liver. In the current study, we investigated whether Notch signal transduction is involved in PaSC activation in PDAC. The expression of Notch signaling pathway components in human PDAC was examined via immunohistochemical staining and assessed in mouse PaSCs using RT-qPCR and western blotting. Notch3 expression in both PDAC stromal cells and activated mouse PaSCs was evaluated using immunofluorescence, RT-qPCR and western blotting. The impact of siRNA-mediated Notch3 knockdown on PaSC activation was detected with RT-qPCR and western blotting, and the impact on PaSC proliferation and migration was detected using CCK-8 assays and scratch experiments. The effect of conditioned medium from PaSCs activated with Notch3 siRNA on pancreatic cancer (LTPA) cells was also detected with CCK-8 assays and scratch experiments. The data were analyzed for statistical significance using Student's t-test. Notch3 was overexpressed in both human PDAC stromal cells and activated mouse PaSCs, and Notch3 knockdown with Notch3 siRNA decreased the proliferation and migration of mouse PaSCs. The levels of markers related to PaSC activation, such as α-smooth muscle actin (α-SMA), collagen I and fibronectin, decreased in response to Notch3 knockdown, indicating that Notch3 plays an important role in PaSC activation. Furthermore, we confirmed that inhibition of PaSC activation via Notch3 siRNA reduced the proliferation and migration of PaSC-induced mouse pancreatic cancer (LTPA) cells. Notch3 inhibition in PaSCs can inhibit the activation, proliferation and migration of PaSCs and reduce the Pa

  19. Sensitivity and specificity of western blot testing of cerebrospinal fluid and serum for diagnosis of equine protozoal myeloencephalitis in horses with and without neurologic abnormalities.

    PubMed

    Daft, Barbara M; Barr, Bradd C; Gardner, Ian A; Read, Deryck; Bell, William; Peyser, Karen G; Ardans, Alex; Kinde, Hailu; Morrow, Jennifer K

    2002-10-01

    To determine sensitivity and specificity of western blot testing (WBT) of CSF and serum for diagnosis of equine protozoal myeloencephalitis (EPM) in horses with and without neurologic abnormalities. Prospective investigation. 65 horses with and 169 horses without neurologic abnormalities. CSF and serum from horses submitted for necropsy were tested for Sarcocystis neurona-specific antibody with a WBT. Results of postmortem examination were used as the gold standard against which results of the WBT were compared. Sensitivity of WBT of CSF was 87% for horses with and 88% for horses without neurologic abnormalities. Specificity of WBT of CSF was 44% for horses with and 60% for horses without neurologic abnormalities. Regardless of whether horses did or did not have neurologic abnormalities, sensitivity and specificity of WBT of serum were not significantly different from values for WBT of CSF. Ninety-four horses without EPM had histologic evidence of slight CNS inflammation. The low specificity of WBT of CSF indicated that it is inappropriate to diagnose EPM on the basis of a positive test result alone because of the possibility of false-positive test results. The high sensitivity, however, means that a negative result is useful in ruling out EPM. There was no advantage in testing CSF versus serum in horses without neurologic abnormalities. Slight CNS inflammation was common in horses with and without S neurona-specific antibodies in the CSF and should not be considered an indication of CNS infection with S neurona.

  20. Failure analysis of blots for diesel engine intercooler

    NASA Astrophysics Data System (ADS)

    Ren, Ping; Li, Zongquan; Wu, Jiangfei; Guo, Yibin; Li, Wanyou

    2017-05-01

    In diesel generating sets, it will lead to the abominable working condition if the fault couldn’t be recovered when the bolt of intercooler cracks. This paper aims at the fault of the blots of diesel generator intercooler and completes the analysis of the static strength and fatigue strength. Static intensity is checked considering blot preload and thermal stress. In order to obtain the thermal stress of the blot, thermodynamic of intercooler is calculated according to the measured temperature. Based on the measured vibration response and the finite element model, using dynamic load identification technique, equivalent excitation force of unit was solved. In order to obtain the force of bolt, the excitation force is loaded into the finite element model. By considering the thermal stress and preload as the average stress while the mechanical stress as the wave stress, fatigue strength analysis has been accomplished. Procedure of diagnosis is proposed in this paper. Finally, according to the result of intensity verification the fatigue failure is validation. Thereby, further studies are necessary to verification the result of the intensity analysis and put forward some improvement suggestion.

  1. Identification of a protein associated with the activity of cytokine-induced killer cells

    PubMed Central

    Cao, Jingsong; Chen, Cong; Gao, Yongqiang; Hu, Li; Liang, Yu; Xiao, Jianhua

    2017-01-01

    Cytokine-induced killer cells (CIKs) adoptive immunotherapy for efficient antitumor ability is used clinically, but details regarding the proteins associated with CIK activity remain unclear. In the current study, the cytotoxicity of CIKs on hepatoma was identified to be significantly downregulated by 1.61-fold following gentamincin treatment. Further research revealed that a differentially expressed protein (P43) was significantly downregulated by 1.22-fold using one-dimensional gel electrophoresis analysis. Of these, the P43 was identified as human haptoglobin using liquid chromatography-mass spectrometry. Western blotting demonstrated that the haptoglobin specifically reacted with rabbit anti-human-haptoglobin. Furthermore, western blotting results verified that the haptoglobin was significantly downregulated by 1.17-fold compared with the control group. In addition, the expression of haptoglobin mRNA was significantly downregulated by 1.73-fold following gentamincin treatment. Taken together, the results of the present study demonstrated that the expression of haptoglobin protein was associated with the activity of CIKs, and the results will be beneficial to the further investigation of CIK activity-enhancement mechanism. PMID:29163711

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

    PubMed

    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.

  3. Evaluation of IgE reactivity of active and thermally inactivated actinidin, a biomarker of kiwifruit allergy.

    PubMed

    Grozdanovic, Milica; Popovic, Milica; Polovic, Natalija; Burazer, Lidija; Vuckovic, Olga; Atanaskovic-Markovic, Marina; Lindner, Buko; Petersen, Arnd; Gavrovic-Jankulovic, Marija

    2012-03-01

    Actinidin, an abundant cysteine protease from kiwifruit, is a specific biomarker of isolated allergy to kiwifruit. This study evaluates the IgE-binding properties of biologically active and thermally inactivated actinidin. Employing two different activity assays (caseinolytic assay and zymogram with gelatin) we showed that actinidin obtained from kiwifruit extract under native conditions represents a mixture of inactive and active enzyme. The structural integrity of actinidin was confirmed by SDS-PAGE, Edman degradation, mass fingerprint and Western blot with polyclonal antibodies. Although it was capable of inducing positive skin prick test reactions, we failed to detect IgE reactivity of active actinidin in Western blot with patient sera. Thermally inactivated actinidin exhibited IgE reactivity both in vivo and in vitro, indicating that heat processed kiwifruit products may induce clinical reactivity. These findings imply that apart from the allergenic epitopes on its surface, actinidin also contains hidden epitopes inside the protein which become accessible to IgE upon thermal treatment. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Warming and earlier spring increase Western U.S. forest wildfire activity

    Treesearch

    A.L. Westerling; H.G. Hidalgo; D.R. Cayan; T.W. Swetnam

    2006-01-01

    Western United States forest wildfire activity is widely thought to have increased in recent decades, yet neither the extent of recent changes nor the degree to which climate may be driving regional changes in wildfire has been systematically documented. Much of the public and scientific discussion of changes in western United States wildfire has focused instead on...

  5. [Scientific Evaluation of Crude Drugs and Kampo Medicines Using the Eastern Blotting Method and Its Application to Biological Metabolic Studies].

    PubMed

    Morinaga, Osamu

    2018-01-01

     The scientific evaluation of crude drugs and kampo medicines (KMs) was demonstrated using the eastern blotting method with monoclonal antibodies (MAbs) against bioactive natural compounds. Scutellariae radix is one of the most important crude drugs used in KMs. Part of its pharmaceutical properties is due to the flavone glycoside baicalin (BI). A quantitative analysis method based on eastern blotting was developed for BI using an anti-BI MAb. A rapid, simple, sensitive, specific analytical system was subsequently established for BI with the eastern blotting technique using dot-blot and chemiluminescent methods. This system was useful as a high-throughput analytical method for the determination of BI in KMs as well as HPLC and enzyme-linked immunosorbent assay systems. Furthermore, an eastern blotting method was applied to the biological metabolic study of glycyrrhizic acid (GL), the major active constituent of licorice, for investigation of metabolites of GL such as 3-monoglucuronyl-glycyrrhetinic acid (3MGA) because licorice causes pseudoaldosteronism as a side effect. This approach may make it possible to determine the pathogenic agents of licorice-induced pseudoaldosteronism.

  6. Autophagy promotes metastasis and glycolysis by upregulating MCT1 expression and Wnt/β-catenin signaling pathway activation in hepatocellular carcinoma cells.

    PubMed

    Fan, Qing; Yang, Liang; Zhang, Xiaodong; Ma, Yingbo; Li, Yan; Dong, Lei; Zong, Zhihong; Hua, Xiangdong; Su, Dongming; Li, Hangyu; Liu, Jingang

    2018-01-19

    Autophagy is a dynamic physiological process that can generate energy and nutrients for cell survival during stress. Autophagy can regulate the migration and invasive ability in cancer cells. However, the connection between autophagy and metabolism is unclear. Monocarboxylate transporter 1 (MCT1) plays an important role in lactic acid transport and H + clearance in cancer cells, and Wnt/β-catenin signaling can increase cancer cell glycolysis. We investigated whether autophagy promotes glycolysis in hepatocellular carcinoma (HCC) cells by activating the Wnt/β-catenin signaling pathway, accompanied by MCT1 upregulation. Autophagic activity was evaluated using western blotting, immunoblotting, and transmission electron microscopy. The underlying mechanisms of autophagy activation on HCC cell glycolysis were studied via western blotting, and Transwell, lactate, and glucose assays. MCT1 expression was detected using quantitative reverse transcription-PCR (real-time PCR), western blotting, and immunostaining of HCC tissues and the paired adjacent tissues. Autophagy promoted HCC cell glycolysis accompanied by MCT1 upregulation. Wnt/β-catenin signaling pathway activation mediated the effect of autophagy on HCC cell glycolysis. β-Catenin downregulation inhibited the autophagy-induced glycolysis in HCC cells, and reduced MCT1 expression in the HCC cells. MCT1 was highly expressed in HCC tissues, and high MCT1 expression correlated positively with the expression of microtubule-associated protein light chain 3 (LC3). Activation of autophagy can promote metastasis and glycolysis in HCC cells, and autophagy induces MCT1 expression by activating Wnt/β-catenin signaling. Our study describes the connection between autophagy and glucose metabolism in HCC cells and may provide a potential therapeutic target for HCC treatment.

  7. Bicentric evaluation of six anti-toxoplasma immunoglobulin G (IgG) automated immunoassays and comparison to the Toxo II IgG Western blot.

    PubMed

    Maudry, Arnaud; Chene, Gautier; Chatelain, Rémi; Patural, Hugues; Bellete, Bahrie; Tisseur, Bernard; Hafid, Jamal; Raberin, Hélène; Beretta, Sophie; Sung, Roger Tran Manh; Belot, Georges; Flori, Pierre

    2009-09-01

    A comparative study of the Toxoplasma IgG(I) and IgG(II) Access (Access I and II, respectively; Beckman Coulter Inc.), AxSYM Toxo IgG (AxSYM; Abbott Diagnostics), Vidas Toxo IgG (Vidas; bioMerieux, Marcy l'Etoile, France), Immulite Toxo IgG (Immulite; Siemens Healthcare Diagnostics Inc.), and Modular Toxo IgG (Modular; Roche Diagnostics, Basel, Switzerland) tests was done with 406 consecutive serum samples. The Toxo II IgG Western blot (LDBio, Lyon, France) was used as a reference technique in the case of intertechnique discordance. Of the 406 serum samples tested, the results for 35 were discordant by the different techniques. Using the 175 serum samples with positive results, we evaluated the standardization of the titrations obtained (in IU/ml); the medians (second quartiles) obtained were 9.1 IU/ml for the AxSYM test, 21 IU/ml for the Access I test, 25.7 IU/ml for the Access II test, 32 IU/ml for the Vidas test, 34.6 IU/ml for the Immulite test, and 248 IU/ml for the Modular test. For all the immunoassays tested, the following relative sensitivity and specificity values were found: 89.7 to 100% for the Access II test, 89.7 to 99.6% for the Immulite test, 90.2 to 99.6% for the AxSYM test, 91.4 to 99.6% for the Vidas test, 94.8 to 99.6% for the Access I test, and 98.3 to 98.7% for the Modular test. Among the 406 serum samples, we did not find any false-positive values by two different tests for the same serum sample. Except for the Modular test, which prioritized sensitivity, it appears that the positive cutoff values suggested by the pharmaceutical companies are very high (either for economical or for safety reasons). This led to imperfect sensitivity, a large number of unnecessary serological follow-ups of pregnant women, and difficulty in determining the serological status of immunosuppressed individuals.

  8. Coriolus versicolor mushroom polysaccharides exert immunoregulatory effects on mouse B cells via membrane Ig and TLR-4 to activate the MAPK and NF-κB signaling pathways.

    PubMed

    Yang, Shu-fa; Zhuang, Tai-feng; Si, Yan-mei; Qi, Ke-yan; Zhao, Juan

    2015-03-01

    This study aimed to characterize the immunopotentiating effects and immune receptors for Coriolus versicolor mushroom polysaccharides (CVP), a Chinese medicinal fungus that exerts anti-tumor activities by enhancing host immunity. Proliferation assays were used to determine whether CVP could activate splenocytes. Flow cytometry analysis and IgM and IgG detection were used to characterize CVP-binding cells. Immune receptors were analyzed in immunoprecipitation and western blot assays. The downstream signaling pathways were identified by western blotting or immunostaining. CVP significantly stimulated the proliferation of mouse splenocytes. Fluorescence-labeled CVP (fl-CVP) selectively stained mouse B cells, but not T cells. CVP induced the production of IgM and IgG1 with or without exogenous IL-4. Membrane Ig (B cell antigen-receptor, BCR) was identified as a CVP-binding protein in immunoprecipitation and western blot experiments. CVP-induced B cell proliferation could be significantly inhibited by anti-mouse immunoglobulin (Ig) blocking antibody (Fab) or in cells from TLR4-mutant mice (C3H/HeJ). Phosphorylation of ERK-1/2 and p38 MAPK were clearly increased in a time-dependent manner, as was the nuclear translocation of the cytosolic NF-κB p65 subunit after CVP stimulation. Together, we demonstrate that CVP can bind and induce B cell activation using membrane Ig and TLR-4 as potential immune receptors. CVP activates mouse B cells through the MAPK and NF-κB signaling pathways. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. The upregulation of receptor activator NF-kappaB ligand expression by interleukin-1alpha and Porphyromonas endodontalis in human osteoblastic cells.

    PubMed

    Chen, S-C; Huang, F-M; Lee, S-S; Li, M-Z; Chang, Y-C

    2009-04-01

    To investigate the receptor activator of nuclear factor-kappa B (NF-kappaB) ligand (RANKL) in osteoblastic cells stimulated with inflammatory mediators. The expression of RANKL in human osteoblastic cell line U2OS stimulated by pro-inflammatory cytokine interleukin (IL)-1alpha and black-pigmented bacteria Porphyromonas endodontalis was investigated by Western blot and enzyme-linked immunosorbent assay (ELISA). The significance of the results obtained from control and treated groups was statistically analysed by the paired Student's t-test. IL-1alpha was found to upregulate RANKL production in U2OS cells (P < 0.05). Investigations of the time dependence of RANKL expression in IL-1alpha-treated cells revealed a rapid accumulation of RANKL protein after 1 h of exposure; it remained elevated throughout the 24-h incubation period shown by Western blot and ELISA. In addition, P. endodontalis also increased RANKL expression in U2OS cells after 4-h incubation period demonstrated by Western blot and ELISA (P < 0.05). IL-1alpha and P. endodontalis may be involved in developing apical periodontitis through the stimulation of RANKL production.

  10. Detection of multiple retroviral infections in cattle and cross-reactivity of bovine immunodeficiency-like virus and human immunodeficiency virus type 1 proteins using bovine and human sera in a western blot assay.

    PubMed Central

    Jacobs, R M; Smith, H E; Gregory, B; Valli, V E; Whetstone, C A

    1992-01-01

    Bovine antibovine immunodeficiency-like virus (BIV) antibodies were detected by Western blot analysis (WBA) using a chemiluminescence protocol. Bovine sera with anti-BIV activity, obtained from cows in two dairy herds, had antibodies directed against a variety of BIV-specific antigens indicating chronic infections. These sera were also tested for serological reactivity against bovine leukemia virus (BLV) and bovine syncytial virus (BSV). Cows most commonly had anti-BSV antibodies (12 of 39). Evidence for infection with BSV and BIV or BSV and BLV occurred with almost equal frequency (5 of 39 and 4 of 39, respectively) while only one instance of BIV and BLV coseropositivity was detected. The high prevalence of BSV seropositivity is consistent with a relatively infectious virus, which, as is known, may be transferred congenitally. Similar rates of coseropositivity of BIV or BLV with BSV in this population suggest that BIV is no more infectious than BLV and probably requires prolonged close contact for transmission. Seven of nine cows with anti-BIV antibodies detected primarily human immunodeficiency virus type 1 (HIV-1) p51 and p63 antigens by WBA using an alkaline phosphatase detection system, suggesting that HIV-1 proteins have potential usefulness in screening cattle for BIV seropositivity. Six human sera that showed strong reactivity against multiple HIV-1 proteins and the serum from one of three patients considered to be an "indeterminate" HIV-1 reactor, cross-reacted primarily with BIV p26. This is the first report of human sera with antibody to BIV-specific proteins.(ABSTRACT TRUNCATED AT 250 WORDS) Images Fig. 1. Fig. 2. Fig. 3. PMID:1335835

  11. Involvement of sigma-1 receptor in astrocyte activation induced by methamphetamine via up-regulation of its own expression.

    PubMed

    Zhang, Yuan; Lv, Xuan; Bai, Ying; Zhu, Xinjian; Wu, Xiaodong; Chao, Jie; Duan, Ming; Buch, Shilpa; Chen, Ling; Yao, Honghong

    2015-02-17

    Although it has been documented that methamphetamine induces astrocyte activation, the mechanism(s) underlying this effect remain poorly understood. We thus sought to examine the molecular mechanisms involved in methamphetamine-mediated activation of astrocytes with a focus on the role of sigma-1 receptor (σ-1R) in this process. The expression of σ-1R and glial fibrillary acidic protein (GFAP) was examined by reverse transcription PCR (RT-PCR), real-time PCR, Western blot, and immunofluorescent staining; phosphorylation of cell signaling pathways was detected by Western blot analysis. Immunoprecipitation was used to determine the interaction between σ-1R and p-Src. Chromatin immunoprecipitation (ChIP) assay was employed to discern the binding of cAMP-response element-binding protein (CREB) with the promoter of σ-1R. The role of σ-1R in astrocyte activation was further validated in σ-1R knockout (KO) mice by Western blot combined with immunofluorescent staining. Exposure of primary rat astrocytes to methamphetamine increased the expression of σ-1R via the activation of Src, ERK mitogen-activated protein kinase, and downstream CREB pathways. Subsequently, CREB translocated into nucleus and interacted with the promoter of σ-1R resulting in increased expression of σ-1R with a concomitant increase in expression of GFAP. This effect was inhibited in cells treated with the σ-1R antagonist-BD1047, thereby implicating the role of σ-1R in the activation of astrocytes. In vivo relevance of these findings was further corroborated in σ-1R KO mice that were administered methamphetamine. In the methamphetamine administered mice, there was a failure of the drug to induce activation of astrocytes, an effect that was evident in wild-type (WT) mice exposed to methamphetamine. The study presented herein demonstrates that methamphetamine-mediated activation of astrocytes involved up-regulation of σ-1R through a positive-feedback mechanism. Understanding the regulation of

  12. CYP1A protein expression and catalytic activity in double-crested cormorants experimentally exposed to Deepwater Horizon Mississippi Canyon 252 oil

    USGS Publications Warehouse

    Alexander, Courtney R.; Hooper, Michael J.; Cacela, Dave; Smelker, Kim D.; Calvin, Caleshia S.; Dean, Karen M.; Bursian, Steve J.; Cunningham, Fred L.; Hanson-Dorr, Katie C.; Horak, Katherine E.; Isanhart, John P.; Link, Jane E.; Shriner, Susan A.; Godard-Codding, Céline A.J.

    2017-01-01

    Double-crested cormorants (Phalacrocorax auritus, DCCO) were orally exposed to Deepwater Horizon Mississippi Canyon 252 (DWH) oil to investigate oil-induced toxicological impacts. Livers were collected for multiple analyses including cytochrome P4501A (CYP1A) enzymatic activity and protein expression. CYP1A enzymatic activity was measured by alkoxyresorufin O-dealkylase (AROD) assays. Activities specific to the O-dealkylation of four resorufin ethers are reported: benzyloxyresorufin O-debenzylase (BROD), ethoxyresorufin O-deethylase (EROD), methoxyresorufin O-demethylase (MROD), and pentoxyresorufin O-depentylase (PROD). CYP1A protein expression was measured by western blot analysis with a CYP1A1 mouse monoclonal antibody. In study 1, hepatic BROD, EROD, and PROD activities were significantly induced in DCCO orally exposed to 20 ml/kg body weight (bw) oil as a single dose or daily for 5 days. Western blot analysis revealed hepatic CYP1A protein induction in both treatment groups. In study 2 (5 ml/kg bw oil or 10 ml/kg bw oil, 21 day exposure), all four hepatic ARODs were significantly induced. Western blots showed an increase in hepatic CYP1A expression in both treatment groups with a significant induction in birds exposed to 10 ml/kg oil. Significant correlations were detected among all 4 AROD activities in both studies and between CYP1A protein expression and both MROD and PROD activities in study 2. EROD activity was highest for both treatment groups in both studies while BROD activity had the greatest fold-induction. While PROD activity values were consistently low, the fold-induction was high, usually 2nd highest to BROD activity. The observed induced AROD profiles detected in the present studies suggest both CYP1A4/1A5 DCCO isoforms are being induced after MC252 oil ingestion. A review of the literature on avian CYP1A AROD activity levels and protein expression after exposure to CYP1A inducers highlights the need for species-specific studies to

  13. A novel domain of amino-Nogo-A protects HT22 cells exposed to oxygen glucose deprivation by inhibiting NADPH oxidase activity.

    PubMed

    Guo, Fan; Wang, Huiwen; Li, Liya; Zhou, Heng; Wei, Haidong; Jin, Weilin; Wang, Qiang; Xiong, Lize

    2013-04-01

    This study aimed to investigate the protective effect of the M9 region (residues 290-562) of amino-Nogo-A fused to the human immunodeficiency virus trans-activator TAT in an in vitro model of ischemia-reperfusion induced by oxygen-glucose deprivation (OGD) in HT22 hippocampal neurons, and to investigate the role of NADPH oxidase in this protection. Transduction of TAT-M9 was analyzed by immunofluorescence staining and western blot. The biologic activity of TAT-M9 was assessed by its effects against OGD-induced HT22 cell damage, compared with a mutant M9 fusion protein or vehicle. Cellular viability and lactate dehydrogenase (LDH) release were assessed. Neuronal apoptosis was evaluated by flow cytometry. The Bax/Bcl-2 ratio was determined by western blotting. Reactive oxygen species (ROS) levels and NADPH oxidase activity were also measured in the presence or absence of an inhibitor or activator of NADPH oxidase. Our results confirmed the delivery of the protein into HT22 cells by immunofluorescence and western blot. Addition of 0.4 μmol/L TAT-M9 to the culture medium effectively improved neuronal cell viability and reduced LDH release induced by OGD. The fusion protein also protected HT22 cells from apoptosis, suppressed overexpression of Bax, and inhibited the reduction in Bcl-2 expression. Furthermore, TAT-M9, as well as apocynin, decreased NADPH oxidase activity and ROS content. The protective effects of the TAT-M9 were reversed by TBCA, an agonist of NADPH oxidase. In conclusion, TAT-M9 could be successfully transduced into HT22 cells, and protected HT22 cells against OGD damage by inhibiting NADPH oxidase-mediated oxidative stress. These findings suggest that the TAT-M9 protein may be an efficient therapeutic agent for neuroprotection.

  14. Influence of the Western Pacific teleconnection pattern on Western North Pacific tropical cyclone activity

    NASA Astrophysics Data System (ADS)

    Choi, Ki-Seon; Moon, Il-Ju

    2012-09-01

    This study analyzes the characteristics of Western North Pacific (WNP) tropical cyclone (TC) activity and large-scale environments according to the Western Pacific (WP) teleconnection pattern in summer. In the positive WP phase, an anomalous cyclone and an anomalous anticyclone develop in the low and middle latitudes of the East Asia area, respectively. As a result, southeasterlies are reinforced in the northeast area of East Asia (including Korea and Japan), which facilitates the movement of TC to this area, whereas northwesterlies are reinforced in the southwest area of East Asia (including southern China and the Indochina Peninsula) which blocks the movement of TC to that area. Due to the spatial distribution of this reinforced pressure system, TCs that develop during the positive WP phase move and turn more to the northeast of the WNP than TCs which develop during the negative WP phase. The characteristics of this TC activity during the positive WP phase are associated with the upper tropospheric jet being located farther to the northeast. TCs during the negative WP phase mainly move to the west from the Philippines toward southern China and the Indochina Peninsula. Due to the terrain effect caused by the passage of TCs in mainland China, the intensity of TCs during the negative WP phase is weaker than those during the positive WP phase.

  15. Lectin-Array Blotting.

    PubMed

    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.

  16. Heterologous expression of laccase cDNA from Ceriporiopsis subvermispora yields copper-activated apoprotein and complex isoform patterns

    Treesearch

    Luis F. Larrondo; Marcela Avila; Loreto Salas; Dan Cullen; Rafael Vicuna

    2003-01-01

    Analysis of genomic clones encoding a putative laccase in homokaryon strains of Ceriporiopsis subvermispora led to the identification of an allelic variant of the previously described lcs-1 gene. A cDNA clone corresponding to this gene was expressed in Aspergillus nidulans and in Aspergillus niger. Enzyme assays and Western blots showed that both hosts secreted active...

  17. Line scanning system for direct digital chemiluminescence imaging of DNA sequencing blots

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Karger, A.E.; Weiss, R.; Gesteland, R.F.

    A cryogenically cooled charge-coupled device (CCD) camera equipped with an area CCD array is used in a line scanning system for low-light-level imaging of chemiluminescent DNA sequencing blots. Operating the CCD camera in time-delayed integration (TDI) mode results in continuous data acquisition independent of the length of the CCD array. Scanning is possible with a resolution of 1.4 line pairs/mm at the 50% level of the modulation transfer function. High-sensitivity, low-light-level scanning of chemiluminescent direct-transfer electrophoresis (DTE) DNA sequencing blots is shown. The detection of DNA fragments on the blot involves DNA-DNA hybridization with oligonucleotide-alkaline phosphatase conjugate and 1,2-dioxetane-based chemiluminescence.more » The width of the scan allows the recording of up to four sequencing reactions (16 lanes) on one scan. The scan speed of 52 cm/h used for the sequencing blots corresponds to a data acquisition rate of 384 pixels/s. The chemiluminescence detection limit on the scanned images is 3.9 [times] 10[sup [minus]18] mol of plasmid DNA. A conditional median filter is described to remove spikes caused by cosmic ray events from the CCD images. 39 refs., 9 refs.« less

  18. Lipid A binding proteins in macrophages detected by ligand blotting

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hampton, R.Y.; Golenbock, D.T.; Raetz, C.R.H.

    1987-05-01

    Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with /sup 32/P/sub i/ (10/sup 9/ dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with (/sup 32/P)-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessedmore » using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS.« less

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

    PubMed

    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.

  20. Chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulation in rats.

    PubMed

    Han, Xun; Ran, Ye; Su, Min; Liu, Yinglu; Tang, Wenjing; Dong, Zhao; Yu, Shengyuan

    2017-01-01

    Background Preclinical experimental studies revealed an acute alteration of pituitary adenylate cyclase-activating polypeptide in response to a single activation of the trigeminovascular system, which suggests a potential role of pituitary adenylate cyclase-activating polypeptide in the pathogenesis of migraine. However, changes in pituitary adenylate cyclase-activating polypeptide after repeated migraine-like attacks in chronic migraine are not clear. Therefore, the present study investigated chronic changes in pituitary adenylate cyclase-activating polypeptide and related receptors in response to repeated chemical dural stimulations in the rat. Methods A rat model of chronic migraine was established by repeated chemical dural stimulations using an inflammatory soup for a different numbers of days. The pituitary adenylate cyclase-activating polypeptide levels were quantified in plasma, the trigeminal ganglia, and the trigeminal nucleus caudalis using radioimmunoassay and Western blotting in trigeminal ganglia and trigeminal nucleus caudalis tissues. Western blot analysis and real-time polymerase chain reaction were used to measure the protein and mRNA expression of pituitary adenylate cyclase-activating polypeptide-related receptors (PAC1, VPAC1, and VPAC2) in the trigeminal ganglia and trigeminal nucleus caudalis to identify changes associated with repetitive applications of chemical dural stimulations. Results All rats exhibited significantly decreased periorbital nociceptive thresholds to repeated inflammatory soup stimulations. Radioimmunoassay and Western blot analysis demonstrated significantly decreased pituitary adenylate cyclase-activating polypeptide levels in plasma and trigeminal ganglia after repetitive chronic inflammatory soup stimulation. Protein and mRNA analyses of pituitary adenylate cyclase-activating polypeptide-related receptors demonstrated significantly increased PAC1 receptor protein and mRNA expression in the trigeminal ganglia, but not

  1. Triton-polyacrylamide gel electrophoresis and leucine aminopeptidase activity staining detect Triton-slowed bands including high-molecular-mass aminopeptidase N (CD13) isoform in cholestatic patient sera.

    PubMed

    Kawai, Makoto; Hara, Yukichi

    2006-02-01

    Western blotting of aminopeptidase N (APN) detects a high-molecular-mass isoform (260 kDa) [M. Kawai, Y. Otake, Y. Hara High-molecular-mass isoform of aminopeptidase N/CD13 in serum from cholestatic patients. Clin Chim Acta 330 (2003) 141-149] in cholestatic patient serum but is time-consuming. Human sera were electrophoresed on polyacrylamide gel containing Triton-X100 (Triton-PAGE) and stained with leucine-B-naphthylamide (LAP-staining). The stained bands were eluted from the gel, treated with N- and O-glycosidase if necessary, and analyzed by Western blotting [M. Kawai, Y. Otake, Y. Hara High-molecular-mass isoform of aminopeptidase N/CD13 in serum from cholestatic patients. Clin Chim Acta 330 (2003) 141-149]. Triton-PAGE and LAP-staining clearly detected fast bands in all the sera examined. Almost parallel with leucine aminopeptidase activity, slow bands were strongly stained in all 11 cholestatic patients but clearly stained in 3 out of 14 patients with hepatobiliary diseases other than cholestasis. PAGE with various concentrations of Triton showed that Triton slows down slow bands but not fast bands. Western blotting showed that Triton-PAGE-slow bands of cholestasis contained 140 and 260-kDa APN and that fast bands were slightly smaller than monomer-size slow bands after glycosidase treatment. Less time-consuming than Western blotting, Triton-PAGE and LAP-staining detect novel APN bands slowed by Triton and partly composed of the high-molecular-mass isoform in cholestasis. The slow bands seem to be homodimers of APN with transmembrane anchors. The polypeptide of the fast band seems to be processed differently from that of the slow band.

  2. Evaluation of Line Immunoassay to Detect HTLV-1 Infection in an Endemic Area, Southwestern Japan; Comparison with Polymerase Chain Reaction and Western Blot.

    PubMed

    Umeki, Kazumi; Umekita, Kunihiko; Hashikura, Yuuki; Yamamoto, Ikuo; Kubo, Kazuyoshi; Nagatomo, Yasuhiro; Okayama, Akihiko

    2017-02-01

    Human T-lymphotropic virus type 1 (HTLV-1) has been recognized as a cause of adult T-cell leukemia/lymphoma, HTLV-1-associated myelopathy/tropical spastic paraparesis, and HTLV-1-associated uveitis. HTLV-1 infection is normally detected by screening for HTLV-1 antibodies, and positive samples are confirmed by Western blot (WB). However, WB fails to confirm some samples that were positive for HTLV-1 antibodies on screening. Line immunoassay (LIA) is commonly used in Europe and Brazil, but not in Japan. Therefore, we evaluated the performance of LIA as a method of confirming HTLV-1 antibodies using samples in Japan. LIA was compared with polymerase chain reaction (PCR) and WB using 50 negative and 70 positive samples tested by chemiluminescent enzyme immunoassay (CLEIA) in Miyazaki, Japan, an HTLV-1 endemic area. LIA (INNO-LIA HTLVI/II Score) and WB (Problot HTLV-I) were performed according to the manufacturer's instructions. Real-time PCR for HTLV-1 pX region was performed using DNA derived from white blood cells. The samples that tested negative by real-time PCR were further tested by nested PCR. All 50 CLEIA negative samples were determined to be negative by LIA and PCR. Of the 70 positive samples, 66 tested positive by both of LIA and PCR. Three samples tested negative by LIA and PCR, and the remaining sample (PCR negative) showed non-specific staining in LIA and WB. WB showed more indeterminate results than LIA. Gp21 antibody in LIA demonstrated a high ability to discriminate between positive and negative PCR results. Furthermore, the degree of gp21 antibody reaction by LIA showed correlation with HTLV-1 proviral loads (PVLs). Our results indicate that LIA performs well in confirming HTLV-1 seropositivity by showing a low incidence of indeterminate results and good agreement with PCR using samples in Japan, although the number of samples tested was small. In addition, semi-quantitative antibody titer to gp21 correlated well with HTLV-1 PVLs. Further study

  3. Anti-inflammatory and anti-cancer activity of mulberry (Morus alba L.) root bark

    PubMed Central

    2014-01-01

    Background Root bark of mulberry (Morus alba L.) has been used in herbal medicine as anti-phlogistic, liver protective, kidney protective, hypotensive, diuretic, anti-cough and analgesic agent. However, the anti-cancer activity and the potential anti-cancer mechanisms of mulberry root bark have not been elucidated. We performed in vitro study to investigate whether mulberry root bark extract (MRBE) shows anti-inflammatory and anti-cancer activity. Methods In anti-inflammatory activity, NO was measured using the griess method. iNOS and proteins regulating NF-κB and ERK1/2 signaling were analyzed by Western blot. In anti-cancer activity, cell growth was measured by MTT assay. Cleaved PARP, ATF3 and cyclin D1 were analyzed by Western blot. Results In anti-inflammatory effect, MRBE blocked NO production via suppressing iNOS over-expression in LPS-stimulated RAW264.7 cells. In addition, MRBE inhibited NF-κB activation through p65 nuclear translocation via blocking IκB-α degradation and ERK1/2 activation via its hyper-phosphorylation. In anti-cancer activity, MRBE deos-dependently induced cell growth arrest and apoptosis in human colorectal cancer cells, SW480. MRBE treatment to SW480 cells activated ATF3 expression and down-regulated cyclin D1 level. We also observed that MRBE-induced ATF3 expression was dependent on ROS and GSK3β. Moreover, MRBE-induced cyclin D1 down-regulation was mediated from cyclin D1 proteasomal degradation, which was dependent on ROS. Conclusions These findings suggest that mulberry root bark exerts anti-inflammatory and anti-cancer activity. PMID:24962785

  4. Anti-inflammatory and anti-cancer activity of mulberry (Morus alba L.) root bark.

    PubMed

    Eo, Hyun Ji; Park, Jae Ho; Park, Gwang Hun; Lee, Man Hyo; Lee, Jeong Rak; Koo, Jin Suk; Jeong, Jin Boo

    2014-06-25

    Root bark of mulberry (Morus alba L.) has been used in herbal medicine as anti-phlogistic, liver protective, kidney protective, hypotensive, diuretic, anti-cough and analgesic agent. However, the anti-cancer activity and the potential anti-cancer mechanisms of mulberry root bark have not been elucidated. We performed in vitro study to investigate whether mulberry root bark extract (MRBE) shows anti-inflammatory and anti-cancer activity. In anti-inflammatory activity, NO was measured using the griess method. iNOS and proteins regulating NF-κB and ERK1/2 signaling were analyzed by Western blot. In anti-cancer activity, cell growth was measured by MTT assay. Cleaved PARP, ATF3 and cyclin D1 were analyzed by Western blot. In anti-inflammatory effect, MRBE blocked NO production via suppressing iNOS over-expression in LPS-stimulated RAW264.7 cells. In addition, MRBE inhibited NF-κB activation through p65 nuclear translocation via blocking IκB-α degradation and ERK1/2 activation via its hyper-phosphorylation. In anti-cancer activity, MRBE deos-dependently induced cell growth arrest and apoptosis in human colorectal cancer cells, SW480. MRBE treatment to SW480 cells activated ATF3 expression and down-regulated cyclin D1 level. We also observed that MRBE-induced ATF3 expression was dependent on ROS and GSK3β. Moreover, MRBE-induced cyclin D1 down-regulation was mediated from cyclin D1 proteasomal degradation, which was dependent on ROS. These findings suggest that mulberry root bark exerts anti-inflammatory and anti-cancer activity.

  5. 2000-2002 Sultandağı-Afyon Earthquake Activity in Western Anatolia, Turkey

    NASA Astrophysics Data System (ADS)

    Kalafat, D.

    2016-12-01

    Western Anatolia is one of the seismically active region in Turkey. The high seismic activity is a result of the complex tectonic deformation of the Anatolian plate which has been dominated by the N-S extensional tectonic regime in the western edge. This extensional tectonic regime is partially maintained by a relative movement of the African-Arabian plates to north, average 2.5 cm per year. In western Turkey, relatively 3 major earthquakes (Mw≥6.0) were identified on the Sultandağı Fault zone (Afyon-Akşehir Graben) between years of 2000-2002. First event occurred at the year of 2000 (Eber-Sultandagi Earthquake, Mw=6.0) , and both events were occurred at February 3, 2002 Sultandağı (Mw=6.5) and Cay-Sultandagi (Mw=6.0). In this study, mentioned local earthquake activity, have been investigated to understand their nature and relation of the regional seismic activity and tectonic deformation on the Sultandağı Fault Zone (Afyon-Akşehir Graben) in western Anatolia. At first, we analyzed the distribution of mainshock and aftershocks of the two earthquakes which occurred in February 3, 2002 in the region. Fault mechanism solutions of the selected earthquakes and detailed stress regime analyses performed for the mainshock and aftershock sequences of two earthquakes. In regard with mentioned earthquakes, the identified surface ruptures have been investigated by detailed geological field study in the region. Also source mechanism solutions of the selected 17 regional earthquakes between years of 2000 and 2009 years in the region provided to understand the relation of the Sultandagi earthquakes sequences and regional seismic activity. Regional and local seismic investigations shows that, consecutive seismic activity is a result of the disturbance of stress balance in the region which has been triggered by sequentially occuring of earthquakes and triggering in short interval in years of 2000-2002. Also all seismic source studies approved that extensional deformation

  6. Activation of PPARγ mediates icaritin-induced cell cycle arrest and apoptosis in glioblastoma multiforme.

    PubMed

    Liu, Yongji; Shi, Ling; Liu, Yuan; Li, Peng; Jiang, Guoping; Gao, Xiaoning; Zhang, Yongbin; Jiang, Chuanwu; Zhu, Weiping; Han, Hongxing; Ju, Fang

    2018-04-01

    Glioblastoma multiforme (GBM) is the most prevalent primary malignancy of the brain. This study was designed to investigate whether icaritin exerts anti-neoplastic activity against GBM in vitro. Cell Counting Kit-8 (CCK-8) assay was utilized to examine the viability of GBM cells. The apoptotic cell population was measured by flow cytometry analysis. Cell cycle distribution was detected by flow cytometry as well. Western blot analysis was performed to examine the level of biomarker proteins in GBM cells. Levels of PPARγ mRNA and protein were detected by qPCR and western blot analysis, respectively. To examine the role of PPARγ in the anti-neoplastic activity of icaritin, PPARγ antagonist GW9662 or PPARγ siRNA was used. The activity of PPARγ was determined by DNA binding and luciferase assays. Our findings revealed that icaritin markedly suppresses cell growth in a dose-dependent and time-dependent fashion. The cell population at the G0/G1 phase of the cell cycle was significantly increased following icaritin treatment. Meanwhile, icaritin promoted apoptotic cell death in T98G and U87MG cells. Further investigation showed upregulation of PPARγ played a key role in the anti-neoplastic activities of icaritin. Moreover, our result demonstrated activation of AMPK signaling by icaritin mediated the modulatory effect of icaritin on PPARγ. Our results suggest the PPARγ may mediate anti-neoplastic activities against GBM. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  7. Anti-Inflammatory Activities of Cinnamomum cassia Constituents In Vitro and In Vivo

    PubMed Central

    Liao, Jung-Chun; Deng, Jeng-Shyan; Chiu, Chuan-Sung; Hou, Wen-Chi; Huang, Shyh-Shyun; Shie, Pei-Hsin; Huang, Guang-Jhong

    2012-01-01

    We have investigated the anti-inflammatory effects of Cinnamomum cassia constituents (cinnamic aldehyde, cinnamic alcohol, cinnamic acid, and coumarin) using lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW264.7) and carrageenan (Carr)-induced mouse paw edema model. When RAW264.7 macrophages were treated with cinnamic aldehyde together with LPS, a significant concentration-dependent inhibition of nitric oxide (NO), tumor necrosis factor (TNF-α), and prostaglandin E2 (PGE2) levels productions were detected. Western blotting revealed that cinnamic aldehyde blocked protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), nuclear transcription factor kappa B (NF-κB), and IκBα, significantly. In the anti-inflammatory test, cinnamic aldehyde decreased the paw edema after Carr administration, and increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the paw tissue. We also demonstrated cinnamic aldehyde attenuated the malondialdehyde (MDA) level and myeloperoxidase (MPO) activity in the edema paw after Carr injection. Cinnamic aldehyde decreased the NO, TNF-α, and PGE2 levels on the serum level after Carr injection. Western blotting revealed that cinnamic aldehyde decreased Carr-induced iNOS, COX-2, and NF-κB expressions in the edema paw. These findings demonstrated that cinnamic aldehyde has excellent anti-inflammatory activities and thus has great potential to be used as a source for natural health products. PMID:22536283

  8. Degalactosylated/desialylated human serum containing GcMAF induces macrophage phagocytic activity and in vivo antitumor activity.

    PubMed

    Kuchiike, Daisuke; Uto, Yoshihiro; Mukai, Hirotaka; Ishiyama, Noriko; Abe, Chiaki; Tanaka, Daichi; Kawai, Tomohito; Kubo, Kentaro; Mette, Martin; Inui, Toshio; Endo, Yoshio; Hori, Hitoshi

    2013-07-01

    The group-specific component protein-derived macrophage-activating factor (GcMAF) has various biological activities, such as macrophage activation and antitumor activity. Clinical trials of GcMAF have been carried out for metastatic breast cancer, prostate cancer, and metastatic colorectal cancer. In this study, despite the complicated purification process of GcMAF, we used enzymatically-treated human serum containing GcMAF with a considerable macrophage-stimulating activity and antitumor activity. We detected GcMAF in degalactosylated/desialylated human serum by western blotting using an anti-human Gc globulin antibody, and Helix pomatia agglutinin lectin. We also found that GcMAF-containing human serum significantly enhanced the phagocytic activity of mouse peritoneal macrophages and extended the survival time of mice bearing Ehrlich ascites tumors. We demonstrated that GcMAF-containing human serum can be used as a potential macrophage activator for cancer immunotherapy.

  9. Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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. EarthScope Transportable Array Siting Outreach Activities in Alaska and Western Canada

    NASA Astrophysics Data System (ADS)

    Dorr, P. M.; Gardine, L.; Tape, C.; McQuillan, P.; Cubley, J. F.; Samolczyk, M. A.; Taber, J.; West, M. E.; Busby, R.

    2015-12-01

    The EarthScope Transportable Array is deploying about 260 stations in Alaska and western Canada. IRIS and EarthScope are partnering with the Alaska Earthquake Center, part of the University of Alaska's Geophysical Institute, and Yukon College to spread awareness of earthquakes in Alaska and western Canada and the benefits of the Transportable Array for people living in these regions. We provide an update of ongoing education and outreach activities in Alaska and Canada as well as continued efforts to publicize the Transportable Array in the Lower 48. Nearly all parts of Alaska and portions of western Canada are tectonically active. The tectonic and seismic variability of Alaska, in particular, requires focused attention at the regional level, and the remoteness and inaccessibility of most Alaskan and western Canadian villages and towns often makes frequent visits difficult. When a community is accessible, every opportunity to engage the residents is made. Booths at state fairs and large cultural gatherings, such as the annual convention of the Alaska Federation of Natives, are excellent venues to distribute earthquake information and to demonstrate a wide variety of educational products and web-based applications related to seismology and the Transportable Array that residents can use in their own communities. Meetings and interviews with Alaska Native Elders and tribal councils discussing past earthquakes has led to a better understanding of how Alaskans view and understand earthquakes. Region-specific publications have been developed to tie in a sense of place for residents of Alaska and the Yukon. The Alaska content for IRIS's Active Earth Monitor emphasizes the widespread tectonic and seismic features and offers not just Alaska residents, but anyone interested in Alaska, a glimpse into what is going on beneath their feet. The concerted efforts of the outreach team will have lasting effects on Alaskan and Canadian understanding of the seismic hazard and

  12. Liver X receptor activation inhibits PC-3 prostate cancer cells via the beta-catenin pathway.

    PubMed

    Youlin, Kuang; Li, Zhang; Weiyang, He; Jian, Kang; Siming, Liang; Xin, Gou

    2017-03-01

    Liver X receptors (LXRs) are nuclear receptors family of ligand-dependent transcription factors that play a crucial role in regulating cholesterol metabolism and inflammation. Recent studies show that LXR agonists exhibit anti-cancer activities in a variety of cancer cell lines including prostate. To further identify the potential mechanisms of LXRα activation on prostate cancer, we investigated the effect of LXR agonist T0901317 on PC3 prostate cancer cell and in which activity of beta-catenin pathway involved. Prostate cancer PC3 cells were transfected with LXR-a siRNA and treated with LXR activator T0901317. qRT-PCR and western blot were used to detect the LXR-a expression. beta-catenin, cyclin D1 and c-MYC were analyzed by western blot. Cell apoptosis was examined by flow cytometry and Cell proliferation was assessed by Cell Counting Kit-8 assay. Cell migration was detected by Transwell chambers. Data showed that T0901317 significantly inhibited PC3 cell proliferation as well as invasion and increased apoptosis in vitro. Furthermore, we found that LXRα activation induced the reduction of beta-catenin expression in PC3 cells, and this inhibitory effect could be totally abolished when cells were treated with LXRα. Meanwhile, the expression of beta-catenin target gene cyclin D1 and c-MYC were also decreased. This study provided additional evidence that LXR activation inhibited PC-3 prostate cancer cells via suppressing beta-catenin pathway. Copyright © 2016 Elsevier GmbH. All rights reserved.

  13. Blotting from PhastGel to Membranes by Ultrasound.

    PubMed

    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).

  14. Blotting from PhastGel to membranes by ultrasound.

    PubMed

    Kost, Joseph

    2009-01-01

    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 (14)C-labeled Rainbow protein molecular weight markers (14,300-200,000 Da).

  15. Tanshinone IIA protects H9c2 cells from oxidative stress-induced cell death via microRNA-133 upregulation and Akt activation.

    PubMed

    Gu, Yunfei; Liang, Zhuo; Wang, Haijun; Jin, Jun; Zhang, Shouyan; Xue, Shufeng; Chen, Jianfeng; He, Huijuan; Duan, Kadan; Wang, Jing; Chang, Xuewei; Qiu, Chunguang

    2016-08-01

    The aim of the present study was to investigate the cardioprotective effect of tanshinone IIA and the underlying molecular mechanisms. An in vitro model of oxidative stress injury was established in cardiac H9c2 cells, and the effects of tanshinone IIa were investigated using cell viability, reverse transcription-quantitative polymerase chain reaction and western blotting assays. The results demonstrated that tanshinone IIA protects H9c2 cells from H 2 O 2 -induced cell death in a concentration-dependent manner, via a mechanism involving microRNA-133 (miR-133), and that treatment with TIIA alone exerted no cytotoxic effects on H9c2. In order to further elucidate the mechanisms underlying the actions of TIIA, reverse transcription-quantitative polymease chain reaction and western blot analysis were performed. Reductions in miR-133 expression levels induced by increasing concentrations of H 2 O 2 were reversed by treatment with tanshinone IIA. In addition, the inhibition of miR-133 by transfection with an miR-133 inhibitor abolished the cardioprotective effects of tanshinone IIA against H 2 O 2 -induced cell death. Furthermore, western blot analysis demonstrated that tanshinone IIA activated Akt kinase via the phosphorylation of serine 473. Inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway by pretreatment with the PI3K specific inhibitors wortmannin and LY294002 also eliminated the cardioprotective effects of tanshinone IIA against H 2 O 2 -induced cell death. Western blot analysis demonstrated that H 2 O 2 -induced reductions in B cell lymphoma 2 (Bcl-2) expression levels were reversed by tanshinone IIA. In addition, the effect of tanshinone IIA on Bcl-2 protein expression level in an oxidative environment was suppressed by a PI3K inhibitor, wortmannin, indicating that tanshinone IIA exerts cardioprotective effects against H 2 O 2 -induced cell death via the activation of the PI3K/Akt signal transduction pathway and the consequent

  16. Role of connexin 43 in the maintenance of spontaneous activity in the guinea pig prostate gland

    PubMed Central

    Dey, Anupa; Kusljic, Snezana; Lang, Richard J; Exintaris, Betty

    2010-01-01

    BACKGROUND AND PURPOSE To investigate the role of connexin 43 in the maintenance of spontaneous activity in prostate tissue from young and old guinea pigs. EXPERIMENTAL APPROACH Conventional intracellular microelectrode and tension recording techniques, coupled with Western blot analysis and immunohistochemistry for connexin 43 (CX43) were used. The effects of three gap junction uncouplers, 18β glycyrrhetinic acid (10 µM, 40 µM), carbenoxolone (10 µM, 50 µM) and octanol (0.5 mM, 1 mM), were studied in cells displaying slow wave activity and on spontaneously contracting tissue from prostate glands of young (2–5 months) and old (9–16 months) guinea pigs. KEY RESULTS 18β Glycyrrhetinic acid (40 µM), carbenoxolone (50 µM) or octanol (0.5 mM) abolished slow wave activity in prostate tissue from young and old guinea pigs and depolarized membrane potential by approximately 5 mV. These treatments also abolished all contractions in both sets of prostate tissue. These effects were reversed upon washout. Western blot analysis and CX43 immunohistochemistry showed that there was no age-related difference in the expression and distribution of CX43 in prostate tissues. CONCLUSION AND IMPLICATIONS When gap junctional communication via CX43 was disrupted, spontaneous activity was abolished at a cellular and whole tissue level; CX43 is therefore essential for the maintenance of spontaneous slow wave activity and subsequent contractile activity in the guinea pig prostate gland. PMID:20735413

  17. NF-kappaB mediates mitogen-activated protein kinase pathway-dependent iNOS expression in human melanoma.

    PubMed

    Uffort, Deon G; Grimm, Elizabeth A; Ellerhorst, Julie A

    2009-01-01

    Tumor expression of inducible nitric oxide synthase (iNOS) predicts poor outcomes for melanoma patients. We have reported the regulation of melanoma iNOS by the mitogen-activated protein kinase (MAPK) pathway. In this study, we test the hypothesis that NF-kappaB mediates this regulation. Western blotting of melanoma cell lysates confirmed the constitutive expression of iNOS. Western blot detected baseline levels of activated nuclear extracellular signal-regulated kinase and NF-kappaB. Indirect immunofluorescence confirmed the presence of NF-kappaB p50 and p65 in melanoma cell nuclei, with p50 being more prevalent. Electrophoretic mobility shift assay demonstrated baseline NF-kappaB activity, the findings confirmed by supershift analysis. Treatment of melanoma cells with the MEK inhibitor U0126 decreased NF-kappaB binding to its DNA recognition sequence, implicating the MAPK pathway in NF-kappaB activation. Two specific NF-kappaB inhibitors suppressed iNOS expression, demonstrating regulation of iNOS by NF-kappaB. Several experiments indicated the presence of p50 homodimers, which lack a transactivation domain and rely on the transcriptional coactivator Bcl-3 to carry out this function. Bcl-3 was detected in melanoma cells and co-immunoprecipitated with p50. These data suggest that the constitutively activated melanoma MAPK pathway stimulates activation of NF-kappaB hetero- and homodimers, which, in turn, drive iNOS expression and support melanoma tumorigenesis.

  18. Deregulation of miRNAs Contributes to Development and Progression of Prostate Cancer

    DTIC Science & Technology

    2012-09-01

    p14ARF gene were co-transfected with miR-125b into LNCaP cells. Cotransfection resulted in approximately 50% reduction of the enzyme activity (Fig...Figure3. Downregulation of miR-125b activity induces apoptosis in p53-null CaP cells. A) Western blot analysis of p14ARF and...miR-124-mediated downregulation of the AR affects the AR activity , both AR- positive LNCaP and C4-2B were treated with miR-124 mimic. Western blot

  19. PCR colorimetric dot-blot assay and clinical pretest probability for diagnosis of Pulmonary Tuberculosis in Smear-Negative patients

    PubMed Central

    Scherer, Luciene Cardoso; Sperhacke, Rosa Dea; Jarczewski, Carla; Cafrune, Patrícia I; Minghelli, Simone; Ribeiro, Marta Osório; Mello, Fernanda CQ; Ruffino-Netto, Antonio; Rossetti, Maria LR; Kritski, Afrânio L

    2007-01-01

    Background Smear-negative pulmonary tuberculosis (SNPTB) accounts for 30% of Pulmonary Tuberculosis (PTB) cases reported annually in developing nations. Polymerase chain reaction (PCR) may provide an alternative for the rapid detection of Mycobacterium tuberculosis (MTB); however little data are available regarding the clinical utility of PCR in SNPTB, in a setting with a high burden of TB/HIV co-infection. Methods To evaluate the performance of the PCR dot-blot in parallel with pretest probability (Clinical Suspicion) in patients suspected of having SNPTB, a prospective study of 213 individuals with clinical and radiological suspicion of SNPTB was carried out from May 2003 to May 2004, in a TB/HIV reference hospital. Respiratory specialists estimated the pretest probability of active disease into high, intermediate, low categories. Expectorated sputum was examined by direct microscopy (Ziehl-Neelsen staining), culture (Lowenstein Jensen) and PCR dot-blot. Gold standard was based on culture positivity combined with the clinical definition of PTB. Results In smear-negative and HIV subjects, active PTB was diagnosed in 28.4% (43/151) and 42.2% (19/45), respectively. In the high, intermediate and low pretest probability categories active PTB was diagnosed in 67.4% (31/46), 24% (6/25), 7.5% (6/80), respectively. PCR had sensitivity of 65% (CI 95%: 50%–78%) and specificity of 83% (CI 95%: 75%–89%). There was no difference in the sensitivity of PCR in relation to HIV status. PCR sensitivity and specificity among non-previously TB treated and those treated in the past were, respectively: 69%, 43%, 85% and 80%. The high pretest probability, when used as a diagnostic test, had sensitivity of 72% (CI 95%:57%–84%) and specificity of 86% (CI 95%:78%–92%). Using the PCR dot-blot in parallel with high pretest probability as a diagnostic test, sensitivity, specificity, positive and negative predictive values were: 90%, 71%, 75%, and 88%, respectively. Among non

  20. Fault activation by hydraulic fracturing in western Canada.

    PubMed

    Bao, Xuewei; Eaton, David W

    2016-12-16

    Hydraulic fracturing has been inferred to trigger the majority of injection-induced earthquakes in western Canada, in contrast to the Midwestern United States, where massive saltwater disposal is the dominant triggering mechanism. A template-based earthquake catalog from a seismically active Canadian shale play, combined with comprehensive injection data during a 4-month interval, shows that earthquakes are tightly clustered in space and time near hydraulic fracturing sites. The largest event [moment magnitude (M W ) 3.9] occurred several weeks after injection along a fault that appears to extend from the injection zone into crystalline basement. Patterns of seismicity indicate that stress changes during operations can activate fault slip to an offset distance of >1 km, whereas pressurization by hydraulic fracturing into a fault yields episodic seismicity that can persist for months. Copyright © 2016, American Association for the Advancement of Science.

  1. Diagnostic performance of ELISA, IFAT and Western blot for the detection of anti-Leishmania infantum antibodies in cats using a Bayesian analysis without a gold standard.

    PubMed

    Persichetti, Maria Flaminia; Solano-Gallego, Laia; Vullo, Angela; Masucci, Marisa; Marty, Pierre; Delaunay, Pascal; Vitale, Fabrizio; Pennisi, Maria Grazia

    2017-03-13

    Anti-Leishmania antibodies are increasingly investigated in cats for epidemiological studies or for the diagnosis of clinical feline leishmaniosis. The immunofluorescent antibody test (IFAT), the enzyme-linked immunosorbent assay (ELISA) and western blot (WB) are the serological tests more frequently used. The aim of the present study was to assess diagnostic performance of IFAT, ELISA and WB to detect anti-L. infantum antibodies in feline serum samples obtained from endemic (n = 76) and non-endemic (n = 64) areas and from cats affected by feline leishmaniosis (n = 21) by a Bayesian approach without a gold standard. Cut-offs were set at 80 titre for IFAT and 40 ELISA units for ELISA. WB was considered positive in presence of at least a 18 KDa band. Statistical analysis was performed through a written routine with MATLAB software in the Bayesian framework. The latent data and observations from the joint posterior were simulated in the Bayesian approach by an iterative Markov Chain Monte Carlo technique using the Gibbs sampler for estimating sensitivity and specificity of the three tests. The median seroprevalence in the sample used for evaluating the performance of tests was estimated at 0.27 [credible interval (CI) = 0.20-0.34]. The median sensitivity of the three different methods was 0.97 (CI: 0.86-1.00), 0.75 (CI: 0.61-0.87) and 0.70 (CI: 0.56-0.83) for WB, IFAT and ELISA, respectively. Median specificity reached 0.99 (CI: 0.96-1.00) with WB, 0.97 (CI: 0.93-0.99) with IFAT and 0.98 (CI: 0.94-1.00) with ELISA. IFAT was more sensitive than ELISA (75 vs 70%) for the detection of subclinical infection while ELISA was better for diagnosing clinical leishmaniosis when compared with IFAT (98 vs 97%). The overall performance of all serological techniques was good and the most accurate test for anti-Leishmania antibody detection in feline serum samples was WB.

  2. 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.

    PubMed

    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.

  3. A Low-Level Carbon Dioxide Laser Promotes Fibroblast Proliferation and Migration through Activation of Akt, ERK, and JNK

    PubMed Central

    Shingyochi, Yoshiaki; Kanazawa, Shigeyuki; Tajima, Satoshi; Tanaka, Rica; Mizuno, Hiroshi; Tobita, Morikuni

    2017-01-01

    Background Low-level laser therapy (LLLT) with various types of lasers promotes fibroblast proliferation and migration during the process of wound healing. Although LLLT with a carbon dioxide (CO2) laser was also reported to promote wound healing, the underlying mechanisms at the cellular level have not been previously described. Herein, we investigated the effect of LLLT with a CO2 laser on fibroblast proliferation and migration. Materials and Methods Cultured human dermal fibroblasts were prepared. MTS and cell migration assays were performed with fibroblasts after LLLT with a CO2 laser at various doses (0.1, 0.5, 1.0, 2.0, or 5.0 J/cm2) to observe the effects of LLLT with a CO2 laser on the proliferation and migration of fibroblasts. The non-irradiated group served as the control. Moreover, western blot analysis was performed using fibroblasts after LLLT with a CO2 laser to analyze changes in the activities of Akt, extracellular signal-regulated kinase (ERK), and Jun N-terminal kinase (JNK), which are signaling molecules associated with cell proliferation and migration. Finally, the MTS assay, a cell migration assay, and western blot analysis were performed using fibroblasts treated with inhibitors of Akt, ERK, or JNK before LLLT with a CO2 laser. Results In MTS and cell migration assays, fibroblast proliferation and migration were promoted after LLLT with a CO2 laser at 1.0 J/cm2. Western blot analysis revealed that Akt, ERK, and JNK activities were promoted in fibroblasts after LLLT with a CO2 laser at 1.0 J/cm2. Moreover, inhibition of Akt, ERK, or JNK significantly blocked fibroblast proliferation and migration. Conclusions These findings suggested that LLLT with a CO2 laser would accelerate wound healing by promoting the proliferation and migration of fibroblasts. Activation of Akt, ERK, and JNK was essential for CO2 laser-induced proliferation and migration of fibroblasts. PMID:28045948

  4. Atmospheric conditions associated with extreme fire activity in the Western Mediterranean region.

    PubMed

    Amraoui, Malik; Pereira, Mário G; DaCamara, Carlos C; Calado, Teresa J

    2015-08-15

    Active fire information provided by TERRA and AQUA instruments on-board sun-synchronous polar MODIS platform is used to describe fire activity in the Western Mediterranean and to identify and characterize the synoptic patterns of several meteorological fields associated with the occurrence of extreme fire activity episodes (EEs). The spatial distribution of the fire pixels during the period of 2003-2012 leads to the identification of two most affected sub-regions, namely the Northern and Western parts of the Iberian Peninsula (NWIP) and Northern Africa (NAFR). The temporal distribution of the fire pixels in these two sub-regions is characterized by: (i) high and non-concurrent inter- and intra-annual variability with maximum values during the summer of 2003 and 2005 in NWIP and 2007 and 2012 in NAFR; and, (ii) high intra-annual variability dominated by a prominent annual cycle with a main peak centred in August in both sub-regions and a less pronounced secondary peak in March only evident in NWIP region. The 34 EEs identified were grouped according to the location, period of occurrence and spatial configuration of the associated synoptic patterns into 3 clusters (NWIP-summer, NWIP-winter and NAFR-summer). Results from the composite analysis reveal similar fire weather conditions (statistically significant positive anomalies of air temperature and negative anomalies of air relative humidity) but associated with different circulation patterns at lower and mid-levels of the atmosphere associated with the occurrence of EEs in each cluster of the Western Mediterranean region. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Activated platelet-derived growth factor β receptor and Ras-mitogen-activated protein kinase pathway in natural bovine urinary bladder carcinomas.

    PubMed

    Corteggio, Annunziata; Di Geronimo, Ornella; Roperto, Sante; Roperto, Franco; Borzacchiello, Giuseppe

    2012-03-01

    Bovine papillomavirus types 1 or 2 (BPV-1/2) are involved in the aetiopathogenesis of bovine urinary bladder cancer. BPV-1/2 E5 activates the platelet-derived growth factor β receptor (PDGFβR). The aim of this study was to analyse the Ras/mitogen-activated protein kinase (MAPK) pathway in relation to activation of PDGFβR in natural bovine urinary bladder carcinomas. Co-immunoprecipitation and Western blot analysis demonstrated that recruitment of growth factor receptor bound protein 2 (GRB-2) and Sos-1 to the activated PDGFβR was increased in carcinomas compared to normal tissues. Higher grade bovine urinary bladder carcinomas were associated with activation of Ras, but not with activation of downstream mitogen-activated protein kinase/extracellular signal-regulated kinase (Mek 1/2) or extracellular signal-regulated kinase (Erk 1/2). Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Egypt western desert activity hits high gear

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Petzet, G.A.

    1996-11-04

    Exploration in Egypt`s western desert is turning up large oil and gas reserves in several sub-basins. Though not a threat to the Gulf of Suez`s oil production dominance, the western desert`s output is climbing. And gas production is also set to rise as more pipelines are built. The paper describes the geology, Qarun field production, further exploration at Qarun and the Khalda/Obaiyed gas.

  7. [Effect of NF-κB activation on the radiation response of esophageal cancer cells].

    PubMed

    Li, Baozhong; Chen, Zhaoli; Zhou, Fang; He, Jie

    2014-07-01

    To investigate the effect of NF-κB activation on radiation response of esophageal carcinoma. The expression of NF-κB was detected in pretreatment and posttreatment specimens of patients with ESCC by immunohistochemistry. Electrophoretic mobility shift assay (EMSA) and Western blot were used to detect the activation of NF-κB in esophageal cancer cell line KYSE150 cells. SN50, a specific NF-κB inhibitor, was applied to inhibit the activation of NF-κB. Clone formation test was used to detect the radiosensitivity of esophageal cancer cells. The median survival time of patients with activated and inactivated NF-κB in the pretreatment specimens were 16 and 19 months, respectively, with a non-significant difference between the two groups (P > 0.05). As to the patients with activated and inactivated NF-κB in posttreatment specimens, the median survival times were 13 and 35 months, respectively, with a significant difference (P < 0.01) between them. Western blot showed that the cytoplasmic expression of NF-κB was reduced with increasing radiation dose at 1.5 and 3 hours after radiation treatment. However, the expression of NF-κB in the cell nuclei was increased under the same condition, showing a trend of increased nucleus/cytoplasm ratio. The clone number in SN50 group was 96.66, 64.66, 76.66 and 10.00 under 0, 2, 4 and 12 Gy irradiation, which demonstrated a significant difference compared with the control groups (P < 0.001). Our results show that activation of NF-κB is induced by radiotherapy. Activation of NF-κB reduces the outcome of radiation treatment of esophageal cancer patients.

  8. A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro.

    PubMed

    Jia, Zhaofeng; Liang, Yujie; Ma, Bin; Xu, Xiao; Xiong, Jianyi; Duan, Li; Wang, Daping

    2017-05-17

    The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation. Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC's wider application. In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N + membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

  9. Active Magmatic Underplating in Western Eger Rift, Central Europe

    NASA Astrophysics Data System (ADS)

    Hrubcová, Pavla; Geissler, Wolfram H.; Bräuer, Karin; Vavryčuk, Václav; Tomek, Čestmír.; Kämpf, Horst

    2017-12-01

    The Eger Rift is an active element of the European Cenozoic Rift System associated with intense Cenozoic intraplate alkaline volcanism and system of sedimentary basins. The intracontinental Cheb Basin at its western part displays geodynamic activity with fluid emanations, persistent seismicity, Cenozoic volcanism, and neotectonic crustal movements at the intersections of major intraplate faults. In this paper, we study detailed geometry of the crust/mantle boundary and its possible origin in the western Eger Rift. We review existing seismic and seismological studies, provide new interpretation of the reflection profile 9HR, and supplement it by new results from local seismicity. We identify significant lateral variations of the high-velocity lower crust and relate them to the distribution and chemical status of mantle-derived fluids and to xenolith studies from corresponding depths. New interpretation based on combined seismic and isotope study points to a local-scale magmatic emplacement at the base of the continental crust within a new rift environment. This concept of magmatic underplating is supported by detecting two types of the lower crust: a high-velocity lower crust with pronounced reflectivity and a high-velocity reflection-free lower crust. The character of the underplated material enables to differentiate timing and tectonic setting of two episodes with different times of origin of underplating events. The lower crust with high reflectivity evidences magmatic underplating west of the Eger Rift of the Late Variscan age. The reflection-free lower crust together with a strong reflector at its top at depths of 28-30 km forms a magma body indicating magmatic underplating of the late Cenozoic (middle and upper Miocene) to recent. Spatial and temporal relations to recent geodynamic processes suggest active magmatic underplating in the intracontinental setting.

  10. Pituitary adenylate cyclase-activating polypeptide type 1 (PAC1) receptor is expressed during embryonic development of the earthworm.

    PubMed

    Boros, Akos; Somogyi, Ildikó; Engelmann, Péter; Lubics, Andrea; Reglodi, Dóra; Pollák, Edit; Molnár, László

    2010-03-01

    Pituitary adenylate cyclase activating polypeptide (PACAP)-like molecules have been shown to be present in cocoon albumin and in Eisenia fetida embryos at an early developmental stage (E1) by immunocytochemistry and radioimmunoassay. Here, we focus on detecting the stage at which PAC1 receptor (PAC1R)-like immunoreactivity first appears in germinal layers and structures, e.g., various parts of the central nervous system (CNS), in developing earthworm embryos. PAC1R-like immunoreactivity was revealed by Western blot and Far Western blot as early as the E2 developmental stage, occurring in the ectoderm and later in specific neurons of the developing CNS. Labeled CNS neurons were first seen in the supraesophageal ganglion (brain) and subsequently in the subesophageal and ventral nerve cord ganglia. Ultrastructurally, PAC1Rs were located mainly on plasma membranes and intracellular membranes, especially on cisternae of the endoplasmic reticulum. Therefore, PACAP-like compounds probably influence the differentiation of germinal layers (at least the ectoderm) and of some neurons and might act as signaling molecules during earthworm embryonic development.

  11. Blockade of NMDA receptors decreased spinal microglia activation in bee venom induced acute inflammatory pain in rats.

    PubMed

    Li, Li; Wu, Yongfang; Bai, Zhifeng; Hu, Yuyan; Li, Wenbin

    2017-03-01

    Microglial cells in spinal dorsal horn can be activated by nociceptive stimuli and the activated microglial cells release various cytokines enhancing the nociceptive transmission. However, the mechanisms underlying the activation of spinal microglia during nociceptive stimuli have not been well understood. In order to define the role of NMDA receptors in the activation of spinal microglia during nociceptive stimuli, the present study was undertaken to investigate the effect of blockade of NMDA receptors on the spinal microglial activation induced by acute peripheral inflammatory pain in rats. The acute inflammatory pain was induced by subcutaneous bee venom injection to the plantar surface of hind paw of rats. Spontaneous pain behavior, thermal withdrawal latency and mechanical withdrawal threshold were rated. The expression of specific microglia marker CD11b/c was assayed by immunohistochemistry and western blot. After bee venom treatment, it was found that rats produced a monophasic nociception characterized by constantly lifting and licking the injected hind paws, decreased thermal withdrawal latency and mechanical withdrawal threshold; immunohistochemistry displayed microglia with enlarged cell bodies, thickened, extended cellular processes with few ramifications, small spines, and intensive immunostaining; western blot showed upregulated expression level of CD11b/c within the period of hyperalgesia. Prior intrathecal injection of MK-801, a selective antagonist of NMDA receptors, attenuated the pain behaviors and suppressed up-regulation of CD11b/c induced by bee venom. It can be concluded that NMDA receptors take part in the mediation of spinal microglia activation in bee venom induced peripheral inflammatory pain and hyperalgesia in rats.

  12. Quantitation of Protein Carbonylation by Dot Blot

    PubMed Central

    Wehr, Nancy B.; Levine, Rodney L.

    2012-01-01

    Protein carbonylation is the most commonly used measure of oxidative modification of proteins. It is frequently measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent, 2,4-dinitrophenylhydrazine. We developed an immunochemical dot blot method for quantitation of protein carbonylation in homogenates or purified proteins. Dimethyl sulfoxide was employed as the solvent because it very efficiently extracts proteins from tissues and keeps them soluble. It also readily dissolves 2,4-dinitrophenylhydrazine and wets PVDF membranes. The detection limit is 0.19 ± 0.04 pmol carbonyl. Sixty ng protein is sufficient to measure protein carbonyl content. This level of sensitivity allowed measurement of protein carbonylation in individual Drosophila. PMID:22326366

  13. [ERK activation effects on GABA secretion inhibition induced by SDF-1 in hippocampal neurons of rats].

    PubMed

    Zhang, Zi-juan; Guo, Mei-xia; Xing, Ying

    2015-09-01

    To investigate the effect of extracellular regulating kinase (ERK) signaling pathway on the secretion of gamma-aminobutyric acid (GABA) in cultured rat hippocampal neurons induced by stromal cell derived factor-1 (SDF-1). The hippocampal neurons of newborn SD rats were cultured and identified in vitro; the phosphorylation level of ERK1/2 was examined by Western blot; ELISA was used to detect the effect of PD98059, a ERK1/2 specific blocker on GABA secretion of cultured hippocampal neurons and Western blot were adopted to measure the protein expression levels of glutamate decarboxylase (GAD65/67) and gamma aminobutyric acid transporter (GAT); after blocking ERK1/2 signaling pathway with PD98059; RT-PCR was used to detect the mRNA expression levels of GAT-1 and GAD65 after treated with PD98059. The levels of ERKl/2 phosphorylation were increased significantly by SDF1 acting on hippocampal neurons, and CX-CR4 receptor blocker AMD3100, could inhibit SDF-1 induced ERK1/2 activation; SDF-1 could inhibit the secretion of GABA in cultured hippocampal neurons, and ERK1/2 specific inhibitor PD98059, could partly reverse the inhibition of GABA secretion by SDF-1. The effects of SDF-1 on cultured hippocampal neurons was to decrease the mRNA genesis of glutamic acid decarboxylase GAD65 and GABA transporter GAT-1, besides, ERK inhibitor PD98059 could effectively flip the effect of SDF-1. The results of Western blot showed that SDF-1 could inhibit the protein expression of GAT-1 and GAD65/67 in hippocampal neurons and the inhibition of GAT-1 and GAD65/67 protein expression could be partially restored by ERK1/2 blocker. SDF-1 acts on the CXCR4 of hippocampal neurons in vitro, and inhibits the expression of GAD by activating the ERK1/2 signaling pathway, and this may represent one possible pathway of GABA secretion inhibition.

  14. Northern Blot Detection of Virus-Derived Small Interfering RNAs in Caenorhabditis elegans Using Nonradioactive Oligo Probes.

    PubMed

    Long, Tianyun; Lu, Rui

    2017-01-01

    Northern blot analysis has been widely used as a tool for detection and characterization of specific RNA molecules. When coupled with radioactive probe northern blot allows for robust detection and characterization of small RNA molecules of trace amount. Here, we describe the detection and size characterization of virus-derived small interfering RNAs (vsiRNAs) in C. elegans using nonradioactive DNA oligo probes in northern blotting. Our protocol allows for the detection and characterization of not only primary vsiRNAs but also secondary vsiRNAs, a class of single-stranded vsiRNAs that has distinct migration pattern, and can be easily adapted to the detection of vsiRNAs in other organisms.

  15. Role of high-mobility group box 1 in methamphetamine-induced activation and migration of astrocytes.

    PubMed

    Zhang, Yuan; Zhu, Tiebing; Zhang, Xiaotian; Chao, Jie; Hu, Gang; Yao, Honghong

    2015-09-04

    Mounting evidence has indicated that high-mobility group box 1 (HMGB1) is involved in cell activation and migration. Our previous study demonstrated that methamphetamine mediates activation of astrocytes via sigma-1 receptor (σ-1R). However, the elements downstream of σ-1R in this process remain poorly understood. Thus, we examined the molecular mechanisms involved in astrocyte activation and migration induced by methamphetamine. The expression of HMGB1, σ-1R, and glial fibrillary acidic protein (GFAP) was examined by western blot and immunofluorescent staining. The phosphorylation of cell signaling pathways was detected by western blot, and cell migration was examined using a wound-healing assay in rat C6 astroglia-like cells transfected with lentivirus containing red fluorescent protein (LV-RFP) as well as in primary human astrocytes. The role of HMGB1 in astrocyte activation and migration was validated using a siRNA approach. Exposure of C6 cells to methamphetamine increased the expression of HMGB1 via the activation of σ-1R, Src, ERK mitogen-activated protein kinase, and downstream NF-κB p65 pathways. Moreover, methamphetamine treatment resulted in increased cell activation and migration in C6 cells and primary human astrocytes. Knockdown of HMGB1 in astrocytes transfected with HMGB1 siRNA attenuated the increased cell activation and migration induced by methamphetamine, thereby implicating the role of HMGB1 in the activation and migration of C6 cells and primary human astrocytes. This study demonstrated that methamphetamine-mediated activation and migration of astrocytes involved HMGB1 up-regulation through an autocrine mechanism. Targeting HMGB1 could provide insights into the development of a potential therapeutic approach for alleviation of cell activation and migration of astrocytes induced by methamphetamine.

  16. [Prokaryotic expression and immunological activity of human neutrophil gelatinase associated lipocalin].

    PubMed

    Wu, Jianwei; Cai, Lei; Qian, Wei; Jiao, Liyuan; Li, Jiangfeng; Song, Xiaoli; Wang, Jihua

    2015-07-01

    To construct a prokaryotic expression vector of human neutrophil gelatinase associated lipocalin (NGAL) and identify the bioactivity of the fusion protein. The cDNA of human NGAL obtained from GenBank was linked to a cloning vector to construct the prokaryotic expression vector pCold-NGAL. Then the vector was transformed into E.coli BL21(DE3) plysS. Under the optimal induction condition, the recombinant NGAL (rNGAL) was expressed and purified by Ni Sepharose 6 Fast Flow affinity chromatography. The purity and activity of the rNGAL were respectively identified by SDS-PAGE and Western blotting combined with NGAL reagent (Latex enhanced immunoturbidimetry). Restriction enzyme digestion and nucleotide sequencing proved that the expression vector pCold-NGAL was successfully constructed. Under the optimal induction condition that we determined, the rNGAL was expressed in soluble form in E.coli BL21(DE3) plysS. The relative molecular mass of the rNGAL was 25 000, and its purity was more than 98.0%. Furthermore, Western blotting and immunoturbidimetry indicated that the rNGAL reacted with NGAL mAb specifically. Human rNGAL of high purity and bioactivity was successfully constructed in E.coli BL21(DE3) plysS using the expression vector pCold-NGAL.

  17. Metabolic profile in two physically active Inuit groups consuming either a western or a traditional Inuit diet

    PubMed Central

    Munch-Andersen, Thor; Olsen, David B.; Søndergaard, Hans; Daugaard, Jens R.; Bysted, Anette; Christensen, Dirk L.; Saltin, Bengt; Helge, Jørn W.

    2012-01-01

    Objectives To evaluate the effect of regular physical activity on metabolic risk factors and blood pressure in Inuit with high BMI consuming a western diet (high amount of saturated fatty acids and carbohydrates with a high glycemic index). Study design Cross sectional study, comparing Inuit eating a western diet with Inuit eating a traditional diet. Methods Two physically active Greenland Inuit groups consuming different diet, 20 eating a traditional diet (Qaanaaq) and 15 eating a western diet (TAB), age (mean (range)); 38, (22–58) yrs, BMI; 28 (20–40) were subjected to an oral glucose tolerance test (OGTT), blood sampling, maximal oxygen uptake test, food interview/collection and monitoring of physical activity. Results All Inuit had a normal OGTT. Fasting glucose (mmol/l), HbA1c (%), total cholesterol (mmol/l) and HDL-C (mmol/l) were for Qaanaaq women: 4.8±0.2, 5.3±0.1, 4.96±0.42, 1.34±0.06, for Qaanaaq men: 4.9±0.1, 5.7±0.1, 5.08±0.31, 1.28±0.09, for TAB women: 5.1±0.2, 5.3±0.1, 6.22±0.39, 1.86±0.13, for TAB men: 5.1±0.2, 5.3±0.1, 6.23±0.15, 1.60±0.10. No differences were found in systolic or diastolic blood pressure between the groups. There was a more adverse distribution of small dense LDL-C particles and higher total cholesterol and HDL-C concentration in the western diet group. Conclusions Diabetes or impaired glucose tolerance was not found in the Inuit consuming either the western or the traditional diet, and this could, at least partly, be due to the high amount of regular daily physical activity. However, when considering the total cardio vascular risk profile the Inuit consuming a western diet had a less healthy profile than the Inuit consuming a traditional diet. PMID:22456044

  18. Metabolic profile in two physically active Inuit groups consuming either a western or a traditional Inuit diet.

    PubMed

    Munch-Andersen, Thor; Olsen, David B; Søndergaard, Hans; Daugaard, Jens R; Bysted, Anette; Christensen, Dirk L; Saltin, Bengt; Helge, Jørn W

    2012-03-19

    To evaluate the effect of regular physical activity on metabolic risk factors and blood pressure in Inuit with high BMI consuming a western diet (high amount of saturated fatty acids and carbohydrates with a high glycemic index). Cross sectional study, comparing Inuit eating a western diet with Inuit eating a traditional diet. Two physically active Greenland Inuit groups consuming different diet, 20 eating a traditional diet (Qaanaaq) and 15 eating a western diet (TAB), age (mean (range)); 38, (22-58) yrs, BMI; 28 (20-40) were subjected to an oral glucose tolerance test (OGTT), blood sampling, maximal oxygen uptake test, food interview/collection and monitoring of physical activity. All Inuit had a normal OGTT. Fasting glucose (mmol/l), HbA1c (%), total cholesterol (mmol/l) and HDL-C (mmol/l) were for Qaanaaq women: 4.8±0.2, 5.3±0.1, 4.96±0.42, 1.34±0.06, for Qaanaaq men: 4.9±0.1, 5.7±0.1, 5.08±0.31, 1.28±0.09, for TAB women: 5.1±0.2, 5.3±0.1, 6.22±0.39, 1.86±0.13, for TAB men: 5.1±0.2, 5.3±0.1, 6.23±0.15, 1.60±0.10. No differences were found in systolic or diastolic blood pressure between the groups. There was a more adverse distribution of small dense LDL-C particles and higher total cholesterol and HDL-C concentration in the western diet group. Diabetes or impaired glucose tolerance was not found in the Inuit consuming either the western or the traditional diet, and this could, at least partly, be due to the high amount of regular daily physical activity. However, when considering the total cardio vascular risk profile the Inuit consuming a western diet had a less healthy profile than the Inuit consuming a traditional diet.

  19. Monoclonal antibodies against the native urease of Helicobacter pylori: synergistic inhibition of urease activity by monoclonal antibody combinations.

    PubMed Central

    Nagata, K; Mizuta, T; Tonokatu, Y; Fukuda, Y; Okamura, H; Hayashi, T; Shimoyama, T; Tamura, T

    1992-01-01

    Monoclonal antibodies (MAbs) against the native urease of Helicobacter pylori NCTC 11637 were found to clearly inhibit the urease activity. Interestingly, synergistic inhibition by two MAbs recognizing different subunits was also observed. Ten MAbs were produced and classified as two isotypes of the immunoglobulin G (IgG) subclass, IgG1, and IgG2a. Western blot (immunoblot) analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that five MAbs recognized the large subunit and the other five recognized the small subunit of the urease. Among the MAbs, L2 and S2, which recognized the large and the small subunits, respectively, were also able to inhibit the urease activity of clinical isolates from H. pylori-infected patients. The combination of L2 and S2 led to augmented synergistic inhibition. L2, but not S2, could also inhibit the urease activity from Helicobacter mustelae; enzyme-linked immunosorbent assay and Western blot analysis showed that L2 cross-reacted with this urease. These results suggested that the epitope recognized by L2 had a structure common to both Helicobacter species and may be involved in the active site of the urease. In contrast to the MAbs, a polyclonal antibody in sera from mice immunized with H. pylori urease did not have the ability to inhibit H. pylori urease activity. However, the polyclonal antibody retained the ability to abolish the inhibitory action of these MAbs. Moreover, other MAbs which could not inhibit H. pylori urease activity also abolished the inhibitory action. Images PMID:1383158

  20. Relationships Between Global Warming and Tropical Cyclone Activity in the Western North Pacific

    DTIC Science & Technology

    2007-09-01

    In this work, we investigate the relationships between global warming and tropical cyclone activity in the Western North Pacific (WNP). Our...hypothesis is that global warming impacts on TC activity occur through changes in the large scale environmental factors (LSEFs) known to be important in...averages. Using a least squares fit, we identify global warming signals in both the SST and vertical wind shear data across the WNP. These signals vary

  1. Muscarinic Acetylcholine Receptor Localization and Activation Effects on Ganglion Response Properties

    PubMed Central

    Renna, Jordan M.; Amthor, Franklin R.; Keyser, Kent T.

    2010-01-01

    Purpose. The activation and blockade of muscarinic acetylcholine receptors (mAChRs) affects retinal ganglion cell light responses and firing rates. This study was undertaken to identify the full complement of mAChRs expressed in the rabbit retina and to assess mAChR distribution and the functional effects of mAChR activation and blockade on retinal response properties. Methods. RT-PCR, Western blot analysis, and immunohistochemistry were used to identify the complement and distribution of mAChRs in the rabbit retina. Extracellular electrophysiology was used to determine the effects of the activation or blockade of mAChRs on ganglion cell response properties. Results. RT-PCR of whole neural retina resulted in the amplification of mRNA transcripts for the m1 to m5 mAChR subtypes. Western blot and immunohistochemical analyses confirmed that all five mAChR subtypes were expressed by subpopulations of bipolar, amacrine, and ganglion cells in the rabbit retina, including subsets of cells in cholinergic and glycinergic circuits. Nonspecific muscarinic activation and blockade resulted in the class-specific modulation of maintained ganglion cell firing rates and light responses. Conclusions. The expression of mAChR subtypes on subsets of bipolar, amacrine, and ganglion cells provides a substrate for both enhancement and suppression of retinal responses via activation by cholinergic agents. Thus, the muscarinic cholinergic system in the retina may contribute to the modulation of complex stimuli. Understanding the distribution and function of mAChRs in the retina has the potential to provide important insights into the visual changes that are caused by decreased ACh in the retinas of Alzheimer's patients and the potential visual effects of anticholinergic treatments for ocular diseases. PMID:20042645

  2. Adult-onset hyperthyroidism impairs spatial learning: possible involvement of mitogen-activated protein kinase signaling pathways.

    PubMed

    Bitiktaş, Soner; Kandemir, Başak; Tan, Burak; Kavraal, Şehrazat; Liman, Narin; Dursun, Nurcan; Dönmez-Altuntaş, Hamiyet; Aksan-Kurnaz, Işil; Suer, Cem

    2016-08-03

    Given evidence that mitogen-activated protein kinase (MAPK) activation is part of the nongenomic actions of thyroid hormones, we investigated the possible consequences of hyperthyroidism for the cognitive functioning of adult rats. Young adult rats were treated with L-thyroxine or saline. Twenty rats in each group were exposed to Morris water maze testing, measuring their performance in a hidden-platform spatial task. In a separate set of rats not exposed to Morris water maze testing (untrained rats), the expression and phosphorylated levels of p38-MAPK and of its two downstream effectors, Elk-1 and cAMP response element-binding protein, were evaluated using quantitative reverse transcriptase-PCR and western blotting. Rats with hyperthyroidism showed delayed acquisition of learning compared with their wild-type counterparts, as shown by increased escape latencies and distance moved on the last two trials of daily training in the water maze. The hyperthyroid rats, however, showed no difference during probe trials. Western blot analyses of the hippocampus showed that hyperthyroidism increased phosphorylated p38-MAPK levels in untrained rats. Although our study is correlative in nature and does not exclude the contribution of other molecular targets, our findings suggest that the observed impairments in acquisition during actual learning in rats with hyperthyroidism may result from the increased phosphorylation of p38-MAPK.

  3. Activation of p44/42 MAPK plays a role in the TBT-induced loss of human natural killer (NK) cell function.

    PubMed

    Dudimah, Fred D; Griffey, Denisha; Wang, Xiaofei; Whalen, Margaret M

    2010-10-01

    Natural killer (NK) cells destroy (lyse) tumor cells, virally infected cells, and antibody-coated cells. Previous studies indicated that exposure to the environmental contaminant tributyltin (TBT) decreases the lytic function of NK cells and activates mitogen-activated protein kinases (MAPK), including p44/42 (Aluoch and Whalen Toxicology 209:263-277, 2005). If activation of p44/42 is required for TBT-induced decreases of lytic function, then activation of p44/42 to similar extents by pharmacological agents such as phorbol 12-myristate 13-acetate (PMA) should mimic to some extent changes induced in NK cells with TBT exposures. NK cells were exposed to PMA concentrations between 0.25 and 10 nM for 10 min, 1 h, and 6 h before determining the lytic function ((51)Cr release assay) and phosphorylation state of MAPKs (Western blot). A 1-h exposure of NK cells to 5 nM PMA resulted in a loss of lytic function of 47%. Western blot analysis showed that a 1-h exposure to 5 nM PMA caused a sixfold increase in phospho-p44/42 levels. Previous studies showed a fivefold increase in phospho-p44/42 in response to a 1-h exposure to 300 nM TBT. Exposure to 300 nM TBT caused about a 40% decrease in lytic function. This study supports the hypothesis that p44/42 activation (as seen with TBT exposures) can cause a loss of NK-cell lytic function.

  4. Activation of p44/42 MAPK Plays a Role in the TBT-induced Loss of Human Natural Killer (NK) Cell Function

    PubMed Central

    Dudimah, Fred D.; Griffey, Denisha; Wang, Xiaofei; Whalen, Margaret M.

    2009-01-01

    Natural Killer (NK) cells destroy (lyse) tumor cells, virally infected cells and antibody-coated cells. Previous studies indicated that exposure to the environmental contaminant tributyltin (TBT) decreases the lytic function of NK cells and activates mitogen activated protein kinases (MAPK), including p44/42 (Aluoch and Whalen, 2005). If activation of p44/42 is required for TBT-induced decreases of lytic function, then activation of p44/42 to similar extents by pharmacological agents such as Phorbol 12-myristate 13-acetate (PMA) should mimic to some extent changes induced in NK cells with TBT exposures. NK cells were exposed to PMA concentrations between 0.25 and 10 nM for 10 min, 1 h, and 6 h before determining the lytic function (51Cr release assay) and phosphorylation state of MAPKs (Western blot). A 1 h exposure of NK cells to 5 nM PMA resulted in a loss of lytic function of 47%. Western blot analysis showed that a 1 h exposure to 5 nM PMA caused a 6 fold increase in phospho-p44/42 levels. Previous studies showed a 5 fold increase in phospho-p44/42 in response to a 1 h exposure to 300 nM TBT. Exposure to 300 nM TBT caused about a 40% decrease in lytic function. This study supports the hypothesis that p44/42 activation (as seen with TBT exposures) can cause a loss of NK-cell lytic function. PMID:20213532

  5. Quantitation of protein carbonylation by dot blot.

    PubMed

    Wehr, Nancy B; Levine, Rodney L

    2012-04-15

    Protein carbonylation is the most commonly used measure of oxidative modification of proteins. It is frequently measured spectrophotometrically or immunochemically by derivatizing proteins with the classical carbonyl reagent, 2,4-dinitrophenylhydrazine. We developed an immunochemical dot blot method for quantitation of protein carbonylation in homogenates or purified proteins. Dimethyl sulfoxide was employed as the solvent because it very efficiently extracts proteins from tissues and keeps them soluble. It also readily dissolves 2,4-dinitrophenylhydrazine and wets polyvinylidene difluoride (PVDF) membranes. The detection limit is 0.19 ± 0.04 pmol of carbonyl, and 60 ng of protein is sufficient to measure protein carbonyl content. This level of sensitivity allowed measurement of protein carbonylation in individual Drosophila. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Initial analysis of peripheral lymphocytic extracellular signal related kinase activation in autism.

    PubMed

    Erickson, Craig A; Ray, Balmiki; Wink, Logan K; Bayon, Baindu L; Pedapati, Ernest V; Shaffer, Rebecca; Schaefer, Tori L; Lahiri, Debomoy K

    2017-01-01

    Dysregulation of extracellular signal-related kinase (ERK) activity has been potentially implicated in the pathophysiology of autistic disorder (autism). ERK is part of a central intracellular signaling cascade responsible for a myriad of cellular functions. ERK is expressed in peripheral blood lymphocytes, and measurement of activated (phosphorylated) lymphocytic ERK is commonly executed in many areas of medicine. We sought to conduct the first study of ERK activation in humans with autism by utilizing a lymphocytic ERK activation assay. We hypothesized that ERK activation would be enhanced in peripheral blood lymphocytes from persons with autism compared to those of neurotypical control subjects. We conducted an initial study of peripheral lymphocyte ERK activation in 45 subjects with autism and 26 age- and gender-matched control subjects (total n = 71). ERK activation was measured using a lymphocyte counting method (primary outcome expressed as lymphocytes staining positive for cytosolic phosphorylated ERK divided by total cells counted) and additional Western blot analysis of whole cell phosphorylated ERK adjusted for total ERK present in the lymphocyte lysate sample. Cytosolic/nuclear localization of pERK activated cells were increased by almost two-fold in the autism subject group compared to matched neurotypical control subjects (cell count ratio of 0.064 ± 0.044 versus 0.034 ± 0.031; p = 0.002). Elevated phosphorylated ERK levels in whole cell lysates also showed increased activated ERK in the autism group compared to controls (n = 54 total) in Western blot analysis. The results of this first in human ERK activation study are consistent with enhanced peripheral lymphocytic ERK activation in autism, as well as suggesting that cellular compartmentalization of activated ERK may be altered in this disorder. Future work will be required to explore the impact of concomitant medication use and other subject characteristics such as level of cognitive

  7. Initial analysis of peripheral lymphocytic extracellular signal related kinase activation in autism

    PubMed Central

    Erickson, Craig A.; Ray, Balmiki; Wink, Logan K.; Bayon, Baindu L.; Pedapati, Ernest V.; Shaffer, Rebecca; Schaefer, Tori L.; Lahiri, Debomoy K.

    2018-01-01

    Background Dysregulation of extracellular signal-related kinase (ERK) activity has been potentially implicated in the pathophysiology of autistic disorder (autism). ERK is part of a central intracellular signaling cascade responsible for a myriad of cellular functions. ERK is expressed in peripheral blood lymphocytes, and measurement of activated (phosphorylated) lymphocytic ERK is commonly executed in many areas of medicine. We sought to conduct the first study of ERK activation in humans with autism by utilizing a lymphocytic ERK activation assay. We hypothesized that ERK activation would be enhanced in peripheral blood lymphocytes from persons with autism compared to those of neurotypical control subjects. Method We conducted an initial study of peripheral lymphocyte ERK activation in 45 subjects with autism and 26 age- and gender-matched control subjects (total n = 71). ERK activation was measured using a lymphocyte counting method (primary outcome expressed as lymphocytes staining positive for cytosolic phosphorylated ERK divided by total cells counted) and additional Western blot analysis of whole cell phosphorylated ERK adjusted for total ERK present in the lymphocyte lysate sample. Results Cytosolic/nuclear localization of pERK activated cells were increased by almost two-fold in the autism subject group compared to matched neurotypical control subjects (cell count ratio of 0.064 ± 0.044 versus 0.034 ± 0.031; p = 0.002). Elevated phosphorylated ERK levels in whole cell lysates also showed increased activated ERK in the autism group compared to controls (n = 54 total) in Western blot analysis. Conclusions The results of this first in human ERK activation study are consistent with enhanced peripheral lymphocytic ERK activation in autism, as well as suggesting that cellular compartmentalization of activated ERK may be altered in this disorder. Future work will be required to explore the impact of concomitant medication use and other subject characteristics

  8. Increased expression of activated pSTAT3 and PIM-1 in the pulmonary vasculature of experimental congenital diaphragmatic hernia.

    PubMed

    Hofmann, Alejandro D; Takahashi, Toshiaki; Duess, Johannes; Gosemann, Jan-Hendrik; Puri, Prem

    2015-06-01

    Signal transducer and activator of transcription (STAT) protein family (STAT1-6) regulates diverse cellular processes. Recently, the isoform STAT3 has been implicated to play a central role in the pathogenesis of pulmonary hypertension (PH). In human PH activated STAT3 (pSTAT3) was shown to directly trigger expression of the provirus integration site for Moloney murine leukemia virus (Pim-1), which promotes proliferation and resistance to apoptosis in SMCs. We designed this study to investigate the hypothesis that pSTAT3 and Pim-1 pulmonary vascular expression is increased in nitrofen-induced CDH. Pregnant rats were exposed to nitrofen or vehicle on D9.5. Fetuses were sacrificed on D21 and divided into nitrofen (n=16) and control group (n=16). QRT-PCR, western blotting, and confocal-immunofluorescence were performed to determine pulmonary gene and protein expression levels of pSTAT3 and Pim-1. Pulmonary Pim-1 gene expression was significantly increased in the CDH group compared to controls. Western blotting and confocal-microscopy confirmed increased pulmonary protein expression of Pim-1 and increased activation of pSTAT3 in CDH lungs compared to controls. Markedly increased gene and protein expression of Pim-1 and activated pSTAT3 in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that pSTAT3 and Pim-1 are important mediators of PH in nitrofen-induced CDH. Copyright © 2015. Published by Elsevier Inc.

  9. Substance P provides neuroprotection in cerebellar granule cells through Akt and MAPK/Erk activation: evidence for the involvement of the delayed rectifier potassium current.

    PubMed

    Amadoro, G; Pieri, M; Ciotti, M T; Carunchio, I; Canu, N; Calissano, P; Zona, C; Severini, C

    2007-05-01

    In the current study, we have evaluated the ability of substance P (SP) and other neurokinin 1 receptor (NK1) agonists to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death (S-K5). We also established the presence of SP high affinity NK1 transcripts and the NK1 protein localization in the membrane of a sub-population of CGCs. Moreover, SP significantly and dose-dependently reduced the Akt 1/2 and Erk1/2 dephosphorylation induced by S-K5 conditions, as demonstrated by Western blot analysis. Surprisingly, in SP-treated CGCs caspase-3 activity was not inhibited, while the calpain-1 activity was moderately reduced. Corroborating this result, SP blocked calpain-mediated cleavage of tau protein, as demonstrated by the reduced appearance of a diagnostic fragment of 17 kDa by Western blot analysis. In addition, SP induced a significant reduction of the delayed rectifier K+ currents (Ik) in about 42% of the patched neurons, when these were evoked with depolarizing potential steps. Taken together, the present results demonstrate that the activation of NK1 receptors expressed in CGCs promote the neuronal survival via pathways involving Akt and Erk activation and by inhibition of Ik which can contribute to the neuroprotective effect of the peptide.

  10. Evaluation of Two Commercial Systems for Automated Processing, Reading, and Interpretation of Lyme Borreliosis Western Blots▿

    PubMed Central

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

    2008-01-01

    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. PMID:18463211

  11. Selection of a Clostridium perfringens type D epsilon toxin producer via dot-blot test.

    PubMed

    Gonçalves, Luciana A; Lobato, Zélia I P; Silva, Rodrigo O S; Salvarani, Felipe M; Pires, Prhiscylla S; Assis, Ronnie A; Lobato, Francisco C F

    2009-11-01

    Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin.

  12. Pedometer-determined physical activity of Western Kenyan children.

    PubMed

    Croteau, Karen; Schofield, Grant; Towle, George; Suresh, Vijiayarani

    2011-08-01

    It is speculated that rural Kenyan children are more physically active than those in developed countries. The purpose of this study was to examine pedometer-measured physical activity levels of western Kenyan youth. Participants in this study were children in Levels 3 and 5 who attended a private primary school. The sample (n = 72) consisted of 43 girls and 29 boys (average age = 9.8 ± 1.1, range = 8-12 years). Age, gender, tribe, and height and weight measures were collected. Weight status category was determined according to CDC guidelines. Participants wore a sealed Yamax pedometer for 4 weekdays during the measurement period. Data analysis included descriptive statistics and 2-way ANOVA (age × gender). The total sample averaged 14558 ± 3993 daily steps. There was no significant effect for age [F(4,68) = 1.682, P = .102] nor significant age × gender interaction [F(4,68)=1.956, P = .117]. There was a significant effect for gender [F(1,68) = 4.791, P = .033], with boys (16262 ± 4698) significantly more active than girls (13463 ± 3051). The observed daily steps are higher than those observed in the U.S., similar to samples in other developed countries, but lower than Amish youth.

  13. Nucleotide-binding oligomerization domain 2 (NOD2) activation induces apoptosis of human oral squamous cell carcinoma cells.

    PubMed

    Yoon, Hyo-Eun; Ahn, Mee-Young; Kwon, Seong-Min; Kim, Dong-Jae; Lee, Jun; Yoon, Jung-Hoon

    2016-04-01

    Microbial Pattern-recognition receptors (PRRs), such as nucleotide-binding oligomerization domains (NODs), are essential for mammalian innate immune response. This study was designed to determine the effect of NOD1 and NOD2 agonist on innate immune responses and antitumor activity in oral squamous cell carcinoma (OSCC) cells. NODs expression was examined by RT-PCR, and IL-8 production by NODs agonist was examined by ELISA. Western blot analysis was performed to determine the MAPK activation in response to their agonist. Cell proliferation was determined by MTT assay. Flow cytometry and Western blot analysis were performed to determine the MDP-induced cell death. The levels of NODs were apparently expressed in OSCC cells. NODs agonist, Tri-DAP and MDP, led to the production of IL-8 and MAPK activation. NOD2 agonist, MDP, inhibited the proliferation of YD-10B cells in a dose-dependent manner. Also, the ratio of Annexin V-positive cells and cleaved PARP was increased by MDP treatment in YD-10B cells, suggesting that MDP-induced cell death in YD-10B cells may be owing to apoptosis. Our results indicate that NODs are functionally expressed in OSCC cells and can trigger innate immune responses. In addition, NOD2 agonist inhibited cell proliferation and induced apoptosis. These findings provide the potential value of MDP as novel candidates for antitumor agents of OSCC. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Development of a nanogold slot blot inhibition assay for the detection of antibodies against bovine herpesvirus type 1.

    PubMed

    Japolla, Greice; Cunha-Junior, Jair Pereira; Pajuaba, Ana Claudia Arantes Marquez; Taketomi, Ernesto Akio; Bührer-Sékula, Samira; Bataus, Luiz Artur Mendes; de Souza, Guilherme Rocha Lino

    2018-06-01

    Bovine herpesvirus type 1 (BoHV-1) is recognized as an important pathogen causing respiratory, reproductive, and neurological disorders in cattle and is associated with economic losses to animal industry. Accurate diagnostic methods are needed for prevention of disease transmission. While the virus neutralization test is considered the gold standard method, it requires maintenance of the virus and cell cultures, which is time consuming and expensive. Serological techniques such as enzyme-linked immunosorbent assay (ELISA) are widely applied, as these are easy to perform and provide quick results. In the present study, a nanogold slot blot inhibition assay was developed for the serological diagnosis of BoHV-1 and compared with standard ELISA and horseradish peroxidase (HRP) slot blot assays. Of 42 serum samples tested by ELISA, 32 (76.2%) were positive and 10 (23.8%), were negative. The sensitivity and specificity of the nanogold slot blot inhibition assay was similar to that observed for ELISA and HRP slot blot assays, and a strong correlation was observed between the tests. Thus, the nanogold slot blot inhibition assay may serve as an efficient and rapid alternative to ELISA in settings, where plate-reading equipment is lacking.

  15. Activation of mitochondrial calpain and release of apoptosis-inducing factor from mitochondria in RCS rat retinal degeneration.

    PubMed

    Mizukoshi, Sayuri; Nakazawa, Mitsuru; Sato, Kota; Ozaki, Taku; Metoki, Tomomi; Ishiguro, Sei-ichi

    2010-09-01

    The present study was performed to investigate changes of cytosolic and mitochondrial calpain activities, and effects of intravitreously injected calpain inhibitor on photoreceptor apoptosis in Royal College of Surgeon's (RCS) rats. Time courses of activities for both cytosolic and mitochondrial calpains and amount of calpastatin in RCS rat retina were analyzed by subcellular fractionation, calpain assay and western blotting. Calpain assay was colorimetrically performed using Suc-LLVY-Glo as substrate. Effects of intravitreously injected calpain inhibitor (ALLN and PD150606) on RCS rat retinal degeneration were analyzed by TUNEL staining. Effects of mitochondrial calpain activity on activation and translocation of apoptosis-inducing factor (AIF) were analyzed by western blotting. Mitochondrial calpain started to be significantly activated at postnatal (p) 28 days in RCS rat retina, whereas cytosolic micro-calpain was activated at p 35 days, although specific activity of mitochondrial calpain was 13% compared to cytosolic micro-calpain. Intravitreously injected ALLN and PD150606 effectively inhibited photoreceptor apoptosis only when injected at p 25 days, but did not inhibit photoreceptor apoptosis when injected at p 32 days. Parts of AIF were truncated/activated by mitochondrial calpains and translocated to the nucleus. These results suggest that 1), calpain presents not only in the cytosolic fraction but also in the mitochondrial fraction in RCS rat retina; 2), mitochondrial calpain is activated earlier than cytosolic calpain during retinal degeneration in RCS rats; 3), photoreceptor apoptosis may be regulated by not only calpain systems but also other mechanisms; 4), mitochondrial calpain may activate AIF to induce apoptosis; and 5), calpain inhibitors may be partially effective to inhibit photoreceptor apoptosis in RCS rats. The present study provides new insights into the molecular basis for photoreceptor apoptosis in RCS rats and the future possibility of new

  16. Baicalein reduces lipopolysaccharide-induced inflammation via suppressing JAK/STATs activation and ROS production.

    PubMed

    Qi, Zhilin; Yin, Fei; Lu, Lina; Shen, Lei; Qi, Shimei; Lan, Lei; Luo, Lan; Yin, Zhimin

    2013-09-01

    To investigate the precise molecular mechanisms by which baicalein exerts beneficial biochemical activities in RAW264.7 macrophages treated with LPS. RAW264.7 cells were cultured in the absence or presence of baicalein together with or without LPS. iNOS and COX-2 expression were measured by western blot and RT-PCR analyses. TNF-α, IL-1β, and IL-6 were determined by using double-antibody sandwich ELISA. Phosphorylations of JAK1 and JAK2, and of STAT1 and STAT3 were detected by western blotting. Nuclear translocation of STAT1 and STAT3 was visualized by confocal microscopy. ROS production was detected by ROS assay. Baicalein significantly reduced the phosphorylation of STAT1 and STAT3 and the phosphorylation of JAK1 and JAK2, but without affecting MAPKs phosphorylation in LPS-stimulated RAW264.7 cells. Baicalein suppressed the nuclear translocation of STAT1 and STAT3 and inhibited production of iNOS upon LPS-stimulation, resulting in the inhibition of releases of NO and pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, in a dose-dependent manner. In addition, we found that baicalein reduced the LPS-induced accumulation of ROS, confirming that baicalein serves as an antioxidant. Our results suggested that suppressing JAK/STATs activation and interfering with ROS production might contribute to the anti-inflammatory action of baicalein in macrophages.

  17. The Use of Biotin to Demonstrate Immunohistochemistry, Western Blotting, and Dot Blots in University Practical Classes

    ERIC Educational Resources Information Center

    Millar, Thomas James; Knighton, Ronald; Chuck, Jo-Anne

    2012-01-01

    Immunological detection of proteins is an essential method to demonstrate to undergraduate biology students, however, is often difficult in resource and time poor student laboratory sessions. This method describes a failsafe method to rapidly and economically demonstrate this technique using biotinylated proteins or biotin itself as targets for…

  18. Glial activation in the collagenase model of nociception associated with osteoarthritis.

    PubMed

    Adães, Sara; Almeida, Lígia; Potes, Catarina S; Ferreira, Ana Rita; Castro-Lopes, José M; Ferreira-Gomes, Joana; Neto, Fani L

    2017-01-01

    Background Experimental osteoarthritis entails neuropathic-like changes in dorsal root ganglia (DRG) neurons. Since glial activation has emerged as a key player in nociception, being reported in numerous models of neuropathic pain, we aimed at evaluating if glial cell activation may also occur in the DRG and spinal cord of rats with osteoarthritis induced by intra-articular injection of collagenase. Methods Osteoarthritis was induced by two injections, separated by three days, of 500 U of type II collagenase into the knee joint of rats. Movement-induced nociception was evaluated by the Knee-Bend and CatWalk tests during the following six weeks. Glial fibrillary acidic protein (GFAP) expression in satellite glial cells of the DRG was assessed by immunofluorescence and Western Blot analysis; the pattern of GFAP and activating transcription factor-3 (ATF-3) expression was also compared through double immunofluorescence analysis. GFAP expression in astrocytes and IBA-1 expression in microglia of the L3-L5 spinal cord segments was assessed by immunohistochemistry and Western Blot analysis. The effect of the intrathecal administration of fluorocitrate, an inhibitor of glial activation, on movement-induced nociception was evaluated six weeks after the first collagenase injection. Results GFAP expression in satellite glial cells of collagenase-injected animals was significantly increased six weeks after osteoarthritis induction. Double immunofluorescence showed GFAP upregulation in satellite glial cells surrounding ATF-3-positive neurons. In the spinal cord of collagenase-injected animals, an ipsilateral upregulation of GFAP and IBA-1 was also observed. The inhibition of glial activation with fluorocitrate decreased movement- and loading-induced nociception. Conclusion Collagenase-induced knee osteoarthritis leads to the development of nociception associated with movement of the affected joint and to the activation of glial cells in both the DRG and the spinal cord

  19. Systematic network assessment of the carcinogenic activities of cadmium

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, Peizhan; Duan, Xiaohua; Li, Mian

    Cadmium has been defined as type I carcinogen for humans, but the underlying mechanisms of its carcinogenic activity and its influence on protein-protein interactions in cells are not fully elucidated. The aim of the current study was to evaluate, systematically, the carcinogenic activity of cadmium with systems biology approaches. From a literature search of 209 studies that performed with cellular models, 208 proteins influenced by cadmium exposure were identified. All of these were assessed by Western blotting and were recognized as key nodes in network analyses. The protein-protein functional interaction networks were constructed with NetBox software and visualized with Cytoscapemore » software. These cadmium-rewired genes were used to construct a scale-free, highly connected biological protein interaction network with 850 nodes and 8770 edges. Of the network, nine key modules were identified and 60 key signaling pathways, including the estrogen, RAS, PI3K-Akt, NF-κB, HIF-1α, Jak-STAT, and TGF-β signaling pathways, were significantly enriched. With breast cancer, colorectal and prostate cancer cellular models, we validated the key node genes in the network that had been previously reported or inferred form the network by Western blotting methods, including STAT3, JNK, p38, SMAD2/3, P65, AKT1, and HIF-1α. These results suggested the established network was robust and provided a systematic view of the carcinogenic activities of cadmium in human. - Highlights: • A cadmium-influenced network with 850 nodes and 8770 edges was established. • The cadmium-rewired gene network was scale-free and highly connected. • Nine modules were identified, and 60 key signaling pathways related to cadmium-induced carcinogenesis were found. • Key mediators in the network were validated in multiple cellular models.« less

  20. A rapid chemiluminescent slot blot immunoassay for the detection and quantification of Clostridium botulinum neurotoxin type E, in cultures.

    PubMed

    Cadieux, Brigitte; Blanchfield, Burke; Smith, James P; Austin, John W

    2005-05-01

    A simple, rapid, cost-effective in vitro slot blot immunoassay was developed for the detection and quantification of botulinum neurotoxin type E (BoNT/E) in cultures. Culture supernatants of 36 strains of clostridia, including 12 strains of Clostridium botulinum type E, 12 strains of other C. botulinum neurotoxin serotypes, and 12 strains of other clostridial species were tested. Samples containing BoNT/E were detected using affinity-purified polyclonal rabbit antisera prepared against BoNT/E with subsequent detection of secondary antibodies using chemiluminescence. All strains of C. botulinum type E tested positive, while all non C. botulinum type E strains tested negative. The sensitivity of the slot blot immunoassay for detection of BoNT/E was approximately four mouse lethal doses (MLD). The intensity of chemiluminescence was directly correlated with the concentration of BoNT/E up to 128 MLD, allowing quantification of BoNT/E between 4 and 128 MLD. The slot blot immunoassay was compared to the mouse bioassay for detection of BoNT/E using cultures derived from fish samples inoculated with C. botulinum type E, and cultures derived from naturally contaminated environmental samples. A total of 120 primary enrichment cultures derived from fish samples, of which 103 were inoculated with C. botulinum type E, and 17 were uninoculated controls, were assayed. Of the 103 primary enrichment cultures derived from inoculated fish samples, all were positive by mouse bioassay, while 94 were also positive by slot blot immunoassay, resulting in a 7.5% false-negative rate. All 17 primary enrichment cultures derived from the uninoculated fish samples were negative by both mouse bioassay and slot blot immunoassay. A total of twenty-six primary enrichment cultures derived from environmental samples were tested by mouse bioassay and slot blot immunoassay. Of 13 primary enrichment cultures positive by mouse bioassay, 12 were also positive by slot blot immunoassay, resulting in a 3

  1. Autophagy Inhibition Contributes to ROS-Producing NLRP3-Dependent Inflammasome Activation and Cytokine Secretion in High Glucose-Induced Macrophages.

    PubMed

    Dai, Jiezhi; Zhang, Xiaotian; Li, Li; Chen, Hua; Chai, Yimin

    2017-01-01

    Type 2 diabetes is a persistent inflammatory response that impairs the healing process. We hypothesized that stimulation with high glucose following a pro-inflammatory signal would lead to autophagy inhibition, reactive oxygen species (ROS) production and eventually to the activation of the Nod-like receptor protein (NLRP) -3. Macrophages were isolated from human diabetic wound. We measured the expression of NLRP3, caspase1 and interleukin-1 beta (IL-1β) by western blot and real-time PCR, and the surface markers on cells by flow cytometry. THP-1-derived macrophages exposed to high glucose were applied to study the link between autophagy, ROS and NLRP3 activation. LC3-II, P62, NLRP3 inflammation and IL-1β expression were measured by western blot and real-time PCR. ROS production was measured with a Cellular Reactive Oxygen Species Detection Assay Kit. Macrophages isolated from diabetic wounds exhibited a pro-inflammatory phenotype, including sustained NLRP3 inflammasome activity associated with IL-1β secretion. Our data showed that high glucose inhibited autophagy, induced ROS production, and activated NLRP3 inflammasome and cytokine secretion in THP-1-derived macrophages. To study high glucose-induced NLRP3 inflammasome signalling, we performed studies using an autophagy inducer, a ROS inhibitor and a NLRP3 inhibitor and found that all reduced the NLRP3 inflammasome activation and cytokine secretion. Sustained NLRP3 inflammasome activity in wound-derived macrophages contributes to the hyper-inflammation in human diabetic wounds. Autophagy inhibition and ROS generation play an essential role in high glucose-induced NLRP3 inflammasome activation and cytokine secretion in macrophages. © 2017 The Author(s). Published by S. Karger AG, Basel.

  2. Adenosine 5'-monophosphate-induced hypothermia inhibits the activation of ERK1/2, JNK, p38 and NF-κB in endotoxemic rats.

    PubMed

    Wang, Yunlong; Zhang, Aihua; Lu, Shulai; Pan, Xinting; Jia, Dongmei; Yu, Wenjuan; Jiang, Yanxia; Li, Xinde; Wang, Xuefeng; Zhang, Jidong; Hou, Lin; Sun, Yunbo

    2014-11-01

    Many studies have shown that LPS mainly activates four signal transduction pathways to induce inflammation, namely the p38, ERK1/2, JNK and IKK/NF-κB pathways. Studies have demonstrated that 5'-AMP-induced hypothermia (AIH) exhibits high anti-inflammatory capabilities. In this study, we explore that how AIH inhibits the inflammatory response. Wistar rats were divided into five groups: a control group, an LPS group, a 5'-AMP pre-treatment group, a 5'-AMP post-treatment group and a 5'-AMP group. For each group, plasma and lung were collected from the rats at 6h and 12h after LPS injection. ELISA assays were used to detect plasma levels of CD14, CRP and MCP-1. Inflammatory pathway activation and TLR4 expression were assayed separately by Western blot analysis and immunohistochemistry. Our results showed that rats treated with AIH either before or after an LPS-challenge had a significant decrease in plasma levels of CD14, CRP and TLR4 compared with rats that received LPS only. Western blot analysis showed that AIH inhibited the activation of extracellular signal-regulated kinases (ERK) 1/2, p38, c-Jun N-terminal kinase (JNK) and NF-κB in inflammatory rats. Our study concluded that AIH attenuated LPS-induced inflammation mainly by inhibiting activation on the ERK1/2, p38, JNK and NF-κB signaling pathways. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. TRAF Family Member-Associated NF-κB Activator (TANK) Induced by RANKL Negatively Regulates Osteoclasts Survival and Function

    PubMed Central

    Wu, Mengrui; Wang, Yiping; Deng, Lianfu; Chen, Wei; Li, Yi-Ping

    2012-01-01

    Osteoclasts are the principle bone-resorbing cells. Precise control of balanced osteoclast activity is indispensable for bone homeostasis. Osteoclast activation mediated by RANK-TRAF6 axis has been clearly identified. However, a negative regulation-machinery in osteoclast remains unclear. TRAF family member-associated NF-κB activator (TANK) is induced by about 10 folds during osteoclastogenesis, according to a genome-wide analysis of gene expression before and after osteoclast maturation, and confirmed by western blot and quantitative RT-PCR. Bone marrow macrophages (BMMs) transduced with lentivirus carrying tank-shRNA were induced to form osteoclast in the presence of RANKL and M-CSF. Tank expression was downregulated by 90% by Tank-shRNA, which is confirmed by western blot. Compared with wild-type (WT) cells, osteoclastogenesis of Tank-silenced BMMs was increased, according to tartrate-resistant acid phosphatase (TRAP) stain on day 5 and day 7. Number of bone resorption pits by Tank-silenced osteoclasts was increased by 176% compared with WT cells, as shown by wheat germ agglutinin (WGA) stain and scanning electronic microscope (SEM) analysis. Survival rate of Tank-silenced mature osteoclast is also increased. However, acid production of Tank-knockdown cells was not changed compared with control cells. IκBα phosphorylation is increased in tank-silenced cells, indicating that TANK may negatively regulate NF-κB activity in osteoclast. In conclusion, Tank, whose expression is increased during osteoclastogenesis, inhibits osteoclast formation, activity and survival, by regulating NF-κB activity and c-FLIP expression. Tank enrolls itself in a negative feedback loop in bone resorption. These results may provide means for therapeutic intervention in diseases of excessive bone resorption. PMID:23139637

  4. TRAF family member-associated NF-κB activator (TANK) induced by RANKL negatively regulates osteoclasts survival and function.

    PubMed

    Wu, Mengrui; Wang, Yiping; Deng, Lianfu; Chen, Wei; Li, Yi-Ping

    2012-01-01

    Osteoclasts are the principle bone-resorbing cells. Precise control of balanced osteoclast activity is indispensable for bone homeostasis. Osteoclast activation mediated by RANK-TRAF6 axis has been clearly identified. However, a negative regulation-machinery in osteoclast remains unclear. TRAF family member-associated NF-κB activator (TANK) is induced by about 10 folds during osteoclastogenesis, according to a genome-wide analysis of gene expression before and after osteoclast maturation, and confirmed by western blot and quantitative RT-PCR. Bone marrow macrophages (BMMs) transduced with lentivirus carrying tank-shRNA were induced to form osteoclast in the presence of RANKL and M-CSF. Tank expression was downregulated by 90% by Tank-shRNA, which is confirmed by western blot. Compared with wild-type (WT) cells, osteoclastogenesis of Tank-silenced BMMs was increased, according to tartrate-resistant acid phosphatase (TRAP) stain on day 5 and day 7. Number of bone resorption pits by Tank-silenced osteoclasts was increased by 176% compared with WT cells, as shown by wheat germ agglutinin (WGA) stain and scanning electronic microscope (SEM) analysis. Survival rate of Tank-silenced mature osteoclast is also increased. However, acid production of Tank-knockdown cells was not changed compared with control cells. IκBα phosphorylation is increased in tank-silenced cells, indicating that TANK may negatively regulate NF-κB activity in osteoclast. In conclusion, Tank, whose expression is increased during osteoclastogenesis, inhibits osteoclast formation, activity and survival, by regulating NF-κB activity and c-FLIP expression. Tank enrolls itself in a negative feedback loop in bone resorption. These results may provide means for therapeutic intervention in diseases of excessive bone resorption.

  5. Seasonal prediction of lightning activity in North Western Venezuela: Large-scale versus local drivers

    NASA Astrophysics Data System (ADS)

    Muñoz, Á. G.; Díaz-Lobatón, J.; Chourio, X.; Stock, M. J.

    2016-05-01

    The Lake Maracaibo Basin in North Western Venezuela has the highest annual lightning rate of any place in the world (~ 200 fl km- 2 yr- 1), whose electrical discharges occasionally impact human and animal lives (e.g., cattle) and frequently affect economic activities like oil and natural gas exploitation. Lightning activity is so common in this region that it has a proper name: Catatumbo Lightning (plural). Although short-term lightning forecasts are now common in different parts of the world, to the best of the authors' knowledge, seasonal prediction of lightning activity is still non-existent. This research discusses the relative role of both large-scale and local climate drivers as modulators of lightning activity in the region, and presents a formal predictability study at seasonal scale. Analysis of the Catatumbo Lightning Regional Mode, defined in terms of the second Empirical Orthogonal Function of monthly Lightning Imaging Sensor (LIS-TRMM) and Optical Transient Detector (OTD) satellite data for North Western South America, permits the identification of potential predictors at seasonal scale via a Canonical Correlation Analysis. Lightning activity in North Western Venezuela responds to well defined sea-surface temperature patterns (e.g., El Niño-Southern Oscillation, Atlantic Meridional Mode) and changes in the low-level meridional wind field that are associated with the Inter-Tropical Convergence Zone migrations, the Caribbean Low Level Jet and tropical cyclone activity, but it is also linked to local drivers like convection triggered by the topographic configuration and the effect of the Maracaibo Basin Nocturnal Low Level Jet. The analysis indicates that at seasonal scale the relative contribution of the large-scale drivers is more important than the local (basin-wide) ones, due to the synoptic control imposed by the former. Furthermore, meridional CAPE transport at 925 mb is identified as the best potential predictor for lightning activity in the Lake

  6. Near-infrared laser increases MDPC-23 odontoblast-like cells proliferation by activating redox sensitive pathways.

    PubMed

    Rizzi, Manuela; Migliario, Mario; Rocchetti, Vincenzo; Tonello, Stelvio; Renò, Filippo

    2016-11-01

    Near infrared laser is known to induce biostimulatory effects, resulting in cell proliferation enhancement. Although such positive effect is widely exploited in various clinical applications, molecular mechanisms involved are still poorly understood. The aim of the study was to investigate the ability of laser stimulation to increase cell proliferation through an early activation of three redox sensitive pathways, namely Nrf-2, NF-κB and ERK in a rat odontoblast-like cell line (MDPC-23 cells). MDPC-23 cells were irradiated with different energy settings (0-50J, corresponding to 0-32.47J/cm 2 ) and cell proliferation was evaluated by cell counting. Nrf-2, NF-κB and ERK signaling pathways activation was investigated through Western blot analysis. Our results show that a single 25J laser stimulation is able to increase cell proliferation and that this effect could be increased by repeating the stimulation twice with a time lapse of 24h. Western blot experiments demonstrated that laser stimulation is able to induce an early activation response in intracellular signaling, with an overlapping time pattern between the three considered pathways. Results discussed in this paper reveal a complex mechanism underlying near-infrared induced increase in pre-odontoblasts proliferation, involving three survival pathways that can act both separately or through reciprocal crosstalk. In particular, data presented suggest an important role for ERK pathway that could act directly by stimulating cell proliferation but can also induce both Nrf-2 and NF-κB activation, acting as a critical cellular checkpoint in response to imbalanced redox state generated by a laser induced increase in ROS production. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Dobesilate diminishes activation of the mitogen - activated protein kinase ERK1/2 in glioma cells

    PubMed Central

    Cuevas, P; Diaz-González, Diana; Garcia-Martin-Córdova, C; Sánchez, I; Lozano, Rosa Maria; Giménez-Gallego, G; Dujovny, M

    2006-01-01

    Fibroblast growth factors (FGFs) and their receptors, regularly expressed at high levels in gliomas, are further upregulated during the transition of the tumor from low- to high-grade malignancy, and are essential for glioma progression. FGFs induce upregulation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured glioma cells, which suggests that MAPK pathway participates in the FGF-dependent glioma development. Recently, it has been shown that dobesilate, an inhibitor of FGF mitogenic activity, shows antiproliferative and proapoptotic activities in glioma cell cultures. Accordingly, it should be expected this new synthetic FGF inhibitor to affect the activation levels of MAPK. Here we report that immunocytochemical and Western blot data unequivocally show that treatment of cell cultures with dobesilate causes a significant decrease of the intracellular levels of ERK1/2 activation, one of the components of the MAPK signalling cascade. This finding supports an important role for dobesilate in glioma growth, suggesting that dobesilate should be a treatment to be born in mind for glioma management. PMID:16563234

  8. Specific recognition of hydatid cyst antigens by serum IgG, IgE, and IgA using western blot.

    PubMed

    Sbihi, Y; Janssen, D; Osuna, A

    1997-01-01

    Diagnosis of hydatid disease in humans relies on the detection of specific antibodies against antigens of the metacestode from Echinococcus granulosus. The specificity and sensitivity of current immunological techniques based on specific serum IgG rely on the way antigens are purified. We used Western immunoblotting to detect specific IgG, IgE, and IgA antibodies in serum from patients with hydatid disease using either crude antigen preparations (total hydatid fluid), purified fractions enriched in Antigens 5 and B, and glycoproteins from hydatid fluid. Depending on whether crude HF or purified antigen fractions were used, IgG and IgE recognized specifically low-to-medium MW bands between 12 and 42 kDa. IgA recognized specifically 110 kDa band in crude hydatid fluid and in the glycoprotein fraction of hydatid fluid, and a 42 kDa band in all antigen samples used. Besides the advantage of detecting specific IgA in crude hydatid fluid, these results offer the possibility of simplifying future immunological tests if specific secretory IgA can be similarly detected.

  9. Loss of PTEN as a Predictive Biomarker of Response to Lithium Chloride, A Potential Targeted Treatment for Breast Cancer

    DTIC Science & Technology

    2012-06-01

    infected cells, we were unable to produce HCC712 and HCC1187 cell lines with knocked out PTEN. We hypothesize that this is due to the high level of...Growth Factor Receptor in MCF-10A human breast epithelial cells. Western blot demonstrating levels of total EGFR in parental MCF-10A, and three stably...overexpression of EGFR. We performed western blot analyses to determine the degree of MAPK and PI3K pathway activation by comparing relative levels of

  10. Regulation of synthesis and activity of NAD(+)-dependent 15-hydroxy-prostaglandin dehydrogenase (15-PGDH) by dexamethasone and phorbol ester in human erythroleukemia (HEL) cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xun, C.Q.; Ensor, C.M.; Tai, H.H.

    1991-06-28

    Dexamethasone stimulated 15-PGDH activity in HEL cells in a time and concentration dependent manner. Increase in 15-PGDH activity by dexamethasone was found to be accompanied by an increase in enzyme synthesis as revealed by Western blot and (35S)methionine labeling studies. In addition to dexamethasone, other anti-inflammatory steroids also increased 15-PGDH activity in the order of their glucocorticoid activity. Among sex steroids only progesterone increased significantly 15-PGDH activity. 12-0-Tetradecanoylphorbol-13-acetate (TPA) also induced the synthesis of 15-PGDH but inhibited the enzyme activity. It appears that TPA caused a time dependent inactivation of 15-PGDH by a protein kinase C mediated mechanism.

  11. Plasma cholinesterase activity of rats, western grey kangaroos, alpacas, sheep, cattle, and horses.

    PubMed

    Mayberry, Chris; Mawson, Peter; Maloney, Shane K

    2015-01-01

    Plasma cholinesterase activity levels of various species may be of interest to toxicologists or pathologists working with chemicals that interfere with the activity of plasma cholinesterase. We used a pH titration method to measure the plasma cholinesterase activity of six mammalian species. Plasma cholinesterase activity varied up to 50-fold between species: sheep (88 ± 45 nM acetylcholine degraded per ml of test plasma per minute), cattle (94 ± 35), western grey kangaroos (126 ± 92), alpaca (364 ± 70), rats (390 ± 118) and horses (4539 ± 721). We present a simple, effective technique for the assay of plasma cholinesterase activity levels from a range of species. Although labour-intensive, it requires only basic laboratory equipment. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. The novel curcumin analog FLLL32 decreases STAT3 DNA binding activity and expression, and induces apoptosis in osteosarcoma cell lines

    PubMed Central

    2011-01-01

    Background Curcumin is a naturally occurring phenolic compound shown to have a wide variety of antitumor activities; however, it does not attain sufficient blood levels to do so when ingested. Using structure-based design, a novel compound, FLLL32, was generated from curcumin. FLLL32 possesses superior biochemical properties and more specifically targets STAT3, a transcription factor important in tumor cell survival, proliferation, metastasis, and chemotherapy resistance. In our previous work, we found that several canine and human osteosarcoma (OSA) cell lines, but not normal osteoblasts, exhibit constitutive phosphorylation of STAT3. Compared to curcumin, we hypothesized that FLLL32 would be more efficient at inhibiting STAT3 function in OSA cells and that this would result in enhanced downregulation of STAT3 transcriptional targets and subsequent death of OSA cells. Methods Human and canine OSA cells were treated with vehicle, curcumin, or FLLL32 and the effects on proliferation (CyQUANT®), apoptosis (SensoLyte® Homogeneous AMC Caspase- 3/7 Assay kit, western blotting), STAT3 DNA binding (EMSA), and vascular endothelial growth factor (VEGF), survivin, and matrix metalloproteinase-2 (MMP2) expression (RT-PCR, western blotting) were measured. STAT3 expression was measured by RT-PCR, qRT- PCR, and western blotting. Results Our data showed that FLLL32 decreased STAT3 DNA binding by EMSA. FLLL32 promoted loss of cell proliferation at lower concentrations than curcumin leading to caspase-3- dependent apoptosis, as evidenced by PARP cleavage and increased caspase 3/7 activity; this could be inhibited by treatment with the pan-caspase inhibitor Z-VAD-FMK. Treatment of OSA cells with FLLL32 decreased expression of survivin, VEGF, and MMP2 at both mRNA and protein levels with concurrent decreases in phosphorylated and total STAT3; this loss of total STAT3 occurred, in part, via the ubiquitin-proteasome pathway. Conclusions These data demonstrate that the novel curcumin

  13. Western diet enhances intestinal tumorigenesis in Min/+ mice, associating with mucosal metabolic and inflammatory stress and loss of Apc heterozygosity.

    PubMed

    Niku, Mikael; Pajari, Anne-Maria; Sarantaus, Laura; Päivärinta, Essi; Storvik, Markus; Heiman-Lindh, Anu; Suokas, Santeri; Nyström, Minna; Mutanen, Marja

    2017-01-01

    Western-type diet (WD) is a risk factor for colorectal cancer, but the underlying mechanisms are poorly understood. We investigated the interaction of WD and heterozygous mutation in the Apc gene on adenoma formation and metabolic and immunological changes in the histologically normal intestinal mucosa of Apc Min/+ (Min/+) mice. The diet used was high in saturated fat and low in calcium, vitamin D, fiber and folate. The number of adenomas was twofold higher in the WD mice compared to controls, but adenoma size, proliferation or apoptosis did not differ. The ratio of the Min to wild-type allele was higher in the WD mice, indicating accelerated loss of Apc heterozygosity (LOH). Densities of intraepithelial CD3ε + T lymphocytes and of mucosal FoxP3 + regulatory T cells were higher in the WD mice, implying inflammatory changes. Western blot analyses from the mucosa of the WD mice showed suppressed activation of the ERK and AKT pathways and a tendency for reduced activation of the mTOR pathway as measured in phosphoS6/S6 levels. The expression of pyruvate dehydrogenase kinase 4 was up-regulated in both mRNA and protein levels. Gene expression analyses showed changes in oxidation/reduction, fatty acid and monosaccharide metabolic pathways, tissue organization, cell fate and regulation of apoptosis. Together, our results suggest that the high-risk Western diet primes the intestine to tumorigenesis through synergistic effects in energy metabolism, inflammation and oxidative stress, which culminate in the acceleration of LOH of the Apc gene. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. The Central-Western Mediterranean: Anomalous igneous activity in an anomalous collisional tectonic setting

    NASA Astrophysics Data System (ADS)

    Lustrino, Michele; Duggen, Svend; Rosenberg, Claudio L.

    2011-01-01

    The central-western Mediterranean area is a key region for understanding the complex interaction between igneous activity and tectonics. In this review, the specific geochemical character of several 'subduction-related' Cenozoic igneous provinces are described with a view to identifying the processes responsible for the modifications of their sources. Different petrogenetic models are reviewed in the light of competing geological and geodynamic scenarios proposed in the literature. Plutonic rocks occur almost exclusively in the Eocene-Oligocene Periadriatic Province of the Alps while relatively minor plutonic bodies (mostly Miocene in age) crop out in N Morocco, S Spain and N Algeria. Igneous activity is otherwise confined to lava flows and dykes accompanied by relatively greater volumes of pyroclastic (often ignimbritic) products. Overall, the igneous activity spanned a wide temporal range, from middle Eocene (such as the Periadriatic Province) to the present (as in the Neapolitan of southern Italy). The magmatic products are mostly SiO 2-oversaturated, showing calcalkaline to high-K calcalcaline affinity, except in some areas (as in peninsular Italy) where potassic to ultrapotassic compositions prevail. The ultrapotassic magmas (which include leucitites to leucite-phonolites) are dominantly SiO 2-undersaturated, although rare, SiO 2-saturated (i.e., leucite-free lamproites) appear over much of this region, examples being in the Betics (southeast Spain), the northwest Alps, northeast Corsica (France), Tuscany (northwest Italy), southeast Tyrrhenian Sea (Cornacya Seamount) and possibly in the Tell region (northeast Algeria). Excepted for the Alpine case, subduction-related igneous activity is strictly linked to the formation of the Mediterranean Sea. This Sea, at least in its central and western sectors, is made up of several young (< 30 Ma) V-shaped back-arc basins plus several dispersed continental fragments, originally in crustal continuity with the European

  15. A novel activity for substance P: stimulation of peroxisome proliferator-activated receptor-γ protein expression in human monocytes and macrophages

    PubMed Central

    Amoruso, A; Bardelli, C; Gunella, G; Ribichini, F; Brunelleschi, S

    2008-01-01

    Background and purpose: Substance P (SP) and peroxisome proliferator-activated receptor-γ (PPAR-γ) play important roles in different inflammatory conditions and are both expressed in human monocytes and macrophages. However, it is not known whether or not they interact. This study was undertaken to evaluate the effects of SP on PPAR-γ protein expression in monocytes and macrophages (MDMs: monocyte-derived macrophages) from healthy smokers and non-smokers. Experimental approach: PPAR-γ protein was detected by western blot and quantified by calculating the ratio between PPAR-γ and β-actin protein expression. Constitutive tachykinin NK1 receptor expression in monocytes and MDMs from healthy smokers and non-smokers was evaluated by western blot. Cytokine release was evaluated by ELISA. Key results: In the concentration range 10−10–10−6 M, SP stimulated PPAR-γ protein expression in monocytes and MDMs, being more effective in cells from healthy smokers. Moreover, in these cells there was a constitutively increased expression of NK1 receptors. SP-induced expression of the PPAR-γ protein was receptor-mediated, as it was reproduced by the NK1 selective agonist [Sar9Met(O2)11]SP and reversed by the competitive NK1 antagonist GR71251. SP-induced maximal effects were similar to those evoked by 15-deoxy-Δ12,14-prostaglandin J2; an endogenous PPAR-γ agonist, and were significantly reduced by a PPAR-γ antagonist. NK1 and PPAR-γ agonists exerted opposite effects on TNF-α release from monocytes and MDMs. Conclusions and implications: Enhancement of PPAR-γ protein expression represents a novel activity for SP, which could contribute to a range of chronic inflammatory disorders. PMID:18278062

  16. Intramuscular renin-angiotensin system is activated in human muscular dystrophy.

    PubMed

    Sun, Guilian; Haginoya, Kazuhiro; Dai, Hongmei; Chiba, Yoko; Uematsu, Mitsugu; Hino-Fukuyo, Naomi; Onuma, Akira; Iinuma, Kazuie; Tsuchiya, Shigeru

    2009-05-15

    To investigate the role of the muscular renin-angiotensin system (RAS) in human muscular dystrophy, we used immunohistochemistry and Western blotting to examine the cellular localization of angiotensin-converting enzyme (ACE), the angiotensin II type 1 receptor (AT1) and the angiotensin II type 2 receptor (AT2) in muscle biopsies from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and congenital muscular dystrophy (CMD). In normal muscle, ACE was expressed in vascular endothelial cells and neuromuscular junctions (NMJs), whereas AT1 was immunolocalized to the smooth muscle cells of blood vessels and intramuscular nerve twigs. AT2 was immunolocalized in the smooth muscle cells of blood vessels. These findings suggest that the RAS has a functional role in peripheral nerves and NMJs. ACE and AT1, but AT2 immunoreactivity were increased markedly in dystrophic muscle as compared to controls. ACE and the AT1 were strongly expressed in the cytoplasm and nuclei of regenerating muscle fibers, fibroblasts, and in macrophages infiltrating necrotic fibers. Double immunolabeling revealed that activated fibroblasts in the endomysium and perimysium of DMD and CMD muscle were positive for ACE and AT1. Triple immunolabeling demonstrated that transforming growth factor-beta1 (TGF-beta1) and ACE were colocalized on the cytoplasm of activated fibroblasts in dystrophic muscle. Furthermore, Western blotting showed increases in the expression of AT1 and TGF-beta1 protein in dystrophic muscle, which coincided with our immunohistochemical results. The overexpression of ACE and AT1 in dystrophic muscle would likely result in the increased production of Ang II, which may act on these cells in an autocrine manner via AT1. The activation of AT1 may induce fibrous tissue formation through overexpression of TGF-beta1, which potently activates fibrogenesis and suppresses regeneration. In conclusion, our results imply that the intramuscular RAS-TGF-beta1 pathway

  17. Live-cell Imaging with Genetically Encoded Protein Kinase Activity Reporters.

    PubMed

    Maryu, Gembu; Miura, Haruko; Uda, Youichi; Komatsubara, Akira T; Matsuda, Michiyuki; Aoki, Kazuhiro

    2018-04-25

    Protein kinases play pivotal roles in intracellular signal transduction, and dysregulation of kinases leads to pathological results such as malignant tumors. Kinase activity has hitherto been measured by biochemical methods such as in vitro phosphorylation assay and western blotting. However, these methods are less useful to explore spatial and temporal changes in kinase activity and its cell-to-cell variation. Recent advances in fluorescent proteins and live-cell imaging techniques enable us to visualize kinase activity in living cells with high spatial and temporal resolutions. Several genetically encoded kinase activity reporters, which are based on the modes of action of kinase activation and phosphorylation, are currently available. These reporters are classified into single-fluorophore kinase activity reporters and Förster (or fluorescence) resonance energy transfer (FRET)-based kinase activity reporters. Here, we introduce the principles of genetically encoded kinase activity reporters, and discuss the advantages and disadvantages of these reporters.Key words: kinase, FRET, phosphorylation, KTR.

  18. Interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway in renal carcinogenesis of uninephrectomized rats.

    PubMed

    Yang, Ke-Ke; Sui, Yi; Zhou, Hui-Rong; Zhao, Hai-Lu

    2017-05-01

    Renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway both play important roles in carcinogenesis, but the interplay of renin-angiotensin system and adenosine monophosphate-activated protein kinase in carcinogenesis is not clear. In this study, we researched the interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase in renal carcinogenesis of uninephrectomized rats. A total of 96 rats were stratified into four groups: sham, uninephrectomized, and uninephrectomized treated with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker. Renal adenosine monophosphate-activated protein kinase and its downstream molecule acetyl coenzyme A carboxylase were detected by immunohistochemistry and western blot at 10 months after uninephrectomy. Meanwhile, we examined renal carcinogenesis by histological transformation and expressions of Ki67 and mutant p53. During the study, fasting lipid profiles were detected dynamically at 3, 6, 8, and 10 months. The results indicated that adenosine monophosphate-activated protein kinase expression in uninephrectomized rats showed 36.8% reduction by immunohistochemistry and 89.73% reduction by western blot. Inversely, acetyl coenzyme A carboxylase expression increased 83.3% and 19.07% in parallel to hyperlipidemia at 6, 8, and 10 months. The histopathology of carcinogenesis in remnant kidneys was manifested by atypical proliferation and carcinoma in situ, as well as increased expressions of Ki67 and mutant p53. Intervention with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker significantly prevented the inhibition of adenosine monophosphate-activated protein kinase signaling pathway and renal carcinogenesis in uninephrectomized rats. In conclusion, the novel findings suggest that uninephrectomy-induced disturbance in adenosine monophosphate-activated protein kinase signaling pathway resulted in hyperlipidemia and

  19. Carbonylation Contributes to SERCA2a Activity Loss and Diastolic Dysfunction in a Rat Model of Type 1 Diabetes

    PubMed Central

    Shao, Chun Hong; Capek, Haley L.; Patel, Kaushik P.; Wang, Mu; Tang, Kang; DeSouza, Cyrus; Nagai, Ryoji; Mayhan, William; Periasamy, Muthu; Bidasee, Keshore R.

    2011-01-01

    OBJECTIVE Approximately 25% of children and adolescents with type 1 diabetes will develop diastolic dysfunction. This defect, which is characterized by an increase in time to cardiac relaxation, results in part from a reduction in the activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a), the ATP-driven pump that translocates Ca2+ from the cytoplasm to the lumen of the sarcoplasmic reticulum. To date, mechanisms responsible for SERCA2a activity loss remain incompletely characterized. RESEARCH DESIGN AND METHODS The streptozotocin (STZ)-induced murine model of type 1 diabetes, in combination with echocardiography, high-speed video detection, confocal microscopy, ATPase and Ca2+ uptake assays, Western blots, mass spectrometry, and site-directed mutagenesis, were used to assess whether modification by reactive carbonyl species (RCS) contributes to SERCA2a activity loss. RESULTS After 6–7 weeks of diabetes, cardiac and myocyte relaxation times were prolonged. Total ventricular SERCA2a protein remained unchanged, but its ability to hydrolyze ATP and transport Ca2+ was significantly reduced. Western blots and mass spectroscopic analyses revealed carbonyl adducts on select basic residues of SERCA2a. Mutating affected residues to mimic physio-chemical changes induced on them by RCS reduced SERCA2a activity. Preincubating with the RCS, methylglyoxal (MGO) likewise reduced SERCA2a activity. Mutating an impacted residue to chemically inert glutamine did not alter SERCA2a activity, but it blunted MGO's effect. Treating STZ-induced diabetic animals with the RCS scavenger, pyridoxamine, blunted SERCA2a activity loss and minimized diastolic dysfunction. CONCLUSIONS These data identify carbonylation as a novel mechanism that contributes to SERCA2a activity loss and diastolic dysfunction during type 1 diabetes. PMID:21300842

  20. Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

    PubMed Central

    Abdolalizadeh, Jalal; Majidi Zolbanin, Jafar; Nouri, Mohammad; Baradaran, Behzad; Movassaghpour, AliAkbar; Farajnia, Safar; Omidi, Yadollah

    2013-01-01

    Purpose: Recombinant tumor necrosis factor-alpha (TNF-α) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods:In this study, we examined the potential of our produced anti-TNF-α scFv fragments for purification of TNF-α produced by Raji cells. The Raji cells were induced by lipopolysaccharides (LPS) to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS) flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications. PMID:24312807

  1. [Purification of human goose-type lysozyme 2 (HLysG2) from human seminal plasma and analysis of its enzymatic properties].

    PubMed

    Huang, Peng; Yang, Zhifang; Bao, Jianying; Zhang, Ning; Li, Wenshu

    2017-03-01

    Objective To purify human goose-type lysozyme 2 (HLysG2) from human seminal plasma by chromatography and analyze its enzymatic properties. Methods The distribution of HLysG2 in semen was analyzed by Western blot analysis. Seminal plasma was subjected to the separation of target protein using cation-exchange chromatography, chitin affinity chromatography and size-exclusion chromatography. The purified product was identified by Western blot analysis and mass spectrometry (MS).The purity was analyzed by high performance liquid chromatography (HPLC). Then, the optimum pH, ion concentration and temperature of HLysG2 and its standard activity were determined by the turbidimetric assay. The bactericidal activity of HLysG2 was assessed by the colony-forming assay. Results The existence of HLysG2 in seminal plasma was confirmed by Western blot analysis. A protein of about 21.5 kDa was purified from seminal plasma by the three kinds of chromatography and identified as HLysG2 by Western blot analysis and MS. The final purity of the purified product was above 99.0% and the peak enzymatic activity reached 13 800 U/mg under the condition of pH 6.4, 0.09 mol/L Na + , 30DegreesCelsius. In vitro assay indicated that HLysG2 had a significant killing effect on Micrococcus lysodeikticus, Bacillus subtilis and Staphylococcus aureus, but not on Pseudomonas aeruginosa and Escherichia coli. Conclusion Native HLysG2 can be obtained from seminal plasma by chromatography. It has in vitro bactericidal activity against Gram-positive bacteria, suggesting that it might play a role in innate immunity of the male reproductive system.

  2. Significant Centers of Tectonic Activity as Identified by Wrinkle Ridges for the Western Hemisphere of Mars

    NASA Technical Reports Server (NTRS)

    Anderson, R.C.; Haldemann, A. F. C.; Golombek, M. P.; Franklin, B. J.; Dohm, J. M.; Lias, J.

    2000-01-01

    The western hemisphere region of Mars has been the site of numerous scientific investigations regarding its tectonic evolution. For this region of Mars, the dominant tectonic region is the Tharsis province. Tharsis is characterized by an enormous system of radiating grabens and a circumferential system of wrinkle ridges. Past investigations of grabens associated with Tharsis have identified specific centers of tectonic activity. A recent structural analysis of the western hemisphere region of Mars which includes the Tharsis region, utilized 25,000 structures to determine the history of local and regional centers of tectonic activity based primarily on the spatial and temporal relationships of extensional features. This investigation revealed that Tharsis is more structurally complex (heterogeneous) than has been previously identified: it consists of numerous regional and local centers of tectonic activity (some are more dominant and/or more long lived than others). Here we use the same approach as Anderson et al. to determine whether the centers of tectonic activity that formed the extensional features also contributed to wrinkle ridge (compressional) formation.

  3. Expression of retinoblastoma gene product (pRb) in mantle cell lymphomas. Correlation with cyclin D1 (PRAD1/CCND1) mRNA levels and proliferative activity.

    PubMed Central

    Jares, P.; Campo, E.; Pinyol, M.; Bosch, F.; Miquel, R.; Fernandez, P. L.; Sanchez-Beato, M.; Soler, F.; Perez-Losada, A.; Nayach, I.; Mallofré, C.; Piris, M. A.; Montserrat, E.; Cardesa, A.

    1996-01-01

    Mantle cell lymphomas (MCLs) are molecularly characterized by bcl-1 rearrangement and constant cyclin D1 (PRAD-1/CCND1) gene overexpression. Cyclin D1 is a G1 cyclin that participates in the control of the cell cycle progression by interacting with the retinoblastoma gene product (pRb). Inactivation of the Rb tumor suppressor gene has been implicated in the development of different types of human tumors including some high grade non-Hodgkin's lymphomas. To determine the role of the retinoblastoma gene in the pathogenesis of MCLs and its possible interaction with cyclin D1, pRb expression was examined in 23 MCLs including 17 typical and 6 blastic variants by immunohistochemistry and Western blot. Rb gene structure was studied in 13 cases by Southern blot. Cytogenetic analysis was performed in 5 cases. The results were compared with the cyclin D1 mRNA levels examined by Northern analysis, and the proliferative activity of the tumors was measured by Ki-67 growth fraction and flow cytometry. pRb was expressed in all MCLs. The expression varied from case to case (mean, 14.1% of positive cells; range, 1.3 to 42%) with a significant correlation with the proliferative activity of the tumors (mitotic index r = 0.85; Ki-67 r = 0.7; S phase = 0.73). Blastic variants showed higher numbers of pRb-positive cells (mean, 29%) than the typical cases (10%; P < 0.005) by immunohistochemistry and, concordantly, higher levels of expression by Western blot. In addition, the blastic cases also had an increased expression of the phosphorylated protein. No alterations in Rb gene structure were observed by Southern blot analysis. Cyclin D1 mRNA levels were independent of pRb expression and the proliferative activity of the tumors. These findings suggest that pRb in MCLs is normally regulated in relation to the proliferative activity of the tumors. Cyclin D1 overexpression may play a role in the maintenance of cell proliferation by overcoming the suppressive growth control of pRb. Images

  4. Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells.

    PubMed

    Xiao, W; Li, C Q; Xiao, X P; Lin, F Z

    2013-12-16

    Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

  5. Activation of angiotensin-converting enzyme 2 (ACE2) attenuates allergic airway inflammation in rat asthma model

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Dhawale, Vaibhav Shrirang; Amara, Venkateswara Rao

    Angiotensin-I converting enzyme (ACE) is positively correlated to asthma, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS) and is highly expressed in lungs. ACE2, the counteracting enzyme of ACE, was proven to be protective in pulmonary, cardiovascular diseases. In the present study we checked the effect of ACE2 activation in animal model of asthma. Asthma was induced in male wistar rats by sensitization and challenge with ovalbumin and then treated with ACE2 activator, diminazene aceturate (DIZE) for 2 weeks. 48 h after last allergen challenge, animals were anesthetized, blood, BALF, femoral bone marrow lavage were collected for leucocytemore » count; trachea for measuring airway responsiveness to carbachol; lungs and heart were isolated for histological studies and western blotting. In our animal model, the characteristic features of asthma such as altered airway responsiveness to carbachol, eosinophilia and neutrophilia were observed. Western blotting revealed the increased pulmonary expression of ACE1, IL-1β, IL-4, NF-κB, BCL2, p-AKT, p-p38 and decreased expression of ACE2 and IκB. DIZE treatment prevented these alterations. Intraalveolar interstitial thickening, inflammatory cell infiltration, interstitial fibrosis, oxidative stress and right ventricular hypertrophy in asthma control animals were also reversed by DIZE treatment. Activation of ACE2 by DIZE conferred protection against asthma as evident from biochemical, functional, histological and molecular parameters. To the best of our knowledge, we report for the first time that activation of ACE2 by DIZE prevents asthma progression by altering AKT, p38, NF-κB and other inflammatory markers. - Highlights: • Diminazene aceturate (DIZE), an ACE2 activator prevents ovalbumin-induced asthma. • DIZE acted by upregulating ACE2, downregulating ACE1, MAPKs, markers of inflammation, apoptosis. • DIZE reduced airway inflammation, fibrosis, right ventricular

  6. Anti-adenovirus activities of shikonin, a component of Chinese herbal medicine in vitro.

    PubMed

    Gao, Hong; Liu, Lei; Qu, Zhang-Yi; Wei, Feng-Xiang; Wang, Shu-Qiu; Chen, Guang; Qin, Le; Jiang, Fu-Yang; Wang, Ying-Chen; Shang, Lei; Gao, Chun-Yan

    2011-01-01

    Radix Lithosperm eyrthrorhizon is a common prescription compound in traditional Chinese medicine. Shikonin is a major component of Radix Lithospermi and has various biological activities. We have investigated the inhibitory effect of shikonin on the growth of adenovirus type 3 (AdV3) in vitro. The antiviral function of shikonin against AdV3 and its virus inhibition ratio were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method (MTT). The expression of hexon protein in AdV3 was determined by immunofluorescence assay using laser scanning confocal microscopy (LSCM) and Western blot analysis. In addition, the rate of apoptosis in cells infected by AdV3 was determined by flow cytometry. Shikonin (0.0156-1 µM) inhibited growth of AdV3 in a concentration-dependent manner with a virus inhibition rate of 23.8-69.1%. Expression of hexon protein in AdV3 was higher in the virus control group than in the shikonin-treated groups as determined by immunofluorescence assay and Western blotting (p<0.05). The rate of shikonin-treated HeLa cell apoptosis had a statistically significant decrease with increasing concentration of drug (p<0.05). Our data demonstrate that shikonin possesses anti-AdV3 capabilities and that the potential antiviral mechanism might involve inhibiting the degree of apoptosis and hexon protein expression of AdV.

  7. Inhibitory effect of ginsenoside Rg1 on extracellular matrix production via extracellular signal-regulated protein kinase/activator protein 1 pathway in nasal polyp-derived fibroblasts.

    PubMed

    Cho, Jung-Sun; Moon, You-Mi; Um, Ji-Young; Moon, Jun-Hyeok; Park, Il-Ho; Lee, Heung-Man

    2012-06-01

    Nasal polyps are associated with chronic inflammation of the sinonasal mucosa and are involved in myofibroblast differentiation and extracellular matrix (ECM) accumulation. Ginsenoside Rg1, a compound derived from Panax ginseng, shows antifibrotic and anticancer effects. However, the molecular effects of Rg1 on myofibroblast differentiation and ECM production remain unknown. The aims of this study were to investigate the effect of Rg1 on transforming growth factor (TGF)-β1-induced myofibroblast differentiation and ECM production and to determine the molecular mechanism of Rg1 in nasal polyp-derived fibroblasts (NPDFs). NPDFs were isolated from nasal polyps of seven patients who had chronic rhinosinusitis with nasal polyp. NPDFs were exposed to TGF-β1 with or without Rg1. Expression levels of α-smooth muscle actin (SMA), fibronectin and collagen type Iα1 were determined by reverse transcription polymerase chain reaction, Western blot and immunofluorescent staining. TGF-β1 signaling molecules, including Smad2/3, extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 were analyzed by Western blotting. Transcription factors involved with TGF-β1 signaling, nuclear factor (NF)-κB and activator protein 1 (AP-1) were also assessed by Western blot. The cytotoxic effect of Rg1 was measured by an established viability assay. The mRNA and protein expression levels of α-SMA, fibronectin and collagen type Iα1 were increased in TGF-β1-induced NPDFs. Rg1 inhibited these effects. The inhibitory molecular mechanism of Rg1 was involved in the ERK pathway. Rg1 inhibited the transcription factor activation of AP-1. Rg1 itself was not cytotoxic. The ginsenoside Rg1 has inhibitory effects on myofibroblast differentiation and ECM production. The inhibitory mechanism of Rg1 is involved with the ERK and AP-1 signaling pathways. Rg1 may be useful as an inhibitor of ECM deposition, and has potential to be used as a novel treatment option for nasal

  8. Rubus idaeus L. reverses epithelial-to-mesenchymal transition and suppresses cell invasion and protease activities by targeting ERK1/2 and FAK pathways in human lung cancer cells.

    PubMed

    Hsieh, Yih-Shou; Chu, Shu-Chen; Hsu, Li-Sung; Chen, Kuo-Shuen; Lai, Ming-Tsung; Yeh, Chia-Heng; Chen, Pei-Ni

    2013-12-01

    Epithelial to mesenchymal transition (EMT) has been considered essential for cancer metastasis, a multistep complicated process including local invasion, intravasation, extravasation, and proliferation at distant sites. Herein we provided molecular evidence associated with the antimetastatic effect of Rubus idaeus L. extracts (RIE) by showing a nearly complete inhibition on the invasion (p<0.001) of highly metastatic A549 cells via reduced activities of matrix metalloproteinase-2 (MMP-2) and urokinasetype plasminogen activator (u-PA). We performed Western blot to find that RIE could induce up-regulation of epithelial marker such as E-cadherin and α-catenin and inhibit the mesenchymal markers such as N-cadherin, fibronectin, snail-1, and vimentin. Selective snail-1 inhibition by snail-1-specific-siRNA also showed increased E-cadherin expression in A549 cells suggesting a possible involvement of snail-1 inhibition in RIE-caused increase in E-cadherin level. RIE also inhibited p-FAK, p-paxillin and AP-1 by Western blot analysis, indicating the anti-EMT effect of RIE in human lung carcinoma. Importantly, an in vivo BALB/c nude mice xenograft model showed that RIE treatment reduced tumor growth by oral gavage, and RIE represent promising candidates for future phytochemical-based mechanistic pathway-targeted cancer prevention strategies. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Preliminary cellular-automata forecast of permit activity from 1998 to 2010, Idaho and Western Montana

    USGS Publications Warehouse

    Raines, G.L.; Zientek, M.L.; Causey, J.D.; Boleneus, D.E.

    2002-01-01

    For public land management in Idaho and western Montana, the U.S. Forest Service (USFS) has requested that the U.S. Geological Survey (USGS) predict where mineral-related activity will occur in the next decade. Cellular automata provide an approach to simulation of this human activity. Cellular automata (CA) are defined by an array of cells, which evolve by a simple transition rule, the automaton. Based on exploration trends, we assume that future exploration will focus in areas of past exploration. Spatial-temporal information about mineral-related activity, that is permits issued by USFS and Bureau of Land Management (BLM) in the last decade, and spatial information about undiscovered resources, provide a basis to calibrate a CA. The CA implemented is a modified annealed voting rule that simulates mineral-related activity with spatial and temporal resolution of 1 mi2 and 1 year based on activity from 1989 to 1998. For this CA, the state of the economy and exploration technology is assumed constant for the next decade. The calibrated CA reproduces the 1989-1998-permit activity with an agreement of 94%, which increases to 98% within one year. Analysis of the confusion matrix and kappa correlation statistics indicates that the CA underestimates high activity and overestimates low activity. Spatially, the major differences between the actual and calculated activity are that the calculated activity occurs in a slightly larger number of small patches and is slightly more uneven than the actual activity. Using the calibrated CA in a Monte Carlo simulation projecting from 1998 to 2010, an estimate of the probability of mineral activity shows high levels of activity in Boise, Caribou, Elmore, Lincoln, and western Valley counties in Idaho and Beaverhead, Madison, and Stillwater counties in Montana, and generally low activity elsewhere. ?? 2002 International Association for Mathematical Geology.

  10. The detection of hemorrhagic proteins in snake venoms using monoclonal antibodies against Virginia opossum (Didelphis virginiana) serum.

    PubMed

    Sánchez, E E; García, C; Pérez, J C; De La Zerda, S J

    1998-10-01

    Most snakes and a few warm-blooded animals have a resistance to snake venoms because of naturally occurring antihemorrhagins found in their sera. The antihemorrhagins in serum of Virginia opossum (Didelphis virginiana) neutralize hemorrhagic activity by binding to hemorrhagins in snake venoms. The binding characteristic of antihemorrhagins in D. virginiana serum was used to develop a five-step western blot. The detection of hemorrhagic proteins were measured indirectly with antihemorrhagins in Virginia opossum serum and with DV-2LD#2, a monoclonal antibody specific for Virginia opossum antihemorrhagins. Snake venoms were separated by native-PAGE, transferred to a Millipore Immobilon-P membrane and then incubated with crude Virginia opossum serum. The hemorrhagins in snake venom bind to antihemorrhagins in Virginia opossum serum which react with DV-2LD#2 a monoclonal antibody that is specific for Virginia opossum antihemorrhagins. DV-2LD#2 monoclonal antibody inhibits antihemorrhagic activity in Virginia opossum serum when mixed in equal amounts. The inhibition of antihemorrhagins by DV-2LD#2 monoclonal antibody suggests specificity. DV-2LD#2 monoclonal antibody does not recognize antihemorrhagins in gray woodrat (Neotoma micropus) serum. The five-step western blot reveals two well-defined bands which represent hemorrhagins found in Western diamondback rattlesnake (Crotalus atrox) venom. Venoms from 15 different snake species were examined to determine the usefulness of the five-step western blot. Other hemorrhagic venoms (Great Basin rattlesnake (C. viridis lutosus), Prairie rattlesnake (C. viridis viridis), Tancitaran dusky rattlesnake (C. pusillus), Northern Mojave rattlesnake (C. scutulatus scutulatus type B) and Northern Pacific rattlesnake (C. v. oreganus)) had one single band in the five-step western blot. DV-2LD#2 did not bind to the non-hemorrhagic venoms and reacted with 50% of the hemorrhagic venoms used in this study. The monoclonal antibody, CAH

  11. Fire activity and severity in the western US vary along proxy gradients representing fuel amount and fuel moisture.

    PubMed

    Parks, Sean A; Parisien, Marc-André; Miller, Carol; Dobrowski, Solomon Z

    2014-01-01

    Numerous theoretical and empirical studies have shown that wildfire activity (e.g., area burned) at regional to global scales may be limited at the extremes of environmental gradients such as productivity or moisture. Fire activity, however, represents only one component of the fire regime, and no studies to date have characterized fire severity along such gradients. Given the importance of fire severity in dictating ecological response to fire, this is a considerable knowledge gap. For the western US, we quantify relationships between climate and the fire regime by empirically describing both fire activity and severity along two climatic water balance gradients, actual evapotranspiration (AET) and water deficit (WD), that can be considered proxies for fuel amount and fuel moisture, respectively. We also concurrently summarize fire activity and severity among ecoregions, providing an empirically based description of the geographic distribution of fire regimes. Our results show that fire activity in the western US increases with fuel amount (represented by AET) but has a unimodal (i.e., humped) relationship with fuel moisture (represented by WD); fire severity increases with fuel amount and fuel moisture. The explicit links between fire regime components and physical environmental gradients suggest that multivariable statistical models can be generated to produce an empirically based fire regime map for the western US. Such models will potentially enable researchers to anticipate climate-mediated changes in fire recurrence and its impacts based on gridded spatial data representing future climate scenarios.

  12. Fire Activity and Severity in the Western US Vary along Proxy Gradients Representing Fuel Amount and Fuel Moisture

    PubMed Central

    Parks, Sean A.; Parisien, Marc-André; Miller, Carol; Dobrowski, Solomon Z.

    2014-01-01

    Numerous theoretical and empirical studies have shown that wildfire activity (e.g., area burned) at regional to global scales may be limited at the extremes of environmental gradients such as productivity or moisture. Fire activity, however, represents only one component of the fire regime, and no studies to date have characterized fire severity along such gradients. Given the importance of fire severity in dictating ecological response to fire, this is a considerable knowledge gap. For the western US, we quantify relationships between climate and the fire regime by empirically describing both fire activity and severity along two climatic water balance gradients, actual evapotranspiration (AET) and water deficit (WD), that can be considered proxies for fuel amount and fuel moisture, respectively. We also concurrently summarize fire activity and severity among ecoregions, providing an empirically based description of the geographic distribution of fire regimes. Our results show that fire activity in the western US increases with fuel amount (represented by AET) but has a unimodal (i.e., humped) relationship with fuel moisture (represented by WD); fire severity increases with fuel amount and fuel moisture. The explicit links between fire regime components and physical environmental gradients suggest that multivariable statistical models can be generated to produce an empirically based fire regime map for the western US. Such models will potentially enable researchers to anticipate climate-mediated changes in fire recurrence and its impacts based on gridded spatial data representing future climate scenarios. PMID:24941290

  13. Schisantherin A Attenuates Neuroinflammation in Activated Microglia: Role of Nrf2 Activation Through ERK Phosphorylation.

    PubMed

    Li, Chuwen; Chen, Tongkai; Zhou, Hefeng; Zhang, Chao; Feng, Yu; Tang, Fan; Hoi, Maggie Pui-Man; He, Chengwei; Zheng, Ying; Lee, Simon Ming-Yuen

    2018-06-28

    In the present study, we investigated whether schisantherin A (StA) had anti-inflammatory effects under neuroinflammatory conditions. The effects of StA and its underlying mechanisms were examined in lipopolysaccharide (LPS)-activated BV-2 microglial cells by ELISA, qPCR, EMSA, Western blot, and IHC. Firstly, we found that StA inhibited the inflammatory response in LPS-activated BV-2 microglia. Secondly, we found that StA suppressed LPS-induced activation of NF-κB via interfering with degradation of IκB and phosphorylation of IκB, IKK, PI3K/Akt, JNK, and p38 MAPK. Thirdly, StA conferred indirect antioxidative effects via quenching ROS and promoted expression of antioxidant enzymes, including HO-1 and NQO-1, via stimulating activation of Nrf2 pathways. Finally, we demonstrated that anti-neuroinflammatory actions of StA were dependent on ERK phosphorylation-mediated Nrf2 activation. StA induced ERK phosphorylation-mediated Nrf2 activation, which contributed to its anti-inflammation and anti-oxidation. The anti-neuroinflammatory and anti-oxidative effects of StA may show preventive therapeutic potential for various neuroinflammatory disorders. © 2018 The Author(s). Published by S. Karger AG, Basel.

  14. Whey peptide Isoleucine-Tryptophan inhibits expression and activity of matrix metalloproteinase-2 in rat aorta.

    PubMed

    Kopaliani, Irakli; Martin, Melanie; Zatschler, Birgit; Müller, Bianca; Deussen, Andreas

    2016-08-01

    Aortic stiffness is an independent risk factor for development of cardiovascular diseases. Activation of renin-angiotensin-aldosterone system (RAAS) including angiotensin converting enzyme (ACE) activity leads to overproduction of angiotensin II (ANGII) from its precursor angiotensin I (ANGI). ANGII leads to overexpression and activation of matrix metalloproteinase-2 (MMP2), which is critically associated with pathophysiology of aortic stiffness. We previously reported that the whey peptide Isoleucine-Tryptophan (IW) acts as a potent ACE inhibitor. Herein, we critically elucidate the mechanism of action by which IW causes inhibition of expression and activity of MMP2 in aortic tissue. Effects of IW on expression and activity of MMP2 were assessed on endothelial and smooth muscle cells (ECs and SMCs) in vitro and ex vivo (isolated rat aorta). As controls we used the pharmaceutical ACE inhibitor - captopril and the ANGII type 1 receptor blocker - losartan. In vitro, both ANGII and ANGI stimulation significantly (P<0.01) increased expression of MMP2 assessed with western blot. Similarly, to captopril IW significantly (P<0.05) inhibited ANGI, but not ANGII mediated increase in expression of MMP2, while losartan also blocked effects of ANGII. Signaling pathways regulating MMP2 expression in ECs and SMCs were similarly inhibited after treatment with IW or captopril. In ECs IW significantly (P<0.05) inhibited JNK pathway, whereas in SMCs JAK2/STAT3 pathway, assessed with western blot. In vitro findings were fully consistent with results in isolated rat aorta ex vivo. Moreover, IW not only inhibited the MMP2 expression, but also its activation assessed with gelatin zymography. Our findings demonstrate that IW effectively inhibits expression and activation of MMP2 in rat aorta by decreasing local conversion of ANGI to ANGII. Thus, similar to pharmaceutical ACE inhibitor captopril the dipeptide IW may effectively inhibit ACE activity and prevent the age and hypertension

  15. [Detection of stable expression of human interlukin-2 gene in transfected keratinocytes].

    PubMed

    Liao, W; Liu, Y; Ye, L

    1999-09-01

    To investigate the stable expression and secretion of human interlukin-2 gene in transfected keratinocytes. Keratinocytes were transfected with lipofectamine and selected by G418. Then the samples were analyzed with the techniques of DNA dot blot, RNA dot blot, hybridization in situ, immunohistochemistry, Western blot and MTT. The positive signals were observed in transfected keratinocytes by DNA dot blot, RNA dot blot, hybridization in situ and immunohistochemistry. With Western blot analysis, a specific band exhibiting a molecular weight of 15,000 was detected in transfected keratinocytes, which was in acordance with that of IL-2. The expression of IL-2 can maintain for up to 1 month. The amounts of IL-2 in the supernatants of two and four passages transfected keratinocytes were 27.7 U/ml and 15.0 U/ml, respectively. Keratinocytes have the potential for stable gene expression and secretion of active transgene products. Thus, it is possible to use keratinocytes as a target cell for gene transfection, gene expression and even gene therapy.

  16. Resveratrol protects bupivacaine-induced neuro-apoptosis in dorsal root ganglion neurons via activation on tropomyosin receptor kinase A.

    PubMed

    Guo, Zhiliang; Liu, Yuanyuan; Cheng, Min

    2018-07-01

    General anesthesia in spinal cord may lead to unexpected but irreversible neurotoxicity. We investigated whether resveratrol (RSV) may protect bupivacaine (BUP)-induced neuro-apoptosis in spinal cord dorsal root ganglia (DRG). Mouse DRG cells were cultured in vitro, pre-treated with RSV and then 5 mM BUP. A concentration-dependent effect of RSV on reducing BUP-induced apoptosis of DRG neurons (DRGNs) was evaluated using a TUNEL assay. QRT-PCR and western blot assays were also conducted to evaluate gene and protein expressions of tropomyosin receptor kinase A/B/C (TrkA/B/C) and activated (phosphorylated) Trk receptors, phospho-TrkA/B/C. In addition, a functional TrkA blocking antibody MNAC13 was applied in DRG culture to further measure the functional role of Trk receptor in RSV-initiated apoptotic protection on BUP-damaged DRGNs. BUP promoted significant apoptosis in DRG. RSV exhibited protective effects against BUP-induced neuro-apoptosis in a concentration-dependent manner. qRT-PCR and western blot showed that RSV did not alter TrkA/B/C gene or protein expression, but significantly upregulated phospho-TrkA. Conversely, application of MNAC13 decreased phospho-TrkA and reversed RSV-initiated neuro-protection on BUP-induced DRGN apoptosis. Resveratrol may protect anesthesia-induced DRG neuro-apoptosis, and activation of TrkA signaling pathway may be the underlying mechanism in this process. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  17. Reproduction and seasonal activity of silver-haired bats (Lasionycteris noctivagans) in western Nebraska

    USGS Publications Warehouse

    Geluso, Keith; Huebschman, J.J.; White, J.A.; Bogan, M.A.

    2004-01-01

    Silver-haired bats (Lasionycteris noctivagans) were thought only to migrate through Nebraska; however, recent surveys in eastern Nebraska report summer records of females and their young. Our study in western Nebraska also shows that silver-haired bats are summer residents. We discovered the 1st reproductively active L. noctivagans in this part of the state. We caught lactating females and volant young in riparian forests along the North Platte River and in forested areas of the Pine Ridge. Previously, adult males were not known from Nebraska in summer, and only 4 records of L. noctivagans were known from western Nebraska during migration. On 28 July we captured an adult male in a coniferous forest of the Wildcat Hills, and we have more than 100 records of migrating individuals. Lastly, an obese L. noctivagans captured on 4 November may represent an individual preparing to hibernate in the state.

  18. Openness as a predictor of political orientation and conventional and unconventional political activism in Western and Eastern Europe.

    PubMed

    Roets, Arne; Cornelis, Ilse; Van Hiel, Alain

    2014-01-01

    This study provides a comprehensive investigation of the relationship between Openness and political orientation and activism in Europe. Analyses were conducted on the 4 waves of the European Social Survey, including large representative samples in up to 26 European countries (total N > 175,000). In line with previous studies, a robust, positive relationship between Openness and left-wing political orientation was obtained in Western Europe. However, in Eastern Europe, the relationship between Openness and political orientation was weaker, and reversed in 3 out of 4 waves. Moreover, Openness yielded significant positive relationships with unconventional activism and to a lesser degree with conventional activism. The magnitude of the relationship between Openness and activism was dependent on political orientation and region. Stronger associations between Openness and activism were found for those having a left-wing orientation in Western Europe, whereas in Eastern Europe, Openness was somewhat stronger related to activism for those having a right-wing orientation. In the discussion we elaborate on the role of the geopolitical context in the relationship between Openness and political variables.

  19. Peroxisome proliferator-activated receptor (PPAR)gamma is highly expressed in normal human pituitary gland.

    PubMed

    Bogazzi, F; Russo, D; Locci, M T; Chifenti, B; Ultimieri, F; Raggi, F; Viacava, P; Cecchetti, D; Cosci, C; Sardella, C; Acerbi, G; Gasperi, M; Martino, E

    2005-11-01

    Expression of peroxisome proliferator-activated receptor (PPAR)gamma in normal pituitary seems to be restricted to ACTH-secreting cells. The aim of the study was to evaluate the expression of PPARgamma in normal human pituitary tissue and to study its localization in the pituitary secreting cells. Normal pituitary tissue samples were obtained form 11 patients with non-secreting adenoma who underwent surgical excision of the tumor. Expression of PPARgamma was evaluated by immunostaining and western blotting; localization of PPARgamma in each pituitary secreting cell lineage was evaluated by double immunofluorescence using confocal microscopy. Pituitary non-functioning adenomas served as Controls. PPARgamma was highly expressed in all pituitary samples with a (mean +/- SD) 81 +/- 6.5% of stained cells; expression of PPARgamma was confirmed by western blotting. Non-functioning pituitary adenomas had 74 +/- 11% PPARgamma positive cells. Expression of PPARy was either in cytoplasm or nuclei. In addition, treatment of GH3 cells, with a PPARgamma ligand was associated with traslocation of the receptor from cytoplasm into the nucleus. Double immunostaining revealed that every pituitary secreting cell (GH, TSH, LH, FSH, PRL and ACTH) had PPARgamma expressed. The present study demonstrated that PPARgamma is highly expressed in every normal pituitary secreting cell lineage. It can translocate into the nucleus by ligand binding; however, its role in pituitary hormone regulation remains to be elucidated.

  20. Seasonal Activity, Density, and Collection Efficiency of the Blacklegged Tick (Ixodes scapularis) (Acari: Ixodidae) in Mid-Western Pennsylvania.

    PubMed

    Simmons, T W; Shea, J; Myers-Claypole, M A; Kruise, R; Hutchinson, M L

    2015-11-01

    Although Pennsylvania has recently reported the greatest number of Lyme disease cases in the United States, with the largest increase for PA occurring in its western region, the population biology of the blacklegged tick (Ixodes scapularis Say) has not been adequately characterized in western PA. We studied the seasonal activity of host-seeking I. scapularis larvae, nymphs, and adults in mid-western PA over the course of a year, including a severe winter, and determined their absolute densities and collection efficiencies using replicated mark-release-recapture or removal methods. Our results are compared to those from similar studies conducted in the highly Lyme disease endemic Hudson Valley region of southeastern New York State. The seasonal activity of I. scapularis was intermediate between patterns observed in the coastal northeastern and upper Midwestern United States. Only one peak of larval activity was observed, which was later than the major peak in the Midwest, but earlier than in the northeast. Seasonal synchrony of larvae and nymphs was similar to the northeast, but the activity peaks were much closer together, although not completely overlapping as in the Midwest. Pre- and postwinter relative densities of questing adult I. scapularis were not significantly different from one another. The absolute densities and collection efficiencies of larvae, nymphs, and adults were comparable to results from classic research conducted at the Louis Calder Center in Westchester County, NY. We conclude that the population biology of I. scapularis in mid-western PA is similar to southeastern NYS contributing to a high acarological Lyme disease risk. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  1. Stilbene Glucoside, a Putative Sleep Promoting Constituent from Polygonum multiflorum Affects Sleep Homeostasis by Affecting the Activities of Lactate Dehydrogenase and Salivary Alpha Amylase.

    PubMed

    Wei, Qian; Ta, Guang; He, Wenjing; Wang, Wei; Wu, Qiucheng

    2017-01-01

    Chinese herbal medicine (CHM) has been used for treating insomnia for centuries. The most used CHM for insomnia was Polygonum multiflorum. However, the molecular mechanism for CHM preventing insomnia is unknown. Stilbene glucoside (THSG), an important active component of P. multiflorum, may play an important role for treating insomnia. To test the hypothesis, Kunming mice were treated with different dosages of THSG. To examine the sleep duration, a computer-controlled sleep-wake detection system was implemented. Electroencephalogram (EEG) and electromyogram (EMG) electrodes were implanted to determine sleep-wake state. RT-PCR and Western blot was used to measure the levels of lactate dehydrogenase (LDH) and saliva alpha amylase. Spearman's rank correlation coefficient was used to identify the strength of correlation between the variables. The results showed that THSG significantly prolonged the sleep time of the mice (p<0.01). THSG changed sleep profile by reducing wake and rapid eye movement (REM) period, and increasing non-REM period. RT-PCR and Western blot analysis showed that THSG could down-regulate the levels of LDH and saliva alpha amylase (p<0.05). The level of lactate and glucose was positively related with the activity of LDH and saliva alpha amylase (p<0.05), respectively. On the other hand, the activities of LDH and amylase were negatively associated with sleep duration (p<0.05). The levels of lactate and glucose affect sleep homeostasis. Thus, THSG may prevent insomnia by regulating sleep duration via LDH and salivary alpha amylase.

  2. Modifications of Western-type diet regarding protein, fat and sucrose levels as modulators of steroid metabolism and activity in liver.

    PubMed

    Krawczyńska, Agata; Herman, Andrzej P; Antushevich, Hanna; Bochenek, Joanna; Dziendzikowska, Katarzyna; Gajewska, Alina; Gromadzka-Ostrowska, Joanna

    2017-01-01

    The aim of the study was to evaluate whether the modification of the Western-type diet (high-fat, high-sucrose diet rich in saturated fatty acids) considering macronutrients content would influence hepatic metabolism and activity of steroids. For 3 weeks Wistar rat were fed the Western-type diet (21% fat, 35% sucrose, 19% protein, lard) and its modifications regarding dietary protein (10 and 19%), fat (5 and 21%) and sucrose (0 and 35%) levels. The steroid 5α-reductase type 1 (Srd5a1) and androgen receptor (Ar) gene expression as well as testosterone (T) conversion towards 5α-reduced derivatives in liver were positively correlated with body weight gain. The Western-type diets with decreased protein content regardless of the sucrose level exerted the most negative effect on the antioxidant system decreasing catalase (Cat), sodium dismutase (Sod1) and glutathione peroxidase (Gpx1) gene expression as well as Cat and Gpx activity and total antioxidant status, simultaneously intensifying lipid peroxidation. The impaired antioxidant system was accompanied by decreased level of hepatic T metabolism towards estrogens: 17β-estradiol (E2) and estriol, and increased estrogen receptor type 1 (Esr1) gene expression. Liver Esr1 mRNA level was differently correlated with T (positively) and E2 (negatively) plasma levels. Whereas the fat reduction in Western-type diet restored the plasma proportion between T and E2. In conclusion it could be stated that Western-type diet modification relating to protein, sucrose and fat content can influence hepatic steroid metabolism and activity; however the estrogens and androgens metabolism in liver would be connected with impairment of liver function or catabolic activity, respectively. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Essential oil of Artemisia argyi suppresses inflammatory responses by inhibiting JAK/STATs activation.

    PubMed

    Chen, Lin-Lin; Zhang, Hao-Jun; Chao, Jung; Liu, Jun-Feng

    2017-05-23

    Artemisia argyi is a herbal medicine traditionally used in Asia for the treatment of bronchitis, dermatitis and arthritis. Recent studies revealed the anti-inflammatory effect of essential oil in this plant. However, the mechanisms underlying the therapeutic potential have not been well elucidated. The present study is aimed to verify its anti-inflammatory effect and investigate the probable mechanisms. The essential oil from Artemisia argyi (AAEO) was initially tested against LPS-induced production of inflammatory mediators and cytokines in RAW264.7 macrophages. Protein and mRNA expressions of iNOS and COX-2 were determined by Western blotting and RT-PCR analysis, respectively. The effects on the activation of MAPK/NF-κB/AP-1 and JAK/STATs pathway were also investigated by western blot. Meanwhile, in vivo anti-inflammatory effect was examined by histologic and immunohistochemical analysis in TPA-induced mouse ear edema model. The results of in vitro experiments showed that AAEO dose-dependently suppressed the release of pro-inflammatory mediators (NO, PGE 2 and ROS) and cytokines (TNF-α, IL-6, IFN-β and MCP-1) in LPS-induced RAW264.7 macrophages. It down-regulated iNOS and COX-2 protein and mRNA expression but did not affect the activity of these two enzymes. AAEO significantly inhibited the phosphorylation of JAK2 and STAT1/3, but not the activation of MAPK and NF-κB cascades. In animal model, oral administration of AAEO significantly attenuated TPA-induced mouse ear edema and decreased the protein level of COX-2. AAEO suppresses inflammatory responses via down-regulation of the JAK/STATs signaling and ROS scavenging, which could contribute, at least in part, to the anti-inflammatory effect of AAEO. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

  4. 17β-estradiol activates mTOR in chondrocytes by AKT-dependent and AKT-independent signaling pathways

    PubMed Central

    Tao, Yulei; Sun, Haibiao; Sun, Hongyan; Qiu, Xianxing; Xu, Changbo; Shi, Changxiu; Du, Jiahui

    2015-01-01

    To confirm whether 17β-estradiol (E2) activates mammalian target of rapamycin (mTOR) signaling pathway in chondrocytes and in what way activates mTOR. Human immortalized chondrocytes cell lines TC28a2 and C28/I2 were subjected to incubate with or without E2, LY294002 (the inhibitor of PI3K), rapamycin (the inhibitor of mTOR), or E2 in combination with LY294002 or rapamycin. Thereafter, protein levels of S6K1, p-S6K1, protein kinase B (AKT), and p-AKT were determined by Western blot analysis. Matrix metallopeptidase (MMP) 3 or MMP13 mRNA levels were evaluated by quantitative real-time PCR (qRT-PCR). Co-immunoprecipitation and Western blot analysis were performed to verify the interaction between ERα and mTOR. Both p-S6K1 and p-AKT protein levels in TC28a2 and C28/I2E2 cells were significantly increased by incubation with E2 (0.5 h and 1 h) (P < 0.05). Rapamycin did not affect the levels of p-AKT, but were significantly reduced by LY294002 or E2 in combination with LY294002. The levels of p-S6K1 were significantly decreased by incubation with LY294002, but the effect could be reversed by E2 in combination with LY294002. Rabbit anti-mTOR antibody was able to immunoprecipitate ERα after incubation with E2. Moreover, E2 inhibited the mRNA levels of MMP3 and MMP13 by mTOR pathway. E2 actives mTOR in chondrocytes through AKT-dependent and independent ways. PMID:26884863

  5. Black tea and D. candidum extracts play estrogenic activity via estrogen receptor α-dependent signaling pathway

    PubMed Central

    Wang, Yongsen; Sun, Jing; Zhang, Kun; Hu, Xin; Sun, Yuchu; Sheng, Jun; Fu, Xueqi

    2018-01-01

    In recent years, phytoestrogens have been shown as useful selective estrogen receptor modulators. The estrogen-like effects of black tea (BT) and D. candidum (DC), as well as the combination of the two herbs, have remained largely elusive. This study aims to investigate the phytoestrogenic effect of BT and DC extract, and the possible mechanism. The effects on T47D (ER+ cell line) proliferation were evaluated by using MTT assay. The S phase proportion of ER+ cells was determined by using flow cytometry. The estrogen antagonist ICI 182,780 was applied to block the ER function. The activation of ER-mediated PI3K/AKT and ERK signal pathways were observed by using western blot. Expression of ERα and PGR, as well as PS2 and Cyclin D1 were detected by using western blot and real-time quantitative PCR. Firstly, our results found that BT and DC extracts promoted cell proliferation in ER-positive cells, and this effect was ER-dependent. Besides, BT and DC extracts increased the S-phase cell number. Next, PI3K, AKT and ERK pathways below ER were activated by phytoestrogen treatment, and this activation was blocked by the ER antagonist. Moreover, prolonged BT and DC treatments increased the expression of ESR1 and PGR. Consistently, the mRNA levels of not only ESR1 and PGR but also estrogen-dependent effectors ps2 and cyclin D1, were increased by phytoestrogens and blocked by ICI 182,780. Taken Together, BT and DC extracts have phytoestrogenic effects, and this may provide new ideas and experimental basis for the development and application of phytoestrogens. PMID:29422998

  6. CAPN 7 promotes the migration and invasion of human endometrial stromal cell by regulating matrix metalloproteinase 2 activity.

    PubMed

    Liu, Hongyu; Jiang, Yue; Jin, Xiaoyan; Zhu, Lihua; Shen, Xiaoyue; Zhang, Qun; Wang, Bin; Wang, Junxia; Hu, Yali; Yan, Guijun; Sun, Haixiang

    2013-07-15

    Matrix metalloproteinase 2 (MMP-2) has been reported to be an important regulator of cell migration and invasion through degradation of the extracellular matrix (ECM) in many diseases, such as cancer and endometriosis. Here, we found calcium-activated neutral protease 7 (CAPN 7) expression was markedly upregulated in the eutopic endometrium and endometrial stromal cells of women diagnosed with endometriosis. Our studies were carried out to detect the effects of CAPN 7 on human endometrial stromal cell (hESC) migration and invasion. Western blotting and quantitative real-time PCR were used to detect the expression of CAPN 7 in endometriosis patients and normal fertile women. Scratch-wound-healing and invasion chamber assay were used to investigate the role of CAPN 7 in hESC migration and invasion. Western blotting, quantitative real-time PCR and zymography were carried out to detect the effect of CAPN 7 on the expressions and activity of MMP-2. CAPN 7 was markedly up-regulated in endometriosis, thereby promoting the migration and invasion of hESC. CAPN 7 overexpression led to increased expression of MMP-2 and tissue inhibitor of metalloproteinases 2 (TIMP-2); CAPN 7 knockdown reversed these changes. CAPN 7 increased MMP-2 activity by increasing the ratio of MMP-2 to TIMP-2. We also found that OA-Hy (an MMP-2 inhibitor) decreased the effects of CAPN 7 overexpression on hESC migration and invasion by approximately 50% and 55%, respectively. Additionally, a coimmunoprecipitation assay demonstrated that CAPN 7 interacted with activator protein 2α (AP-2α): an important transcription factor of MMP-2. CAPN 7 promotes hESC migration and invasion by increasing the activity of MMP-2 via an increased ratio of MMP-2 to TIMP-2.

  7. CAPN 7 promotes the migration and invasion of human endometrial stromal cell by regulating matrix metalloproteinase 2 activity

    PubMed Central

    2013-01-01

    Background Matrix metalloproteinase 2 (MMP-2) has been reported to be an important regulator of cell migration and invasion through degradation of the extracellular matrix (ECM) in many diseases, such as cancer and endometriosis. Here, we found calcium-activated neutral protease 7 (CAPN 7) expression was markedly upregulated in the eutopic endometrium and endometrial stromal cells of women diagnosed with endometriosis. Our studies were carried out to detect the effects of CAPN 7 on human endometrial stromal cell (hESC) migration and invasion. Methods Western blotting and quantitative real-time PCR were used to detect the expression of CAPN 7 in endometriosis patients and normal fertile women. Scratch-wound-healing and invasion chamber assay were used to investigate the role of CAPN 7 in hESC migration and invasion. Western blotting, quantitative real-time PCR and zymography were carried out to detect the effect of CAPN 7 on the expressions and activity of MMP-2. Results CAPN 7 was markedly up-regulated in endometriosis, thereby promoting the migration and invasion of hESC. CAPN 7 overexpression led to increased expression of MMP-2 and tissue inhibitor of metalloproteinases 2 (TIMP-2); CAPN 7 knockdown reversed these changes. CAPN 7 increased MMP-2 activity by increasing the ratio of MMP-2 to TIMP-2. We also found that OA-Hy (an MMP-2 inhibitor) decreased the effects of CAPN 7 overexpression on hESC migration and invasion by approximately 50% and 55%, respectively. Additionally, a coimmunoprecipitation assay demonstrated that CAPN 7 interacted with activator protein 2α (AP-2α): an important transcription factor of MMP-2. Conclusions CAPN 7 promotes hESC migration and invasion by increasing the activity of MMP-2 via an increased ratio of MMP-2 to TIMP-2. PMID:23855590

  8. Platelet-activating factor mediates monocyte chemoattractant protein-1 expression in glomerular immune injury.

    PubMed

    Jocks, T; Freudenberg, J; Zahner, G; Stahl, R A

    1998-01-01

    These studies were designed to determine the possible role of platelet-activating factor (PAF) in the production of monocyte chemoattractant protein-1 (MCP-1) in glomerular immune injury. The glomerular lesion was induced in isolated perfused rat kidneys by a rabbit anti-rat-thymocyte serum (ATS) and rat serum (RS) as a complement source. Perfusion of kidneys with ATS and RS results in the selective binding of the antiserum to the glomerular mesangium with consecutive intraglomerular activation of complement. Antibody binding and complement activation induced a significant increase in glomerular MCP-1 mRNA levels when assessed by Northern blotting or RT-PCR. Decomplemented RS or non antibody rabbit IgG had only moderate effects on glomerular MCP-1 mRNA levels. The PAF receptor antagonist WEB 2170 almost completely blocked the ATS and RS induced MCP-1 mRNA levels. Perfusion of control kidneys with PAF increased MCP-1 mRNA expression, an effect which was blocked by WEB 2170. Glomerular MCP-1 protein formation, assessed by Western blotting, was stimulated following ATS and RS and PAF, respectively, was blocked by WEB 2170. These data show that PAF, derived from glomerular resident cells following antibody and complement induced injury, stimulates MCP-1 expression. In addition to the direct effects on leukocyte adhesion and activation PAF may mediate inflammatory cell influx in glomerular injuries due to the release of MCP-1.

  9. Corin mutations K317E and S472G from preeclamptic patients alter zymogen activation and cell surface targeting. [Corrected].

    PubMed

    Dong, Ningzheng; Zhou, Tiantian; Zhang, Yue; Liu, Meng; Li, Hui; Huang, Xiaoyi; Liu, Zhenzhen; Wu, Yi; Fukuda, Koichi; Qin, Jun; Wu, Qingyu

    2014-06-20

    Corin is a membrane-bound serine protease that acts as the atrial natriuretic peptide (ANP) convertase in the heart. Recent studies show that corin also activates ANP in the pregnant uterus to promote spiral artery remodeling and prevent pregnancy-induced hypertension. Two CORIN gene mutations, K317E and S472G, were identified in preeclamptic patients and shown to have reduced activity in vitro. In this study, we carried out molecular modeling and biochemical experiments to understand how these mutations impair corin function. By molecular modeling, the mutation K317E was predicted to alter corin LDL receptor-2 module conformation. Western blot analysis of K317E mutant in HEK293 cells showed that the mutation did not block corin expression on the cell surface but inhibited corin zymogen activation. In contrast, the mutation S472G was predicted to abolish a β-sheet critical for corin frizzled-2 module structure. In Western blot analysis and flow cytometry, S472G mutant was not detected on the cell surface in transfected HEK293 cells. By immunostaining, the S472G mutant was found in the ER, indicating that the mutation S472G disrupted the β-sheet, causing corin misfolding and ER retention. Thus, these results show that mutations in the CORIN gene may impair corin function by entirely different mechanisms. Together, our data provide important insights into the molecular basis underlying corin mutations that may contribute to preeclampsia in patients. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Differential Role of Rapamycin in Epidermis-Induced IL-15-IGF-1 Secretion via Activation of Akt/mTORC2.

    PubMed

    Bai, Yang; Xu, Rui; Zhang, Xueyuan; Zhang, Xiaorong; Hu, Xiaohong; Li, Yashu; Li, Haisheng; Liu, Meixi; Huang, Zhenggen; Yan, Rongshuai; He, Weifeng; Luo, Gaoxing; Wu, Jun

    2017-01-01

    Backgroud/Aims: The effects of rapamycin (RPM) on wound healing have been previously studied. However, reciprocal contradictory data have been reported, and the underlying mechanism remains unclear. This study aims to uncover differential role of RPM in regulation of wound healing and explore the possible mechanism. C57BL/6J mice and epidermal cells were treated with different doses of RPM. The wound re-epithelialization was observed by hematoxylin and eosin (HE) staining. The expression of IL-15 and IGF-1 were detected by immunohistochemistry and quantitative real-time PCR. Epidermal cell survival was determined by CCK-8 assays. Moreover, the mTORC1 and mTORC2 pathway were examined by western blot analysis. This study showed that differential doses of RPM could lead to separate consequences in epidermis. Histological analyses showed that low-dose RPM promoted wound healing, and enhanced the expression of IL-15 and IGF-1. Furthermore, western blot analysis showed that the effect of low-dose RPM in epidermis were not through mTORC1 pathway. Instead, activation of the Akt/mTORC2 pathway was involved in low-dose RPM-induced IL-15 and IGF-1 production in epidermis, while high-dose RPM inhibited the expression of IL-15 and IGF-1 and the activity of mTORC1 and mTORC2 pathway. This study for the first time demonstrated that RPM-mediated wound healing was dose-dependent. © 2017 The Author(s). Published by S. Karger AG, Basel.

  11. (-)-7(S)-hydroxymatairesinol protects against tumor necrosis factor-α-mediated inflammation response in endothelial cells by blocking the MAPK/NF-κB and activating Nrf2/HO-1.

    PubMed

    Yang, Di; Xiao, Chen-Xi; Su, Zheng-Hua; Huang, Meng-Wei; Qin, Ming; Wu, Wei-Jun; Jia, Wan-Wan; Zhu, Yi-Zhun; Hu, Jin-Feng; Liu, Xin-Hua

    2017-08-15

    Endothelial inflammation is an increasingly prevalent condition in the pathogenesis of many cardiovascular diseases. (-)-7(S)-hydroxymatairesinol (7-HMR), a naturally occurring plant lignan, possesses both antioxidant and anti-cancer properties and therefore would be a good strategy to suppress tumor necrosis factor-α (TNF-α)-mediated inflammation in vascular endothelial cells (VECs). The objective of this study is to evaluate for its anti-inflammatory effect on TNF-α-stimulated VECs and underling mechanisms. The effect of the 7-HMR on suppression of TNF-α-induced inflammation mediators in VECs were determined by qRT-PCR and Western blot. MAPKs and phosphorylation of Akt, HO-1 and NF-κB p65 were examined using Western blot. Nuclear localisation of NF-κB was also examined using Western blot and immunofluorescence. Here we found that 7-HMR could suppress TNF-α-induced inflammatory mediators, such as vascularcelladhesion molecule-1, interleukin-6 and inducible nitric oxide synthase expression both in mRNA and protein levels, and concentration-dependently attenuated reactive oxidase species generation. We further identified that 7-HMR remarkably induced superoxide dismutase and heme oxygenase-1 expression associated with degradation of Kelch-like ECH-associated protein 1 (keap1) and up-regulated nuclear factor erythroid 2-related factor 2 (Nrf2). In addition, 7-HMR time- and concentration-dependently attenuated TNF-α-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK) and Akt, but not p38, or c-Jun N-terminal kinase 1/2. Moreover, 7-HMR significantly suppressed TNF-α-mediated nuclear factor-κB (NF-κB) activation by inhibiting phosphorylation and nuclear translocation of NF-κB p65. Our results demonstrated that 7-HMR inhibited TNF-α-stimulated endothelial inflammation, at least in part, through inhibition of NF-κB activation and upregulation of Nrf2-antioxidant response element signaling pathway, suggesting 7-HMR might be used as a

  12. Deep proteomic profiling of vasopressin-sensitive collecting duct cells. I. Virtual Western blots and molecular weight distributions

    PubMed Central

    Yang, Chin-Rang; Tongyoo, Pumipat; Emamian, Milad; Sandoval, Pablo C.; Raghuram, Viswanathan

    2015-01-01

    The mouse mpkCCD cell line is a continuous cultured epithelial cell line with characteristics of renal collecting duct principal cells. This line is widely used to study epithelial transport and its regulation. To provide a data resource useful for experimental design and interpretation in studies using mpkCCD cells, we have carried out “deep” proteomic profiling of these cells using three levels of fractionation (differential centrifugation, SDS-PAGE, and HPLC) followed by tandem mass spectrometry to identify and quantify proteins. The analysis of all resulting samples generated 34.6 gigabytes of spectral data. As a result, we identified 6,766 proteins in mpkCCD cells at a high level of stringency. These proteins are expressed over eight orders of magnitude of protein abundance. The data are provided to users as a public data base (https://helixweb.nih.gov/ESBL/Database/mpkFractions/). The mass spectrometry data were mapped back to their gel slices to generate “virtual Western blots” for each protein. For most of the 6,766 proteins, the apparent molecular weight from SDS-PAGE agreed closely with the calculated molecular weight. However, a substantial fraction (>15%) of proteins was found to run aberrantly, with much higher or much lower mobilities than predicted. These proteins were analyzed to identify mechanisms responsible for altered mobility on SDS-PAGE, including high or low isoelectric point, high or low hydrophobicity, physiological cleavage, residence in the lysosome, posttranslational modifications, and expression of alternative isoforms due to alternative exon usage. Additionally, this analysis identified a previously unrecognized isoform of aquaporin-2 with apparent molecular mass <20 kDa. PMID:26310816

  13. L- and D-lactate enhance DNA repair and modulate the resistance of cervical carcinoma cells to anticancer drugs via histone deacetylase inhibition and hydroxycarboxylic acid receptor 1 activation.

    PubMed

    Wagner, Waldemar; Ciszewski, Wojciech M; Kania, Katarzyna D

    2015-07-25

    The consideration of lactate as an active metabolite is a newly emerging and attractive concept. Recently, lactate has been reported to regulate gene transcription via the inhibition of histone deacetylases (HDACs) and survival of cancer cells via hydroxycarboxylic acid receptor 1 (HCAR1). This study examined the role of L- and D-lactate in the DNA damage response in cervical cancer cells. Three cervical cancer cell lines were examined: HeLa, Ca Ski and C33A. The inhibitory activity of lactate on HDACs was analysed using Western blot and biochemical methods. The lactate-mediated stimulation of DNA repair and cellular resistance to neocarzinostatin, doxorubicin and cisplatin were studied using γ-H2AX, comet and clonogenic assays. HCAR1 and DNA repair gene expression was quantified by real-time PCR. DNA-PKcs activity and HCAR1 protein expression were evaluated via immunocytochemistry and Western blot, respectively. HCAR1 activation was investigated by measuring intracellular cAMP accumulation and Erk phosphorylation. HCAR1 expression was silenced using shRNA. L- and D-lactate inhibited HDACs, induced histone H3 and H4 hyperacetylation, and decreased chromatin compactness in HeLa cells. Treating cells with lactate increased LIG4, NBS1, and APTX expression by nearly 2-fold and enhanced DNA-PKcs activity. Based on γ-H2AX and comet assays, incubation of cells in lactate-containing medium increased the DNA repair rate. Furthermore, clonogenic assays demonstrated that lactate mediates cellular resistance to clinically used chemotherapeutics. Western blot and immunocytochemistry showed that all studied cell lines express HCAR1 on the cellular surface. Inhibiting HCAR1 function via pertussis toxin pretreatment partially abolished the effects of lactate on DNA repair. Down-regulating HCAR1 decreased the efficiency of DNA repair, abolished the cellular response to L-lactate and decreased the effect of D-lactate. Moreover, HCAR1 shRNA-expressing cells produced significantly

  14. TRPM8 Channel Activation Induced by Monoterpenoid Rotundifolone Underlies Mesenteric Artery Relaxation

    PubMed Central

    Silva, Darizy Flavia; de Almeida, Monica Moura; Chaves, Cinthia Guedes; Braz, Ana Letícia; Gomes, Maria Aparecida; Pinho-da-Silva, Leidiane; Pesquero, Jorge Luiz; Andrade, Viviane Aguiar; Leite, Maria de Fátima; de Albuquerque, José George Ferreira; Araujo, Islania Giselia Albuquerque; Nunes, Xirley Pereira; Barbosa-Filho, José Maria; Cruz, Jader dos Santos; Correia, Nadja de Azevedo; de Medeiros, Isac Almeida

    2015-01-01

    In this study, our aims were to investigate transient receptor potential melastatin-8 channels (TRPM8) involvement in rotundifolone induced relaxation in the mesenteric artery and to increase the understanding of the role of these thermosensitive TRP channels in vascular tissue. Thus, message and protein levels of TRPM8 were measured by semi-quantitative PCR and western blotting in superior mesenteric arteries from 12 week-old Spague-Dawley (SD) rats. Isometric tension recordings evaluated the relaxant response in mesenteric rings were also performed. Additionally, the intracellular Ca2+ changes in mesenteric artery myocytes were measured using confocal microscopy. Using PCR and western blotting, both TRPM8 channel mRNA and protein expression was measured in SD rat mesenteric artery. Rotundifolone and menthol induced relaxation in the isolated superior mesenteric artery from SD rats and improved the relaxant response induced by cool temperatures. Also, this monoterpene induced an increase in transient intracellular Ca2+. These responses were significantly attenuated by pretreatment with capsazepine or BCTC, both TRPM8 channels blockers. The response induced by rotundifolone was not significantly attenuated by ruthenium red, a non-selective TRP channels blocker, or following capsaicin-mediated desensitization of TRPV1. Our findings suggest that rotundifolone induces relaxation by activating TRPM8 channels in rat superior mesenteric artery, more selectively than menthol, the classic TRPM8 agonist, and TRPM8 channels participates in vasodilatory pathways in isolated rat mesenteric arteries. PMID:26599698

  15. TRPM8 Channel Activation Induced by Monoterpenoid Rotundifolone Underlies Mesenteric Artery Relaxation.

    PubMed

    Silva, Darizy Flavia; de Almeida, Monica Moura; Chaves, Cinthia Guedes; Braz, Ana Letícia; Gomes, Maria Aparecida; Pinho-da-Silva, Leidiane; Pesquero, Jorge Luiz; Andrade, Viviane Aguiar; Leite, Maria de Fátima; de Albuquerque, José George Ferreira; Araujo, Islania Giselia Albuquerque; Nunes, Xirley Pereira; Barbosa-Filho, José Maria; Cruz, Jader dos Santos; Correia, Nadja de Azevedo; de Medeiros, Isac Almeida

    2015-01-01

    In this study, our aims were to investigate transient receptor potential melastatin-8 channels (TRPM8) involvement in rotundifolone induced relaxation in the mesenteric artery and to increase the understanding of the role of these thermosensitive TRP channels in vascular tissue. Thus, message and protein levels of TRPM8 were measured by semi-quantitative PCR and western blotting in superior mesenteric arteries from 12 week-old Spague-Dawley (SD) rats. Isometric tension recordings evaluated the relaxant response in mesenteric rings were also performed. Additionally, the intracellular Ca2+ changes in mesenteric artery myocytes were measured using confocal microscopy. Using PCR and western blotting, both TRPM8 channel mRNA and protein expression was measured in SD rat mesenteric artery. Rotundifolone and menthol induced relaxation in the isolated superior mesenteric artery from SD rats and improved the relaxant response induced by cool temperatures. Also, this monoterpene induced an increase in transient intracellular Ca2+. These responses were significantly attenuated by pretreatment with capsazepine or BCTC, both TRPM8 channels blockers. The response induced by rotundifolone was not significantly attenuated by ruthenium red, a non-selective TRP channels blocker, or following capsaicin-mediated desensitization of TRPV1. Our findings suggest that rotundifolone induces relaxation by activating TRPM8 channels in rat superior mesenteric artery, more selectively than menthol, the classic TRPM8 agonist, and TRPM8 channels participates in vasodilatory pathways in isolated rat mesenteric arteries.

  16. Inhibition of nitric oxide synthase expression in activated microglia and peroxynitrite scavenging activity by Opuntia ficus indica var. saboten.

    PubMed

    Lee, Ming Hong; Kim, Jae Yeon; Yoon, Jeong Hoon; Lim, Hyo Jin; Kim, Tae Hee; Jin, Changbae; Kwak, Wie-Jong; Han, Chang-Kyun; Ryu, Jae-Ha

    2006-09-01

    Activated microglia by neuronal injury or inflammatory stimulation overproduce nitric oxide (NO) by inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) such as superoxide anion, resulting in neurodegenerative diseases. The toxic peroxynitrite (ONOO-), the reaction product of NO and superoxide anion further contributes to oxidative neurotoxicity. A butanol fraction obtained from 50% ethanol extracts of Opuntia ficus indica var. saboten (Cactaceae) stem (SK OFB901) and its hydrolysis product (SK OFB901H) inhibited the production of NO in LPS-activated microglia in a dose dependent manner (IC50 15.9, 4.2 microg/mL, respectively). They also suppressed the expression of protein and mRNA of iNOS in LPS-activated microglial cells at higher than 30 microg/mL as observed by western blot analysis and RT-PCR experiment. They also inhibited the degradation of I-kappaB-alpha in activated microglia. Moreover, they showed strong activity of peroxynitrite scavenging in a cell free bioassay system. These results imply that Opuntia ficus indica may have neuroprotective activity through the inhibition of NO production by activated microglial cells and peroxynitrite scavenging activity. Copyright (c) 2006 John Wiley & Sons, Ltd.

  17. Chemotactic Activity of Cyclophilin A in the Skin Mucus of Yellow Catfish (Pelteobagrus fulvidraco) and Its Active Site for Chemotaxis

    PubMed Central

    Dawar, Farman Ullah; Tu, Jiagang; Xiong, Yang; Lan, Jiangfeng; Dong, Xing Xing; Liu, Xiaoling; Khattak, Muhammad Nasir Khan; Mei, Jie; Lin, Li

    2016-01-01

    Fish skin mucus is a dynamic barrier for invading pathogens with a variety of anti-microbial enzymes, including cyclophilin A (CypA), a multi-functional protein with peptidyl-prolyl cis/trans isomerase (PPIase) activity. Beside various other immunological functions, CypA induces leucocytes migration in vitro in teleost. In the current study, we have discovered several novel immune-relevant proteins in yellow catfish skin mucus by mass spectrometry (MS). The CypA present among them was further detected by Western blot. Moreover, the CypA present in the skin mucus displayed strong chemotactic activity for yellow catfish leucocytes. Interestingly, asparagine (like arginine in mammals) at position 69 was the critical site in yellow catfish CypA involved in leucocyte attraction. These novel efforts do not only highlight the enzymatic texture of skin mucus, but signify CypA to be targeted for anti-inflammatory therapeutics. PMID:27589721

  18. RTG resource book for western states and provinces: Final proceedings

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    NONE

    The Western Interstate Energy Board held a workshop and liaison activities among western states, provinces, and utilities on the formation of Regional Transmission Groups (RTGs). Purpose of the activities was to examine the policy implications for western states and provinces in the formation of RTGs in the West, the implications for western ratepayers and utilities of the RTG formation and potential impacts of RTGs on the western electricity system. The workshop contributed to fulfilling the transmission access and competition objectives of Title VII of the Energy Policy Act of 1992.

  19. INTERPLAY OF SORBITOL PATHWAY OF GLUCOSE METABOLISM, 12/15-LIPOXYGENASE, AND MITOGEN-ACTIVATED PROTEIN KINASES IN THE PATHOGENESIS OF DIABETIC PERIPHERAL NEUROPATHY

    PubMed Central

    Stavniichuk, Roman; Shevalye, Hanna; Hirooka, Hiroko; Nadler, Jerry L.; Obrosova, Irina G.

    2012-01-01

    The interactions among multiple pathogenetic mechanisms of diabetic peripheral neuropathy largely remain unexplored. Increased activity of aldose reductase, the first enzyme of the sorbitol pathway, leads to accumulation of cytosolic Ca++, essentially required for 12/15-lipoxygenase activation. The latter, in turn, causes oxidative-nitrosative stress, an important trigger of MAPK phosphorylation. This study therefore evaluated the interplay of aldose reductase, 12/15-lipoxygenase, and MAPKs in diabetic peripheral neuropathy. In experiment 1, male control and streptozotocin-diabetic mice were maintained with or without the aldose reductase inhibitor fidarestat, 16 mg kg−1 d−1, for 12 weeks. In experiment 2, male control and streptozotocin-diabetic wild-type (C57Bl6/J) and 12/15-lipoxygenase-deficient mice were used. Fidarestat treatment did not affect diabetes-induced increase in glucose concentrations, but normalized sorbitol and fructose concentrations (enzymatic spectrofluorometric assays) as well as 12(S) hydroxyeicosatetraenoic concentration (ELISA), a measure of 12/15-lipoxygenase activity, in the sciatic nerve and spinal cord. 12/15-lipoxygenase expression in these two tissues (Western blot analysis) as well as dorsal root ganglia (immunohistochemistry) was similarly elevated in untreated and fidarestat-treated diabetic mice. 12/15-lipoxygenase gene deficiency prevented diabetesassociated p38 MAPK and ERK, but not SAPK/JNK, activation in the sciatic nerve (Western blot analysis) and all three MAPK activation in the dorsal root ganglia (immunohistochemistry). In contrast, spinal cord p38 MAPK, ERK, and SAPK/JNK were similarly activated in diabetic wild-type and 12/15-lipoxygenase−/− mice. These findings identify the nature and tissue specificity of interactions among three major mechanisms of diabetic peripheral neuropathy, and suggest that combination treatments, rather than monotherapies, can sometimes be an optimal choice for its management. PMID

  20. Curcumin suppresses colon cancer cell invasion via AMPK-induced inhibition of NF-κB, uPA activator and MMP9.

    PubMed

    Tong, Weihua; Wang, Quan; Sun, Donghui; Suo, Jian

    2016-11-01

    Curcumin, an active nontoxic ingredient of turmeric, possesses potent anti-inflammatory, antioxidant and anti-cancer properties; however, the molecular mechanisms of curcumin are not fully understood. The transcription factor nuclear factor-κB (NF-κB) is key in cellular processes, and the expression/activation of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9) are crucial for cell invasion. The present study investigated the hypothesis that curcumin inhibits colon cancer cell invasion by modulating NF-κB-mediated expression and activation of uPA and MMP9. Human colon cancer SW480 and LoVo cells were treated with various concentrations of curcumin. Curcumin was demonstrated to dose-dependently inhibit the adhesion and proliferation ability of LoVo and SW480 cells using Transwell and MTT assays, respectively. In addition, curcumin activated 5' AMP-activated protein kinase (AMPK) and suppressed p65 NF-κB phosphorylation, as shown by western blot analysis. Compound C, a potent AMPK inhibitor, abolished curcumin-induced inhibition of NF-κB, uPA and MMP9, suggesting that AMPK activation is responsible for curcumin-mediated NF-κB, uPA and MMP9 inhibition. The binding activity of NF-κB to DNA was examined and western blotting and quantitative polymerase reaction was performed to detect the effect of curcumin on the expression of uPA and MMP9. The present results revealed that curcumin significantly decreased the expression of uPA and MMP9 and NF-κB DNA binding activity. Furthermore, curcumin decreased the level of the p65 subunit of NF-κB binding to the promoter of the gene encoding uPA and MMP9, which suppressed transcriptional activation of uPA and MMP9. Overall, the present data suggest that curcumin inhibits colon cancer cell invasion via AMPK activation and subsequent inhibition of p65 NF-κB, uPA and MMP9. The therapeutic potential of curcumin for colon cancer metastasis required additional study.

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

    PubMed

    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.

  2. Western Samoa.

    PubMed

    1985-12-01

    This discussion of Western Samoa, which lies 2575 km northeast of Auckland, New Zealand, focuses on the following: geography; the people; history; government; political conditions; the economy; foreign relations; and relations the US. The population of Western Samoa, as of 1985, totals 163,000 with an annual growth rate of 0.9%. The infant mortality rate is 13/1000; life expectancy is 65 years. The main islands are formed ranges of extinct volcanoes. Volcanic activity last occurred in 1911. More than 2000 years age, waves of Polynesians migrated from Southeast Asia to the Samoan Islands. Samoans are the 2nd largest Polynesian group, after the Maoris of New Zealand, and speak a Polynesian dialect. Samoans have tended to retain their traditional ways despite exposure to European influence for more than 150 years. Most Samoans live within the traditional social system based on an extended family group, headed by a chief. Western Samoans are Christian. Education is free but not compulsory. In 1967, 95% of the children of primary school age attended school. From 1947 to 1961, a series of constitutional advances, assisted by visits from UN missions, brought Western Samoa from dependent status to self-government and finally to independence. The 1960 constitution is based on the British pattern of parliamentary democracy, modified to take Samoan customs into account. The present head of state holds his position for life. Future heads of state will be elected by the Legislative Assembly for 5-year terms. The Parliament consists of the Legislative Assembly and the head of state. The Supreme Court is the superior court of record and has full jurisdiction in civil, criminal, and constitutional matters. The "matai" of chief system still dominates the politics of Western Samoa, although several political parties have been formed and seem to be taking root. The "matai" system is a predominantly conservative force but does provide for change. Western Samoa is predominantly

  3. Igneous activity and related ore deposits in the western and southern Tushar Mountains, Marysvale volcanic field, west-central Utah

    USGS Publications Warehouse

    Steven, Thomas A.

    1984-01-01

    PART A: Igneous activity in the Marysvale volcanic field of western Utah can be separated into many episodes of extrusion, intrusion, and hydrothermal activity. The rocks of the western Tushar Mountains, near the western part of the volcanic field, include intermediate-composition, calc-alkalic volcanic rocks erupted from scattered volcanoes in Oligocene through earliest Miocene time and related monzonitic intrusions emplaced 24-23 m.y. ago. Beginning 22-21 m.y. ago and extending through much of the later Cenozoic, a bimodal basalt-rhyolite assemblage was erupted widely throughout the volcanic field. Only volcanic and intrusive rocks belonging to the rhyolitic end member of this bimodal assemblage are present in the western Tushar Mountains; most of these rocks either fill the Mount Belknap caldera (19 m.y. old) or are part of the rhyolite of Gillies Hill (9---8 m.y. old). Episodic hydrothermal activity altered and mineralized rocks at many places in the western Tushar Mountains during Miocene time. The earliest activity took place in and adjacent to monzonitic calcalkalic intrusions emplaced in the vicinity of Indian Creek and Cork Ridge. These rocks were widely propylitized, and gold-bearing quartz-pyrite-carbonate veins formed in local fractures. Hydrothermal activity associated with the Mount Belknap caldera mobilized and redeposited uranium contained in the caldera-fill rocks and formed primary concentrations of lithophile elements (including molybdenum and uranium) in the vicinity of intrusive bodies. Hydrothermal activity associated with the rhyolite of Gillies Hill altered and mineralized rocks at several places along the fault zone that marks the western margin of the Tushar Mountains; the zoned alunite and gold deposits at Sheep Rock, the gold deposit at the Sunday Mine, and an alunite deposit near Indian Creek were thus produced. Resetting of isotopic ages suggests that another center of hydrothermally altered rocks associated with a buried pluton about

  4. Activation of Müller cells occurs during retinal degeneration in RCS rats.

    PubMed

    Zhao, Tong Tao; Tian, Chun Yu; Yin, Zheng Qin

    2010-01-01

    Müller cells can be activated and included in different functions under many kinds of pathological conditions, however, the status of Müller cells in retinitis pigmentosa are still unknown. Using immunohistochemisty, Western blots and co-culture, we found that Müller cells RCS rats, a classic model of RP, could be activated during the progression of retinal degeneration. After being activated at early stage, Müller cells began to proliferate and hypertrophy, while at later stages, they formed a local 'glial seal' in the subretinal space. As markers of Müller cells activation, the expression of GFAP and ERK increased significantly with progression of retinal degeneration. Co-cultures of normal rat Müller cells and mixed RCS rat retinal cells show that Müller cells significantly increase GFAP and ERK in response to diffusable factors from the degenerting retina, which implies that Müller cells activation is a secondary response to retinal degeneration.

  5. Sun Grant - Western Regional Center | | Oregon State University

    Science.gov Websites

    Services Make a Gift Search Field Search Sun Grant - Western Regional Center Home About Us Mission Western States News Sun Grant Initiative Contacts The Five Regional Centers Center Activities Competitive Grant Territories Sun Grant Initiative Contacts The Five Regional Centers Center Activities Competitive Grant

  6. Identification and characterization of plasmids from the western aster yellows mycoplasmalike organism.

    PubMed Central

    Kuske, C R; Kirkpatrick, B C

    1990-01-01

    Supercoiled double-stranded DNA molecules (plasmids) were isolated from plants infected with three laboratory strains of western aster yellows mycoplasma-like organism (AY-MLO) by using cesium chloride-ethidium bromide density gradients. Southern blot analysis, using plasmids from the severe strain of AY-MLO (SAY-MLO) as the probe, identified at least four plasmids in celery, aster, and periwinkle plants and in Macrosteles severini leafhopper vectors infected with either the dwarf AY-MLO, Tulelake AY-MLO, or SAY-MLO strain. Plasmids were also detected in two California field isolates of AY-MLO but not in plants infected with the beet leafhopper-transmitted virescence agent, western X, or elm yellows MLOs. SAY-MLO plasmids were 5.2, 4.9, 3.4, and 1.7 kilobase pairs in size. Plasmids isolated from dwarf AY- and Tulelake AY-MLOs were 7.4, 5.1, 3.5, and 1.7 kilobase pairs in size. No evidence was obtained for integration of SAY-MLO plasmids into the MLO chromosome. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 PMID:2307660

  7. Chemical Characterization and Antitumor Activities of Polysaccharide Extracted from Ganoderma lucidum

    PubMed Central

    Liang, Zengenni; Yi, Youjin; Guo, Yutong; Wang, Rencai; Hu, Qiulong; Xiong, Xingyao

    2014-01-01

    Ganoderma lucidum polysaccharide (GLP) is a biologically active substance reported to possess anti-tumor ability. Nonetheless, the mechanisms of GLP-stimulated apoptosis are still unclear. This study aims to determine the inhibitory and apoptosis-inducing effects of GLP on HCT-116 cells. We found that GLP reduced cell viability on HCT-116 cells in a time- and dose-dependent manner, which in turn, induced cell apoptosis. The observed apoptosis was characterized by morphological changes, DNA fragmentation, mitochondrial membrane potential decrease, S phase population increase, and caspase-3 and -9 activation. Furthermore, inhibition of c-Jun N-terminal kinase (JNK) by SP600125 led to a dramatic decrease of the GLP-induced apoptosis. Western blot analysis unveiled that GLP up-regulated the expression of Bax/Bcl-2, caspase-3 and poly (ADP-ribose) polymerase (PARP). These results demonstrate that apoptosis stimulated by GLP in human colorectal cancer cells is associated with activation of mitochondrial and mitogen-activated protein kinase (MAPK) pathways. PMID:24857920

  8. Eicosapentaenoic Acid (EPA) Induced Macrophages Activation through GPR120-Mediated Raf-ERK1/2-IKKβ-NF-κB p65 Signaling Pathways

    PubMed Central

    Han, Lirong; Song, Shumin; Niu, Yabing; Meng, Meng; Wang, Chunling

    2017-01-01

    Objectives: To investigate the immunomodulatory effect and molecular mechanisms of Eicosapentaenoic acid (EPA, a typical kind of n-3PUFAs) on RAW264.7 cells. Methods: A variety of research methods, including the RAW264.7 cells culture, cell proliferation assays, morphologic observations, measurements of NO production, cytokine assays, nuclear protein extractions, western blot analyses and NF-κB p65 immunofluorescence assays were used in this study. Results: The results showed that EPA could increase the proliferation index and enhance the release of nitric oxide (NO) and cytokines in RAW264.7 cells. Western blotting results revealed that the protein level of GPR120 increased significantly in RAW264.7 cells after EPA treatment. Meanwhile, EPA elevated the phosphorylation status of Raf, which may act as an upstream regulator of EPA-induced phosphorylated ERK1/2. In addition, the phosphorylated ERK1/2 may then promote IKKβ in endochylema and translocate the NF-κB p65 subunit into the nucleus, thus regulating the production of inducible nitric oxide synthase (iNOS) and cytokines. Conclusions: EPA (0.6–3.0 μmol) activates RAW264.7 cells through GPR120-mediated Raf-ERK1/2-IKKβ-NF-κB p65 signaling pathways. PMID:28841192

  9. Eicosapentaenoic Acid (EPA) Induced Macrophages Activation through GPR120-Mediated Raf-ERK1/2-IKKβ-NF-κB p65 Signaling Pathways.

    PubMed

    Han, Lirong; Song, Shumin; Niu, Yabing; Meng, Meng; Wang, Chunling

    2017-08-25

    Objectives: To investigate the immunomodulatory effect and molecular mechanisms of Eicosapentaenoic acid (EPA, a typical kind of n-3PUFAs) on RAW264.7 cells. Methods: A variety of research methods, including the RAW264.7 cells culture, cell proliferation assays, morphologic observations, measurements of NO production, cytokine assays, nuclear protein extractions, western blot analyses and NF-κB p65 immunofluorescence assays were used in this study. Results: The results showed that EPA could increase the proliferation index and enhance the release of nitric oxide (NO) and cytokines in RAW264.7 cells. Western blotting results revealed that the protein level of GPR120 increased significantly in RAW264.7 cells after EPA treatment. Meanwhile, EPA elevated the phosphorylation status of Raf, which may act as an upstream regulator of EPA-induced phosphorylated ERK1/2. In addition, the phosphorylated ERK1/2 may then promote IKKβ in endochylema and translocate the NF-κB p65 subunit into the nucleus, thus regulating the production of inducible nitric oxide synthase (iNOS) and cytokines. Conclusions: EPA (0.6-3.0 μmol) activates RAW264.7 cells through GPR120-mediated Raf-ERK1/2-IKKβ-NF-κB p65 signaling pathways.

  10. Effect of the Gc-derived macrophage-activating factor precursor (preGcMAF) on phagocytic activation of mouse peritoneal macrophages.

    PubMed

    Uto, Yoshihiro; Yamamoto, Syota; Takeuchi, Ryota; Nakagawa, Yoshinori; Hirota, Keiji; Terada, Hiroshi; Onizuka, Shinya; Nakata, Eiji; Hori, Hitoshi

    2011-07-01

    The 1f1f subtype of the Gc protein (Gc(1f1f) protein) was converted into Gc-derived macrophage-activating factor (GcMAF) by enzymatic processing in the presence of β-galactosidase of an activated B-cell and sialidase of a T-cell. We hypothesized that preGc(1f1f)MAF, the only Gc(1f1f) protein lacking galactose, can be converted to GcMAF in vivo because sialic acid is cleaved by residual sialidase. Hence, we investigated the effect of preGc(1f1f)MAF on the phagocytic activation of mouse peritoneal macrophages. We examined the sugar moiety of preGc(1f1f)MAF with a Western blot using peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA) lectin. We also found that preGc(1f1f)MAF significantly enhanced phagocytic activity in mouse peritoneal macrophages but only in the presence of the mouse peritoneal fluid; the level of phagocytic activity was the same as that observed for GcMAF. PreGc(1f1f)MAF can be used as an effective macrophage activator in vivo.

  11. 78 FR 6120 - Government-Owned Inventions; Availability for Licensing

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-01-29

    ..., including immunoprecipitation, western blot analysis, immunohistochemistry, ELISA, etc. Competitive... immunoprecipitation, western blot analysis, immunohistochemistry, ELISA, etc. Competitive Advantages: Binding of a new...

  12. Anti-proliferative and pro-apoptotic activities of Alpinia oxyphylla on HepG2 cells through ROS-mediated signaling pathway.

    PubMed

    Zhang, Qiao; Cui, Can; Chen, Cong-Qin; Hu, Xiao-Long; Liu, Ya-Hui; Fan, Yan-Hua; Meng, Wei-Hong; Zhao, Qing-Chun

    2015-07-01

    Fructus Alpiniae oxyphyllae (A. oxyphylla) is a traditional herb which is widely used in East Asian for the treatment of dyspepsia, diarrhea, abdominal pain, poor memory, inflammatory conditions and cancer. The cytotoxic activities of ethanol extract (EE) and five extract layers including petroleum ether (PE), dichloromethane (DCLM), acetoacetate (EtOAc), n-Butanol (n-Bu) and water fractions (WF) of A. oxyphylla were tested on HepG2, SW480, MCF-7, K562 and HUVEC cell lines using MTT assay and LDH release assay. The component analysis was performed on HPLC with gradient elution. Hoechst 33342 staining, DCFH-DA fluorescence microscopy, flow cytometry analysis, western blot and migration assays were carried out to determine the anti-cancer mechanisms of PE. MTT analysis showed that EE, PE and DCLM could inhibit cell proliferation on HepG2, SW480, MCF-7, K562 and HUVEC cell lines, especially PE fraction. HPLC analysis pointed out five main components which may contribute to the anti-proliferative activity of PE. Further study showed that PE increased LDH release, induced apoptosis, disrupted mitochondrial membrane potential and elevated intracellular reactive oxygen species (ROS) in HepG2 cells, whereas the antioxidant N-acetyl-l-cysteine (NAC) prevented PE-induced ROS generation. The results of western blot revealed that PE induced apoptosis in HepG2 cells by enhancing Bax/Bcl-2 ratio, increasing cytochrome c in cytosol and activating caspase-3/9. Meanwhile, high levels of ROS could induce DNA damage-mediated protein expression, AKT, ERK inactivation and SAPKs activation. Furthermore, PE conspicuously blocked the migration of HUVEC cells. The present results demonstrated that PE induced apoptosis in HepG2 cells may be via a ROS-mediated signaling pathway. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  13. Dual effects of ouabain on the regulation of proliferation and apoptosis in human umbilical vein endothelial cells: involvement of Na(+)-K(+)-ATPase α-subunits and NF-κB.

    PubMed

    Ren, Yan-Ping; Zhang, Ming-Juan; Zhang, Ting; Huang, Ruo-Wen

    2014-01-01

    To elucidate the effect of ouabain on the regulation of proliferation and apoptosis of HUVECs and involvement of different Na(+)-K(+)-ATPase α-subunits and NF-κB. HUVECs were isolated by collagenase perfusion, and MTT assays and cell cycle analysis were performed to study proliferation. NF-κB expression and function were examined by immunohistochemical staining and western blotting. Na(+)-K(+)-ATPase activity was determined by measuring released ouabain inhibitable inorganic phosphate (Pi). The expression of different α-subunits was investigated by real RT-PCR, western blotting and cell immunofluorescence. 0.3 nM ouabain treatment for 0.5 h triggered the proliferation of HUVECs, peaking at 1-2 h. At 1.8 nM for 0.5 h, ouabain induced an increase of cell proliferation for a short time, and then triggered a decrease after 1 h. Cell cycle analysis show that 37% of HUVECs were in G2/M phase of the cell cycle following incubation with 1.8 nM ouabain, compared with 18% with 0.3 nM ouabain. NF-κB activity was assessed by western blot analysis of IκB expression, which was significantly reduced with 0.3 nM ouabain treatment; there was no different between 1.8 nM ouabain treatment and untreated cells. Na(+)-K(+)-ATPase activity in HUVECs was markedly reduced after treatment with 0.3 nM and 1.8 nM ouabain. Real RT-PCR and western blotting indicated that Na(+)-K(+)-ATPase α1-subunit mRNA expression levels increased after 0.3 nM ouabain treatment and decreased after 1.8 nM ouabain treatment. However, α2- and α3-subunit mRNA decreased after 0.3 nM ouabain treatment and increased after 1.8 nM ouabain treatment. Ouabain at different concentrations caused dual effects on proliferation and apoptosis in HUVECs.

  14. Albumin Overload and PINK1/Parkin Signaling-Related Mitophagy in Renal Tubular Epithelial Cells.

    PubMed

    Tan, Jin; Xie, Qi; Song, Shuling; Miao, Yuyang; Zhang, Qiang

    2018-03-01

    BACKGROUND Albumin, as a major urinary protein component, is a risk factor for chronic kidney disease progression. Mitochondrial dysfunction is one of the main causes of albumin-induced proximal tubule cells injury. Mitophagy is considered as a pivotal protective mechanism for the elimination of dysfunctional mitochondria. The objective of this research was to determine whether albumin overload-induced mitochondrial dysfunction can activate PINK1/Parkin-mediated mitophagy in renal tubular epithelial cells (TECs). MATERIAL AND METHODS Immunofluorescence assay and Western blot assay were used to detect the effects of albumin overload on autophagy marker protein LC3. Transmission electron microscopy and Western blot assay were used to investigate the role of albumin in mitochondrial injury. Western blot assay and co-localization of acidic lysosomes and mitochondria assay were employed to detect the activation of mitophagy induced by albumin. Finally, we explored the role of PINK1/Parkin signaling in albumin-induced mitophagy by inhibiting mitophagy by knockdown of PARK2 (Parkin) level. RESULTS Immunofluorescence and Western blot results showed that the expression level of LC3-II increased, and the maximum increase point was observed after 8 h of albumin treatment. Transmission electron microscopy results demonstrated that albumin overload-induced mitochondrial injury and quantity of autophagosomes increased. Additionally, expression of PINK1 and cytosolic cytochrome C increased and mitochondria cytochrome C decreased in the albumin group. The co-localization of acidic lysosomes and mitochondria demonstrated that the number of albumin overload-induced mitophagy-positive dots increased. The transient transfection of PARK2 siRNA result showed knockdown of the expression level of PARK2 can inhibit mitophagy induced by albumin. CONCLUSIONS In conclusion, our study suggests that mitochondrial dysfunction activates the PINK1/Parkin signaling and mitophagy in renal tubular

  15. Betaine supplement enhances skeletal muscle differentiation in murine myoblasts via IGF-1 signaling activation.

    PubMed

    Senesi, Pamela; Luzi, Livio; Montesano, Anna; Mazzocchi, Nausicaa; Terruzzi, Ileana

    2013-07-19

    Betaine (BET) is a component of many foods, including spinach and wheat. It is an essential osmolyte and a source of methyl groups. Recent studies have hypothesized that BET might play a role in athletic performance. However, BET effects on skeletal muscle differentiation and hypertrophy are still poorly understood. We examined BET action on neo myotubes maturation and on differentiation process, using C2C12 murine myoblastic cells. We used RT2-PCR array, Western blot and immunofluorescence analysis to study the BET effects on morphological features of C2C12 and on signaling pathways involved in muscle differentiation and hypertrophy. We performed a dose-response study, establishing that 10 mM BET was the dose able to stimulate morphological changes and hypertrophic process in neo myotubes. RT2-PCR array methodology was used to identify the expression profile of genes encoding proteins involved in IGF-1 pathway. A dose of 10 mM BET was found to promote IGF-1 receptor (IGF-1 R) expression. Western blot and immunofluorescence analysis, performed in neo myotubes, pointed out that 10 mM BET improved IGF-1 signaling, synthesis of Myosin Heavy Chain (MyHC) and neo myotubes length. Our findings provide the first evidence that BET could promote muscle fibers differentiation and increase myotubes size by IGF-1 pathway activation, suggesting that BET might represent a possible new drug/integrator strategy, not only in sport performance but also in clinical conditions characterized by muscle function impairment.

  16. IL-9 exhibits elevated expression in osteonecrosis of femoral head patients and promotes cartilage degradation through activation of JAK-STAT signaling in vitro.

    PubMed

    Geng, Wei; Zhang, Wen; Ma, Jinzhu

    2018-05-15

    Osteonecrosis of the femoral head (ONFH) often causes severe symptoms in young people and limits the mobility of the hip joint. Interleukin-9 (IL-9) is a multi-functional inflammatory factor that participates in lumbar disk herniation and arthritis and has been reported in many studies. However, the correlation between IL-9 and ONFH is unclear. The present study aimed to determine the role of IL-9 in the pathogenetic mechanism of osteonecrosis. To assess IL-9 expression in ONFH and femoral neck fracture patients, cartilage tissue was examined through western blot analysis and immunohistochemistry. Human primary chondrocytes were stimulated with IL-9, and inflammation-related cytokines and cartilage matrix-degrading enzymes were assessed via real-time PCR. After being treated with IL-9, Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling were tested through western blot analysis. Our results showed a significant increase in the expression of IL-9 in ONFH patients. IL-9 raised the level of inflammation-related cytokines and cartilage matrix-degrading enzymes and enhanced the activation of JAK-STAT signaling. Furthermore, blocking the JAK-STAT signaling pathway reduced the secretion of inflammation-related cytokines and cartilage matrix-degrading enzymes and markedly alleviated the degradation of the cartilage matrix. These findings provide new insights into the role that IL-9 plays in the pathogenetic mechanism of osteonecrosis and also provide a potential treatment for ONFH. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. Attenuation of ischemia-reperfusion injury by sevoflurane postconditioning involves protein kinase B and glycogen synthase kinase 3 beta activation in isolated rat hearts.

    PubMed

    Fang, Neng-Xin; Yao, Yun-Tai; Shi, Chun-Xia; Li, Li-Huan

    2010-12-01

    Volatile anesthetic ischemic postconditioning reduces infarct size following ischemia/reperfusion. Whether phosphorylation of protein kinase B (PKB/Akt) and glycogen synthase kinase 3 beta (GSK3β) is causal for cardioprotection by postconditioning is controversial. We therefore investigated the impact of PKB/Akt and GSK3β in isolated perfused rat hearts subjected to 40 min of ischemia followed by 1 h of reperfusion. 2.0% sevoflurane (1.0 minimum alveolar concentration) was administered at the onset of reperfusion in 15 min as postconditioning. Western blot analysis was used to determine phosphorylation of PKB/Akt and its downstream target GSK3β after 1 h of reperfusion. Mitochondrial and cytosolic content of cytochrome C checked by western blot served as a marker for mitochondrial permeability transition pore opening. Sevoflurane postconditioning significantly improved functional cardiac recovery and decreased infarct size in isolated rat hearts. Compared with unprotected hearts, sevoflurane postconditioning-induced phosphorylation of PKB/Akt and GSK3β were significantly increased. Increase of cytochrome C in mitochondria and decrease of it in cytosol is significant when compared with unprotected ones which have reversal effects on cytochrome C. The current study presents evidence that sevoflurane-induced cardioprotection at the onset of reperfusion are partly through activation of PKB/Akt and GSK3β.

  18. Chitinolytic activities in Bacillus thuringiensis and their synergistic effects on larvicidal activity.

    PubMed

    Liu, M; Cai, Q X; Liu, H Z; Zhang, B H; Yan, J P; Yuan, Z M

    2002-01-01

    To investigate the distribution of chitinase in Bacillus thuringiensis strains, and the enhancing effects of the chitinase-producing B. thuringiensis strains on insecticidal toxicity of active B. thuringiensis strain against Spodoptera exigua larvae. The chitinolytic activities of B.thuringiensis strains representing the 70 serotypes were investigated by the whitish opaque halo and the colorimetric method. Thirty-eight strains produced different levels of chitinase at pH 7.0, and so did 17 strains at pH 10.0. The strain T04A001 exhibited the highest production, reaching a specific activity of 355 U ml(-1) in liquid medium. SDS-PAGE and Western blotting showed that the chitinase produced by some B. thuringiensis strains had a molecular weight of about 61 kDa. The bioassay results indicated that the chitinase-producing B. thuringiensis strains could enhance the insecticidal activity of B. thuringiensis strain DL5789 against S. exigua larvae, with an enhancing ratio of 2.35-fold. This study demonstrated that chitinase was widely produced in B. thuringiensis strains and some of the strains could enhance the toxicity of active B. thuringiensis strain. This is the first investigation devoted exclusively to analyse the distribution of chitinase in B. thuringiensis. It infers that the chitinase produced by B. thuringiensis might play a role in the activity of the biopesticide.

  19. Pathogenic activity of circulating anti-desmoglein-3 autoantibodies isolated from pemphigus vulgaris patients

    PubMed Central

    Boncela, Joanna; Smolarczyk, Katarzyna; Kowalewski, Cezary; Wozniak, Katarzyna; Torzecka, Jolanta Dorota; Sysa-Jedrzejowska, Anna; Cierniewski, Czesław S.; Lesiak, Aleksandra

    2012-01-01

    Introduction There are scarce data on immunochemical properties of pemphigus antibodies detected in clinical remission in pemphigus vulgaris (PV) patients. The aim of the study was to compare biological activity of anti-Dsg3 autoantibodies purified from the sera of PV patients in active stage and in clinical remission. Material and methods The effect of purified antibodies on expression of procaspase-3, Bax, Bcl-2, uPAR, IL-1β, IL-6, and TNF-α mRNAs in the HaCaT keratinocytes was evaluated by Western blot and RT-PCR method. Results Incubation of HaCaT cells with anti-Dsg-3 autoantibodies caused their binding to cell membranes surfaces. Anti-Dsg3 autoantibodies isolated from the patients in active stage and clinical remission showed proapoptotic effect, caused enhanced expression of analyzed proinflammatory cytokines’ mRNAs and uPAR mRNA. Conclusions Our data revealed similar pathogenic activity of anti Dsg-3 autoantibodies isolated from active and clinical remission PV patients. PMID:22662010

  20. [Significant increase of glucose transport activity in breast cancer].

    PubMed

    Li, Juan; Yang, Shou-jing; Zhao, Xi-long; Zhang, Ya-qing; Li, Kai-nan; Cui, Ji-hong; Li, Jing

    2008-02-01

    To study the expression level and significance of glucose transporter 1 (Glut-1) in normal breast tissue, adenosis, adenoma and breast carcinoma. A total of 147 cases of female breast tissue samples, including 92 cases of invasive ductal carcinoma, 26 cases of breast fibroadenoma, 24 cases of breast adenosis and 5 cases of normal breast tissues, were collected for quantitative detection of the expression of Glut-1 protein by immunohistochemistry (EnVision method) and Western blot, and its mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR). In normal breast tissue and benign lesions of the breast, Glut-1 was undetectable or only weakly detectable in cytoplasm of ductal and acinar epithelia. In contrast, the intensity of Glut-1 staining was significantly higher in invasive ductal carcinomas (P = 0.0002) with protein expression predominantly in cellular membrane and lesser in cytoplasm. Western blot and RT-PCR analyses showed that the expression of Glut-1 protein and mRNA were significantly increased in invasive ductal carcinoma than fibroadenoma (P =0.001 for protein; P <0.05 for mRNA) and adenosis (P =0.001 for protein; P < 0.05 for mRNA). There was a significant difference among groups (P = 0.0002 for protein; P = 0.0001 for mRNA). Glucose transport activity, as indicated by Glut-1 protein and its mRNA expression, significantly increases in breast carcinoma than non-cancerous lesions. The over-expression of Glut-1 in breast carcinoma is tightly coupled with tumor cell proliferation, invasion and metastasis, implying that Glut-1 may serve as a new marker in the early diagnosis and prognostication of breast malignancy as well as a new therapeutic target.

  1. The use of circulation weather types to predict upwelling activity along the Western Iberian Peninsula coast

    NASA Astrophysics Data System (ADS)

    Ramos, Alexandre M.; Cordeiro Pires, Ana; Sousa, Pedro M.; Trigo, Ricardo M.

    2013-04-01

    Coastal upwelling is a phenomenon that occurs in most western oceanic coasts due to the presence of mid-latitude high-pressure systems that generate equatorward winds along the coast and consequent offshore displacement of surface waters that in turn cause deeper, colder, nutrient-rich waters to arise. In western Iberian Peninsula (IP) the high-pressure system associated to northerly winds occurs mainly during spring and summer. Upwelling systems are economically relevant, being the most productive regions of the world ocean and crucial for fisheries. In this work, we evaluate the intra- and inter-annual variability of the Upwelling Index (UI) off the western coast of the IP considering four locations at various latitudes: Rias Baixas, Aveiro, Figueira da Foz and Cabo da Roca. In addition, the relationship between the variability of the occurrence of several circulation weather types (Ramos et al., 2011) and the UI variability along this coast was assessed in detail, allowing to discriminate which types are frequently associated with strong and weak upwelling activity. It is shown that upwelling activity is mostly driven by wind flow from the northern quadrant, for which the obtained correlation coefficients (for the N and NE types) are higher than 0.5 for the four considered test locations. Taking into account these significant relationships, we then developed statistical multi-linear regression models to hindcast upwelling series (April to September) at the four referred locations, using monthly frequencies of circulation weather types as predictors. Modelled monthly series reproduce quite accurately observational data, with correlation coefficients above 0.7 for all locations, and relatively small absolute errors. Ramos AM, Ramos R, Sousa P, Trigo RM, Janeira M, Prior V (2011) Cloud to ground lightning activity over Portugal and its association with Circulation Weather Types. Atmospheric Research 101:84-101. doi: 10.1016/j.atmosres.2011.01

  2. Geothermic analysis of high temperature hydrothermal activities area in Western plateau of Sichuan province, China

    NASA Astrophysics Data System (ADS)

    Zhang, J.

    2016-12-01

    There is a high temperature hydrothermal activity area in the western plateau of Sichuan. More than 200 hot springs points have been found in the region, including 11 hot spring water temperature above local boiling point. Most of these distribute along Jinshajjiang fracture, Dege-Xiangcheng fracture, Ganzi-Litang fracture as well as Xianshuihe fracture, and form three high-temperature hydrothermal activity strips in the NW-SE direction. Using gravity, magnetic, seismic and helium isotope data, this paper analyzed the crust-mantle heat flow structure, crustal heat source distribution and water heating system. The results show that the geothermal activity mainly controlled by the "hot" crust. The ratio of crustal heat flow and surface heat flow is higher than 60%. In the high temperature hydrothermal activities area, there is lower S wave velocity zone with Vs<3.2 km/s in 15 30 km depth in middle and lower crust. Basing on the S wave velocity inversion temperature of crust-mantle, it has been found that there is a high temperature layer with 850 1000 ° in 20 40 km depth. It is the main heat source of high temperature hydrothermal activity area of western Sichuan. Our argument is that atmospheric precipitation, surface water infiltrated along the fault fracture into the crustal deep, heating by crustal hot source, and circulation to surface become high temperature hot water. Geothermal water mainly reserve in the Triassic strata of the containing water good carbonate rocks, and in the intrusive granite which is along the fault zone. The thermal energy of Surface heat thermal activities mainly comes from the high-temperature hot source which is located in the middle and lower crust. Being in the deep crustal fracture, the groundwater infiltrated to the deep crust and absorbed heat, then, quickly got back to the surface and formed high hot springs.

  3. Detection of Dystrophin Dp71 in Human Skeletal Muscle Using an Automated Capillary Western Assay System.

    PubMed

    Kawaguchi, Tatsuya; Niba, Emma Tabe Eko; Rani, Abdul Qawee Mahyoob; Onishi, Yoshiyuki; Koizumi, Makoto; Awano, Hiroyuki; Matsumoto, Masaaki; Nagai, Masashi; Yoshida, Shinobu; Sakakibara, Sachiko; Maeda, Naoyuki; Sato, Osamu; Nishio, Hisahide; Matsuo, Masafumi

    2018-05-23

    Dystrophin Dp71 is one of the isoforms produced by the DMD gene which is mutated in patients with Duchenne muscular dystrophy (DMD). Although Dp71 is expressed ubiquitously, it has not been detected in normal skeletal muscle. This study was performed to assess the expression of Dp71 in human skeletal muscle. Human skeletal muscle RNA and tissues were obtained commercially. Mouse skeletal muscle was obtained from normal and DMD mdx mice. Dp71 mRNA and protein were determined by reverse-transcription PCR and an automated capillary Western assay system, the Simple Western, respectively. Dp71 was over-expressed or suppressed using a plasmid expressing Dp71 or antisense oligonucleotide, respectively. Full-length Dp71 cDNA was PCR amplified as a single product from human skeletal muscle RNA. A ca. 70 kDa protein peak detected by the Simple Western was determined as Dp71 by over-expressing Dp71 in HEK293 cells, or suppressing Dp71 expression with antisense oligonucleotide in rhabdomyosarcoma cells. The Simple Western assay detected Dp71 in the skeletal muscles of both normal and DMD mice. In human skeletal muscle, Dp71 was also detected. The ratio of Dp71 to vinculin of human skeletal muscle samples varied widely, indicating various levels of Dp71 expression. Dp71 protein was detected in human skeletal muscle using a highly sensitive capillary Western blotting system.

  4. Dot-Blot Immunoassay of Fasciola gigantica Infection using 27 kDa and Adult Worm Regurge Antigens in Egyptian Patients

    PubMed Central

    Kamel, Hanan H.; Saad, Ghada A.

    2013-01-01

    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. PMID:23710084

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

    PubMed

    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.

  6. MicroRNA-21 promotes bone mesenchymal stem cells migration in vitro by activating PI3K/Akt/MMPs pathway.

    PubMed

    Lv, Chen; Yang, Shengwu; Chen, Xin; Zhu, Xiongbai; Lin, Wenjun; Wang, Lu; Huang, Zhengxiang; Wang, Mingyue; Tu, Guanjun

    2017-12-01

    MicroRNA-21 (miR-21) contributes to anti-apoptosis in bone marrow mesenchymal stem cells (BMSC), but its role in the migration of BMSCs remains vague. The aim of this study was to determine the possible effect of miR-21 on regulating BMSCs directional migration and the expression of MMP-2/MMP-9 in BMSCs in vitro. BMSCs were successfully infected with miR-21-up lentivirus. Cell migration using Transwell assay indicated that upregulated expression of miR-21 could significantly promote BMSCs migration. Western blot analysis indicated that miR-21 significantly upregulated the expression of MMP-2 and MMP-9, which were related to metastasis-associated genes. GM6001, the specific MMPs inhibitor, abrogated the upregulated expression of MMP-2/MMP-9 and abolished the positive effect of miR-21 on promoting BMSCs migration. Meanwhile, miR-21 significantly enhanced Akt phosphorylation, as measured by Western blot analysis. LY294002, an inhibitor of Akt activation, abrogated the phosphorylation of Akt and abolished the positive effect of miR-21 on promoting BMSCs migration and upregulating MMP-2/MMP-9 expression. These results suggest that miR-21 contributes to BMSCs migration by upregulating MMP-2/MMP-9, potentially via the PI3K/Akt pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Nrf2 activator ameliorates hemorrhagic transformation in focal cerebral ischemia under warfarin anticoagulation.

    PubMed

    Imai, Takahiko; Takagi, Toshinori; Kitashoji, Akira; Yamauchi, Keita; Shimazawa, Masamitsu; Hara, Hideaki

    2016-05-01

    Oxidative stress has been reported to be a main cause of neuronal cell death in ischemia reperfusion injury (IRI). Nuclear factor-erythroid 2-related factor 2 (Nrf2) is an important factor involved in anti-oxidative responses. We previously reported that bardoxolone methyl (BARD), an Nrf2 activator, prevented damage induced by IRI. In this study, we investigated the effect of BARD on hemorrhagic transformation in the context of blood brain barrier (BBB) protection. Mice received pre-treatment with warfarin (4.0 mg/kg, p.o.). IRI was subsequently induced 18 h after the warfarin administration by transient middle cerebral artery occlusion (MCAO) for 6 h. BARD (0.06, 0.2, 0.6 or 2.0 mg/kg) or saline was injected intravenously immediately after reperfusion. The infarct volume, neurological score, intracranial hemorrhage volume, and BBB permeability were evaluated 24 h after MCAO. The survival rate and behavioral functional recovery were evaluated for 7 days following IRI. Furthermore, the effects of BARD on BBB components were investigated by western blotting and immunostaining analysis. BARD suppressed warfarin-mediated increases in the intracranial hemorrhage volume without affecting the infarct volume. BBB permeability was also suppressed by administration of BARD. Western blotting showed that BARD increased expression of BBB components such as endothelial cells, pericytes, and tight junction proteins. Furthermore, immunostaining showed that BARD induced localization of Nrf2 to endothelial cells and pericytes. BARD suppressed the exacerbation hemorrhage caused by warfarin pretreatment and ameliorated BBB disruption by protecting endothelial cells, pericytes, and tight junction protein expressions. These results indicate that Nrf2 activators may be an effective therapy against hemorrhagic transformation caused by anticoagulant drugs. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Activation of the nuclear factor E2-related factor 2/anitioxidant response element alleviates the nitroglycerin-induced hyperalgesia in rats.

    PubMed

    Di, Wei; Shi, Xiaolei; Lv, Hua; Liu, Jun; Zhang, Hong; Li, Zhiwei; Fang, Yannan

    2016-12-01

    Antioxidants have been proven to weaken hyperalgesia in neuropathic pain. Endogenous antioxidant defense system may have a role in the prevention of hyperalgesia in migraine. In this study, we aimed to evaluate the role of nuclear factor E2-related factor 2/antioxidant response element (Nrf2/ARE) pathway in regulating the activation of the trigeminovascular system (TGVS) and hypersensitivity in nitroglycerin (NTG)-induced hyperalgesia rats. The expression levels of Nrf2, HO, HO1, and NQO1 in the trigeminal nucleus caudalis (TNC) were detected by western blot. Immunofluorescence was used to demonstrate the cell-specific localization of Nrf2 in TNC. Sulforaphane, a Nrf2 activator, was administered to NTG-induced rats. Then, the number of c-Fos- and nNOS-immunoreactive neurons in TNC was evaluated using immunofluorescence, and c-Fos and nNOS protein levels were quantified using western blot. Von Frey hair testing was used to evaluate the tactile thresholds of rats at different time points in different groups. Total cellular and nuclear levels of the proteins Nrf2, HO1, and NQO1 were elevated in TNC after NTG injection, and Nrf2 was found to be located in the nucleus and cytoplasm of the neurons. Sulforaphane pretreatment significantly increased the nuclear Nrf2, HO1, and NQO1 levels in TNC. In addition, sulforaphane exposure effectively inhibited the expression of nNOS and c-Fos, reduced the number of nNOS and c-Fos immunoreactive neurons in TNC, and attenuated the tactile thresholds induced by NTG injection. Oxidative stress was involved in nitroglycerin-induced hyperalgesia. Activation of the Nrf2/ARE pathway inhibited the activation of TGVS and prevented the induction of hyperalgesia. Sulforaphane might therefore be an effective agent for hyperalgesia. Further studies are needed to discover the underlying mechanisms of the process.

  9. Seco-pregnane steroidal glycosides from the roots of Cynanchum atratum and their anti-TMV activity.

    PubMed

    Yan, Ying; Zhang, Jian-xin; Liu, Kai-xing; Huang, Tao; Yan, Chen; Huang, Lie-jun; Liu, Sheng; Mu, Shu-zhen; Hao, Xiao-jiang

    2014-09-01

    Fifteen new seco-pregnane steroidal glycosides cynanosides A-O (1-15) together with twenty-seven known ones were isolated from the roots of Cynanchum atratum. The structures of 1-15 were determined by extensive analysis of spectroscopic data. The anti-tobacco mosaic virus (TMV) activity of these steroidal glycosides was screened by the conventional half-leaf method, enzyme-linked immunosorbent assay, and Western blot methods, most of them showed potent anti-TMV activity. Among them, compounds 1, 7, 13, 28 and 31 showed significantly anti-TMV activity with an IC50 value of 20.5, 18.6, 22.0, 19.2 and 22.2 μg/mL, respectively, and were much more effective than the positive control, ningnanmycin (IC50=49.6 μg/mL). Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Anti-Inflammatory Activities of Inotilone from Phellinus linteus through the Inhibition of MMP-9, NF-κB, and MAPK Activation In Vitro and In Vivo

    PubMed Central

    Huang, Guan-Jhong; Huang, Shyh-Shyun; Deng, Jeng-Shyan

    2012-01-01

    Inotilone was isolated from Phellinus linteus. The anti-inflammatory effects of inotilone were studied by using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells and λ-carrageenan (Carr)-induced hind mouse paw edema model. Inotilone was tested for its ability to reduce nitric oxide (NO) production, and the inducible nitric oxide synthase (iNOS) expression. Inotilone was tested in the inhibitor of mitogen-activated protein kinase (MAPK) [extracellular signal-regulated protein kinase (ERK), c-Jun NH2-terminal kinase (JNK), p38], and nuclear factor-κB (NF-κB), matrix-metalloproteinase (MMP)-9 protein expressions in LPS-stimulated RAW264.7 cells. When RAW264.7 macrophages were treated with inotilone together with LPS, a significant concentration-dependent inhibition of NO production was detected. Western blotting revealed that inotilone blocked the protein expression of iNOS, NF-κB, and MMP-9 in LPS-stimulated RAW264.7 macrophages, significantly. Inotilone also inhibited LPS-induced ERK, JNK, and p38 phosphorylation. In in vivo tests, inotilone decreased the paw edema at the 4th and the 5th h after Carr administration, and it increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx). We also demonstrated that inotilone significantly attenuated the malondialdehyde (MDA) level in the edema paw at the 5th h after Carr injection. Inotilone decreased the NO and tumor necrosis factor (TNF-α) levels on serum at the 5th h after Carr injection. Western blotting revealed that inotilone decreased Carr-induced iNOS, cyclooxygenase-2 (COX-2), NF-κB, and MMP-9 expressions at the 5th h in the edema paw. An intraperitoneal (i.p.) injection treatment with inotilone diminished neutrophil infiltration into sites of inflammation, as did indomethacin (Indo). The anti-inflammatory activities of inotilone might be related to decrease the levels of MDA, iNOS, COX-2, NF-κB, and MMP-9 and increase the activities of CAT

  11. Synthesis and Structure activity relationships of EGCG Analogues, A Recently Identified Hsp90 Inhibitor

    PubMed Central

    Khandelwal, Anuj; Hall, Jessica

    2014-01-01

    Epigallocatechin-3-gallate (EGCG), the principal polyphenol isolated from green tea, was recently shown to inhibit Hsp90, however structure-activity relationships for this natural product have not yet been produced. Herein, we report the synthesis and biological evaluation of EGCG analogues to establish structure-activity relationships between EGCG and Hsp90. All four rings as well as the linker connecting the C- and the D-rings were systematically investigated, which led to the discovery of compounds that inhibit Hs90 and display improvement in efficacy over EGCG. Anti-proliferative activity of all the analogues was determined against MCF-7 and SKBr3 cell lines and Hsp90 inhibitory activity of four most potent analogues was further evaluated by western blot analyses and degradation of Hsp90-dependent client proteins. Prenyl substituted aryl ester of 3,5-dihydroxychroman-3-ol ring system was identified as novel scaffold that exhibit Hsp90 inhibitory activity. PMID:23834230

  12. Anticancer activity of Astragalus polysaccharide in human non-small cell lung cancer cells.

    PubMed

    Wu, Chao-Yan; Ke, Yuan; Zeng, Yi-Fei; Zhang, Ying-Wen; Yu, Hai-Jun

    2017-01-01

    We have reported that Chinese herbs Astragalus polysaccharide (APS) can inhibit nuclear factor kappaB (NF-κB) activity during the development of diabetic nephropathy in mice. NF-κB plays important roles in genesis, growth, development and metastasis of cancer. NF-κB is also involved in the development of treatment resistance in tumors. Here we investigated the antitumor activity of APS in human non-small cell lung cells (A549 and NCI-H358) and the related mechanisms of action. The dose-effect and time-effect of antitumor of APS were determined in human lung cancer cell line A549 and NCI-H358. The inhibition effect of APS on the P65 mRNA and protein was detected by reverse transcriptase-PCR (RT-PCR) and Western blot in A549 cells respectively. The inhibition effect of APS on the p50, CyclinD1 and Bcl-xL protein was detected by Western blot in A549 cells respectively. The effect of APS on NF-κB transcription activity was measured with NF-κB luciferase detection. Finally, the nude mice A549 xenograft was introduced to confirm the antitumor activity of APS in vivo. Cell viability detection results indicated that APS can inhibit the proliferation of human lung cancer cell line A549 and NCI-H358 in the concentration of 20 and 40 mg/mL. NF-κB activator Phorbol 12-myristate13-acetate (PMA) can attenuate the antitumor activity of APS in both cell lines, but NF-κB inhibitor BAY 11-7082 (Bay) can enhance the effect of APS in both cell lines. In vivo APS can delay the growth of A549 xenograft in BALB/C nude mice. APS can down-regulate the expression of P65 mRNA and protein of A549 cells and decrease the expression of p50, CyclinD1 and Bcl-xL protein. The luciferase detection showed that the APS could reduce the P65 transcription activity in A549 cells. PMA can partially alleviate the inhibition activity of P65 transcription activity of APS in A549 cells, and Bay can enhance the down-regulation of the P65 transcription activity induced by APS in A549 cells. APS has a

  13. Thrombin-induced apoptosis in neurons through activation of c-Jun-N-terminal kinase.

    PubMed

    Bao, Lei; Zu, Jie; He, Qianqian; Zhao, Hui; Zhou, Su; Ye, Xinchun; Yang, Xinxin; Zan, Kun; Zhang, Zuohui; Shi, Hongjuan; Cui, Guiyun

    2017-01-01

    Studies have shown that thrombin activation played a central role in cell injuries associated with intracerebral hemorrhage (ICH). Here, our study investigated the cytotoxicity of thrombin on neurons, and determined the involvement of JNK pathways in thrombin-induced neuronal apoptosis. Primary cultured neurons were treated with different doses of thrombin. Some neurons were given either SP600125 or vehicle. LDH release assay and flow cytometry were used to measure neuronal apoptosis caused by thrombin. The activation of JNK and capases-3 were measured by Western blot. Our results showed large doses of thrombin that increased the LDH release, the level of cleaved caspase-3 and apoptosis rate of neurons. JNK was activated by thrombin in a time-dependent manner. Administration of SP600125 protects neurons from thrombin-induced apoptosis. These data indicate that the activation of JNK is crucial for thrombin-induced neuronal apoptosis, and inhibition of JNK may be a potential therapeutic target for ICH.

  14. Rock-slope failure activity and geological crises in western Norway

    NASA Astrophysics Data System (ADS)

    Hilger, Paula; Hermanns, Reginald L.; Myhra, Kristin S.; Gosse, John C.; Ivy-Ochs, Susan; Etzelmüller, Bernd

    2017-04-01

    In Norway a compilation of terrestrial cosmogenic nuclide (TCN) ages of rock-avalanche deposits suggests a close link of rock-slope failures related to deglaciation. Although ages spread over several thousand years at the end of the Late Pleistocene, 50% of all documented events occurred within 1000 years after deglaciation. It is therefore likely that debuttressing triggered most of the events. The same data set suggests that 25% of the events occurred during a period stretching until the Holocene thermal maximum (HTM). These events might be interpreted as possible reactions to additional factors such as the thawing of high-altitude permafrost. An example of a geological crisis following deglaciation and before the HTM are seven lobate rock-avalanche deposits mapped under the slope of the Vora mountain (1450 m asl.) in the Nordfjord area of western Norway. Three events of this rock-slope failure cluster date within a short time period of 2000 years, where modelling studies indicate that high-altitude permafrost was present. After the HTM rock-slope failures are distributed temporally and spatially rather evenly throughout the Holocene and western Norway. But there are two independent local clusters with frequent rock slides during a short time span. (1) At the active Mannen rock-slope instability several rock-avalanche and rockslide deposits were mapped on the valley bottom. Stratigraphic relations combined with TCN dating suggest that at least one event occurred when the valley bottom was below the marine limit. TCN ages of further four lobes cluster around 5.2 ka BP, which does not coincide with any other rock-avalanche occurrence in the region. The top of the north facing 1295 m high unstable slope concurs with the currently estimated permafrost boundary. Preliminary TCN ages of the sliding surface indicate that larger parts of the mountain did not become active until the climate maximum. It is likely that due to structural complexity not allowing for any easy

  15. Active rollback in the Gibraltar Arc: Evidences from CGPS data in the western Betic Cordillera

    NASA Astrophysics Data System (ADS)

    Gonzalez-Castillo, L.; Galindo-Zaldivar, J.; de Lacy, M. C.; Borque, M. J.; Martinez-Moreno, F. J.; García-Armenteros, J. A.; Gil, A. J.

    2015-11-01

    The Gibraltar Arc, located in the western Mediterranean Sea, is an arcuate Alpine orogen formed by the Betic and Rif Cordilleras, separated by the Alboran Sea. New continuous GPS data (2008-2013) obtained in the Topo-Iberia stations of the western Betic Cordillera allow us to improve the present-day deformation pattern related to active tectonics in this collision area between the Eurasian and African plates. These data indicate a very consistent westward motion of the Betic Cordillera with respect to the relatively stable Iberian Massif foreland. The displacement in the Betics increases toward the south and west, reaching maximum values in the Gibraltar Strait area (4.27 mm/yr in Ceuta, CEU1, and 4.06 mm/yr in San Fernando, SFER), then progressively decreasing toward the northwestern mountain front. The recent geological structures and seismicity evidence moderate deformation in a roughly NW-SE to WNW-ESE compressional stress setting in the mountain frontal areas, and moderate extension toward the internal part of the cordillera. The mountain front undergoes progressive development of folds affecting at least up to Pliocene deposits, with similar recent geological and geodetical rates. This folded strip helps to accommodate the active deformation with scarce associated seismicity. The displacement pattern is in agreement with the present-day clockwise rotation of the tectonic units in the northern branch of the Gibraltar Arc. Our data support that the westward emplacement of the Betic Cordillera continues to be active in a rollback tectonic scenario.

  16. 7, 8, 3′-Trihydroxyflavone Promotes Neurite Outgrowth and Protects Against Bupivacaine-Induced Neurotoxicity in Mouse Dorsal Root Ganglion Neurons

    PubMed Central

    Shi, Haohong; Luo, Xingjing

    2016-01-01

    Background 7, 8, 3′-trihydroxyflavone (THF) is a novel pro-neuronal small molecule that acts as a TrkB agonist. In this study, we examined the effect of THF on promoting neuronal growth and protecting anesthetics-induced neurotoxicity in dorsal root ganglion (DRG) neurons in vitro. Material/Methods Neonatal mouse DRG neurons were cultured in vitro and treated with various concentrations of THF. The effect of THF on neuronal growth was investigated by neurite outgrowth assay and Western blot. In addition, the protective effects of THF on bupivacaine-induced neurotoxicity were investigated by apoptosis TUNEL assay, neurite outgrowth assay, and Western blot, respectively. Results THF promoted neurite outgrowth of DRG neurons in dose-dependent manner, with an EC50 concentration of 67.4 nM. Western blot analysis showed THF activated TrkB signaling pathway by inducing TrkB phosphorylation. THF also rescued bupivacaine-induced neurotoxicity by reducing apoptosis and protecting neurite retraction in DRG neurons. Furthermore, the protection of THF in bupivacaine-injured neurotoxicity was directly associated with TrkB phosphorylation in a concentration-dependent manner in DRG neurons. Conclusions THF has pro-neuronal effect on DRG neurons by promoting neurite growth and protecting against bupivacaine-induced neurotoxicity, likely through TrkB activation. PMID:27371503

  17. Teaching Western Literature to Non-Western Students

    ERIC Educational Resources Information Center

    Pollock, Eric J.; Chun, Hye Won; Kim, Chung Ah

    2008-01-01

    This paper explores ways in which Western literature can be taught to Non-western students. This paper demonstrates that non-western values do not have to be overcome but rather Western values can be highlighted and reinforced to deal with literary complexity. Values and ideals such as freedom, self-identity, religion, feminism, and equality are…

  18. The recombinant expression and activity detection of MAF-1 fusion protein.

    PubMed

    Fu, Ping; Wu, Jianwei; Gao, Song; Guo, Guo; Zhang, Yong; Liu, Jian

    2015-10-01

    This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.

  19. Substance P modulation of TRPC3/7 channels improves respiratory rhythm regularity and ICAN-dependent pacemaker activity

    PubMed Central

    Ben-Mabrouk, Faiza; Tryba, Andrew Kieran

    2011-01-01

    Neuromodulators, such as Substance P (SubP) play an important role in modulating many rhythmic activities driven by central pattern generators (e.g., locomotion, respiration). However, the mechanism by which SubP enhances breathing regularity has not been determined. Here, we used mouse brainstem slices containing the pre-Bötzinger Complex (Pre-BötC) to demonstrate, for the first time, that SubP activates transient receptor protein canonical (TRPC) channels to enhance respiratory rhythm regularity. Moreover, SubP enhancement of network regularity is accomplished via selective enhancement of ICAN-dependent intrinsic bursting properties. In contrast to INaP-dependant pacemakers, ICAN-dependant pacemaker bursting activity is TRPC dependent. Western Blots reveal TRPC3 and TRPC7 channels are expressed in rhythmically active ventral respiratory group (VRG) island preparations. Taken together, these data suggest that SubP-mediated activation of TRPC3/7 channels underlies rhythmic ICAN-dependent pacemaker activity and enhances the regularity of respiratory rhythm activity. PMID:20345918

  20. Substance P modulation of TRPC3/7 channels improves respiratory rhythm regularity and ICAN-dependent pacemaker activity.

    PubMed

    Ben-Mabrouk, Faiza; Tryba, Andrew K

    2010-04-01

    Neuromodulators, such as substance P (SubP), play an important role in modulating many rhythmic activities driven by central pattern generators (e.g. locomotion, respiration). However, the mechanism by which SubP enhances breathing regularity has not been determined. Here, we used mouse brainstem slices containing the pre-Bötzinger complex to demonstrate, for the first time, that SubP activates transient receptor protein canonical (TRPC) channels to enhance respiratory rhythm regularity. Moreover, SubP enhancement of network regularity is accomplished via selective enhancement of ICAN (inward non-specific cation current)-dependent intrinsic bursting properties. In contrast to INaP (persistent sodium current)-dependent pacemakers, ICAN-dependent pacemaker bursting activity is TRPC-dependent. Western Blots reveal TRPC3 and TRPC7 channels are expressed in rhythmically active ventral respiratory group island preparations. Taken together, these data suggest that SubP-mediated activation of TRPC3/7 channels underlies rhythmic ICAN-dependent pacemaker activity and enhances the regularity of respiratory rhythm activity.

  1. The activation of G protein-coupled estrogen receptor induces relaxation via cAMP as well as potentiates contraction via EGFR transactivation in porcine coronary arteries

    PubMed Central

    Yu, Xuan; Stallone, John N.; Heaps, Cristine L.

    2018-01-01

    Estrogen exerts protective effects against cardiovascular diseases in premenopausal women, but is associated with an increased risk of both coronary heart disease and stroke in older postmenopausal women. Studies have shown that activation of the G-protein-coupled estrogen receptor 1 (GPER) can cause either relaxation or contraction of arteries. It is highly likely that these dual actions of GPER may contribute to the seemingly paradoxical effects of estrogen in regulating coronary artery function. The objective of this study was to test the hypothesis that activation of GPER enhances agonist-stimulated porcine coronary artery contraction via epidermal growth factor receptor (EGFR) transactivation and its downstream extracellular signal-regulated kinases (ERK1/2) pathway. Isometric tension studies and western blot were performed to determine the effect of GPER activation on coronary artery contraction. Our findings demonstrated that G-1 caused concentration-dependent relaxation of ET-1-induced contraction, while pretreatment of arterial rings with G-1 significantly enhanced ET-1-induced contraction. GPER antagonist, G-36, significantly inhibited both the G-1-induced relaxation effect and G-1-enhanced ET-1 contraction. Gallein, a Gβγ inhibitor, significantly increased G-1-induced relaxation, yet inhibited G-1-enhanced ET-1-mediated contraction. Similarly, inhibition of EGFR with AG1478 or inhibition of Src with phosphatase 2 further increased G-1-induced relaxation responses in coronary arteries, but decreased G-1-enhanced ET-1-induced contraction. Western blot experiments in porcine coronary artery smooth muscle cells (PCASMC) showed that G-1 increased tyrosine phosphorylation of EGFR, which was inhibited by AG-1478. Furthermore, enzyme-linked immunosorbent assays showed that the level of heparin-binding EGF (HB-EGF) released by ET-1 treatment increased two-fold; whereas pre-incubation with G-1 further increased ET-1-induced HB-EGF release to four-fold over

  2. The activation of G protein-coupled estrogen receptor induces relaxation via cAMP as well as potentiates contraction via EGFR transactivation in porcine coronary arteries.

    PubMed

    Yu, Xuan; Stallone, John N; Heaps, Cristine L; Han, Guichun

    2018-01-01

    Estrogen exerts protective effects against cardiovascular diseases in premenopausal women, but is associated with an increased risk of both coronary heart disease and stroke in older postmenopausal women. Studies have shown that activation of the G-protein-coupled estrogen receptor 1 (GPER) can cause either relaxation or contraction of arteries. It is highly likely that these dual actions of GPER may contribute to the seemingly paradoxical effects of estrogen in regulating coronary artery function. The objective of this study was to test the hypothesis that activation of GPER enhances agonist-stimulated porcine coronary artery contraction via epidermal growth factor receptor (EGFR) transactivation and its downstream extracellular signal-regulated kinases (ERK1/2) pathway. Isometric tension studies and western blot were performed to determine the effect of GPER activation on coronary artery contraction. Our findings demonstrated that G-1 caused concentration-dependent relaxation of ET-1-induced contraction, while pretreatment of arterial rings with G-1 significantly enhanced ET-1-induced contraction. GPER antagonist, G-36, significantly inhibited both the G-1-induced relaxation effect and G-1-enhanced ET-1 contraction. Gallein, a Gβγ inhibitor, significantly increased G-1-induced relaxation, yet inhibited G-1-enhanced ET-1-mediated contraction. Similarly, inhibition of EGFR with AG1478 or inhibition of Src with phosphatase 2 further increased G-1-induced relaxation responses in coronary arteries, but decreased G-1-enhanced ET-1-induced contraction. Western blot experiments in porcine coronary artery smooth muscle cells (PCASMC) showed that G-1 increased tyrosine phosphorylation of EGFR, which was inhibited by AG-1478. Furthermore, enzyme-linked immunosorbent assays showed that the level of heparin-binding EGF (HB-EGF) released by ET-1 treatment increased two-fold; whereas pre-incubation with G-1 further increased ET-1-induced HB-EGF release to four-fold over

  3. Seismic imaging of the geodynamic activity at the western Eger rift in central Europe

    NASA Astrophysics Data System (ADS)

    Mullick, N.; Buske, S.; Hrubcova, P.; Ruzek, B.; Shapiro, S.; Wigger, P.; Fischer, T.

    2015-04-01

    The western Eger rift at the Czech-German border in central Europe is an important geodynamically active area within the European Cenzoic rift system (ECRS) in the forelands of the Alps. Along with two other active areas of the ECRS, the French Massif Central and the east and west Eifel volcanic fields, it is characterized by numerous CO2-rich fluid emission points and frequent micro-seismicity. Existence of a plume(s) is indicated in the upper mantle which may be responsible for these observations. Here we reprocess a pre-existing deep seismic reflection profile '9HR' and interpret the subsurface structures as mapped by seismic reflectivity with previous findings, mainly from seismological and geochemical studies, to investigate the geodynamic activity in the subsurface. We find prominent hints of pathways which may allow magmatic fluids originating in the upper mantle to rise through the crust and cause the observed fluid emanations and earthquake activity.

  4. Oridonin stabilizes retinoic acid receptor alpha through ROS-activated NF-κB signaling.

    PubMed

    Cao, Yang; Wei, Wei; Zhang, Nan; Yu, Qing; Xu, Wen-Bin; Yu, Wen-Jun; Chen, Guo-Qiang; Wu, Ying-Li; Yan, Hua

    2015-04-10

    Retinoic acid receptor alpha (RARα) plays an essential role in the regulation of many biological processes, such as hematopoietic cell differentiation, while abnormal RARα function contributes to the pathogenesis of certain diseases including cancers, especially acute promyelocytic leukemia (APL). Recently, oridonin, a natural diterpenoid isolated from Rabdosia rubescens, was demonstrated to regulate RARα by increasing its protein level. However, the underlying molecular mechanism for this action has not been fully elucidated. In the APL cell line, NB4, the effect of oridonin on RARα protein was analyzed by western blot and real-time quantitative RT-PCR analyses. Flow cytometry was performed to detect intracellular levels of reactive oxygen species (ROS). The association between nuclear factor-kappa B (NF-κB) signaling and the effect of oridonin was assessed using specific inhibitors, shRNA gene knockdown, and immunofluorescence assays. In addition, primary leukemia cells were treated with oridonin and analyzed by western blot in this study. RARα possesses transcriptional activity in the presence of its ligand, all-trans retinoic acid (ATRA). Oridonin remarkably stabilized the RARα protein, which retained transcriptional activity. Oridonin also moderately increased intracellular ROS levels, while pretreatment with the ROS scavenger, N-acetyl-l-cysteine (NAC), dramatically abrogated RARα stabilization by oridonin. More intriguingly, direct exposure to low concentrations of H2O2 also increased RARα protein but not mRNA levels, suggesting a role for ROS in oridonin stabilization of RARα protein. Further investigations showed that NAC antagonized oridonin-induced activation of NF-κB signaling, while the NF-κB signaling inhibitor, Bay 11-7082, effectively blocked the oridonin increase in RARα protein levels. In line with this, over-expression of IκΒα (A32/36), a super-repressor form of IκΒα, or NF-κB-p65 knockdown inhibited oridonin or H2O2-induced

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

    NASA Astrophysics Data System (ADS)

    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.

  6. Mechanism of the melanogenesis stimulation activity of (-)-cubebin in murine B16 melanoma cells.

    PubMed

    Hirata, Noriko; Naruto, Shunsuke; Ohguchi, Kenji; Akao, Yukihiro; Nozawa, Yoshinori; Iinuma, Munekazu; Matsuda, Hideaki

    2007-07-15

    (-)-Cubebin showed a melanogenesis stimulation activity in a concentration-dependent manner in murine B16 melanoma cells without any significant effects on cell proliferation. Tyrosinase activity was increased at 24-72 h after addition of cubebin to B16 cells, and then intracellular melanin amount was increased at 48-96 h after the treatment. The expression levels of tyrosinase were time-dependently enhanced after the treatment with cubebin. At the same time, the expression levels of tyrosinase mRNA were also increased after addition of cubebin. Furthermore Western blot analysis revealed that cubebin elevated the level of phosphorylation of p38 mitogen-activated protein kinase (MAPK). SB203580, a selective inhibitor of p38 MAPK, completely blocked cubebin-induced expression of tyrosinase mRNA in B16 cells. These results suggested that cubebin increased melanogenesis in B16 cells through the enhancement of tyrosinase expression mediated by activation of p38 MAPK.

  7. Development and application of a quantitative multiplexed small GTPase activity assay using targeted proteomics.

    PubMed

    Zhang, Cheng-Cheng; Li, Ru; Jiang, Honghui; Lin, Shujun; Rogalski, Jason C; Liu, Kate; Kast, Juergen

    2015-02-06

    Small GTPases are a family of key signaling molecules that are ubiquitously expressed in various types of cells. Their activity is often analyzed by western blot, which is limited by its multiplexing capability, the quality of isoform-specific antibodies, and the accuracy of quantification. To overcome these issues, a quantitative multiplexed small GTPase activity assay has been developed. Using four different binding domains, this assay allows the binding of up to 12 active small GTPase isoforms simultaneously in a single experiment. To accurately quantify the closely related small GTPase isoforms, a targeted proteomic approach, i.e., selected/multiple reaction monitoring, was developed, and its functionality and reproducibility were validated. This assay was successfully applied to human platelets and revealed time-resolved coactivation of multiple small GTPase isoforms in response to agonists and differential activation of these isoforms in response to inhibitor treatment. This widely applicable approach can be used for signaling pathway studies and inhibitor screening in many cellular systems.

  8. Secreted phospholipase A2 of Clonorchis sinensis activates hepatic stellate cells through a pathway involving JNK signalling.

    PubMed

    Wu, Yinjuan; Li, Ye; Shang, Mei; Jian, Yu; Wang, Caiqin; Bardeesi, Adham Sameer A; Li, Zhaolei; Chen, Tingjin; Zhao, Lu; Zhou, Lina; He, Ai; Huang, Yan; Lv, Zhiyue; Yu, Xinbing; Li, Xuerong

    2017-03-16

    Secreted phospholipase A2 (sPLA2) is a protein secreted by Clonorchis sinensis and is a component of excretory and secretory products (CsESPs). Phospholipase A2 is well known for its role in liver fibrosis and inhibition of tumour cells. The JNK signalling pathway is involved in hepatic stellate cells (HSCs) activation. Blocking JNK activity with SP600125 inhibits HSCs activation. In a previous study, the protein CssPLA2 was expressed in insoluble inclusion bodies. Therefore, it's necessary to express CssPLA2 in water-soluble form and determine whether the enzymatic activity of CssPLA2 or cell signalling pathways is involved in liver fibrosis caused by clonorchiasis. Balb/C mice were given an abdominal injection of MBP-CssPLA2. Liver sections with HE and Masson staining were observed to detect accumulation of collagen. Western blot of mouse liver was done to detect the activation of JNK signalling pathway. In vitro, HSCs were incubated with MBP-CssPLA2 to detect the activation of HSCs as well as the activation of JNK signalling pathway. The mutant of MBP-CssPLA2 without enzymatic activity was constructed and was also incubated with HSCs to check whether activation of the HSCs was related to the enzymatic activity of MBP-CssPLA2. The recombinant protein MBP-CssPLA2 was expressed soluble and of good enzymatic activity. A mutant of CssPLA2, without enzymatic activity, was also constructed. In vivo liver sections of Balb/C mice that were given an abdominal injection of 50 μg/ml MBP-CssPLA2 showed an obvious accumulation of collagen and a clear band of P-JNK1 could be seen by western blot of the liver tissue. In vitro, MBP-CssPLA2, as well as the mutant, was incubated with HSCs and it was proved that activation of HSCs was related to activation of the JNK signalling pathway instead of the enzymatic activity of MBP-CssPLA2. Activation of HSCs by CssPLA2 is related to the activation of the JNK signalling pathway instead of the enzymatic activity of CssPLA2. This finding

  9. Negative pressure wound therapy inhibits inflammation and upregulates activating transcription factor-3 and downregulates nuclear factor-κB in diabetic patients with foot ulcerations.

    PubMed

    Wang, T; He, R; Zhao, J; Mei, J C; Shao, M Z; Pan, Y; Zhang, J; Wu, H S; Yu, M; Yan, W C; Liu, L M; Liu, F; Jia, W P

    2017-05-01

    Negative pressure wound therapy (NPWT) is one of the most important treatments for diabetic foot, but the underlying mechanisms of its benefits still remain elusive. This study aims to evaluate the inflammatory signals involved in the effects of negative pressure therapy on diabetic foot ulcers. We enrolled 22 patients with diabetic foot ulceration, 11 treated with NPWT and the other 11 treated with traditional debridement. All patients were treated and observed for 1 week. Granulation tissues were harvested and analyzed in both groups, and then were histologically and immunohistochemically analyzed. Enzyme-linked immunosorbent assay, Western blot analysis, and real-time PCR were performed to evaluate the expression of interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), inducible nitric oxide synthase (iNOS), nuclear factor-κB (NF-κB) p65, I k B-α, and activating transcription factor-3 (ATF-3). After 7 days of treatment, NPWT could obviously promote diabetic wound healing because of the mild inflammation and the dense cell-deposited matrix. Meanwhile, NPWT significantly decreased the expression of TNF-α, IL-6, and iNOS (all P < .05). The result of Western blotting and real-time PCR indicated that NPWT obviously decreased the level of I k B-α and NF-κB p65, and increased the level of ATF-3 (all P < .05). NPWT exerts an anti-inflammatory effect, possibly through the suppression of proinflammatory enzymes and cytokines resulting from I k B-α inhibition and ATF-3 activation, which may prevent the activation of the NF-κB pathway in human diabetic foot wounds. Copyright © 2016 John Wiley & Sons, Ltd.

  10. A water-soluble polysaccharide from Grifola frondosa induced macrophages activation via TLR4-MyD88-IKKβ-NF-κB p65 pathways

    PubMed Central

    Hou, Lihua; Meng, Meng; Chen, Yuanyuan; Wang, Chunling

    2017-01-01

    Here, the immunomodulatory effects of water-soluble polysaccharide from Grifola frondosa on RAW264.7 macrophages and its molecular mechanisms were investigated. G. frondosa polysaccharide could obviously enhance immunostimulatory activity such as the release of nitric oxide and cytokine production. Western blotting results showed that G. frondosa polysaccharide elevated the TLR4, which might act as an upstream regulator of MyD88 induced G. frondosa polysaccharide. MyD88 promoted IKKβ in endochylema and translocate NF-κB p65 subunit into the nucleus which increased the NO production and cytokine/chemokines level. The results suggested that G. frondosa polysaccharide activated macrophages through TLR4-MyD88-IKKβ-NF-κBp65 signaling pathways. PMID:29156820

  11. Development of Monoclonal Antibodies Recognizing Linear Epitope: Illustration by Three Bacillus thuringiensis Crystal Proteins of Genetically Modified Cotton, Maize, and Tobacco.

    PubMed

    Cao, Zhen; Zhang, Wei; Ning, Xiangxue; Wang, Baomin; Liu, Yunjun; Li, Qing X

    2017-11-22

    Bacillus thuringiensis Cry1Ac, Cry1Ia1, and Cry1Ie are δ-endotoxin insecticidal proteins widely implemented in genetically modified organisms (GMO), such as cotton, maize, and potato. Western blot assay integrates electrophoresis separation power and antibody high specificity for monitoring specific exogenous proteins expressed in GMO. Procedures for evoking monoclonal antibody (mAb) for Western blot were poorly documented. In the present study, Cry1Ac partially denatured at 100 °C for 5 min was used as an immunogen to develop mAbs selectively recognizing a linear epitope of Cry1Ac for Western blot. mAb 5E9C6 and 3E6E2 selected with sandwich ELISA strongly recognized the heat semidenatured Cry1Ac. Particularly, 3E6E2 recognized both E. coli and cotton seed expressed Cry1Ac in Western blot. Such strategy of using partially denatured proteins as immunogens and using sandwich ELISA for mAb screening was also successfully demonstrated with production of mAbs against Cry1Ie for Western blot assay in maize.

  12. An Overview of Recent Geostationary Fire Monitoring Activities and Applications in the Western Hemisphere

    NASA Astrophysics Data System (ADS)

    McRae, D. J.; Conard, S. G.; Ivanova, G. A.; Sukhinin, A. I.; Hao, W. M.; Koutzenogii, K. P.; Prins, E. M.; Schmidt, C. C.; Feltz, J. M.

    2002-05-01

    Over the past twenty years the international scientific research and environmental monitoring communities have recognized the vital role environmental satellites can play in detecting and monitoring active fires both regionally and around the globe for hazards applications and to better understand the extent and impact of biomass burning on the global environment. Both groups have stressed the importance of utilizing operational satellites to produce routine fire products and to ensure long-term stable records of fire activity for applications such as land-use/land cover change analyses and global climate change research. The current NOAA GOES system provides the unique opportunity to detect fires throughout the Western Hemisphere every half-hour from a series of nearly identical satellites for a period of 15+ years. This presentation will provide an overview of the GOES biomass burning monitoring program at UW-Madison Cooperative Institute for Meteorological Satellite Studies (CIMSS) with an emphasis on recent applications of the new GOES Wildfire Automated Biomass Burning Algorithm (WF_ABBA). For the past 8 years, CIMSS has utilized the GOES-8 imager to monitor biomass burning trends in South America. Since September 2000, CIMSS has been producing half-hourly fire products in real-time for most of the Western Hemisphere. The WF_ABBA half-hourly fire product is providing new insights into diurnal, spatial, seasonal and interannual fire dynamics in North, Central, and South America. In North America these products are utilized to detect and monitor wildfires in northerly and remote locations. In South America the diurnal GOES fire product is being used as an indicator of land-use and land-cover change and carbon dynamics along the borders between Brazil, Peru, and Bolivia. The Navy is assimilating the Wildfire ABBA fire product into the Navy Aerosol Analysis and Prediction System (NAAPS) to analyze and predict aerosol loading and transport as part of the NASA

  13. [Effect of GPER on the activation of PI3K/Akt induced by 17β-estradiol in endometrial carcinoma cells].

    PubMed

    Zhang, Yan-Cai; Guo, Rui-Xia; Ge, Xin; Qiao, Yu-Huan

    2012-04-01

    To investigate the expression of G protein-coupled ER (GPER) and ER in the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) induced by 17β-estradiol (17β-E(2))in endometrial carcinoma cells, Ishikawa and HEC-1A. Expressions of GPER, ERα and ERβ protein in Ishikawa and HEC-1A cells were detected by immunohistochemical SP method. Levels of GPER, ERα and ERβ were examined by western blot in Ishikawa and HEC-1A cells after treated with 1×10(-6) mol/L 17β-E(2) at different time (0, 15, 30, 60, 120 minutes). GPER was positive expressed in Ishikawa and HEC-1A cells. ERα and ERβ were both positive expressed in Ishikawa cells. While, ERα was weakly expressed and ERβ was almost negatively expressed in HEC-1A cells. Western blot analysis showed that 1×10(-6) mol/L 17β-E(2) treatment, the Ishikawa and HEC-1A cells GPER protein level for 15 minutes markedly increased (P < 0.05), which Ishikawa 30 minutes, when cells reached the highest level (0.192 ± 0.004), HEC-1A cells for 15 minutes and reached the highest level (0.184 ± 0.006); Ishikawa and HEC-1A cells, Akt, activation of 15 minutes from the treatment start was significantly increased (P < 0.05), which Ishikawa cells for 30 minutes and reached the highest level (0.666 ± 0.021), HEC-1A cells for 15 minutes and reached maximum (0.788 ± 0.035); Ishikawa and HEC-1A cells, ERα and ERβ protein expression did not change significantly (P > 0.05). GPER likely involved in non-nuclear activation of PI3K/Akt signaling pathways in endometrial carcinoma cells, Ishikawa and HEC-1A.

  14. Febuxostat Prevents Renal Interstitial Fibrosis by the Activation of BMP-7 Signaling and Inhibition of USAG-1 Expression in Rats.

    PubMed

    Cao, Jing; Li, Yong; Peng, Yingxian; Zhang, Yaqian; Li, Huanhuan; Li, Ran; Xia, Anzhou

    2015-01-01

    Renal interstitial fibrosis (RIF) is a common pathology associated with end-stage renal diseases. The activation of bone morphogenetic protein-7 (BMP-7)-Smad1/5/8 pathway seems to alleviate RIF. Uterine sensitization-associated gene-1 (USAG-1), a kidney-specific BMPs antagonist, is associated with the development and prognosis of several renal diseases. Febuxostat is a xanthine oxidase inhibitor that can attenuate the renal dysfunction of patients. The purpose of this study was to investigate the effects of febuxostat on renal fibrosis and to clarify the mechanisms underlying these effects. Rats were randomly divided into 6 groups termed a sham-operated group, a unilateral ureteral obstruction (UUO) group, 3 doses of febuxostat groups (low, intermediate and high doses) and a sham group treated with high-dose febuxostat. After 14 days, renal function, relative kidney weight, accumulation of glycogen and collagens were examined by different methods. Expression of α-SMA, transforming growth factor-β1 (TGF-β1), BMP-7 and USAG-1 was detected by western blotting and RT-PCR, respectively. The phosphorylation level of Smad1/5/8 was also quantified by western blotting. The renal function was declined, and large amounts of glycogen and collagens were deposited in the kidneys of UUO rats compared with the rats in the sham group. Besides, expression of α-SMA and USAG-1 in these kidneys was elevated, and the TGF-β1 was also activated, while the BMP-7-Smad1/5/8 pathway was inhibited. Febuxostat reversed the changes stated earlier, exhibiting protective effects on RIF induced by UUO. Febuxostat was able to attenuate RIF caused by UUO, which was associated with the activation of BMP-7-Smad1/5/8 pathway and the inhibition of USAG-1 expression in the kidneys of UUO rats. © 2015 S. Karger AG, Basel.

  15. Up-regulation of IL-23 expression in human dental pulp fibroblasts by IL-17 via activation of the NF-κB and MAPK pathways.

    PubMed

    Wei, L; Liu, M; Xiong, H; Peng, B

    2017-11-06

    To investigate the effects of the pro-inflammatory and Th17-polarizing mediator IL-17 on HDPFs-mediated IL-23 production and the molecular mechanism involved. Interleukin (IL)-17R expression was determined by semi-quantitative reverse transcriptase-polymerase chain reaction and Western blot in cultured human dental pulp fibroblasts (HDPFs). Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay were used to determine IL-23 mRNA and protein levels in IL-17-stimulated HDPFs, respectively. The nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signalling pathways that mediate the IL-17-stimulated production of IL-23 was investigated using Western blot and specific signalling inhibitor analyses. Statistical analyses were performed using Kruskal-Wallis tests followed by the Mann-Whitney U-test. Statistical significance was considered when the P value < 0.05. Primary HDPFs steadily expressed IL-17R mRNA and surface-bound protein. IL-17 stimulated the expression of IL-23 mRNA and protein in cultured human dental pulp fibroblasts, which was attenuated by IL-17 or IL-17R neutralizing antibodies. In accordance with the enhanced expression of IL-23, IL-17 stimulation resulted in rapid activation of p38 MAPK, extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK) and NF-κB in HDPFs. Inhibitors of p38 MAPK, ERK 1/2 or NF-κB significantly suppressed, whereas blocking JNK substantially augmented IL-23 production from IL-17-stimulated HDPFs. HDPFs expressed IL-17R and responded to IL-17 to produce IL-23 via the activation of the NF-κB and MAPK signalling pathways. The findings provide insights into the cellular mechanisms of the participation of IL-17 in the activation of HDPFs in inflamed pulp tissue. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  16. [Preparation and application of monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit].

    PubMed

    Yan, Xiaofei; Wu, Litao; DU, Xiaojuan; Li, Jing; Zhang, Fujun; Han, Yan; Lyu, Shemin; Li, Dongmin

    2016-12-01

    Objective To prepare monoclonal antibodies against DR region (897DVEDSYGQQWTYEQR911) of Na + -K + -ATPase α1 subunit and identify their properties. Methods BALB/c mice were immunized with DR-keyholelimpet hemocyanin (KLH). Splenocytes from the immunized mice were collected and subsequently fused with SP2/0 mouse myeloma cells. Positive hybridoma clones were obtained after cell fusion and selection. ELISA was used to detect DR antibody titer in the cell supernatants. DR region-specific monoclonal antibodies were analyzed by dot blotting, Western blotting and immunofluorescence assay. Na + -K + -ATPase activity was detected by SensoLyte R FDP Protein Phosphatase Assay Kit and the protective effect of the monoclonal antibody against high glucose-induced cell injury was assessed in H9c2 cells. Results Three hybridoma cell lines which secreted stable DR monoclonal antibody were obtained. The strongest positive cell line, named DRm217, was selected to prepare ascites. Dot blotting, Western blotting and immunofluorescence assay showed that DRm217 recognized specially DR region of Na + -K + -ATPase and bound on H9c2 cell membranes. DRm217 stimulated Na + -K + -ATPase activity and alleviated high glucose-induced H9c2 cells injury. Conclusion The monoclonal antibodies against DR region of Na + -K + -ATPase α1 subunit is prepared.

  17. Combination of quercetin, cinnamaldehyde and hirudin protects rat dorsal root ganglion neurons against high glucose-induced injury through Nrf-2/HO-1 activation and NF-κB inhibition.

    PubMed

    Shi, Yue; Liang, Xiao-Chun; Zhang, Hong; Sun, Qing; Wu, Qun-Li; Qu, Ling

    2017-09-01

    To examine the effects of the combination of quercetin (Q), cinnamaldehyde (C) and hirudin (H), a Chinese medicine formula on high glucose (HG)-induced apoptosis of cultured dorsal root ganglion (DRG) neurons. DRG neurons exposed to HG (45 mmol/L) for 24 h were employed as an in vitro model of diabetic neuropathy. Cell viability, reactive oxygen species (ROS) level and apoptosis were determined. The expression of nuclear factor of Kappa B (NF-κB), inhibitory kappa Bα(IκBα), phosphorylated IκBα and Nf-E2 related factor 2 (Nrf2) were examined using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay. The expression of hemeoxygenase-1 (HO-1), interleukin-6 (IL-6), tumor necrosis factor (TNF-α) and caspase-3 were also examined by RT-PCR and Western blot assay. HG treatment markedly increased DRG neuron apoptosis via increasing intracellular ROS level and activating the NF-κB signaling pathway (P<0.05). Co-treatment with Q, C, H and their combination decreased HG-induced caspase-3 activation and apoptosis (P<0.05 or P<0.01). The expressions of NF-κB, IL-6 and TNF-α were down-regulated, and Nrf2/HO-1 expression was up-regulated (P<0.05 or P<0.01). QCH has better effect in scavenging ROS, activating Nrf-2/HO-1, and down-regulating the NF-κB pathway than other treatment group. DRG neurons' apoptosis was increased in diabetic conditions, which was reduced by QCH formula treatment. The possible reason could be activating Nrf-2/HO-1 pathway, scavenging ROS, and inhibition of NF-κB activation. The effect of QCH combination was better than each monomer or the combination of the two monomers.

  18. The active principle region of Buyang Huanwu decoction induced differentiation of bone marrow-derived mesenchymal stem cells into neural-like cells

    PubMed Central

    Zheng, Jinghui; Wan, Yi; Chi, Jianhuai; Shen, Dekai; Wu, Tingting; Li, Weimin; Du, Pengcheng

    2012-01-01

    The present study induced in vitro-cultured passage 4 bone marrow-derived mesenchymal stem cells to differentiate into neural-like cells with a mixture of alkaloid, polysaccharide, aglycone, glycoside, essential oils, and effective components of Buyang Huanwu decoction (active principle region of decoction for invigorating yang for recuperation). After 28 days, nestin and neuron-specific enolase were expressed in the cytoplasm. Reverse transcription-PCR and western blot analyses showed that nestin and neuron-specific enolase mRNA and protein expression was greater in the active principle region group compared with the original formula group. Results demonstrated that the active principle region of Buyang Huanwu decoction induced greater differentiation of rat bone marrow-derived mesenchymal stem cells into neural-like cells in vitro than the original Buyang Huanwu decoction formula. PMID:25806066

  19. Fluocinolone acetonide partially restores the mineralization of LPS-stimulated dental pulp cells through inhibition of NF-κB pathway and activation of AP-1 pathway

    PubMed Central

    Liu, Zhongning; Jiang, Ting; Wang, Xinzhi; Wang, Yixiang

    2013-01-01

    BACKGROUND AND PURPOSE Fluocinolone acetonide (FA) is commonly used as a steroidal anti-inflammatory drug. We recently found that in dental pulp cells (DPCs) FA has osteo-/odonto-inductive as well as anti-inflammatory effects. However, the mechanism by which FA induces these effects in DPCs is poorly understood. EXPERIMENTAL APPROACH The effect of FA on the mineralization of DPCs during inflammatory conditions and the underlying mechanism were investigated by real-time PCR, Western blot, EMSA, histochemical staining, immunostaining and pathway blockade assays. KEY RESULTS FA significantly inhibited the inflammatory response in LPS-treated DPCs not only by down-regulating the expression of pro–inflammation-related genes, but also by up-regulating the expression of the anti-inflammatory gene PPAR-γ and mineralization-related genes. Moreover, histochemical staining and immunostaining showed that FA could partially restore the expressions of alkaline phosphatase, osteocalcin and dentin sialophosphoprotein (DSPP) and mineralization in LPS-stimulated DPCs. Real-time PCR and Western blot analysis revealed that FA up-regulated DSPP and runt-related transcription factor 2 expression by inhibiting the expression of phosphorylated-NF-κB P65 and activating activator protein-1 (AP-1) (p-c-Jun and Fra-1). These results were further confirmed through EMSA, by detection of NF-κB DNA-binding activity and pathway blockade assays using a NF-κB pathway inhibitor, AP-1 pathway inhibitor and glucocorticoid receptor antagonist. CONCLUSIONS AND IMPLICATIONS Inflammation induced by LPS suppresses the mineralization process in DPCs. FA partially restored this osteo-/odonto-genesis process in LPS-treated DPCs and had an anti-inflammatory effect through inhibition of the NF-κB pathway and activation of the AP-1 pathway. Hence, FA is a potential new treatment for inflammation-associated bone/teeth diseases. PMID:24024985

  20. TNF-α mediates choroidal neovascularization by upregulating VEGF expression in RPE through ROS-dependent β-catenin activation.

    PubMed

    Wang, Haibo; Han, Xiaokun; Wittchen, Erika S; Hartnett, M Elizabeth

    2016-01-01

    Inflammation, oxidative stress, and angiogenesis have been proposed to interact in age-related macular degeneration. It has been postulated that external stimuli that cause oxidative stress can increase production of vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells. In this study, we tested the hypothesis that the inflammatory cytokine, tumor necrosis factor alpha (TNF-α), contributed to choroidal neovascularization (CNV) by upregulating VEGF in RPE through intracellular reactive oxygen species (ROS)-dependent signaling and sought to understand the mechanisms involved. In a murine laser-induced CNV model, 7 days after laser treatment and intravitreal neutralizing mouse TNF-α antibody or isotype immunoglobulin G (IgG) control, the following measurements were made: 1) TNF-α protein and VEGF protein in RPE/choroids with western blot, 2) CNV volume in RPE/choroidal flatmounts, and 3) semiquantification of oxidized phospholipids stained with E06 antibody within CNV with immunohistochemistry (IHC). In cultured human RPE cells treated with TNF-α or PBS control, 1) ROS generation was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence assay, and 2) NOX4 protein and VEGF protein or mRNA were measured with western blot or quantitative real-time PCR in cells pretreated with apocynin or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, VAS 2870, or transfected with p22phox siRNA, and each was compared to its appropriate control. Western blots of phosphorylated p65 (p-p65), total p65 and β-actin, and quantitative real-time PCR of VEGF mRNA were measured in human RPE cells treated with TNF-α and pretreatment with the nuclear factor kappa B inhibitor, Bay 11-7082 or control. Western blots of β-catenin, VEGF, and p22phox and coimmunoprecipitation of β-catenin and T-cell transcriptional factor were performed in human RPE cells treated with TNF-α following pretreatment with

  1. Adenosine monophosphate-activated protein kinase activation and suppression of inflammatory response by cell stretching in rabbit synovial fibroblasts.

    PubMed

    Kunanusornchai, Wanlop; Muanprasat, Chatchai; Chatsudthipong, Varanuj

    2016-12-01

    Joint mobilization is known to be beneficial in osteoarthritis (OA) patients. This study aimed to investigate the effect of stretching on adenosine monophosphate-activated protein kinase (AMPK) activity and its role in modulating inflammation in rabbit synovial fibroblasts. Uniaxial stretching of isolated rabbit synovial fibroblasts for ten min was performed. Stretching-induced AMPK activation, its underlying mechanism, and its anti-inflammatory effect were investigated using Western blot. Static stretching at 20 % of initial length resulted in AMPK activation characterized by expression of phosphorylated AMPK and phosphorylated acetyl-Co A carboxylase. AMP-activated protein kinase phosphorylation peaked 1 h after stretching and declined toward resting activity. Using cell viability assays, static stretching did not appear to cause cellular damage. Activation of AMPK involves Ca 2+ influx via a mechanosensitive L-type Ca 2+ channel, which subsequently raises intracellular Ca 2+ and activates AMPK via Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ). Interestingly, stretching suppressed TNFα-induced expression of COX-2, iNOS, and phosphorylated NF-κB. These effects were prevented by pretreatment with compound C, an AMPK inhibitor. These results suggest that mechanical stretching suppressed inflammatory responses in synovial fibroblasts via a L-type Ca 2+ -channel-CaMKKβ-AMPK-dependent pathway which may underlie joint mobilization's ability to alleviate OA symptoms.

  2. Antiviral activity of silymarin against chikungunya virus

    PubMed Central

    Lani, Rafidah; Hassandarvish, Pouya; Chiam, Chun Wei; Moghaddam, Ehsan; Chu, Justin Jang Hann; Rausalu, Kai; Merits, Andres; Higgs, Stephen; Vanlandingham, Dana; Abu Bakar, Sazaly; Zandi, Keivan

    2015-01-01

    The mosquito-borne chikungunya virus (CHIKV) causes chikungunya fever, with clinical presentations such as severe back and small joint pain, and debilitating arthritis associated with crippling pains that persist for weeks and even years. Although there are several studies to evaluate the efficacy of drugs against CHIKV, the treatment for chikungunya fever is mainly symptom-based and no effective licensed vaccine or antiviral are available. Here, we investigated the antiviral activity of three types of flavonoids against CHIKV in vitro replication. Three compounds: silymarin, quercetin and kaempferol were evaluated for their in vitro antiviral activities against CHIKV using a CHIKV replicon cell line and clinical isolate of CHIKV of Central/East African genotype. A cytopathic effect inhibition assay was used to determine their activities on CHIKV viral replication and quantitative reverse transcription PCR was used to calculate virus yield. Antiviral activity of effective compound was further investigated by evaluation of CHIKV protein expression using western blotting for CHIKV nsP1, nsP3, and E2E1 proteins. Briefly, silymarin exhibited significant antiviral activity against CHIKV, reducing both CHIKV replication efficiency and down-regulating production of viral proteins involved in replication. This study may have important consequence for broaden the chance of getting the effective antiviral for CHIKV infection. PMID:26078201

  3. Rapid elevation of sodium transport through insulin is mediated by AKT in alveolar cells

    PubMed Central

    Mattes, Charlott; Laube, Mandy; Thome, Ulrich H.

    2014-01-01

    Abstract Alveolar fluid clearance is driven by vectorial Na+ transport and promotes postnatal lung adaptation. The effect of insulin on alveolar epithelial Na+ transport was studied in isolated alveolar cells from 18–19‐day gestational age rat fetuses. Equivalent short‐circuit currents (ISC) were measured in Ussing chambers and different kinase inhibitors were used to determine the pathway of insulin stimulation. In Western Blot measurements the activation of mediators stimulated by insulin was analyzed. The ISC showed a fast dose‐dependent increase by insulin, which could be attributed to an increased ENaC (epithelial Na+ channel) activity in experiments with permeabilized apical or basolateral membrane. 5‐(N‐Ethyl‐N‐isopropyl)amiloride inhibition of ISC was not affected, however, benzamil‐sensitive ISC was increased in insulin‐stimulated monolayers. The application of LY‐294002 and Akti1/2 both completely blocked the stimulating effect of insulin on ISC. PP242 partly blocked the effect of insulin, whereas Rapamycin evoked no inhibition. Western Blot measurements revealed an increased phosphorylation of AKT after insulin stimulation. SGK1 activity was also increased by insulin as shown by Western Blot of pNDRG1. However, in Ussing chamber measurements, GSK650394, an inhibitor of SGK1 did not prevent the increase in ISC induced by insulin. The application of IGF‐1 mimicked the effect of insulin and increased the ENaC activity. In addition, an increased autophosphorylation of the IGF‐1R/IR was observed after insulin stimulation. We conclude that insulin rapidly increases epithelial Na+ transport by enhancing the activity of endogenous ENaC through activation of PI3K/AKT in alveolar cells. PMID:24760523

  4. [Lactobacillus rhamnosus GG conditioned medium prevents E. coli meningitis by inhibiting nuclear factor-κB pathway].

    PubMed

    Zeng, Qing; He, Xiao-Long; Xiao, Han-Sheng; DU, Lei; Li, Yu-Jing; Chen, Le-Cheng; Tian, Hui-Wen; Huang, Sheng-He; Cao, Hong

    2017-01-20

    To investigate whether Lactobacillus rhamnosus GG conditioned medium(LGG-CM)has preventive effect against E. coli K1-induced neuropathogenicity in vitro by inhibiting nuclear factor-κB (NF-κB) signaling pathway. An in vitro blood-brain barrier (BBB) model was constructed using human brain microvascular endothelial cells (HBMECs). The effect of LGG-CM on E. coli-actived NF-κB signaling pathway was assayed using Western blotting. Invasion assay and polymorphonuclear leukocyte (PMN) transmigration assay were performed to explore whether LGG-CM could inhibit E. coli invasion and PMN transmigration across the BBB in vitro. The expressions of ZO-1 and CD44 were detected using Western blotting and immunofluorescence. The changes of trans-epithelial electric resistance (TEER) and bacterial translocation were determined to evaluate the BBB permeability. Pre-treament with LGG-CM inhibited E. coli-activated NF-κB signaling pathway in HBMECs and decreased the invasion of E. coli K1 and transmigration of PMN. Western blotting showed that LGG-CM could alleviate E. coli-induced up-regulation of CD44 and down-regulation of ZO-1 expressions in HBMECs. In addition, pre-treatment with LGG-CM alleviated E. coli K1-induced reduction of TEER and suppressed bacterial translocation across the BBB in vitro. LGG-CM can block E. coli-induced activation of NF-κB signaling pathway and thereby prevents E. coli K1-induced neuropathogenicity by decreasing E. coli K1 invasion rates and PMN transmigration.

  5. Deletion detection for diagnosis of Duchenne muscular dystrophy in the Japanese population--comparison between the polymerase chain reaction and the Southern blot analysis.

    PubMed

    Katayama, S; Takeshita, N; Yano, T; Ubagai, T; Qiu, X J; Katagiri, Y; Kubo, H; Hirakawa, S

    1993-06-01

    We compared the efficacy of the multiplex PCR with that of the cDNA analysis for detection of deletions of the DMD gene in the Japanese patients. Thirty males with DMD from 27 Japanese families were studied by the multiplex PCR, and 24 of them were also investigated by Southern blot analysis. We used five dystrophin cDNA probes for deletion analysis. A total of 19 regions were amplified by the PCR to detect deletions, 9 regions by the method of Chamberlain et al. and another 10 regions by the method of Beggs et al. Deletions were detected in 14 (52%) out of 27 DMD families by the PCR. Southern blot analysis detected deletions in 14 (64%) out of 22 families. Thirteen (93%) of the 14 DMD families with deletions detected by Southern blotting were also confirmed by the multiplex PCR. Provided care is taken in cases where the deletion is limited to a single exon, the multiplex PCR appears to be an efficient and useful alternative to conventional Southern blot analysis for detecting deletions during the prenatal and postnatal diagnosis of DMD.

  6. Direct analysis of the secretions of the potato cyst nematode Globodera rostochiensis.

    PubMed

    Robertson, L; Robertson, W M; Jones, J T

    1999-08-01

    Secretions were induced from second (invasive) stage juveniles (J2s) of the potato cyst nematode Globodera rostochiensis by exposing them to 5-methoxy-N,N-dimethyl tryptamine oxalate (DMT). Secretions were collected from J2s in sufficient quantity to allow direct analysis. Gel electrophoresis followed by monochromatic silver staining demonstrated the presence of at least 10 proteins. The presence of several enzymes, including superoxide dismutase and proteases, was demonstrated using Western blots and activity assays. Antisera raised against the secretions recognized bands on Western blots consistent in molecular mass with those identified on silver stained gels. The antisera recognized structures implicated in the production of secretions including the subventral gland cells and surface of J2s.

  7. Activation of monoamine oxidase isotypes by prolonged intake of aluminum in rat brain.

    PubMed

    Huh, Jae-Wan; Choi, Myung-Min; Lee, Jang Han; Yang, Seung-Ju; Kim, Mi Jung; Choi, Jene; Lee, Kwan Ho; Lee, Jong Eun; Cho, Sung-Woo

    2005-10-01

    Rats were fed 100 microM aluminum maltolate for one year in their drinking water. Brain aluminum contents have increased 4.2-fold in the aluminum-treated group, whereas no significant changes in the body weight, brain weight, and brain protein content were observed. Long-term aluminum feeding induced apoptosis as assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method and showed activatory effects on the catalytic efficiency (kcat/KM) of monoamine oxidase-A and monoamine oxidase-B up to 1.9- and 3.8-fold, respectively. The expression level of monoamine oxidase isotypes on the Western blot remained unchanged between the two groups, suggesting a change in post-translational regulation of the activities of monoamine oxidase isotypes by long-term aluminum feeding.

  8. Lactobacillus acidophilus Suppresses Colitis-Associated Activation of the IL-23/Th17 Axis

    PubMed Central

    Chen, Linlin; Zou, Yiyou; Peng, Jie; Lu, Fanggen; Yin, Yani; Li, Fujun; Yang, Junwen

    2015-01-01

    The aim of this paper is to determine the modulatory effects of Lactobacillus acidophilus on the IL-23/Th17 immune axis in experimental colitis. DSS-induced mouse models of UC were to be saline, hormones, and different concentrations of Lactobacillus acidophilus intervention. The expression of interleukin- (IL-) 17, tumor necrosis factor α (TNFα), IL-23, transforming growth factor β1 (TGFβ1), signal transducer and activator of transcription 3 (STAT3), and phosphorylated (p)-STAT3 was examined by RT-PCR, Western blotting, and immunohistochemical analysis. And the results showed that administration of L. acidophilus suppressed Th17 cell-mediated secretion of proinflammatory cytokine IL-17 through downregulation of IL-23 and TGFβ1 expression and downstream phosphorylation of p-STAT3. PMID:25973440

  9. Matrine attenuates focal cerebral ischemic injury by improving antioxidant activity and inhibiting apoptosis in mice

    PubMed Central

    ZHAO, PENG; ZHOU, RU; ZHU, XIAO-YUN; HAO, YIN-JU; LI, NAN; WANG, JIE; NIU, YANG; SUN, TAO; LI, YU-XIANG; YU, JIAN-QIANG

    2015-01-01

    Matrine, an active constituent of the Chinese herb, Sophora flavescens Ait., and it is known for its antioxidant, anti-inflammatory and antitumor activities. It has been demonstrated that matrine exerts protective effects against heart failure by decreasing the expression of caspase-3 and Bax, and increasing Bcl-2 levels. In this study, we aimed to determine whether these protective effects of matrine can be applied to cerebral ischemia. Following 7 successive days of treatment with matrine (7.5, 15 and 30 mg/kg) and nimodipine (1 mg/kg) by intraperitoneal injection, male Institute of Cancer Research (ICR) mice were subjected to middle cerebral artery occlusion (MCAO). Following reperfusion, the neurobehavioral score and brain infarct volume were estimated, and morphological changes were analyzed by hematoxylin and eosin (H&E) staining and electron microscopy. The percentage of apoptotic neurons was determined by flow cytometry. The levels of oxidative stress were assessed by measuring the levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and the total antioxidant capacity (T-AOC). Western blot analysis and immunofluorescence staining were used to examine the expression of the apoptosis-related proteins, caspase-3, Bax and Bcl-2. Our results revealed that pre-treatment with matrine significantly decreased the infarct volume and improved the neurological scores. Matrine also reduced the percentage of apoptotic neurons and relieved neuronal morphological damage. Furthermore, matrine markedly decreased the MDA levels, and increased SOD, GSH-Px and CAT activity, and T-AOC. Western blot analysis and immunofluorescence staining revealed a marked decrease in caspase-3 expression and an increase in the Bcl-2/Bax ratio in the group pre-treated with matrine (30 mg/kg) as compared with the vehicle-treated group. The findings of the present study demonstrate that matrine exerts neuroprotective effects against

  10. Degalactosylated/Desialylated Bovine Colostrum Induces Macrophage Phagocytic Activity Independently of Inflammatory Cytokine Production.

    PubMed

    Uto, Yoshihiro; Kawai, Tomohito; Sasaki, Toshihide; Hamada, Ken; Yamada, Hisatsugu; Kuchiike, Daisuke; Kubo, Kentaro; Inui, Toshio; Mette, Martin; Tokunaga, Ken; Hayakawa, Akio; Go, Akiteru; Oosaki, Tomohiro

    2015-08-01

    Colostrum contains antibodies, such as immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM), and, therefore, has potent immunomodulating activity. In particular, IgA has an O-linked sugar chain similar to that in the group-specific component (Gc) protein, a precursor of the Gc protein-derived macrophage-activating factor (GcMAF). In the present study, we investigated the macrophage-activating effects of degalactosylated/desialylated bovine colostrum. We detected the positive band in degalactosylated/ desialylated bovine colostrum by western blotting using Helix pomatia agglutinin lectin. We also found that degalactosylated/ desialylated bovine colostrum could significantly enhance the phagocytic activity of mouse peritoneal macrophages in vitro and of intestinal macrophages in vivo. Besides, degalactosylated/desialylated bovine colostrum did not mediate the production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). Similar to the use of GcMAF, degalactosylated/desialylated bovine colostrum can be used as a potential macrophage activator for various immunotherapies. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  11. Indeterminate HIV-1 Western Blots: Etiology, Natural History and Psychological Reactions

    DTIC Science & Technology

    1991-09-24

    All 6 seroconverters reported HIV risk behaviors , all had p24 bands prior to seroconversion, and 5 of the 6 seroconverted. within one month. HIV-1...tuberculosis or a positive skin test for TB (purified protein derivative or PPD), parity, immunization and transfusion history, past STDs, and risk behaviors ...He reported ongoing high-risk sexual behavior during the study period. Estimation of the sensitivity of the supplhmental HIIV-1 to-ts is not reliable

  12. Western blotting of high and low molecular weight proteins using heat.

    PubMed

    Kurien, Biji T; Scofield, R Hal

    2015-01-01

    A method for the electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes following sodium dodecyl sulfate (SDS) polyacrylamide gel is described here. The transfer was performed with heated (70-75 °C) normal transfer buffer from which methanol had been omitted. Complete transfer of high and low molecular weight antigens (molecular weight protein standards, a purified protein, and proteins from a human tissue extract) could be carried out in 10 min for a 7 % (0.75 mm) SDS polyacrylamide gel. For 10 and 12.5 % gels (0.75 mm) the corresponding time was 15 min. A complete transfer could be carried out in 20 min for 7, 10, and 12.5 % gels (1.5 mm gels). The permeability of the gel is increased by heat, such that the proteins trapped in the polyacrylamide gel matrix can be easily transferred to the membrane. The heat mediated transfer method was compared with a conventional transfer protocol, under similar conditions. The conventional method transferred minimal low molecular weight proteins while retaining most of the high molecular weight proteins in the gel. In summary, this procedure is particularly useful for the transfer of high molecular weight proteins, very rapid, and avoids the use of methanol.

  13. Indeterminate HIV-1 Western Blots: Etiology, Natural History, and Psychological Reactions

    DTIC Science & Technology

    1992-09-16

    cases and for a subset of the controls. The screen for other infectious diseases included serum VDRL, 7 antibody to herpes simplex types 1 and 2 by...ME.1O Skin diseases 0-no: l-seborrhea; 2-recurrent staph: 3-psoriasis: 4-vitil igo 5-other. . .. . .. ... 6-multiple ME. iP Mononucleosis 0-no: 1...correspondence: Dr. Connie Celum. Division of Infectious Diseases. Harborview Medical Center ZA-09, 325 9th Ave.. Seattle, WA clearer estimation of the risk of

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

    PubMed

    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.

  15. Increased activation of NADPH oxidase 4 in the pulmonary vasculature in experimental diaphragmatic hernia.

    PubMed

    Gosemann, Jan-H; Friedmacher, Florian; Hunziker, Manuela; Alvarez, Luis; Corcionivoschi, Nicolae; Puri, Prem

    2013-01-01

    Persistent pulmonary hypertension remains a major cause of mortality and morbidity in congenital diaphragmatic hernia (CDH). NADPH oxidases (Nox) are the main source of superoxide production in vasculature. Nox4 is highly expressed in the smooth muscle and endothelial cells of the vascular wall and increased activity has been reported in the pulmonary vasculature of both experimental and human pulmonary hypertension. Peroxisome proliferator-activated receptor (PPARγ) is a key regulator of Nox4 expression. Targeted depletion of PPARγ results in pulmonary hypertension phenotype whereas activation of PPARγ attenuates pulmonary hypertension and reduces Nox4 production. The nitrofen-induced CDH model is an established model to study the pathogenesis of pulmonary hypertension in CDH. It has been previously reported that PPARγ-signaling is disrupted during late gestation and H(2)O(2) production is increased in nitrofen-induced CDH. We designed this study to investigate the hypothesis that Nox4 expression and activation is increased and vascular PPARγ is decreased in nitrofen-induced CDH. Pregnant rats were treated with either nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into control and CDH. RT-PCR, western blotting and confocal-immunofluorescence-double-staining were performed to determine pulmonary expression levels of PPARγ, Nox4 and Nox4-activation (p22(phox)). There was a marked increase in medial and adventitial thickness in pulmonary arteries of all sizes in CDH compared to controls. Pulmonary Nox4 levels were significantly increased whereas PPARγ levels were decreased in nitrofen-induced CDH compared to controls. Western blotting revealed increased pulmonary protein expression of the Nox4-activating subunit p22(phox) and decreased protein expression of PPARγ in CDH compared to controls. Confocal-microscopy confirmed markedly increased pulmonary expression of the Nox4 activating subunit p22(phox) accompanied by

  16. Epsilon-aminocaproic acid prevents high glucose and insulin induced-invasiveness in MDA-MB-231 breast cancer cells, modulating the plasminogen activator system.

    PubMed

    Viedma-Rodríguez, Rubí; Martínez-Hernández, María Guadalupe; Flores-López, Luis Antonio; Baiza-Gutman, Luis Arturo

    2018-01-01

    Obesity and type II diabetes mellitus have contributed to the increase of breast cancer incidence worldwide. High glucose concentration promotes the proliferation of metastatic cells, favoring the activation of the plasminogen/plasmin system, thus contributing to tumor progression. The efficient formation of plasmin is dependent on the binding of plasminogen to the cell surface. We studied the effect of ε-aminocaproic acid (EACA), an inhibitor of the binding of plasminogen to cell surface, on proliferation, migration, invasion, epithelial-mesenchymal transition (EMT), and plasminogen activation system, in metastatic MDA-MB-231 breast cancer cells grown in a high glucose microenvironment and treated with insulin. MDA-MB-231 cells were treated with EACA 12.5 mmol/L under high glucose 30 mmol/L (HG) and high glucose and insulin 80 nmol/L (HG-I) conditions, evaluating: cell population growth, % of viability, migratory, and invasive abilities, as well as the expression of uPA, its receptor (uPAR), and its inhibitor (PAI-1), by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, MMP-2 and MMP-9 mRNAs were evaluated by RT-PCR. Markers of EMT were evaluated by Western blot. Additionally, the presence of active uPA was studied by gel zymography, using casein-plasminogen as substrates. EACA prevented the increase in cell population, migration and invasion induced by HG and insulin, which was associated with the inhibition of EMT and the attenuation of HG- and insulin-dependent expression of uPA, uPAR, PAI-1, MMP-2, MMP-9, α-enolase (ENO A), and HCAM. The interaction of plasminogen to the cell surface and plasmin formation are mediators of the prometastasic action of hyperglycemia and insulin, potentially, EACA can be employed in the prevention and as adjuvant treatment of breast tumorigenesis promoted by hyperglycemia and insulin.

  17. Modulation of transglutaminase 2 activity in H9c2 cells by PKC and PKA signalling: a role for transglutaminase 2 in cytoprotection

    PubMed Central

    Almami, Ibtesam; Dickenson, John M; Hargreaves, Alan J; Bonner, Philip L R

    2014-01-01

    BACKGROUND AND PURPOSE Tissue transglutaminase (TG2) has been shown to mediate cell survival in many cell types. In this study, we investigated whether the role of TG2 in cytoprotection was mediated by the activation of PKA and PKC in cardiomyocyte-like H9c2 cells. EXPERIMENTAL APPROACH H9c2 cells were extracted following stimulation with phorbol-12-myristate-13-acetate (PMA) and forskolin. Transglutaminase activity was determined using an amine incorporating and a protein crosslinking assay. The presence of TG isoforms (TG1, 2, 3) was determined using Western blot analysis. The role of TG2 in PMA- and forskolin-induced cytoprotection was investigated by monitoring H2O2-induced oxidative stress in H9c2 cells. KEY RESULTS Western blotting showed TG2 >> TG1 protein expression but no detectable TG3. The amine incorporating activity of TG2 in H9c2 cells increased in a time and concentration-dependent manner following stimulation with PMA and forskolin. PMA and forskolin-induced TG2 activity was blocked by PKC (Ro 31-8220) and PKA (KT 5720 and Rp-8-Cl-cAMPS) inhibitors respectively. The PMA- and forskolin-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Immunocytochemistry revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (β-tubulin) and novel (α-actinin) protein substrates for TG2. Pretreatment with PMA and forskolin reversed H2O2-induced decrease in MTT reduction and release of LDH. TG2 inhibitors R283 and Z-DON blocked PMA- and forskolin-induced cytoprotection. CONCLUSIONS AND IMPLICATIONS TG2 activity was stimulated via PKA- and PKC-dependent signalling pathways in H9c2 cells These results suggest a role for TG2 in cytoprotection induced by these kinases. PMID:24821315

  18. Regulation of human corneal epithelial mucins by rebamipide.

    PubMed

    Itoh, Shinsaku; Itoh, Kuni; Shinohara, Hisashi

    2014-02-01

    Membrane-associated mucins (MAMs) play important roles in barrier function and tear stability, and their expression on the ocular surface is altered in dry eye disease. Rebamipide is a mucin secretagogue that promotes the production of mucin-like glycoproteins in human corneal epithelial (HCE) cells. However, the expression of MAMs on the corneal epithelia (MUC1, MUC4, MUC16), which is induced by rebamipide, is poorly understood. In this study, we investigated the effect of rebamipide on the regulation of MAM expression in HCE cells. MUC16, Ki67 and PCNA expression levels in HCE cells isolated at confluence and at 24 hours after confluence were examined by Western blotting to assess cell proliferation. HCE cells isolated at 24 hours after confluence were cultured in medium supplemented with 1-10 µM rebamipide or 0.3-30 nM of epidermal growth factor (EGF). Real-time PCR (RT-PCR) and Western blot analysis of MAMs were performed to evaluate the effect of rebamipide. Western blot analysis of cells treated with an EGF receptor inhibitor (AG1478) or MEK1/2 inhibitor (U0126) was performed to reveal the relationship between EGF receptor activation and rebamipide-induced MAM expression. HCE cells isolated at 24 hours after confluence had lower cell proliferation activity and increased MUC16 expression compared with cells isolated at confluence. RT-PCR and Western blot analysis revealed that rebamipide increased MAM gene expression for 2 hours and protein expression for 24 hours in HCE cells. EGF inhibitor treatment led to reduced levels of all three MAMs that are normally induced by rebamipide, whereas EGF induced the expression of all three MAMs. We suggested that rebamipide increased MUC1, MUC4 and MUC16 expression levels through signals involved in EGF receptor activation in the human corneal epithelia. These data suggest that rebamipide may improve subjective symptoms of dry eye disease by upregulating MAM expression.

  19. "In Vitro" Synthesis and Activity of Reporter Proteins in an "Escherichia coli" S30 Extract System: An Undergraduate Experiment

    ERIC Educational Resources Information Center

    Higgins, Pamela J.

    2005-01-01

    This undergraduate laboratory experiment integrates multiple techniques ("in vitro" synthesis, enzyme assays, Western blotting) to determine the production and detection sensitivity of two common reporter proteins (beta-galactosidase and luciferase) within an "Escherichia coli" S30 transcription/translation extract. Comparison of the data suggests…

  20. The effect of mayfly (Hexagenia spp.) burrowing activity on sediment oxygen demand in western Lake Erie

    USGS Publications Warehouse

    Edwards, William J.; Soster, Frederick M.; Matisoff, Gerald; Schloesser, Donald W.

    2009-01-01

    Previous studies support the hypothesis that large numbers of infaunal burrow-irrigating organisms in the western basin of Lake Erie may increase significantly the sediment oxygen demand, thus enhancing the rate of hypolimnetic oxygen depletion. We conducted laboratory experiments to quantify burrow oxygen dynamics and increased oxygen demand resulting from burrow irrigation using two different year classes of Hexagenia spp. nymphs from western Lake Erie during summer, 2006. Using oxygen microelectrodes and hot film anemometry, we simultaneously determined oxygen concentrations and burrow water flow velocities. Burrow oxygen depletion rates ranged from 21.7 mg/nymph/mo for 15 mm nymphs at 23 °C to 240.7 mg/nymph/mo for 23 mm nymphs at 13 °C. Sealed microcosm experiments demonstrated that mayflies increase the rate of oxygen depletion by 2-5 times that of controls, depending on size of nymph and water temperature, with colder waters having greater impact. At natural population densities, nymph pumping activity increased total sediment oxygen demand 0.3-2.5 times compared to sediments with no mayflies and accounted for 22-71% of the total sediment oxygen demand. Extrapolating laboratory results to the natural system suggest that Hexagenia spp. populations may exert a significant control on oxygen depletion during intermittent stratification. This finding may help explain some of the fluctuations in Hexagenia spp. population densities in western Lake Erie and suggests that mayflies, by causing their own population collapse irrespective of other environmental conditions, may need longer term averages when used as a bio-indicator of the success of pollution-abatement programs in western Lake Erie and possibly throughout the Great Lakes.

  1. Saw palmetto extract suppresses insulin-like growth factor-I signaling and induces stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation in human prostate epithelial cells.

    PubMed

    Wadsworth, Teri L; Carroll, Julie M; Mallinson, Rebecca A; Roberts, Charles T; Roselli, Charles E

    2004-07-01

    A common alternative therapy for benign prostatic hyperplasia (BPH) is the extract from the fruit of saw palmetto (SPE). BPH is caused by nonmalignant growth of epithelial and stromal elements of the prostate. IGF action is important for prostate growth and development, and changes in the IGF system have been documented in BPH tissues. The main signaling pathways activated by the binding of IGF-I to the IGF-I receptor (IGF-IR) are the ERK arm of the MAPK cascade and the phosphoinositol-3-kinase (PI3K)/protein kinase B (PKB/Akt) cascade. We tested the hypothesis that SPE suppresses growth and induces apoptosis in the P69 prostate epithelial cell line by inhibiting IGF-I signaling. Treatment with 150 microg/ml SPE for 24 h decreased IGF-I-induced proliferation of P69 cells and induced cleavage of the enzyme poly(ADP-ribose)polymerase (PARP), an index of apoptosis. Treatment of serum-starved P69 cells with 150 microg/ml SPE for 6 h reduced IGF-I-induced phosphorylation of Akt (assessed by Western blot) and Akt activity (assessed by an Akt kinase assay). Western blot analysis showed that SPE reduced IGF-I-induced phosphorylation of the adapter protein insulin receptor substrate-1 and decreased downstream effects of Akt activation, including increased cyclin D1 levels and phosphorylation of glycogen synthase kinase-3 and p70(s6k). There was no effect on IGF-I-induced phosphorylation of MAPK, IGF-IR, or Shc. Treatment of starved cells with SPE alone induced phosphorylation the proapoptotic protein JNK. SPE treatment may relieve symptoms of BPH, in part, by inhibiting specific components of the IGF-I signaling pathway and inducing JNK activation, thus mediating antiproliferative and proapoptotic effects on prostate epithelia.

  2. In vitro detection of canine distemper virus nucleic acid with a virus-specific cDNA probe by dot-blot and in situ hybridization.

    PubMed

    Oglesbee, M; Jackwood, D; Perrine, K; Axthelm, M; Krakowka, S; Rice, J

    1986-11-01

    A cDNA library was prepared from canine distemper viral (CDV) messenger RNA (mRNA) derived from Vero cells lytically infected with the Onderstepoort strain (Ond) of CDV. A 300 base pair insert was identified which, by Northern blot analysis and Sanger sequence data, was shown to be specific to the nucleocapsid gene. The nucleocapsid (NC) clone was radiolabelled with 32P using nick translation and used to detect viral RNA in both dot-blot and in situ preparations of Vero cells lytically infected with Onderstepoort CDV (Ond-CDV) and immortalized mink lung cells persistently infected with racoon origin CDV (CCL64-RCDV). Dot-blot hybridization results paralleled immunofluorescent results in the lytically infected cells. In 18 persistently infected cell lines from the RCDV-CCL64 parental stock, 13 lines were positive and two were negative on both immunofluorescence and dot-blot hybridization analysis for CDV antigen and RNA, respectively. Viral nucleic acid was detected in these persistently infected cells, where as few as 1.9% of the members of a line were positive on immunofluorescence. A dot-blot autoradiographic signal was obtained in three lines which were negative for CDV antigen. CDV RNA was detected in both lytically and persistently infected cell lines by in situ hybridization, where decreasing probe length was important in increasing the sensitivity of this assay. Viral RNA was detected in over 90% of the lytically infected cells, where only 70% were positive for viral antigen by immunofluorescence.

  3. Anti-asthmatic activity of osthole in an ovalbumin-induced asthma murine model.

    PubMed

    Wang, Jingjing; Fu, Yunhe; Wei, Zhengkai; He, Xuexiu; Shi, Mingyu; Kou, Jinhua; Zhou, Ershun; Liu, Weijian; Yang, Zhengtao; Guo, Changming

    2017-05-01

    Osthole, an active coumarin extracted from the dried fruits of Cnidium monnieri (L.) Cusson, is known to possess a variety of pharmacological activities. In the present study, we investigated and illuminated the mechanisms underlying the protective effects of osthole in an experimental model of allergic asthma. Our results show that osthole treatment significantly reduced the OVA-induced increase in serum IgE and inflammatory cytokines (IL-4, IL-5, IL-13) in bronchoalveolar lavage fluid (BALF), and decreased the recruitment of inflammatory cells in BALF and the lung. It also effectively attenuated goblet cell hyperplasia and mucus overproduction in lung tissue. In addition, western blot analysis demonstrated that osthole blocked NF-κB activation, which may be associated with a reduction in inflammatory cytokine production. These data suggest that osthole attenuated OVA-induced allergic asthma inflammation by inhibiting NF-κB activation. The present study identified the molecular mechanisms of action of osthole, which support the potential pharmaceutical application of osthole treatment for asthma and other airway inflammation disorders. Copyright © 2017. Published by Elsevier B.V.

  4. Antiviral activity of polysaccharide extract from Laminaria japonica against respiratory syncytial virus.

    PubMed

    Cao, Yin-Guang; Hao, Yu; Li, Zhi-Hui; Liu, Shun-Tao; Wang, Le-Xin

    2016-12-01

    This study was designed to investigate the inhibition activity of polysaccharide extract from Laminaria japonica against RSV. The polysaccharide from Laminaria japonica was isolated by ethanol precipitation. HEK293 cells were infected with RVS, and the antiviral activity of polysaccharide extract against RSV in host cells was tested. By using ELISA and western blot assay, the expression level of IFN-α and IRF3 and their functional roles in polysaccharide-mediated antiviral activity against RSV were investigated. The polysaccharide extract from Laminaria japonica had low toxicity to HEK293 cell. The TC50 to HEK293 cells was up to 1.76mg/mL. Furthermore, the EC50 of polysaccharide extract to RSV was 5.27μg/mL, and TI was 334. The polysaccharide extract improved IRF-3 expression which promoted the level of IFN-α. Polysaccharide extract from Laminaria japonica elicits antiviral activity against RSV by up-regulation of IRF3 signaling-mediated IFN-α production. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  5. Sucrose, But Not Glucose, Blocks IL1-β-Induced Inflammatory Response in Human Chondrocytes by Inducing Autophagy via AKT/mTOR Pathway.

    PubMed

    Khan, Nazir M; Ansari, Mohammad Y; Haqqi, Tariq M

    2017-03-01

    Pathogenesis of osteoarthritis (OA) is multifactorial but interleukin-1β (IL-1β) is known to be an important mediator of cartilage degradation. Autophagy is an essential cellular homeostasis mechanism and has been proposed to protect against cartilage degradation and chondrocyte death under pathological conditions. We investigated the role of autophagy activated by sucrose, a natural disaccharide, in suppressing inflammatory mediator's expression and cell death under pathological conditions in human chondrocytes. Autophagy activation was investigated by Western blotting for LC3 and Beclin-1, immunofluorescence staining for LC3 puncta, and measuring autophagic flux. Activation of mTOR, AKT, and P70S6K was evaluated by Western blotting. Chondrocyte apoptosis was evaluated by propidium iodide (PI) staining using flowcytometry, expression of Bax by Western blotting, gene expression by TaqMan assays and caspase 3/7 activity was measured using a luminescence-based assay. We found that sucrose-induced active autophagy in OA chondrocytes in vitro was dependent on the activation of AKT/mTOR/P70S6K signaling pathways but was independent of reactive oxygen species (ROS) production. Sucrose activated autophagy blocked IL-1β-induced apoptosis and mRNA expression of MMP-13, COX-2, and IL-6 in human OA chondrocytes. Glucose or fructose, the two metabolites of sucrose, failed to induce autophagy indicating that autophagy was specifically mediated by sucrose. In conclusion, sucrose attenuated IL-1β induced apoptosis and the expression of catabolic mediators by inducing autophagy, and the autophagy in part was mediated through the activation of AKT/mTOR/P70S6K signaling pathway in human OA chondrocytes. J. Cell. Biochem. 118: 629-639, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  6. Identification and characterization of Serpulina hyodysenteriae by restriction enzyme analysis and Southern blot analysis.

    PubMed Central

    Sotiropoulos, C; Coloe, P J; Smith, S C

    1994-01-01

    Chromosomal DNA restriction enzyme analysis and Southern blot hybridization were used to characterize Serpulina hyodysenteriae strains. When chromosomal DNAs from selected strains (reference serotypes) of S. hyodysenteriae were digested with the restriction endonuclease Sau3A and hybridized with a 1.1-kb S. hyodysenteriae-specific DNA probe, a common 3-kb band was always detected in S. hyodysenteriae strains but was absent from Serpulina innocens strains. When the chromosomal DNA was digested with the restriction endonuclease Asp 700 and hybridized with two S. hyodysenteriae-specific DNA probes (0.75 and 1.1 kb of DNA), distinct hybridization patterns for each S. hyodysenteriae reference strain and the Australian isolate S. hyodysenteriae 5380 were detected. Neither the 1.1-kb nor the 0.75-kb DNA probe hybridized with Asp 700- or Sau3A-digested S. innocens chromosomal DNA. The presence of the 3-kb Sau3A DNA fragment in S. hyodysenteriae reference strains from diverse geographical locations shows that this fragment is conserved among S. hyodysenteriae strains and can be used as a species-specific marker. Restriction endonuclease analysis and Southern blot hybridization with these well-defined DNA probes are reliable and accurate methods for species-specific and strain-specific identification of S. hyodysenteriae. Images PMID:7914209

  7. Determining the Localization of Carbohydrate Active Enzymes Within Gram-Negative Bacteria.

    PubMed

    McLean, Richard; Inglis, G Douglas; Mosimann, Steven C; Uwiera, Richard R E; Abbott, D Wade

    2017-01-01

    Investigating the subcellular location of secreted proteins is valuable for illuminating their biological function. Although several bioinformatics programs currently exist to predict the destination of a trafficked protein using its signal peptide sequence, these programs have limited accuracy and often require experimental validation. Here, we present a systematic method to fractionate gram-negative cells and characterize the subcellular localization of secreted carbohydrate active enzymes (CAZymes). This method involves four parallel approaches that reveal the relative abundance of protein within the cytoplasm, periplasm, outer membrane, and extracellular environment. Cytoplasmic and periplasmic proteins are fractionated by lysis and osmotic shock, respectively. Outer membrane bound proteins are determined by comparing cells before and after exoproteolytic digestion. Extracellularly secreted proteins are collected from the media and concentrated. These four different fractionations can then be probed for the presence and quantity of target proteins using immunochemical methods such as Western blots and ELISAs, or enzyme activity assays.

  8. Boehmenan, a lignan from Hibiscus ficulneus, showed Wnt signal inhibitory activity.

    PubMed

    Shono, Takumi; Ishikawa, Naoki; Toume, Kazufumi; Arai, Midori A; Ahmed, Firoj; Sadhu, Samir K; Ishibashi, Masami

    2015-07-15

    The Wnt signal pathway modulates numerous biological processes, and its aberrant activation is related to various diseases. Therefore, inhibition of the Wnt signal may provide an effective (or efficient) strategy for these diseases. Cell-based luciferase assay targeting the Wnt signal (TOP assay) revealed that Hibiscus ficulneus extract inhibited the Wnt signal. The activity-guided isolation of the MeOH extract of H. ficulneus stems yielded four known (1-4) lignans along with myriceric acid (5). Compounds 1-4 potently inhibited the Wnt signal with TOPflash IC50 values of 1.0, 4.5, 6.3, and 1.9 μM, respectively. Compound 1 exhibited cytotoxicity against both Wnt-dependent (HCT116) and Wnt-independent (RKO) cells. Western blot analysis showed that 1 decreased the expression of full, cytosolic and nuclear β-catenin along with c-myc in STF/293 cells. Our results suggested that 1 may have inhibited the Wnt signal by decreasing β-catenin levels. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis

    DOE PAGES

    Weyman, Philip D.; Beeri, Karen; Lefebvre, Stephane C.; ...

    2014-10-10

    Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow onmore » urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.« less

  10. MULTIPLE EPISODES OF IGNEOUS ACTIVITY, MINERALIZATION, AND ALTERATION IN THE WESTERN TUSHAR MOUNTAINS, UTAH.

    USGS Publications Warehouse

    Cunningham, Charles G.; Steven, Thomas A.; Campbell, David L.; Naeser, Charles W.; Pitkin, James A.; Duval, Joseph S.

    1984-01-01

    The report outlines the complex history of igneous activity and associated alteration and mineralization in the western Tushar Mountains, Utah and pointss out implciations for minerals exploration. The area has been subjected to recurrent episodes of igneous intrusion, hydrothermal alteration, and mineralization, and the mineral-resource potential of the different mineralized areas is directly related to local geologic history. The mineral commodities to be expected vary from one hydrothermal system to another, and from one depth to another within any given system. Uranium and molybdenum seem likely to have the greatest economic potential, although significant concentrations of gold may also exist.

  11. EarthScope Transportable Array Siting Outreach Activities in Alaska and Western Canada

    NASA Astrophysics Data System (ADS)

    Gardine, L.; Dorr, P. M.; Tape, C.; McQuillan, P.; Taber, J.; West, M. E.; Busby, R. W.

    2014-12-01

    The EarthScopeTransportable Array is working to locate over 260 stations in Alaska and western Canada. In this region, new tactics and partnerships are needed to increase outreach exposure. IRIS and EarthScope are partnering with the Alaska Earthquake Center, part of University of Alaska Geophysical Institute, to spread awareness of Alaska earthquakes and the benefits of the Transportable Array for Alaskans. Nearly all parts of Alaska are tectonically active. The tectonic and seismic variability of Alaska requires focused attention at the regional level, and the remoteness and inaccessibility of most Alaska villages and towns often makes frequent visits difficult. For this reason, Alaska outreach most often occurs at community events. When a community is accessible, every opportunity to engage the residents is made. Booths at state fairs and large cultural gatherings, such as the annual convention of the Alaska Federation of Natives, are excellent venues to distribute earthquake information and to demonstrate a wide variety of educational products and web-based applications related to seismology and the Transportable Array that residents can use in their own communities. Region-specific publications have been developed to tie in a sense of place for residents of Alaska. The Alaska content for IRIS's Active Earth Monitor will emphasize the widespread tectonic and seismic features and offer not just Alaska residents, but anyone interested in Alaska, a glimpse into what is going on beneath their feet. The concerted efforts of the outreach team will have lasting effects on Alaskan understanding of the seismic hazard and tectonics of the region. Efforts to publicize the presence of the Transportable Array in Alaska, western Canada, and the Lower 48 also continue. There have been recent articles published in university, local and regional newspapers; stories appearing in national and international print and broadcast media; and documentaries produced by some of the world

  12. Interleukin-4 Ameliorates the Functional Recovery of Intracerebral Hemorrhage Through the Alternative Activation of Microglia/Macrophage.

    PubMed

    Yang, Jianjing; Ding, Saidan; Huang, Weilong; Hu, Jiangnan; Huang, Shengwei; Zhang, Yu; Zhuge, Qichuan

    2016-01-01

    Neuro-inflammation plays an important role in the recovery of brain injury after stroke. Microglia/macrophage is the major executor in the neuro-inflammation, which can be polarized into two distinct phenotypes: injurious/toxic classical activation (M1 phenotype) and protective alternative activation (M2 phenotype). Here, we investigated whether intracerebral administration of interleukin-4 (IL-4) at an early stage could affect the activation of microglia/macrophage and the corresponding outcome after intracerebral hemorrhage (ICH). The neuro-behavior was recorded between different groups in the rat ICH model. The M1 and M2 markers were then determined by qRT-PCR, western blotting, ELISA, and immunofluorescence, respectively. We observed aberrant activation of microglia/macrophage after ICH. After intracerebral injection of IL-4, M1 activation was greatly inhibited while M2 activation was enhanced, along with improving neurobehavioral recovery from deficits after ICH. Our study showed that early intracerebral injection of IL-4 potentially promotes neuro-functional recovery, probably through enhancing the alternative activation of microglia/macrophage.

  13. Short term exposure to elevated levels of leptin reduces proximal tubule cell metabolic activity.

    PubMed

    Briffa, Jessica F; Grinfeld, Esther; McAinch, Andrew J; Poronnik, Philip; Hryciw, Deanne H

    2014-01-25

    Leptin plays a pathophysiological role in the kidney, however, its acute effects on the proximal tubule cells (PTCs) are unknown. In opossum kidney (OK) cells in vitro, Western blot analysis identified that exposure to leptin increases the phosphorylation of the mitogen-activated protein kinase (MAPK) p44/42 and the mammalian target of rapamycin (mTOR). Importantly leptin (0.05, 0.10, 0.25 and 0.50 μg/ml) significantly reduced the metabolic activity of PTCs, and significantly decreased protein content per cell. Investigation of the role of p44/42 and mTOR on metabolic activity and protein content per cell, demonstrated that in the presence of MAPK inhibitor U0126 and mTOR inhibitor Ku-63794, that the mTOR pathway is responsible for the reduction in PTC metabolic activity in response to leptin. However, p44/42 and mTOR play no role the reduced protein content per cell in OKs exposed to leptin. Therefore, leptin modulates metabolic activity in PTCs via an mTOR regulated pathway. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  14. Influence of human activity patterns on epidemiology of plague in Western Usambara Mountains, Tanzania.

    PubMed

    Hubeau, Marianne; Gulinck, Hubert; Kimaro, Didas N; Hieronimo, Proches; Meliyo, Joel

    2014-07-01

    Human plague has been a recurring public health threat in some villages in the Western Usambara Mountains, Tanzania, in the period between 1980 and 2004. Despite intensive past biological and medical research, the reasons for the plague outbreaks in the same set of villages remain unknown. Plague research needs to broaden its scope and formulate new hypotheses. This study was carried out to establish relationships between the nature and the spatial extent of selected human activities on one hand, and the reported plague cases on the other hand. Three outdoor activities namely, fetching water, collecting firewood and going to the market, were selected. Through enquiries the activity patterns related to these activities were mapped in 14 villages. Standard deviation ellipses represent the extent of action spaces. Over 130 activity types were identified and listed. Of these, fetching water, collecting firewood and going to the market were used for further analysis. The results indicate a significant correlation between the plague frequency and the size of these action spaces. Different characteristics of land use and related human activities were correlated with the plague frequency at village and hamlet levels. Significant relationships were found between plague frequency and specific sources of firewood and water, and specific market places.

  15. Pirfenidone Inhibits Proliferation and Promotes Apoptosis of Hepatocellular Carcinoma Cells by Inhibiting the Wnt/β-Catenin Signaling Pathway.

    PubMed

    Zou, Wei-Jie; Huang, Zhi; Jiang, Tian-Peng; Shen, Ya-Ping; Zhao, An-Su; Zhou, Shi; Zhang, Shuai

    2017-12-25

    BACKGROUND Hepatocellular carcinoma (HCC) is the most important cause of cancer-related deaths worldwide. Pirfenidone is an orally available small molecule with therapeutic potential for fibrotic diseases. MATERIAL AND METHODS In this study, we analyzed the effects of different pirfenidone concentrations on the proliferation of HepG2 HCC cells using Cell Counting Kit-8 (CCK-8) and colony formation assays. Flow cytometry was performed to measure the apoptotic effects of pirfenidone on HepG2 cells. Western blot analysis was performed to detect the expression of β-catenin and p-β-catenin. RESULTS Pirfenidone inhibited proliferation and promoted HepG2 cell apoptosis. In addition, Western blot results indicated that pirfenidone suppressed b-catenin expression in HepG2 cells. To assess the mechanism, we treated HepG2 cells with pirfenidone, and pirfenidone plus the β-catenin activator, SB-216763. The results revealed that SB-216763 accelerated proliferation and inhibited apoptosis in HepG2 cells treated with pirfenidone. Western blot results showed that SB-216763 upregulated β-catenin expression in HepG2 cells treated with pirfenidone. CONCLUSIONS In conclusions, pirfenidone may be a potential drug for HCC treatment.

  16. Chronic intermittent hypoxia induces changes on the expression and activity of neprilysin (EC 3.4.24.11) in the brain of rats.

    PubMed

    de Oliveira, Renato W; Julian, Guilherme S; Perry, Juliana C; Tufik, Sergio; Chagas, Jair R

    2018-06-21

    Obstructive sleep apnea (OSA) is a frequent sleeping breathing disorder associated with cognitive impairments. Neprilysin (NEP) is responsible for degrading several substrates related to cognition; however, the effect of chronic intermittent hypoxia (CIH) on NEP is still unknown. This study aimed to evaluate the expression and activity of NEP in cognitive-related brain structures of rats submitted to CIH. Western blot, qRT-PCR and enzyme activity assay, demonstrated that a six-week intermittent hypoxia increased NEP expression and activity, selectively in temporal cortex, but not in the hippocampus and frontal cortex. The increase in NEP activity and expression was reverted followed by two weeks recovery in normoxia. These data show that CIH protocol increases the expression and activity of NEP selectively in the temporal cortex. Additional mechanisms must be investigated to elucidate the effects of CIH in cognition. Copyright © 2018 Elsevier B.V. All rights reserved.

  17. BAG3 protects against hyperthermic stress by modulating NF-κB and ERK activities in human retinoblastoma cells.

    PubMed

    Yunoki, Tatsuya; Tabuchi, Yoshiaki; Hayashi, Atsushi; Kondo, Takashi

    2015-03-01

    BCL2-associated athanogene 3 (BAG3), a co-chaperone of HSP70, is a cytoprotective and anti-apoptotic protein that acts against various stresses, including heat stress. Here, we examined the effect of BAG3 on the sensitivity of human retinoblastoma cells to hyperthermia (HT). We examined the effects of BAG3 knockdown on the sensitivity of Y79 and WERI-Rb-1cells to HT (44 °C, 1 h) by evaluating apoptosis and cell proliferation using western blotting, real-time quantitative PCR (qPCR), flow cytometry, and a WST-8 assay kit. Furthermore, we examined the effects of activating nuclear factor-kappa B (NF-κB) and extracellular signal-regulated kinase (ERK) using western blotting and real time qPCR. HT induced considerable apoptosis along with the activation of caspase-3 and chromatin condensation. The sensitivity of Y79 and WERI-Rb-1 cells to HT was significantly enhanced by BAG3 knockdown. Compared to HT alone, the combination of BAG3 knockdown and HT reduced phosphorylation of the inhibitors of kappa B α (IκBα) and p65, a subunit of NF-κB, and degraded IκB kinase γ (IKKγ) during the recovery period after HT. Furthermore, BAG3 knockdown increased the HT-induced phosphorylation of ERK after HT treatment, and the ERK inhibitor U0126 significantly improved the viability of the cells treated with a combination of BAG3 knockdown and HT. The silencing of BAG3 seems to enhance the effects of HT, at least in part, by maintaining HT-induced inactivity of NF-κB and the phosphorylation of ERK. These findings indicate that BAG3 may be a potential molecular target for modifying the outcomes of HT in retinoblastoma.

  18. EGb-761 prevents ultraviolet B-induced photoaging via inactivation of mitogen-activated protein kinases and proinflammatory cytokine expression.

    PubMed

    Chen, Chih-Chiang; Chiang, An-Na; Liu, Han-Nan; Chang, Yun-Ting

    2014-07-01

    EGb-761 is an antioxidant and anticarcinogen; however, its role as a photoprotector remains unknown. To determine whether EGb-761 photoprotects human dermal fibroblasts and BALB/c mice skin against ultraviolet B (UVB) light irradiation. To simulate chronic photodamage, shaved BALB/c mice were exposed to UVB irradiation (90mJ/cm(2)) thrice weekly for 3 months. EGb-761 (2mg/cm(2)) was topically applied 1h before irradiation to evaluate its effect. The mechanisms by which EGb-761 protects the skin from photodamage were evaluated by immunohistochemical analysis, enzyme-linked immunosorbent assay (ELISA), and Western blotting. In BALB/c mice, the signs of photoaging or photodamage, such as coarse wrinkle formation, epidermal hyperplasia, and elastic fiber degeneration, markedly reduced with the topical application of EGb-761. Western blot and ELISA revealed that the activation of MMP-1 in cultured fibroblasts markedly diminished after pretreatment with EGb-761. In addition, EGb-761 inhibited UVB-induced overexpression by the fibroblasts of the proinflammatory cytokines, such as interleukin (IL)-1α, IL-1β, IL-6, and tumor necrosis factor-α. The phosphorylation of the mitogen-activated protein kinase (MAPK) signal transduction pathway components, including extracellular signal-regulated kinase, C-Jun N-terminal kinase, and p38, which are induced by UV irradiation, was significantly inhibited in vivo and in vitro. EGb-761 also diminished the generation of UVB-induced reactive oxygen species (ROS). EGb-761 photoprotects mice and cultured fibroblasts, inhibits the UVB-induced phosphorylation of MAPK pathway components, and reduces the expression of the proinflammatory cytokines by suppressing ROS generation. Thus, topically applied EGb-761 may be a promising photoprotective agent. Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  19. A novel mAb against a human CD34 peptide reacts with the native protein on CD34+ cells.

    PubMed

    Shams, Mahmood; Jeddi-Tehrani, Mahmood; Notash Haghighat, Farzaneh; Bayat, Ali Ahmad; Mahmoudian, Jafar; Rezvani, Mohammad Reza

    2013-12-01

    ‎Human CD34 is a transmembrane glycoprotein which is expressed in human hematopoietic stem ‎cells (HSCs) and the small-‎vessel endothelial cells of a variety of tissues. CD34 plays a critical role as a ‎marker for diagnosis ‎and classification of leukemia. Anti CD34 antibodies are used for isolation and ‎purification ‎of HSCs from bone marrow, peripheral blood and cord blood. To characterize a newly produced monoclonal antibody against a human CD34 peptide. Anti CD34 monoclonal antibody (Clone 2C10-D3) was purified from mouse ascitic fluid and hybridoma cell culture supernatants by affinity chromatography and its immune reactivity was examined by ELISA. The purified antibody was further characterized using Western blot and flow cytometry on TF1 (Human Erythroblast) cell line. ‎ELISA experiment revealed that the antibody recognized CD34 peptide. Westernblot analysis on TF1 ‎cell lysate confirmed the reactivity of the antibody with a 42 KDa protein. Blocking the antibody with a saturating concentration of specific CD34 peptide resulted in loss of its activity with TF1 lysate in Western blot. The 2C10-D3 antibody reacted with TF1 cells in flow cytometry in a similar manner to a commercial anti CD34 monoclonal antibody.‎ ‎Our data suggest that the anti CD34 monoclonal antibody (Clone 2C10-D3) is an appropriate antibody to study the CD34+ cells by flow cytometry and Western blot.

  20. Attenuation of MUC4 potentiates the anticancer activity of auranofin via regulation of the Her2/Akt/FOXO3 pathway in ovarian cancer cells.

    PubMed

    Bae, Jun Sang; Lee, Jongsung; Park, Yoonkook; Park, Kyungmoon; Kim, Jung Ryul; Cho, Dong Hyu; Jang, Kyu Yun; Park, See-Hyoung

    2017-10-01

    Previously, we reported that auranofin induces apoptosis in SKOV3 cells via regulation of the IKKβ/FOXO3 pathway. In the present study, we reveal that the anticancer activity of auranofin in SKOV3 cells could be enhanced by the attenuation of MUC4 through the regulation of the Her2/Akt/FOXO3 pathway. Compared to the control-siRNA, siRNA transfection against MUC4 into SKOV3 cells accelerated the protein degradation of Her2. Under the same conditions, the expression level of phosphorylated Akt was also downregulated leading to an increase of FOXO3 in the nucleus. Notably, auranofin treatment in SKOV3 cells also resulted in the downregulation of the expression levels of both Her2 and phosphorylated Akt. Thus, Her2 was identified as the common molecular target protein by siRNA transfection against MUC4. Western blot analysis of total and nuclear fraction lysates from SKOV3 cells revealed that attenuation of MUC4 combined with auranofin treatment in SKOV3 cells synergistically activated FOXO3 translocation from the cytoplasm to the nucleus through the regulation of the Her2/Akt/FOXO3 pathway. Attenuation of MUC4 by siRNA transfection potentiated the antitumor effect of auranofin which was examined by performing in vitro assays such as WST-1, cell counting, colony formation, TUNEL and Annexin V staining. In addition, western blot analysis of the apoptosis‑related proteins such as PARP1, caspase-3, Bim extra large (EL), Bax and Bcl2 revealed that the attenuation of MUC4 by siRNA transfection potentiates the pro-apoptotic activity of auranofin in SKOV3 cells. Collectively, auranofin could regulate the Her2/Akt/FOXO3 signaling pathway in SKOV3 cells and be used as a potential antitumor agent considering the expression of MUC4 in ovarian cancer patients.

  1. Cytotoxic and apoptosis-inducing activity of C21 steroids from the roots of Cynanchum atratum.

    PubMed

    Zhang, Jian; Ma, Lin; Wu, Zheng-Feng; Yu, Shu-Le; Wang, Lei; Ye, Wen-Cai; Zhang, Qing-Wen; Yin, Zhi-Qi

    2017-06-01

    Two new (1-2) and two known C 21 steroids (3-4) were isolated from the roots of Cynanchum atratum. Their structures were elucidated by detailed 1D and 2D spectroscopic. The MTT assay showed that compounds 1-4 displayed obvious cytotoxic activities against HepG2 cells with IC 50 values ranging from 10.19μM to 76.12μM. Compounds 1-3 also exhibited cytotoxic effects in A549 cells with IC 50 values of 30.87-95.39μM. Compound 3 showed the antiproliferative activity via G0/G1 cell cycle arrest and proapoptosis in HepG2 cells by Flowcytometry analysis. Western blotting analysis revealed that compound 3 could induce HepG2 cell apoptosis via the mitochondrial pathway by downregulating Bcl-2 expression, upregulating Bax protein expression, and activating caspase-9 and caspase-3. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Taurine Protects Mouse Spermatocytes from Ionizing Radiation-Induced Damage Through Activation of Nrf2/HO-1 Signaling.

    PubMed

    Yang, Wenjun; Huang, Jinfeng; Xiao, Bang; Liu, Yan; Zhu, Yiqing; Wang, Fang; Sun, Shuhan

    2017-01-01

    The increasing prevalence of ionizing radiation exposure has inevitably raised public concern over the potential detrimental effects of ionizing radiation on male reproductive system function. The detection of drug candidates to prevent reproductive system from damage caused by ionizing radiation is urgent. We aimed to investigate the protective role of taurine on the injury of mouse spermatocyte-derived cells (GC-2) subjected to ionizing radiation. mouse spermatocytes (GC-2 cells) were exposed to ionizing radiation with or without treatment of Taurine. The effect of ionizing radiation and Taurine treatment on GC-2 cells were evaluated by cell viability assay (CCK8), cell cycle and apoptosis. The relative protein abundance change was determined by Western blotting. The siRNA was used to explore whether Nrf2 signaling was involved in the cytoprotection of Taurine. Taurine significantly inhibited the decrease of cell viability, percentage of apoptotic cells and cell cycle arrest induced by ionizing radiation. Western blot analysis showed that taurine significantly limited the ionizing radiation-induced down-regulation of CyclinB1 and CDK1, and suppressed activation of Fas/FasL system pathway. In addition, taurine treatment significantly increased the expression of Nrf2 and HO-1 in GC-2 cells exposed to ionizing radiation, two components in antioxidant pathway. The above cytoprotection of Taurine was blocked by siNrf2. Our results demonstrate that taurine has the potential to effectively protect GC-2 cells from ionizing radiation- triggered damage via upregulation of Nrf2/HO-1 signaling. © 2017 The Author(s). Published by S. Karger AG, Basel.

  3. Monoclonal Antibodies against Small Molecule Natural Products and Their Applications, Eastern Blotting and Knockout Extract

    PubMed Central

    Shoyama, Yukihiro

    2011-01-01

    To determine the hapten number in hapten-carrier protein conjugate matrix-assisted laser desorption/ionization (MALDI) tof mass spectrometry was applied. Highly specific anti-ginsenoside Rb1 and Rg1 monoclonal antibodies (MAbs) were prepared. Ginsenosides were developed on thin layer chromatography (TLC) plates which were covered by a polyvinylidene difluoride (PVDF) membrane resulting in blotting. The membrane was treated with NaIO4 solution to release the aldehyde group on the sugar moiety of the ginsenosides. By treatment of the membrane with a protein solution the ginsenoside-protein conjugation as a Schiff-base occurred, which can function to fix it to the PVDF membrane. A part of the ginsenoside aglycone was reacted with anti-ginsenoside Rb1 MAb, secondary MAb conjugated with enzyme and finally a substrate was added, resulting in a specific and highly sensitive staining that we named Eastern blotting. Furthermore, it makes one-step isolation of ginsenoside Rb1 possible using an immuno-affinity column conjugated with anti-ginsenoside Rb1 MAb. Furthermore, immunoaffinity concentration was carried out allowing high sensitivity analysis of lower concentrations of ginsenoside Rb1 so that several unknown bands could be structurally determined.

  4. Myo-inositol reduces β-catenin activation in colitis.

    PubMed

    Bradford, Emily M; Thompson, Corey A; Goretsky, Tatiana; Yang, Guang-Yu; Rodriguez, Luz M; Li, Linheng; Barrett, Terrence A

    2017-07-28

    To assess dietary myo-inositol in reducing stem cell activation in colitis, and validate pβ-catenin S552 as a biomarker of recurrent dysplasia. We examined the effects of dietary myo-inositol treatment on inflammation, pβ-catenin S552 and pAkt levels by histology and western blot in IL-10 -/- and dextran sodium sulfate-treated colitic mice. Additionally, we assessed nuclear pβ-catenin S552 in patients treated with myo-inositol in a clinical trial, and in patients with and without a history of colitis-induced dysplasia. In mice, pβ-catenin S552 staining faithfully reported the effects of myo-inositol in reducing inflammation and intestinal stem cell activation. In a pilot clinical trial of myo-inositol administration in patients with a history of low grade dysplasia (LGD), two patients had reduced numbers of intestinal stem cell activation compared to the placebo control patient. In humans, pβ-catenin S552 staining discriminated ulcerative colitis patients with a history of LGD from those with benign disease. Enumerating crypts with increased numbers of pβ-catenin S552 - positive cells can be utilized as a biomarker in colitis-associated cancer chemoprevention trials.

  5. Activation of PKC isoform beta(I) at the blood-brain barrier rapidly decreases P-glycoprotein activity and enhances drug delivery to the brain.

    PubMed

    Rigor, Robert R; Hawkins, Brian T; Miller, David S

    2010-07-01

    P-glycoprotein is an ATP (adenosine triphosphate)-driven drug efflux transporter that is highly expressed at the blood-brain barrier (BBB) and is a major obstacle to the pharmacotherapy of central nervous system diseases, including brain tumors, neuro-AIDS, and epilepsy. Previous studies have shown that P-glycoprotein transport activity in rat brain capillaries is rapidly reduced by the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha) acting through protein kinase C (PKC)-dependent signaling. In this study, we used isolated rat brain capillaries to show that the TNF-alpha-induced reduction of P-glycoprotein activity was prevented by a PKCbeta(I/II) inhibitor, LY333531, and mimicked by a PKCbeta(I/II) activator, 12-deoxyphorbol-13-phenylacetate-20-acetate (dPPA). Western blotting of brain capillary extracts with phospho-specific antibodies showed that dPPA activated PKCbeta(I), but not PKCbeta(II). Moreover, in intact rats, intracarotid infusion of dPPA potently increased brain accumulation of the P-glycoprotein substrate, [(3)H]-verapamil without compromising tight junction integrity. Thus, PKCbeta(I) activation selectively reduced P-glycoprotein activity both in vitro and in vivo. Targeting PKCbeta(I) at the BBB may prove to be an effective strategy for enhancing the delivery of small molecule therapeutics to the brain.

  6. Changes on lipid peroxidation,enzymatic activities and gene expression in planarian (Dugesia japonica) following exposure to perfluorooctanoic acid.

    PubMed

    Yuan, Zuoqing; Miao, Zili; Gong, Xiaoning; Zhao, Baoying; Zhang, Yuanyuan; Ma, Hongdou; Zhang, Jianyong; Zhao, Bosheng

    2017-11-01

    We investigated perfluorooctanoic acid (PFOA)-induced stress response in planarians. We administered different concentrations of PFOA to planarians for up to 10 d. PFOA exposure resulted in significant concentration-dependent elevations in lipid peroxidation, glutathione S-transferase and caspase-3 protease activities, and a significant decline in glutathione peroxidase activities compared with control groups. Exposure to PFOA significantly up-regulated the heat shock proteins hsp70 and hsp90, and p53, and down-regulated hsp40 compared with controls. PFOA exposure also increased HSP70 protein levels, as demonstrated by western blot analysis. These alterations indicated that PFOA exposure induced a stress response and affected the regulation of oxidative stress, enzymatic activities and gene expression. These results suggest that these sensitive parameters, together with other biomarkers, could be used for evaluating toxicity, for ecological risk assessment of PFOA in freshwaters. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. MiR-21 suppresses the anticancer activities of curcumin by targeting PTEN gene in human non-small cell lung cancer A549 cells.

    PubMed

    Zhang, W; Bai, W; Zhang, W

    2014-08-01

    Curcumin, a natural phytochemical, exhibits potent anticancer activities. Here, we sought to determine the molecular mechanisms underlying the cytotoxic effects of curcumin against human non-small cell lung cancer (NSCLC) cells. MTT assay and annexin-V/PI staining were used to analyze the effects of curcumin on the proliferation and apoptosis of A549 cells. The expression of microRNA-21 in curcumin-treated A549 cells was measured by quantitative real-time polymerase chain reaction assay. The protein level of phosphatase and tensin homolog (PTEN), a putative target of microRNA-21, was determined by Western blot analysis. Transfection of A549 cells with microRNA-21 mimic or PTEN small interfering RNA was performed to modulate the expression of microRNA-21 and PTEN under the treatment of curcumin. Curcumin at 20-40 μM inhibited cell proliferation and induced apoptosis in A549 cells. Curcumin treatment produced a dose-dependent and significant (P < 0.05) suppression of microRNA-21 expression, compared to untreated A549 cells. Moreover, the protein level of PTEN, a putative target of microRNA-21, was significantly elevated in curcumin-treated A549 cells, as determined by Western blot analysis. Transfection of A549 cells with microRNA-21 mimic or PTEN small interfering RNA significantly (P < 0.05) reversed the growth suppression and apoptosis induction by curcumin, compared to corresponding controls. Our data suggest a novel molecular mechanism in which inhibition of microRNA-21 and upregulation of PTEN mediate the anticancer activities of curcumin in NSCLC cells. Suppression of microRNA-21 may thus have therapeutic benefits against this malignancy.

  8. Structural characterization and immunomodulating activity of polysaccharide from Dendrobium officinale.

    PubMed

    He, Tao-Bin; Huang, Yan-Ping; Yang, Liu; Liu, Ti-Ti; Gong, Wan-Ying; Wang, Xuan-Jun; Sheng, Jun; Hu, Jiang-Miao

    2016-02-01

    A neutral heteropolysaccharide (DOP-1-1) consisted by mannose and glucose (5.9:1) with an average molecular weight at about 1.78×10(5) Da was purified from Dendrobium officinale. Based on Fourier transform infrared spectrum (FT-IR) and nuclear magnetic resonance (NMR) spectra, it suggested that partial structure of DOP-1-1 is an O-acetylated glucomannan with β-d configuration in pyranose sugar forms. The immunomodulatory activity of DOP-1-1 was evaluated by secretion level of cytokine (interleukin (IL)-1β and IL-10) and tumor necrosis factor (TNF)-α in vitro. Our results suggested that DOP-1-1 could stimulate cytokine production (TNF-α, IL-1β) in cells. These findings demonstrated that the purified polysaccharide from D. officinale presented significant immune-modulating activities. Furthermore, by Western-blot we can found that the signaling pathways of DOP-1-1 induced immune activities involving ERK1/2 and NF-кB. As to antioxidant activity, DOP-1-1 hadn't showed remarkable scavenging capacity of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) in contrast with other studies of polysaccharides from D. officinale. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Activation of histamine H4 receptor inhibits TNFα/IMD-0354-induced apoptosis in human salivary NS-SV-AC cells.

    PubMed

    Stegajev, Vasili; Kouri, Vesa-Petteri; Salem, Abdelhakim; Rozov, Stanislav; Stark, Holger; Nordström, Dan C E; Konttinen, Yrjö T

    2014-12-01

    Apoptosis is involved in the pathogenesis of Sjögren's syndrome (SS), an autoimmune disease affecting exocrine glands. Our recent studies revealed diminished histamine H4 receptor (H₄R) expression and impaired histamine transport in the salivary gland epithelial cells in SS. The aim was now to test if nanomolar histamine and high-affinity H₄R signaling affect apoptosis of human salivary gland epithelial cell. Simian virus 40-immortalized acinar NS-SV-AC cells were cultured in serum-free keratinocyte medium ± histamine H₄R agonist HST-10. Expression and internalization of H₄R were studied by immunofluorescence staining ± clathrin inhibitor methyl-β-cyclodextrin (MβCD). Apoptosis induced using tumor necrosis factor-α with nuclear factor-κB inhibitor IMD-0354 was studied using phase contrast microscopy, Western blot, flow cytometry and polymerase chain reaction (qRT-PCR). HST-10-stimulated H₄R internalization was inhibited by MβCD. Western blotting revealed diminished phosphorylated c-Jun N-terminal kinase JNK, but unchanged levels of phosphorylated extracellular signal regulated kinase pERK1/2 in H₄R-stimulated samples compared to controls. qRT-PCR showed up-regulated expression of anti-apoptotic B cell lymphoma-extra large/Bcl-xL mRNAs and proteins, whereas pro-apoptotic Bcl-2-associated X protein/BAX remained unchanged in H4R-stimulated samples. H₄R stimulation diminished cleavage of PARP and flow cytometry showed significant dose-dependent inhibitory effect of H₄R stimulation on apoptosis. As far as we know this is the first study showing inhibitory effect of H₄R activation on apoptosis of human salivary gland cells. Diminished H₄R-mediated activation may contribute to loss of immune tolerance in autoimmune diseases and in SS in particular.

  10. Synthesis, Protein Levels, Activity and Phosphorylation State of Tyrosine Hydroxylase in Mesoaccumbens and Nigrostriatal Dopamine Pathways of Chronically Food-restricted Rats

    PubMed Central

    Pan, Yan; Berman, Yemiliya; Haberny, Sandra; Meller, Emanuel; Carr, Kenneth D.

    2006-01-01

    Chronic food restriction (FR) enhances the rewarding and motor-activating effects of abused drugs, and is accompanied by changes in dopamine (DA) dynamics and increased D-1 DA receptor-mediated cell signaling and transcriptional responses in nucleus accumbens (NAc). However, little is known about effects of FR on DA synthetic activity in the mesoaccumbens and nigrostriatal pathways. In Experiment 1 of the present study, tyrosine hydroxylase (TH) gene expression was measured in ventral tegmental area and substantia nigra, using real time RT-PCR and in situ hybridization; no differences were observed between FR and ad libitum fed (AL) rats. In Experiment 2, TH protein levels, determined by Western blot, were found to be elevated in NAc and caudate-putamen (CPu) of FR relative to AL rats. In the absence of increased transcription, this may reflect a slowing of TH degradation. In Experiments 3 and 4, DA synthetic activity was assessed by Western blot measurement of TH phosphorylation at Ser-40, and HPLC measurement of in vivo tyrosine hydroxylation rate, as reflected by DOPA accumulation following administration of a decarboxylase inhibitor (NSD-1015; 100 mg/kg, i.p.). Basal phospho-Ser(40)-TH levels did not differ between groups but DOPA accumulation was decreased by FR. Decreased DOPA synthesis, despite increased levels of TH protein, may reflect the inhibitory effect of increased DA binding to TH protein or decreased concentrations of cofactor tetrahydrobiopterin. Finally, in response to d-amphetamine (0.5 and 5.0 mg/kg, i.p.), phospho-Ser(40)-TH was selectively decreased in NAc of FR rats. This suggests increased feedback inhibition of DA synthesis - a possible consequence of postsynaptic receptor hypersensitivity, or increased extracellular DA concentration. These results indicate that FR increases TH protein levels, but may decrease the capacity for DA synthesis by decreasing TH activity. According to this scheme, the previously observed upregulation of striatal

  11. Cardiac-specific overexpression of insulin-like growth factor I (IGF-1) rescues lipopolysaccharide-induced cardiac dysfunction and activation of stress signaling in murine cardiomyocytes.

    PubMed

    Zhao, Peng; Turdi, Subat; Dong, Feng; Xiao, Xiaoyan; Su, Guohai; Zhu, Xinglei; Scott, Glenda I; Ren, Jun

    2009-07-01

    Lipopolysaccharide (LPS), a component of the outer membrane of Gram-negative bacteria, plays a key role in cardiac dysfunction in sepsis. Low circulating levels of insulin-like growth factor 1 (IGF-1) are found in sepsis, although the influence of IGF-1 on septic cardiac defect is unknown. This study was designed to examine the impact of IGF-1 on LPS-induced cardiac contractile and intracellular Ca2+ dysfunction, activation of stress signal and endoplasmic reticulum (ER) stress. Mechanical and intracellular Ca2+ properties were examined in cardiomyocytes from Fast Violet B and cardiac-specific IGF-1 overexpression mice treated with or without LPS (4 mg kg(-1), 6 h). Reactive oxygen species (ROS), protein carbonyl formation and apoptosis were measured. Activation of mitogen-activated protein kinase pathways (p38, c-jun N-terminal kinase [JNK] and extracellular signal-related kinase [ERK]), ER stress and apoptotic markers were evaluated using Western blot analysis. Our results revealed decreased peak shortening and maximal velocity of shortening/relengthening and prolonged duration of relengthening in LPS-treated Fast Violet B cardiomyocytes associated with reduced intracellular Ca2+ decay. Accumulation of ROS protein carbonyl and apoptosis were elevated after LPS treatment. Western blot analysis revealed activated p38 and JNK, up-regulated Bax, and the ER stress markers GRP78 and Gadd153 in LPS-treated mouse hearts without any change in ERK and Bcl-2. Total protein expression of p38, JNK, and ERK was unaffected by either LPS or IGF-1. Interestingly, these LPS-induced changes in mechanical and intracellular Ca2+ properties, ROS, protein carbonyl, apoptosis, stress signal activation, and ER stress markers were effectively ablated by IGF-1. In vitro LPS exposure (1 microg mL(-1)) produced cardiomyocyte mechanical dysfunction reminiscent of the in vivo setting, which was alleviated by exogenous IGF-1 (50 nM). These data collectively suggested a beneficial of IGF-1 in

  12. Effects of antioxidant components of AREDS vitamins and zinc ions on endothelial cell activation: implications for macular degeneration.

    PubMed

    Zeng, Shemin; Hernández, Jasmine; Mullins, Robert F

    2012-02-01

    To investigate whether the benefit of Age-Related Eye Disease Study (AREDS) formula multivitamins and zinc in the progression of age-related macular degeneration (AMD) may occur through inhibiting inflammatory events in the choroid. Mouse C166 endothelial cells (ECs) and, for some experiments, human retinal pigment epithelium (RPE)-choroid organ cultures were treated with AREDS multivitamin solution (MVS) or ZnCl(2). The cytotoxicity of MVS was evaluated using a lactate dehydrogenase colorimetric assay. Cell motility was assessed using a scratch assay. Macrophage adhesion to EC monolayers or ICAM-1 protein was determined after MVS and zinc treatment and with or without lipopolysaccharide (LPS). Quantitative reverse transcription PCR and Western blot analysis were used to determine the effects of MVS on the expression of proinflammatory molecules in treated and untreated cells. AREDS MVS and zinc did not affect C166 EC viability until the 56th hour after treatment. Scratch assays showed partial inhibition of MVS and zinc on EC migration. In cell adhesion assays, MVS and zinc decreased the number of macrophages bound to EC and to ICAM-1 protein. Quantitative PCR showed that LPS increased the expression of ICAM-1 in both C166 and human RPE-choroid cultures, which was partially offset by MVS and zinc. MVS and zinc also mitigated LPS-induced ICAM-1 protein expression on Western blot analysis. Treatment with AREDS MVS and zinc may affect both angiogenesis and endothelial-macrophage interactions. These results suggest that AREDS vitamins and zinc ions may slow the progression of AMD, in part through the attenuation of EC activation.

  13. Can Native and Western Wildlife Managers Find Common Ground?

    ERIC Educational Resources Information Center

    Ambler, Marjane

    1996-01-01

    Examines different approaches to environmental activism taken by Native Americans and Westerners as a result of their differing views of nature. Describes historical Western-Indian conflicts related to the preservation of bison in Yellowstone Park. Suggests that there is a growing acknowledgment in the scientific community of traditional…

  14. Progress, opportunities, and key fields for groundwater quality research under the impacts of human activities in China with a special focus on western China.

    PubMed

    Li, Peiyue; Tian, Rui; Xue, Chenyang; Wu, Jianhua

    2017-05-01

    Groundwater quality research is extremely important for supporting the safety of the water supply and human health in arid and semi-arid areas of China. This review article was constructed to report the latest research progress of groundwater quality in western China where groundwater quality is undergoing fast deterioration because of fast economic development and extensive anthropogenic activities. The opportunities brought by increasing public awareness of groundwater quality protection were also highlighted and discussed. To guide and promote further development of groundwater quality research in China, especially in western China, ten key groundwater quality research fields were proposed. The review shows that the intensification of human activities and the associated impacts on groundwater quality in China, especially in western China, has made groundwater quality research increasingly important, and has caught the attention of local, national, and international agencies and scholars. China has achieved some progress in groundwater quality research in terms of national and regional laws, regulations, and financial supports. The future of groundwater quality research in China, especially in western China, is promising reflected by the opportunities highlighted. The key research fields proposed in this article may also inform groundwater quality protection and management at the national and international level.

  15. Influence of thyroid disorders on the kidney expression and plasma activity of aminopeptidase A.

    PubMed

    Wangensteen, R; Segarra, A B; Ramirez-Sanchez, M; Gasparo, M De; Dominguez, G; Banegas, I; Vargas, F; Vives, F; Prieto, I

    2015-04-01

    Thyroid disorders may affect blood pressure and renal function modifying factors of the plasmatic and kidney renin-angiotensin system such as aminopeptidase A (AP A) that metabolizes angiotensin II to angiotensin III. We investigated the expression of AP A in the kidney, as well as its enzymatic activity in the plasma of euthyroid, hyperthyroid, and hypothyroid adult male rats. Hyperthyroidism was induced by daily subcutaneous injections of tetraiodothyronine. Hypothyroid rats were obtained by administration of methimazole in drinking water. Expression of AP A was determined by Western blot analysis. Plasma AP A activity was measured fluorometrically using glutamyl-β-naphthylamide as substrate. While hyperthyroid rats exhibited lower levels of plasma AP A activity than controls, the kidney of hyperthyroid animals expressed significantly higher AP A than controls and hypothyroid animals. A discrepancy between the high expression of AP A in kidney of hyperthyroid rats and the low activity of AP A measured in plasma and kidney of hyperthyroid animals was found. The posttranslational influence of environmental biochemical factors may be in part responsible for that divergence.

  16. Anti-inflammatory activity of fisetin in human gingival fibroblasts treated with lipopolysaccharide.

    PubMed

    Gutiérrez-Venegas, Gloria; Contreras-Sánchez, Anabel; Ventura-Arroyo, Jairo Agustín

    2014-10-01

    Fisetin is an anti-inflammatory flavonoid; however, its anti-inflammatory mechanism is not yet understood. In this study, we evaluated the anti-inflammatory effect of fisetin and its association with mitogen-activated protein kinase (MAPK) and nuclear factor kappa-beta pathways in human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) obtained from Porphyromonas gingivalis. The cell signaling, cell viability, and cyclooxygenase-2 (COX-2) expression of HGFs treated with various concentrations (0, 1, 5, 10, and 15 μM) of fisetin were measured by cell viability assay (MTT), Western blotting, and reverse transcriptase polymerase chain reaction analysis on COX-2. We found that fisetin significantly reduced the synthesis and expression of prostaglandin E2 in HGFs treated with LPS. Activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 MAPK was suppressed consistently by fisetin in HGFs treated with LPS. The data indicate that fisetin inhibits MAPK activation and COX-2 expression without affecting cell viability. These findings may be valuable for understanding the mechanism of the effect of fisetin on periodontal disease.

  17. Cytotoxic macrophage-released tumour necrosis factor-alpha (TNF-α) as a killing mechanism for cancer cell death after cold plasma activation

    NASA Astrophysics Data System (ADS)

    Kaushik, Nagendra Kumar; Kaushik, Neha; Min, Booki; Choi, Ki Hong; Hong, Young June; Miller, Vandana; Fridman, Alexander; Choi, Eun Ha

    2016-03-01

    The present study aims at studying the anticancer role of cold plasma-activated immune cells. The direct anti-cancer activity of plasma-activated immune cells against human solid cancers has not been described so far. Hence, we assessed the effect of plasma-treated RAW264.7 macrophages on cancer cell growth after co-culture. In particular, flow cytometer analysis revealed that plasma did not induce any cell death in RAW264.7 macrophages. Interestingly, immunofluorescence and western blot analysis confirmed that TNF-α released from plasma-activated macrophages acts as a tumour cell death inducer. In support of these findings, activated macrophages down-regulated the cell growth in solid cancer cell lines and induced cell death in vitro. Together our findings suggest plasma-induced reactive species recruit cytotoxic macrophages to release TNF-α, which blocks cancer cell growth and can have the potential to contribute to reducing tumour growth in vivo in the near future.

  18. Detection of anti-salmonella flgk antibodies in chickens by automated capillary immunoassay

    USDA-ARS?s Scientific Manuscript database

    Western blot is a very useful tool to identify specific protein, but is tedious, labor-intensive and time-consuming. An automated "Simple Western" assay has recently been developed that enables the protein separation, blotting and detection in an automatic manner. However, this technology has not ...

  19. SEROEPIDEMIOLOGY

    EPA Science Inventory

    ELIZA and Western blot assays have been used to detect serological responses to Cryptosporidium antigens. 1-16. A Western blot assay htat uses a miniblot was developed by the Loveland Clinic Foundation (LCF) with funding from the U.S. Environmental Protection Agency (EPA). This...

  20. Demethoxycurcumin Suppresses Migration and Invasion of Human Cervical Cancer HeLa Cells via Inhibition of NF-κB Pathways.

    PubMed

    Lin, Chin-Chung; Kuo, Chao-Lin; Huang, Yi-Ping; Chen, Cheng-Yen; Hsu, Ming-Jie; Chu, Yung Lin; Chueh, Fu-Shin; Chung, Jing-Gung

    2018-05-01

    Demethoxycurcumin (DMC), one of the curcuminoids present in turmeric, has been shown to induce cell death in many human cancer cell lines, however, there has not been any investigation on whether DMC inhibits metastatic activity in human cervical cancer cells in vitro. In the present study, DMC at 2.5-15 μM decreased cell number, thus, we used IC 20 (7.5 μM) for further investigation of its anti-metastatic activity in human cervical cancer HeLa cells. The wound healing, migration, invasion, zymography, and western blotting assays were used to investigate the effects of DMC on HeLa cells. The wound healing assay was used to show that DMC suppressed cell movement of HeLa cells. Furthermore, the trans-well chamber assay was used to show that DMC suppressed HeLa cell migration and invasion. Gelatin zymography assay did not show any significant effects of DMC on the gelatinolytic activity (MMP-2 and -9) in conditioned media of HeLa cells treated by DMC. Western blotting showed that DMC significantly reduced protein levels of GRB2, MMP-2, ERK1/2, N-cadherin and Ras but increased the levels of E-cadherin and NF-κB in HeLa cells. Confocal laser microscopy indicated that DMC increased NF-κB in HeLa cells confirming the results from Western blotting. DMC may be used as a novel anti-metastatic agent for the treatment of human cervical cancer in the future. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  1. Safety and efficacy of intravitreal injection of recombinant erythropoietin for protection of photoreceptor cells in a rat model of retinal detachment

    PubMed Central

    Xie, Z; Chen, F; Wu, X; Zhuang, C; Zhu, J; Wang, J; Ji, H; Wang, Y; Hua, X

    2012-01-01

    Purpose To elucidate the safety and efficacy of exogenous erythropoietin (EPO) for the protection of photoreceptor cells in a rat model of retinal detachment (RD). Methods Recombinant rat EPO (400 ng) was injected into the vitreous cavity of normal rats to observe the eye manifestations. Retinal function was assessed by flash electroretinograms. Histopathological examination of retinal tissue was performed at 14 days and 2 months after injection, respectively. To investigate the inhibitory effect of EPO on photoreceptor cell apoptosis in RD rats, 100, 200, or 400 ng EPO was injected into the vitreous cavity immediately after RD model establishment. Apoptosis of photoreceptor cells was determined at 3 days after injection. Caspase-3 activation was measured by western blot analysis and immunofluorescence, respectively, and the level of Bcl-XL expression was analyzed by western blot. Results Intravitreal injection of EPO 400 ng into normal rats had no significant impact on retinal function, morphology, or structure. Apoptosis of retinal photoreceptor cells apparently increased after RD and was significantly reduced following EPO treatment. The thickness of the outer nuclear layer in the RD+400 ng group was significantly thicker than that in other experimental RD groups both at 14 days and at 2 months after RD (P<0.05). Western blot and immunofluorescence analyses showed decreased caspase-3 activation and increased Bcl-XL expression following EPO treatment. Conclusion Intravitreal injection of EPO 400 ng is safe, and EPO may suppress caspase-3 activation and enhance Bcl-XL expression, resulting in inhibition of apoptosis and protection of photoreceptor cells. PMID:22020175

  2. Productive Infection of Bovine Papillomavirus Type 2 in the Placenta of Pregnant Cows Affected with Urinary Bladder Tumors

    PubMed Central

    Roperto, Sante; Borzacchiello, Giuseppe; Esposito, Iolanda; Riccardi, Marita; Urraro, Chiara; Lucà, Roberta; Corteggio, Annunziata; Tatè, Rosarita; Cermola, Michele; Paciello, Orlando; Roperto, Franco

    2012-01-01

    Papillomaviruses (PVs) are believed to be highly epitheliotropic as they usually establish productive infections within stratified epithelia. In vitro, various PVs appear to complete their entire life-cycle in different trophoblastic cell lines. In this study, infection by and protein expression of bovine papillomavirus type 2 (BPV-2) in the uterine and chorionic epithelium of the placenta has been described in four cows suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. E5 oncoprotein was detected both by Western blot analysis and immunohistochemically. It appears to be complexed and perfectly co-localized with the activated platelet-derived growth factor ß receptor (PDGFßR) by laser scanning confocal microscopy. The activated PDGFßR might be involved in organogenesis and neo-angiogenesis rather than in cell transformation during pregnancy. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection has been detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the uterine and chorionic epithelium. Trophoblastic cells appear to be the major target for L1 protein expression. Finally, the early protein E2, required for viral DNA replication and known to be expressed during a productive infection, has been detected by Western blot and immunohistochemically. Electron microscopic investigations detected viral particles in nuclei of uterine and chorionic epithelium. This study shows that both active and productive infections by BPV-2 in the placenta of pregnant cows can occur in vivo. PMID:22479413

  3. Productive infection of bovine papillomavirus type 2 in the placenta of pregnant cows affected with urinary bladder tumors.

    PubMed

    Roperto, Sante; Borzacchiello, Giuseppe; Esposito, Iolanda; Riccardi, Marita; Urraro, Chiara; Lucà, Roberta; Corteggio, Annunziata; Tatè, Rosarita; Cermola, Michele; Paciello, Orlando; Roperto, Franco

    2012-01-01

    Papillomaviruses (PVs) are believed to be highly epitheliotropic as they usually establish productive infections within stratified epithelia. In vitro, various PVs appear to complete their entire life-cycle in different trophoblastic cell lines. In this study, infection by and protein expression of bovine papillomavirus type 2 (BPV-2) in the uterine and chorionic epithelium of the placenta has been described in four cows suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. E5 oncoprotein was detected both by Western blot analysis and immunohistochemically. It appears to be complexed and perfectly co-localized with the activated platelet-derived growth factor ß receptor (PDGFßR) by laser scanning confocal microscopy. The activated PDGFßR might be involved in organogenesis and neo-angiogenesis rather than in cell transformation during pregnancy. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection has been detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the uterine and chorionic epithelium. Trophoblastic cells appear to be the major target for L1 protein expression. Finally, the early protein E2, required for viral DNA replication and known to be expressed during a productive infection, has been detected by Western blot and immunohistochemically. Electron microscopic investigations detected viral particles in nuclei of uterine and chorionic epithelium. This study shows that both active and productive infections by BPV-2 in the placenta of pregnant cows can occur in vivo.

  4. Evidence of a novel aggrecan-degrading activity in cartilage: Studies of mice deficient in both ADAMTS-4 and ADAMTS-5.

    PubMed

    Rogerson, Fraser M; Stanton, Heather; East, Charlotte J; Golub, Suzanne B; Tutolo, Leonie; Farmer, Pamela J; Fosang, Amanda J

    2008-06-01

    To characterize aggrecan catabolism and the overall phenotype in mice deficient in both ADAMTS-4 and ADAMTS-5 (TS-4/TS-5 Delta-cat) activity. Femoral head cartilage from the joints of TS-4/TS-5 Delta-cat mice and wild-type mice were cultured in vitro, and aggrecan catabolism was stimulated with either interleukin-1alpha (IL-1alpha) or retinoic acid. Total aggrecan release was measured, and aggrecanase activity was examined by Western blotting using neoepitope antibodies for detecting cleavage at EGE 373-374 ALG, SELE 1279-1280 GRG, FREEE 1467-1468 GLG, and AQE 1572-1573 AGEG. Aggrecan catabolism in vivo was examined by Western blotting of cartilage that had been extracted immediately ex vivo. TS-4/TS-5 Delta-cat mice were viable, fertile, and phenotypically normal. TS-4/TS-5 Delta-cat cartilage explants did not release aggrecan in response to IL-1alpha, and there was no detectable increase in aggrecanase neoepitopes. TS-4/TS-5 Delta-cat cartilage explants released aggrecan in response to retinoic acid. There was no retinoic acid-stimulated cleavage at either EGE 373-374 ALG or AQE 1572-1573 AGEG. There was a low level of cleavage at SELE 1279-1280 GRG and major cleavage at FREEE 1467-1468 GLG. Ex vivo, cleavage at FREEE 1467-1468 GLG was substantially reduced, but still present, in TS-4/TS-5 Delta-cat mouse cartilage compared with wild-type mouse cartilage. An aggrecanase other than ADAMTS-4 and ADAMTS-5 is expressed in mouse cartilage and is up-regulated by retinoic acid but not IL-1alpha. The novel aggrecanase appears to have different substrate specificity from either ADAMTS-4 or ADAMTS-5, cleaving E-G bonds but not E-A bonds. Neither ADAMTS-4 nor ADAMTS-5 is required for normal skeletal development or aggrecan turnover in cartilage.

  5. Application of phage display for the development of a novel inhibitor of PLA2 activity in Western cottonmouth venom

    PubMed Central

    Titus, James K; Kay, Matthew K; Glaser, CDR Jacob J

    2017-01-01

    Snakebite envenomation is an important global health concern. The current standard treatment approach for snakebite envenomation relies on antibody-based antisera, which are expensive, not universally available, and can lead to adverse physiological effects. Phage display techniques offer a powerful tool for the selection of phage-expressed peptides, which can bind with high specificity and affinity towards venom components. In this research, the amino acid sequences of Phospholipase A2 (PLA2) from multiple cottonmouth species were analyzed, and a consensus peptide synthesized. Three phage display libraries were panned against this consensus peptide, crosslinked to capillary tubes, followed by a modified surface panning procedure. This high throughput selection method identified four phage clones with anti-PLA2 activity against Western cottonmouth venom, and the amino acid sequences of the displayed peptides were identified. This is the first report identifying short peptide sequences capable of inhibiting PLA2 activity of Western cottonmouth venom in vitro, using a phage display technique. Additionally, this report utilizes synthetic panning targets, designed using venom proteomic data, to mimic epitope regions. M13 phages displaying circular 7-mer or linear 12-mer peptides with antivenom activity may offer a novel alternative to traditional antibody-based therapy. PMID:29285351

  6. Application of phage display for the development of a novel inhibitor of PLA2 activity in Western cottonmouth venom.

    PubMed

    Titus, James K; Kay, Matthew K; Glaser, Cdr Jacob J

    2017-01-01

    Snakebite envenomation is an important global health concern. The current standard treatment approach for snakebite envenomation relies on antibody-based antisera, which are expensive, not universally available, and can lead to adverse physiological effects. Phage display techniques offer a powerful tool for the selection of phage-expressed peptides, which can bind with high specificity and affinity towards venom components. In this research, the amino acid sequences of Phospholipase A 2 (PLA 2 ) from multiple cottonmouth species were analyzed, and a consensus peptide synthesized. Three phage display libraries were panned against this consensus peptide, crosslinked to capillary tubes, followed by a modified surface panning procedure. This high throughput selection method identified four phage clones with anti-PLA 2 activity against Western cottonmouth venom, and the amino acid sequences of the displayed peptides were identified. This is the first report identifying short peptide sequences capable of inhibiting PLA 2 activity of Western cottonmouth venom in vitro , using a phage display technique. Additionally, this report utilizes synthetic panning targets, designed using venom proteomic data, to mimic epitope regions. M13 phages displaying circular 7-mer or linear 12-mer peptides with antivenom activity may offer a novel alternative to traditional antibody-based therapy.

  7. Effect of OPC-12759 on EGF receptor activation, p44/p42 MAPK activity, and secretion in conjunctival goblet cells.

    PubMed

    Ríos, J David; Shatos, Marie A; Urashima, Hiroki; Dartt, Darlene A

    2008-04-01

    The purpose of the study was to determine if OPC-12759 stimulates secretion from conjunctival goblet cells in culture and if it activates the EGF receptor (EGFR) and p44/p42 mitogen-activated protein kinase (MAPK) to cause mucin secretion. Conjunctival goblet cells were cultured from pieces of male rat conjunctiva. OPC-12759 was added at increasing concentrations and for varying times to the cultured cells. The cholinergic agonist carbachol was used as a positive control. In selected experiments an inhibitor of the EGFR, AG1478, or an inhibitor of the kinase that activates MAPK, U0126, were added before OPC-12759. Goblet cell secretion of high molecular weight glycoconjugates was measured by an enzyme-linked lectin assay using the lectin UEA-1. Activation of the EGFR and MAPK were determined with Western blotting analysis using antibodies specific to the phosphorylated and the total amounts of these proteins. We found that OPC-12759 induced goblet cell secretion in a time- and concentration-dependent manner. Inhibition of the EGFR with AG1478 blocked secretion stimulated by OPC-12759. Inhibition of MAPK with U0126 also blocked secretion stimulated by OPC-12759. OPC-12759 increased the phosphorylation of the EGFR and MAPK in a time-dependent manner. We concluded that OPC-12759 stimulates secretion from cultured conjunctival goblet cells by activating the EGFR, which then induces MAPK activity.

  8. Flavonoids and biphenylneolignans with anti-inflammatory activity from the stems of Millettia griffithii.

    PubMed

    Tang, Huan; Pei, He-Ying; Wang, Tai-Jin; Chen, Kai; Wu, Bo; Yang, Qiu-Nan; Zhang, Qiang; Yang, Jian-Hong; Wang, Xiao-Yan; Tang, Ming-Hai; Peng, Ai-Hua; Ye, Hao-Yu; Chen, Li-Juan

    2016-09-15

    Five new flavonoids, griffinones A-E (1-5), a new biphenylneolignan, griffilignan A (6) and ten known compounds were isolated from the n-hexane and EtOAc extracts of Millettia griffithii. The structures of the new compounds were determined by 1D and 2D NMR, and by HRMS. The anti-inflammatory activity of the isolated compounds was evaluated on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells. Among the isolated compounds, compounds 1, 2 and 14 showed significant anti-inflammatory activity with IC50 values of 20.4, 2.1 and 35.7μM, respectively and no obvious toxicities were observed at 100μM. Western blot and PCR assay further showed that inhibition of nitric oxide production by compound 2 was associated with suppression of iNOS expression. Modeling studies suggested that the amino group, phenyl ring as well as the isopentenyl tails of compound 2 could help bind to iNOs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. The equine herpesvirus-1 IR3 gene that lies antisense to the sole immediate-early (IE) gene is trans-activated by the IE protein, and is poorly expressed to a protein

    PubMed Central

    Ahn, Byung Chul; Breitenbach, Jonathan E.; Kim, Seong K.; O’Callaghan, Dennis J.

    2007-01-01

    The unique IR3 gene of equine herpesvirus 1 (EHV-1) is expressed as a late 1.0-kb transcript. Previous studies confirmed the IR3 transcription initiation site and tentatively identified other cis-acting elements specific to IR3 such as a TATA box, a 443 base pair 5′untranslated region (UTR), a 285 base pair open reading frame (ORF) and a poly adenylation (A) signal (Holden et al., 1992 DNA Seq 3, 143-52). Transient transfection assays revealed that the IR3 promoter is strongly trans-activated by the IE protein (IEP) and that coexpression of the IEP with the early EICP0 and IR4 regulatory proteins results in maximal trans-activation of the IR3 promoter. Gel shift assays revealed that the IEP directly binds to the IR3 promoter region. Western blot analysis showed that the IR3 protein produced in E. coli was detected by antibodies to IR3 synthetic peptides; however, the IR3 protein was not detected in EHV-1 infected cell extracts by these same anti-IR3 antibodies, even though the IR3 transcript was detected by northern blot. These findings suggest that the IR3 may not be expressed to a protein. Expression of an IR3/GFP fusion gene was not observed, but expression of a GFP/IR3 fusion gene was detected by fluorescent microscopy. In further attempts to detect the IR3/GFP fusion protein using anti-GFP antibody, western blot analysis showed that the IR3/GFP fusion protein was not detected in vivo. Interestingly, a truncated form of the GFP/IR3 protein was synthesized from the GFP/IR3 fusion gene. However, GFP/IR3 and IR3/GFP fusion proteins of the predicted sizes were synthesized by in vitro coupled transcription and translation of the fusion genes, suggesting poor expression of the IR3 protein in vivo. The possible role of the IR3 transcript in EHV-1 infection is discussed. PMID:17306852

  10. The Identification of Novel Protein-Protein Interactions in Liver that Affect Glucagon Receptor Activity

    PubMed Central

    Froese, Sean; Dai, Feihan F.; Robitaille, Mélanie; Bhattacharjee, Alpana; Huang, Xinyi; Jia, Weiping; Angers, Stéphane; Wheeler, Michael B.; Wei, Li

    2015-01-01

    Glucagon regulates glucose homeostasis by controlling glycogenolysis and gluconeogenesis in the liver. Exaggerated and dysregulated glucagon secretion can exacerbate hyperglycemia contributing to type 2 diabetes (T2D). Thus, it is important to understand how glucagon receptor (GCGR) activity and signaling is controlled in hepatocytes. To better understand this, we sought to identify proteins that interact with the GCGR to affect ligand-dependent receptor activation. A Flag-tagged human GCGR was recombinantly expressed in Chinese hamster ovary (CHO) cells, and GCGR complexes were isolated by affinity purification (AP). Complexes were then analyzed by mass spectrometry (MS), and protein-GCGR interactions were validated by co-immunoprecipitation (Co-IP) and Western blot. This was followed by studies in primary hepatocytes to assess the effects of each interactor on glucagon-dependent glucose production and intracellular cAMP accumulation, and then in immortalized CHO and liver cell lines to further examine cell signaling. Thirty-three unique interactors were identified from the AP-MS screening of GCGR expressing CHO cells in both glucagon liganded and unliganded states. These studies revealed a particularly robust interaction between GCGR and 5 proteins, further validated by Co-IP, Western blot and qPCR. Overexpression of selected interactors in mouse hepatocytes indicated that two interactors, LDLR and TMED2, significantly enhanced glucagon-stimulated glucose production, while YWHAB inhibited glucose production. This was mirrored with glucagon-stimulated cAMP production, with LDLR and TMED2 enhancing and YWHAB inhibiting cAMP accumulation. To further link these interactors to glucose production, key gluconeogenic genes were assessed. Both LDLR and TMED2 stimulated while YWHAB inhibited PEPCK and G6Pase gene expression. In the present study, we have probed the GCGR interactome and found three novel GCGR interactors that control glucagon-stimulated glucose production

  11. Production, characterization and application of monoclonal antibody to spherulocytes: A subpopulation of coelomocytes of Apostichopus japonicus

    USDA-ARS?s Scientific Manuscript database

    One monoclonal antibody (mAb 3F6) against coelomocytes of sea cucumber Apostichopus japonicus was developed by immunization of Balb/C mice. Analyzed by indirect immunofluorescence assay test (IIFAT), immunocytochemical assay (ICA),Western blotting and fluorescence-activated cell sorter (FACS), mAb 3...

  12. Identification of Potential Anticancer Activities of Novel Ganoderma lucidum Extracts Using Gene Expression and Pathway Network Analysis

    PubMed Central

    Kao, Chi H.J.; Bishop, Karen S.; Xu, Yuanye; Han, Dug Yeo; Murray, Pamela M.; Marlow, Gareth J.; Ferguson, Lynnette R.

    2016-01-01

    Ganoderma lucidum (lingzhi) has been used for the general promotion of health in Asia for many centuries. The common method of consumption is to boil lingzhi in water and then drink the liquid. In this study, we examined the potential anticancer activities of G. lucidum submerged in two commonly consumed forms of alcohol in East Asia: malt whiskey and rice wine. The anticancer effect of G. lucidum, using whiskey and rice wine-based extraction methods, has not been previously reported. The growth inhibition of G. lucidum whiskey and rice wine extracts on the prostate cancer cell lines, PC3 and DU145, was determined. Using Affymetrix gene expression assays, several biologically active pathways associated with the anticancer activities of G. lucidum extracts were identified. Using gene expression analysis (real-time polymerase chain reaction [RT-PCR]) and protein analysis (Western blotting), we confirmed the expression of key genes and their associated proteins that were initially identified with Affymetrix gene expression analysis. PMID:27006591

  13. Assessing carbon storage in western U.S. ecosystems

    NASA Astrophysics Data System (ADS)

    Balcerak, Ernie

    2013-01-01

    Western U.S. ecosystems have the capacity to sequester about 91 million metric tons of carbon per year, according to a report released 5 December by the Department of the Interior. Entitled "Baseline and Projected Future Carbon Storage and Greenhouse-Gas Fluxes in Ecosystems of the Western United States" and written by U.S. Geological Survey (USGS) scientists, the report came out of a congressionally mandated study. It examines how much carbon can be stored naturally through biological activity in various ecosystems, including forests, grasslands, and wetlands, in the western United States, from the Rocky Mountains to the Pacific.

  14. "All Quiet on the Western Front."

    ERIC Educational Resources Information Center

    Soderquist, Alisa

    Based on Erich Maria Remarque's novel "All Quiet on the Western Front" and other war literature, this lesson plan presents activities designed to help students understand that works of art about war can call up strong emotions in readers; and that the writing process can be applied to writing poems. The main activity of the lesson involves…

  15. L-Cysteine Administration Attenuates Pancreatic Fibrosis Induced by TNBS in Rats by Inhibiting the Activation of Pancreatic Stellate Cell

    PubMed Central

    Hu, GuoYong; Shen, Jie; Wang, Feng; Xu, Ling; Dai, WeiQi; Xiong, Jie; Ni, JianBo; Guo, ChuanYong; Wan, Rong; Wang, XingPeng

    2012-01-01

    Background and Aims Recent studies have shown that activated pancreatic stellate cells (PSCs) play a major role in pancreatic fibrogenesis. We aimed to study the effect of L-cysteine administration on fibrosis in chronic pancreatitis (CP) induced by trinitrobenzene sulfonic acid (TNBS) in rats and on the function of cultured PSCs. Methods CP was induced by TNBS infusion into rat pancreatic ducts. L-cysteine was administrated for the duration of the experiment. Histological analysis and the contents of hydroxyproline were used to evaluate pancreatic damage and fibrosis. Immunohistochemical analysis of α-SMA in the pancreas was performed to detect the activation of PSCs in vivo. The collagen deposition related proteins and cytokines were determined by western blot analysis. DNA synthesis of cultured PSCs was evaluated by BrdU incorporation. We also evaluated the effect of L-cysteine on the cell cycle and cell activation by flow cytometry and immunocytochemistry. The expression of PDGFRβ, TGFβRII, collagen 1α1 and α-SMA of PSCs treated with different concentrations of L-cysteine was determined by western blot. Parameters of oxidant stress were evaluated in vitro and in vivo. Nrf2, NQO1, HO-1, IL-1β expression were evaluated in pancreas tissues by qRT-PCR. Results The inhibition of pancreatic fibrosis by L-cysteine was confirmed by histological observation and hydroxyproline assay. α-SMA, TIMP1, IL-1β and TGF-β1 production decreased compared with the untreated group along with an increase in MMP2 production. L-cysteine suppressed the proliferation and extracellular matrix production of PSCs through down-regulating of PDGFRβ and TGFβRII. Concentrations of MDA+4-HNE were decreased by L-cysteine administration along with an increase in GSH levels both in tissues and cells. In addition, L-cysteine increased the mRNA expression of Nrf2, NQO1 and HO-1 and reduced the expression of IL-1β in L-cysteine treated group when compared with control group. Conclusion L

  16. Effect of antiangiogenic therapy on luciferase activity in a cytomegalovirus- or HSP70-promoter-transfected M21 tumor model

    NASA Astrophysics Data System (ADS)

    Hundt, Walter; Schink, Christian; Steinbach, Silke; O'Connell-Rodwell, Caitlin E.; Kiessling, Andreas; Librizzi, Damiano; Burbelko, Mykhaylo; Guccione, Samira

    2012-06-01

    We investigated the effect of targeted gene therapy on heat shock protein 70 expression (Hsp70) and protein production (HSP70) in a melanoma tumor model (M21; M21-L). M21 and M21-L cells transfected with a plasmid containing the Hsp70 (Hspa1b) or the cytomegalovirus (CMV) promoter and the luciferase reporter gene were injected into mice; the resulting tumors grew to a size of 650 mm3. Mice (five per group) were intravenously treated with an Arg-Gly-Asp peptide-nanoparticle/Raf-1 kinase inhibitor protein complex [RGD-NP/RAF(-)] or with a nanoparticle control. Bioluminescence imaging (IVIS®, Xenogen, USA) was performed at 12, 24, 48, and 72 h after the treatment cycle. Western blot analysis of HSP70 protein was performed to monitor protein expression. The size of the treated M21 tumors remained fairly constant (647.8+/-103.4 mm2 at the beginning versus 704.8+/-94.4 mm3 at the end of the experiment). The size of the M21-L tumors increased, similar to the untreated control tumors. Bioluminescent imaging demonstrated that when transcription was controlled by the CMV promoter, luciferase activity decreased to 17.9%+/-4.3% of baseline values in the treated M21 tumors. When transcription was controlled by the Hsp70 promoter, the highest luciferase activity (4.5+/-0.7-fold increase over base-line values) was seen 24 h after injection in the M21 tumors; however, no luciferase activity was seen in the M21-L tumors. In accordance with bioluminescent imaging, western blot analysis showed a peak in HSP70 production at 24 h after the injection of the RGD-NP/RAF(-) complex in the M21 tumors; however, no HSP70 protein induction was seen in the M21-L tumors. Thus, targeted antiangiogenic therapy can induce Hsp70 expression and HSP70 protein in melanoma tumors.

  17. Dihydroartemisinin sensitizes Lewis lung carcinoma cells to carboplatin therapy via p38 mitogen-activated protein kinase activation

    PubMed Central

    Zhang, Bicheng; Zhang, Zhimin; Wang, Jun; Yang, Bo; Zhao, Yong; Rao, Zhiguo; Gao, Jianfei

    2018-01-01

    Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua, is an effective novel antimalarial agent. Studies have suggested that it also exhibits anticancer effects when administered alone or in combination with conventional chemotherapeutic agents. The present study investigated the therapeutic effect of DHA combined with carboplatin (CBP) on Lewis lung carcinoma (LLC) cells and the possible underlying molecular mechanisms. MTT and clonogenic assays demonstrated that the proliferation activity of LLC cells was inhibited in a dose-dependent manner by DHA combined with CBP. In addition, flow cytometry analysis revealed that cell cycle arrest was induced at the G0/G1 phase and apoptosis was induced following treatment with the combination. When administered in combination with CBP, DHA exhibited more effective anticancer activity compared with DHA or CBP used alone, via increased apoptosis. Following treatment with DHA with or without CBP, the expression of phosphorylated-p38 mitogen-activated protein kinase (MAPK), which can be inhibited with the selective inhibitor SB202190, was detected by western blotting. To summarize, the results of the present study indicated that DHA may sensitize LLC cells to CBP therapy via the activation of p38MAPK, which suggests that a combined treatment of DHA and CBP may be a potential novel therapeutic schedule for lung adenocarcinoma. PMID:29740482

  18. Activated Notch signaling cascade is correlated with stem cell differentiation toward absorptive progenitors after massive small bowel resection in a rat.

    PubMed

    Sukhotnik, Igor; Coran, Arnold G; Pollak, Yulia; Kuhnreich, Eviatar; Berkowitz, Drora; Saxena, Amulya K

    2017-09-01

    Notch signaling is thought to act to drive cell versification in the lining of the small intestine. The purpose of the present study was to evaluate the role of the Notch signaling pathway in stem cell differentiation in the late stages of intestinal adaptation after massive small bowel resection in a rat. Male Sprague-Dawley rats were randomly assigned to one of two experimental groups of eight rats each: Sham rats underwent bowel transection and reanastomosis, while SBS rats underwent 75% small bowel resection. Rats were euthanized on day 14 Illumina's Digital Gene Expression (DGE) analysis was used to determine Notch signaling gene expression profiling. Notch-related gene and protein expression was determined using real-time PCR, Western blot analysis, and immunohistochemistry. From seven investigated Notch-related (by DGE analysis) genes, six genes were upregulated in SBS vs. control animals with a relative change in gene expression level of 20% or more. A significant upregulation of Notch signaling-related genes in resected animals was accompanied by a significant increase in Notch-1 protein levels (Western blot analysis) and a significant increase in the number of Notch1 and Hes1 (target gene)-positive cells (immunohistochemistry) compared with sham animals. Evaluation of cell differentiation has shown a strong increase in total number of absorptive cells (unchanged secretory cells) compared with control rats. In conclusion, 2 wk after bowel resection in rats, stimulated Notch signaling directs the crypt cell population toward absorptive progenitors. NEW & NOTEWORTHY This study provides novel insight into the mechanisms of cell proliferation following massive small bowel resection. We show that 2 wk after bowel resection in rats, enhanced stem cell activity was associated with stimulated Notch signaling pathway. We demonstrate that activated Notch signaling cascade directs the crypt cell population toward absorptive progenitors. Copyright © 2017 the American

  19. Active population growth and immigration hypotheses in Western Europe.

    PubMed

    Feld, S

    2000-03-01

    The paper examines, in respect of 12 Western European countries over a period of 20 years, the widely held view that any decline in their working population should be offset by greater reliance on immigrant labor. This research, based on demographic projections and forecasts regarding labor market participation rates by age and sex for each of the countries concerned, focuses on the two most likely scenarios. It appears that only Italy will be faced with a fall in its working population. All other western countries will either maintain the same level or, more generally, see their workforce grow substantially. Accordingly, the authors may safely assert that there is no risk of a shortage of workers between now and the year 2020, and that an increasing supply of labor will render reliance on a greater influx of immigrant workers unnecessary. The second part analyses changes in the structure of the demand for labor. The authors deal chiefly with the phenomenon of the concentration of foreign manpower in each sector, its flexibility and mobility in a context of unemployment, as well as the impact of new technologies and globalization on the main determinants of international migration of labor.

  20. 6-Shogaol attenuates LPS-induced inflammation in BV2 microglia cells by activating PPAR-γ.

    PubMed

    Han, Qinghe; Yuan, Qinghai; Meng, Xiaolin; Huo, Junyuan; Bao, Yuxin; Xie, Guanghong

    2017-06-27

    6-Shogaol, a pungent agent isolated from Zingiber officinale Roscoe, has been known to have anti-tumor and anti-inflammatory effects. However, the anti-inflammatory effects and biological mechanism of 6-Shogaol in LPS-activated BV2 microglia remains largely unknown. In this study, we evaluated the anti-inflammatory effects of 6-Shogaol in LPS-activated BV2 microglia. 6-Shogaol was administrated 1 h before LPS treatment. The production of inflammatory mediators were detected by ELISA. The expression of NF-κB and PPAR-γ were detected by western blot analysis. Our results revealed that 6-Shogaol inhibited LPS-induced TNF-α, IL-1β, IL-6, and PGE2 production in a concentration dependent manner. Furthermore, 6-Shogaol inhibited LPS-induced NF-κB activation by inhibiting phosphorylation and nuclear translocation of NF-κB p65. In addition, 6-Shogaol could increase the expression of PPAR-γ. Moreover, inhibition of PPAR-γ by GW9662 could prevent the inhibition of 6-Shogaol on LPS-induced inflammatory mediator production. In conclusion, 6-Shogaol inhibits LPS-induced inflammation by activating PPAR-γ.

  1. New arylated benzo[h]quinolines induce anti-cancer activity by oxidative stress-mediated DNA damage.

    PubMed

    Yadav, Dharmendra K; Rai, Reeta; Kumar, Naresh; Singh, Surjeet; Misra, Sanjeev; Sharma, Praveen; Shaw, Priyanka; Pérez-Sánchez, Horacio; Mancera, Ricardo L; Choi, Eun Ha; Kim, Mi-Hyun; Pratap, Ramendra

    2016-12-06

    The anti-cancer activity of the benzo[h]quinolines was evaluated on cultured human skin cancer (G361), lung cancer (H460), breast cancer (MCF7) and colon cancer (HCT116) cell lines. The inhibitory effect of these compounds on the cell growth was determined by the MTT assay. The compounds 3e, 3f, 3h and 3j showed potential cytotoxicity against these human cancer cell lines. Effect of active compounds on DNA oxidation and expression of apoptosis related gene was studied. We also developed a quantitative method to measure the activity of cyclin-dependent kinases-2 (CDK2) by western blotting in the presence of active compound. In addition, molecular docking revealed that benzo[h]quinolines can correctly dock into the hydrophobic pocket of the targets receptor protein aromatase and CDK2, while their bioavailability/drug-likeness was predicted to be acceptable but requires future optimization. These findings reveal that benzo[h]quinolines act as anti-cancer agents by inducing oxidative stress-mediated DNA damage.

  2. The Macrophage Activation Induced by Bacillus thuringiensis Cry1Ac Protoxin Involves ERK1/2 and p38 Pathways and the Interaction with Cell-Surface-HSP70.

    PubMed

    Rubio-Infante, Nestor; Ilhuicatzi-Alvarado, Damaris; Torres-Martínez, Marilu; Reyes-Grajeda, Juan Pablo; Nava-Acosta, Raúl; González-González, Edith; Moreno-Fierros, Leticia

    2018-01-01

    Here, we aimed to further characterize the mechanisms involved in protoxin (p) Cry1Ac-induced macrophage activation. We demonstrated that pCry1Ac induces MAPK ERK1/2, p38, and JNK phosphorylation in RAW264.7 macrophages. Because MAPK activation is mainly triggered via ligand-receptor interactions, we focused on the identification of potential pCry1Ac-receptor proteins. Flow cytometry and confocal analysis showed specific saturable pCry1Ac-binding to the macrophage surface and evidenced its internalization via the clathrin-pathway. We performed immunoprecipitation assays and identified by MALDI-TOF-TOF several possible pCry1Ac-binding proteins, such as heat shock proteins (HSPs), vimentin, α-enolase, and actin; whose interaction and presence was confirmed, respectively, by ligand blot and Western blot assays. We also detected cell-surface (cs) pCry1Ac-HSP70 colocalization, so HSP70 was chosen for further characterization. Co-immunoprecipitation with HSP70 antibodies followed by Western blot confirmed the pCry1Ac-HSP70 interaction. Furthermore, pretreatment of RAW264.7 cells with HSP70 antibodies reduced pCry1Ac-induced ERK1 phosphorylation and MCP-1 production; thus suggest the functional participation of csHSP70 in pCry1Ac-induced macrophage activation. csHSP70 also was evaluated in peritoneal-cavity (PerC) macrophages of untreated BALB/c mice, interestingly it was found that the predominant population namely large-peritoneal-macrophages (LPM) displayed csHSP70 + hi. Furthermore, the dynamics of PerC macrophage subsets, LPM, and small-peritoneal macrophages (SPM) were evaluated in response to in vivo pCry1Ac stimuli in presence or not of phenylethynesulfonamide (PES) a functional HSP70 inhibitor. It was found that pCry1Ac increased the proportion of SPM CD11b + F4/80 + lowMHCII + csHSP70 + low and markedly reduced the amount of LPM CD11b + F4/80 + hiMHCII-csHSP70 + hi; while PES, partially suppressed this pCry1Ac-induced effect

  3. Berberine displays antitumor activity in esophageal cancer cells in vitro.

    PubMed

    Jiang, Shu-Xian; Qi, Bo; Yao, Wen-Jian; Gu, Cheng-Wei; Wei, Xiu-Feng; Zhao, Yi; Liu, Yu-Zhen; Zhao, Bao-Sheng

    2017-04-14

    To investigate the effects of berberine on esophageal cancer (EC) cells and its molecular mechanisms. Human esophageal squamous cell carcinoma cell line KYSE-70 and esophageal adenocarcinoma cell line SKGT4 were used. The effects of berberine on cell proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For cell cycle progression, KYSE-70 cells were stained with propidium iodide (PI) staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min and cell cycle was analyzed using a BD FACSCalibur flow cytometer. For apoptosis assay, cells were stained with an Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using BD ModFit software. Levels of proteins related to cell cycle and apoptosis were examined by western blotting. Berberine treatment resulted in growth inhibition of KYSE-70 and SKGT4 cells in a dose-dependent and time-dependent manner. KYSE-70 cells were more susceptible to the inhibitory activities of berberine than SKGT4 cells were. In KYSE-70 cells treated with 50 μmol/L berberine for 48 h, the number of cells in G 2 /M phase (25.94% ± 5.01%) was significantly higher than that in the control group (9.77% ± 1.28%, P < 0.01), and berberine treatment resulted in p21 up-regulation in KYSE-70 cells. Flow cytometric analyses showed that berberine significantly augmented the KYSE-70 apoptotic population at 12 and 24 h post-treatment, when compared with control cells (0.83% vs 43.78% at 12 h, P < 0.05; 0.15% vs 81.86% at 24 h, P < 0.01), and berberine-induced apoptotic effect was stronger at 24 h compared with 12 h. Western blotting showed that berberine inhibited the phosphorylation of Akt, mammalian target of rapamycin and p70S6K, and enhanced AMP-activated protein kinase phosphorylation in a sustained manner. Berberine is an inhibitor of human EC cell growth and could be considered as a potential drug for the treatment

  4. Berberine displays antitumor activity in esophageal cancer cells in vitro

    PubMed Central

    Jiang, Shu-Xian; Qi, Bo; Yao, Wen-Jian; Gu, Cheng-Wei; Wei, Xiu-Feng; Zhao, Yi; Liu, Yu-Zhen; Zhao, Bao-Sheng

    2017-01-01

    AIM To investigate the effects of berberine on esophageal cancer (EC) cells and its molecular mechanisms. METHODS Human esophageal squamous cell carcinoma cell line KYSE-70 and esophageal adenocarcinoma cell line SKGT4 were used. The effects of berberine on cell proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For cell cycle progression, KYSE-70 cells were stained with propidium iodide (PI) staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min and cell cycle was analyzed using a BD FACSCalibur flow cytometer. For apoptosis assay, cells were stained with an Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using BD ModFit software. Levels of proteins related to cell cycle and apoptosis were examined by western blotting. RESULTS Berberine treatment resulted in growth inhibition of KYSE-70 and SKGT4 cells in a dose-dependent and time-dependent manner. KYSE-70 cells were more susceptible to the inhibitory activities of berberine than SKGT4 cells were. In KYSE-70 cells treated with 50 μmol/L berberine for 48 h, the number of cells in G2/M phase (25.94% ± 5.01%) was significantly higher than that in the control group (9.77% ± 1.28%, P < 0.01), and berberine treatment resulted in p21 up-regulation in KYSE-70 cells. Flow cytometric analyses showed that berberine significantly augmented the KYSE-70 apoptotic population at 12 and 24 h post-treatment, when compared with control cells (0.83% vs 43.78% at 12 h, P < 0.05; 0.15% vs 81.86% at 24 h, P < 0.01), and berberine-induced apoptotic effect was stronger at 24 h compared with 12 h. Western blotting showed that berberine inhibited the phosphorylation of Akt, mammalian target of rapamycin and p70S6K, and enhanced AMP-activated protein kinase phosphorylation in a sustained manner. CONCLUSION Berberine is an inhibitor of human EC cell growth and could be considered as a

  5. MANF prevents traumatic brain injury in rats by inhibiting inflammatory activation and protecting Blood Brain Barrier.

    PubMed

    Li, Qing-Xin; Shen, Yu-Xian; Ahmad, Akhlaq; Shen, Yu-Jun; Zhang, Yi-Quan; Xu, Pei-Kun; Chen, Wei-Wei; Yu, Yong-Qiang

    2018-06-05

    Our previous studies have shown that MANF provides neuroprotective effect against ischemia/reperfusion injury and is also involved in inflammatory disease models. This work investigates the potential role and mechanism of MANF in acute brain damage after traumatic brain injury (TBI). The model of TBI was induced by Feeney free falling methods with male Sprauge-Dawley rats. The expression of MANF, 24 hrs after TBI, was detected by the immunohistochemistry, immunofluorescence, Western blot and Reverse transcription PCR(RT-PCR) techniques. After treatment with recombinant human MANF following TBI, assessment was conducted - 24 hrs later for brain water content(BWC), cerebral edema volume in MRI, neurobehavioral testing and Evans blue extravasation. Moreover, by the techniques of Western blot and RT-PCR, the expression of inflammatory cytokines(IL-1β, TNF-α) and P65 was also analyzed to explore the underlying protective mechanism of MANF. At 24 hrs after TBI, we found that endogenous MANF was widely expressed in the rat's brain tissues and different types of cells. Treatment with high dose of recombinant human MANF(20 μg/20 μL) - significantly increased the modified Garcia score, and reduced BWC as well as cerebral edema volume in MRI. Furthermore, MANF alleviated not only the blood-brain barrier(BBB) permeability, but also the expressions of IL-1β and TNF-α mRNA and protein. Besides, the activation of P65 was also inhibited. These results suggest that MANF provides neuroprotective effect against acute brain injury after TBI, via attenuating BBB disruption and intracranial neuroinflammation, while the inhibition of NF-κB signaling pathway might be a potential mechanism. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Anti-inflammatory effect of garlic 14-kDa protein on LPS-stimulated-J774A.1 macrophages.

    PubMed

    Rabe, Shahrzad Zamani Taghizadeh; Ghazanfari, Tooba; Siadat, Zahra; Rastin, Maryam; Rabe, Shahin Zamani Taghizadeh; Mahmoudi, Mahmoud

    2015-04-01

    Garlic 14-kDa protein is purified from garlic (Allium sativum L.) which is used in traditional medicine and exerts various immunomodulatory activities. The present study investigated the suppressive effect of garlic 14-kDa protein on LPS-induced expression of pro-inflammatory mediators and underlying mechanism in inflammatory macrophages. J774A.1 macrophages were treated with 14-kDa protein (5-30 μg/ml) with/without LPS (1 μg/ml) and the production of inflammatory mediators such as prostaglandin E2 (PGE2), TNF-α, and IL-1β released were measured using ELISA. Nitric oxide (NO) production was determined using the Griess method. The anti-inflammatory activity of 14-kDa protein was examined by measuring inducible nitric oxide synthase and cyclooxygenase-2 proteins using western blot. The expression of nuclear NF-κB p65 subunit was assessed by western blot. Garlic 14-kDa protein significantly inhibited the excessive production of NO, PGE, TNF-α, and IL-1β in lipopolysaccharide (LPS)-activated J774A.1 macrophages in a concentration-related manner without cytotoxic effect. Western blot analysis demonstrated that garlic 14-kDa protein suppressed corresponding inducible NO synthase expression and activated cyclooxygenase-2 protein expression. The inhibitory effect was mediated partly by a reduction in the activity and expression of transcription factor NF-κB protein. Our results suggested, for the first time, garlic 14-kDa protein exhibits anti-inflammatory properties in macrophages possibly by suppressing the inflammatory mediators via the inhibition of transcription factor NF-κB signaling pathway. The traditional use of garlic as anti-inflammatory remedy could be ascribed partly to 14-kDa protein content. This protein might be a useful candidate for controlling inflammatory diseases and further investigations in vivo.

  7. Low-Dose Ethanol Preconditioning Protects Against Oxygen-Glucose Deprivation/Reoxygenation-Induced Neuronal Injury By Activating Large Conductance, Ca2+-Activated K+ Channels In Vitro.

    PubMed

    Su, Fang; Guo, An-Chen; Li, Wei-Wei; Zhao, Yi-Long; Qu, Zheng-Yi; Wang, Yong-Jun; Wang, Qun; Zhu, Yu-Lan

    2017-02-01

    Increasing evidence suggests that low to moderate ethanol ingestion protects against the deleterious effects of subsequent ischemia/reperfusion; however, the underlying mechanism has not been elucidated. In the present study, we showed that expression of the neuronal large-conductance, Ca 2+ -activated K + channel (BK Ca ) α-subunit was upregulated in cultured neurons exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) compared with controls. Preconditioning with low-dose ethanol (10 mmol/L) increased cell survival rate in neurons subjected to OGD/R, attenuated the OGD/R-induced elevation of cytosolic Ca 2+ levels, and reduced the number of apoptotic neurons. Western blots revealed that ethanol preconditioning upregulated expression of the anti-apoptotic protein Bcl-2 and downregulated the pro-apoptotic protein Bax. The protective effect of ethanol preconditioning was antagonized by a BK Ca channel inhibitor, paxilline. Inside-out patches in primary neurons also demonstrated the direct activation of the BK Ca channel by 10 mmol/L ethanol. The above results indicated that low-dose ethanol preconditioning exerts its neuroprotective effects by attenuating the elevation of cytosolic Ca 2+ and preventing neuronal apoptosis, and this is mediated by BK Ca channel activation.

  8. Regulation of glutamate level in rat brain through activation of glutamate dehydrogenase by Corydalis ternata.

    PubMed

    Lee, Kwan Ho; Huh, Jae-Wan; Choi, Myung-Min; Yoon, Seung Yong; Yang, Seung-Ju; Hong, Hea Nam; Cho, Sung-Woo

    2005-08-31

    When treated with protopine and alkalized extracts of the tuber of Corydalis ternata for one year, significant decrease in glutamate level and increase in glutamate dehydrogenase (GDH) activity was observed in rat brains. The expression of GDH between the two groups remained unchanged as determined by Western and Northern blot analysis, suggesting a post-translational regulation of GDH activity in alkalized extracts treated rat brains. The stimulatory effects of alkalized extracts and protopine on the GDH activity was further examined in vitro with two types of human GDH isozymes, hGDH1 (house-keeping GDH) and hGDH2 (nerve-specific GDH). Alkalized extracts and protopine activated the human GDH isozymes up to 4.8-fold. hGDH2 (nerve- specific GDH) was more sensitively affected by 1 mM ADP than hGDH1 (house-keeping GDH) on the activation by alkalized extracts. Studies with cassette mutagenesis at ADP-binding site showed that hGDH2 was more sensitively regulated by ADP than hGDH1 on the activation by Corydalis ternata. Our results suggest that prolonged exposure to Corydalis ternata may be one of the ways to regulate glutamate concentration in brain through the activation of GDH.

  9. Modulation of Subseasonal Tropical Cyclone Genesis In The Western North Pacific By Wave Activities

    NASA Astrophysics Data System (ADS)

    Gao, Jianyun; Cheung, Kevin K. W.

    2017-04-01

    Tropical cyclone (TC) activity is well known to possess variability on multiple timescales, ranging from inter-decadal to intraseasonal. In this study, the subseasonal variability of TC genesis in the western North Pacific (WNP) is examined during summer (May-October) for the period of 1979-2015. In particular, clustering of TC activity within subseasonal timescale is the focus. First, three phases (active, normal and inactive phases) of TC clustering are defined based on the statistics of genesis frequency. Then the modes of subseasonal modulation of these three phases by intraseasonal (30-60-day) oscillation (ISO), biweekly (10-20-day) oscillation (BWO), and the convectively coupled equatorial waves (CCEW), including Rossby, Kelvin, and mixed Rossby-gravity and tropical depression-type waves are considered. It is found that the embedding large-scale circulation is significantly different between the inactive phase and the other phases. Further, the intensities and propagation phases of the ISO, BWO and CCEW play different roles to modulate TC genesis frequency during the active and normal phase. Considering the lag correlation of these subseasonal modulation modes and TC genesis, it is possible to construct a statistical model for the purpose of extended-range forecasting of subseasonal variability of TC occurrence over the WNP.

  10. Increasing western US forest wildfire activity: sensitivity to changes in the timing of spring.

    PubMed

    Westerling, Anthony LeRoy

    2016-06-05

    Prior work shows western US forest wildfire activity increased abruptly in the mid-1980s. Large forest wildfires and areas burned in them have continued to increase over recent decades, with most of the increase in lightning-ignited fires. Northern US Rockies forests dominated early increases in wildfire activity, and still contributed 50% of the increase in large fires over the last decade. However, the percentage growth in wildfire activity in Pacific northwestern and southwestern US forests has rapidly increased over the last two decades. Wildfire numbers and burned area are also increasing in non-forest vegetation types. Wildfire activity appears strongly associated with warming and earlier spring snowmelt. Analysis of the drivers of forest wildfire sensitivity to changes in the timing of spring demonstrates that forests at elevations where the historical mean snow-free season ranged between two and four months, with relatively high cumulative warm-season actual evapotranspiration, have been most affected. Increases in large wildfires associated with earlier spring snowmelt scale exponentially with changes in moisture deficit, and moisture deficit changes can explain most of the spatial variability in forest wildfire regime response to the timing of spring.This article is part of the themed issue 'The interaction of fire and mankind'. © 2016 The Author(s).

  11. Increasing western US forest wildfire activity: sensitivity to changes in the timing of spring

    PubMed Central

    2016-01-01

    Prior work shows western US forest wildfire activity increased abruptly in the mid-1980s. Large forest wildfires and areas burned in them have continued to increase over recent decades, with most of the increase in lightning-ignited fires. Northern US Rockies forests dominated early increases in wildfire activity, and still contributed 50% of the increase in large fires over the last decade. However, the percentage growth in wildfire activity in Pacific northwestern and southwestern US forests has rapidly increased over the last two decades. Wildfire numbers and burned area are also increasing in non-forest vegetation types. Wildfire activity appears strongly associated with warming and earlier spring snowmelt. Analysis of the drivers of forest wildfire sensitivity to changes in the timing of spring demonstrates that forests at elevations where the historical mean snow-free season ranged between two and four months, with relatively high cumulative warm-season actual evapotranspiration, have been most affected. Increases in large wildfires associated with earlier spring snowmelt scale exponentially with changes in moisture deficit, and moisture deficit changes can explain most of the spatial variability in forest wildfire regime response to the timing of spring. This article is part of the themed issue ‘The interaction of fire and mankind’. PMID:27216510

  12. Antibacterial activity and chemical characteristics of several Western Australian honeys compared to manuka honey and pasture honey.

    PubMed

    Roshan, Niloufar; Rippers, Thomas; Locher, Cornelia; Hammer, Katherine A

    2017-03-01

    The physicochemical parameters and antibacterial activity of 10 Western Australian (WA) and two comparator honeys were determined. Honeys showed a pH range of 4.0-4.7, colour range of 41.3-470.7 mAU, methylglyoxal levels ranging from 82.2 to 325.9 mg kg -1 and hydrogen peroxide levels after 2 h of 22.7-295.5 µM. Antibacterial activity was assessed by the disc diffusion assay, phenol equivalence assay, determination of minimum inhibitory and bactericidal concentrations and a time-kill assay. Activity was shown for all honeys by one or more method, however, activity varied according to which assay was used. Minimum inhibitory concentrations for WA honeys against 10 organisms ranged from 4.0 to >32.0% (w/v). Removal of hydrogen peroxide activity by catalase resulted in decreased activity for several honeys. Overall, the data showed that honeys in addition to those derived from Leptospermum spp. have antimicrobial activity and should not be overlooked as potential sources of clinically useful honey.

  13. Design, synthesis, and anti-inflammatory activity of caffeoyl salicylate analogs as NO production inhibitors.

    PubMed

    Yu, Pan; Xia, Chao-Jie; Li, Dong-Dong; Ni, Jun-Jun; Zhao, Lin-Guo; Ding, Gang; Wang, Zhen-Zhong; Xiao, Wei

    2018-05-28

    Chlorogenic acid (CGA) has been reported to exhibit potent anti-inflammatory activity. However, the development of anti-inflammatory agent based on CGA has not been investigated. In this paper, a series of caffeoyl salicylate compounds derived from CGA were designed, synthesized, and evaluated by LPS-induced nitric oxide synthase inhibition and QRT-PCR technique. Most compounds showed modest activity to inhibit production of nitric oxide (NO) in RAW 264.7 cells induced by lipopolysaccharides (LPS). Among these compounds, QRT-PCR and western blotting results indicated that compounds 6b, 6c, 6f, 6g and D104 that possess 5-member ring or 6-member ring caused a significant inhibition against expression of the iNOS2 in LPS-induced macrophages. In addition, cytotoxic assay displayed most derivatives have good safety in vitro. This new promising scaffold could be further exploited for the development of anti-inflammatory agent in the future. Copyright © 2017. Published by Elsevier B.V.

  14. Anticomplementary activity of horse IgG and F(ab')2 antivenoms.

    PubMed

    Squaiella-Baptistão, Carla Cristina; Marcelino, José Roberto; Ribeiro da Cunha, Luiz Eduardo; Gutiérrez, José María; Tambourgi, Denise V

    2014-03-01

    Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several batches of IgG and F(ab')2 antivenoms from the Butantan, Vital Brazil, and Clodomiro Picado Institutes activated the complement classical pathway and induced the production of C3a; however, only those antivenoms from Clodomiro Picado generated C5a. Different protein profiles (IgG heavy chain, protein contaminants, and aggregates) were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses. Our results show that various antivenoms from different producers are able to activate the classical pathway of the complement system and generate anaphylatoxins, and these findings suggest that factors, such as composition, contaminant proteins, and aggregates, may influence the anticomplementary activity of antivenoms in vitro. Therefore, there is a need to further improve antivenom production methods to reduce their anticomplementary activity and potential to cause EARs.

  15. Temporal activation of Nrf2 in the penumbra and Nrf2 activator-mediated neuroprotection in ischemia-reperfusion injury.

    PubMed

    Takagi, Toshinori; Kitashoji, Akira; Iwawaki, Takao; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Yoshimura, Shinichi; Iwama, Toru; Hara, Hideaki

    2014-07-01

    Oxidative stress plays a critical role in mediating tissue injury and neuron death during ischemia-reperfusion injury (IRI). The Keap1-Nrf2 defense pathway serves as a master regulator of endogenous antioxidant defense, and Nrf2 has been attracting attention as a target for the treatment of IRI. In this study, we evaluated Nrf2 expression in IRI using OKD (Keap1-dependent oxidative stress detector) mice and investigated the neuroprotective ability of an Nrf2 activator. We demonstrated temporal changes in Nrf2 expression in the same mice with luciferase assays and an Nrf2 activity time course using Western blotting. We also visualized Nrf2 expression in the ischemic penumbra and investigated Nrf2 expression in mice and humans using immunohistochemistry. Endogenous Nrf2 upregulation was not detected early in IRI, but expression peaked 24h after ischemia. Nrf2 expression was mainly detected in the penumbra, and it was found in neurons and astrocytes in both mice and humans. Intravenous administration of the Nrf2 activator bardoxolone methyl (BARD) resulted in earlier upregulation of Nrf2 and heme oxygenase-1. Furthermore, BARD decreased infarction volume and improved neurological symptoms after IRI. These findings indicate that earlier Nrf2 activation protects neurons, possibly via effects on astrocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. ROLE OF RAS IN METAL-INDUCED EGF RECEPTOR AND NFKB SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    EPA Science Inventory

    We have shown previously that EGF receptor signaling is triggered by some metals associated with ambient air particles. Western blot using phospho-specific antibodies showed that As, Zn and V activated EGF receptor tyrosine kinase and the downstream kinases, MEK1/2 and ERK1/2. Us...

  17. Expression and purification of the antimicrobial peptide GSL1 in bacteria for raising antibodies.

    PubMed

    Meiyalaghan, Sathiyamoorthy; Latimer, Julie M; Kralicek, Andrew V; Shaw, Martin L; Lewis, John G; Conner, Anthony J; Barrell, Philippa J

    2014-11-04

    The Gibberellin Stimulated-Like (GSL) or Snakin peptides from higher plants are cysteine-rich, with broad spectrum activity against a range of bacterial and fungal pathogens. To detect GSL peptides in applications such as western blot analysis and enzyme-linked immunosorbent assays (ELISA), specific antibodies that recognise GSL peptides are required. However, the intrinsic antimicrobial activity of these peptides is likely to prevent their expression alone in bacterial or yeast expression systems for subsequent antibody production in animal hosts. To overcome this issue we developed an Escherichia coli expression strategy based on the expression of the GSL1 peptide as a His-tagged thioredoxin fusion protein. The DNA sequence for the mature GSL1 peptide from potato (Solanum tuberosum L.) was cloned into the pET-32a expression vector to produce a construct encoding N-terminally tagged his6-thioredoxin-GSL1. The fusion protein was overexpressed in E. coli to produce soluble non-toxic protein. The GSL1 fusion protein could be easily purified by using affinity chromatography to yield ~1.3 mg of his6-thioredoxin-GSL1 per L of culture. The fusion protein was then injected into rabbits for antibody production. Western blot analysis showed that the antibodies obtained from rabbit sera specifically recognised the GSL1 peptide that had been expressed in a wheat germ cell-free expression system. We present here the first report of a GSL1 peptide expressed as a fusion protein with thioredoxin that has resulted in milligram quantities of soluble protein to be produced. We have also demonstrated that a wheat germ system can be used to successfully express small quantities of GSL1 peptide useful as positive control in western blot analysis. To our knowledge this is the first report of antibodies being produced against GSL1 peptide. The antibodies will be useful for analysis of GSL1peptides in western blot, localization by immunohistochemistry (IHC) and quantitation by ELISA.

  18. Expression of activation-induced cytidine deaminase is associated with a poor prognosis of diffuse large B cell lymphoma patients treated with CHOP-based chemotherapy.

    PubMed

    Kawamura, Kiyoko; Wada, Akihiko; Wang, Ji-Yang; Li, Quanhai; Ishii, Akihiro; Tsujimura, Hideki; Takagi, Toshiyuki; Itami, Makiko; Tada, Yuji; Tatsumi, Koichiro; Shimada, Hideaki; Hiroshima, Kenzo; Tagawa, Masatoshi

    2016-01-01

    Activation-induced cytidine deaminase (AID) is involved in somatic hypermutation and class switch recombination processes in the antibody formation. The AID activity induces gene mutations and could be associated with transformation processes of B cells. Nevertheless, the relation between AID expression and the prognosis of B cell lymphoma patients remains uncharacterized. We examined expression levels of the AID gene in 89 lymph node specimens from lymphoma and non-lymphoma patients with Northern blot analysis and investigated an association with their survival. The AID gene was preferentially expressed in B cell lymphoma in particular in diffuse large B cell lymphoma and follicular lymphoma. We confirmed AID protein expression in the mRNA-positive but not in the negative specimens with Western blot analysis and immunohistochemical staining. Survival of the patients treated with cyclophosphamide-/doxorubicin-/vincristine-/prednisone-based chemotherapy demonstrated that the prognosis of diffuse large B cell patients was unfavorable in the mRNA-positive group compared with the negative group, and that AID expression levels were correlated with the poor prognosis. In contrast, AID expression was not linked with the prognosis of follicular lymphoma patients. AID expression is a predictive marker for an unfavorable outcome in DLBCL patients treated with the chemotherapy.

  19. Widespread activity of multiple lineages of Usutu virus, western Europe, 2016

    PubMed Central

    Cadar, Daniel; Lühken, Renke; van der Jeugd, Henk; Garigliany, Mutien; Ziegler, Ute; Keller, Markus; Lahoreau, Jennifer; Lachmann, Lars; Becker, Norbert; Kik, Marja; Oude Munnink, Bas B; Bosch, Stefan; Tannich, Egbert; Linden, Annick; Schmidt, Volker; Koopmans, Marion P; Rijks, Jolianne; Desmecht, Daniel; Groschup, Martin H; Reusken, Chantal; Schmidt-Chanasit, Jonas

    2017-01-01

    In the summer of 2016, Belgium, France, Germany and the Netherlands reported widespread Usutu virus (USUV) activity based on live and dead bird surveillance. The causative USUV strains represented four lineages, of which two putative novel lineages were most likely recently introduced into Germany and spread to other western European countries. The spatial extent of the outbreak area corresponded with R0 values > 1. The occurrence of the outbreak, the largest USUV epizootic registered so far in Europe, allowed us to gain insight in how a recently introduced arbovirus with potential public health implications can spread and become a resident pathogen in a naïve environment. Understanding the ecological and epidemiological factors that drive the emergence or re-emergence of USUV is critical to develop and implement timely surveillance strategies for adequate preventive and control measures. Public health authorities, blood transfusion services and clinicians in countries where USUV was detected should be aware of the risk of possible USUV infection in humans, including in patients with unexplained encephalitis or other neurological impairments, especially during late summer when mosquito densities peak. PMID:28181903

  20. Family Instability and Early Initiation of Sexual Activity in Western Kenya

    PubMed Central

    2014-01-01

    Epidemiological, economic, and social forces have produced high levels of volatility in family and household structure for young people growing up in sub-Saharan Africa in recent decades. However, scholarship on the family to date has not examined the influence of this family instability on young people’s well-being. The current study employs unique life history calendar data from Western Kenya to investigate the relationship between instability in caregiving and early initiation of sexual activity. It draws on a body of work on parental union instability in the United States, and examines new dimensions of family change. Analyses reveal a positive association between transitions in primary caregiver and the likelihood of early sexual debut that is rapidly manifested following caregiver change and persists for a short period. The association is strongest at early ages, and there is a cumulative effect of multiple caregiver changes. The results highlight the importance of studying family stability in sub-Saharan Africa, as distinct from family structure, and for attention to dimensions such as age and recency. PMID:23055236

  1. Family instability and early initiation of sexual activity in Western Kenya.

    PubMed

    Goldberg, Rachel E

    2013-04-01

    Epidemiological, economic, and social forces have produced high levels of volatility in family and household structure for young people growing up in sub-Saharan Africa in recent decades. However, scholarship on the family to date has not examined the influence of this family instability on young people's well-being. The current study employs unique life history calendar data from Western Kenya to investigate the relationship between instability in caregiving and early initiation of sexual activity. It draws on a body of work on parental union instability in the United States, and examines new dimensions of family change. Analyses reveal a positive association between transitions in primary caregiver and the likelihood of early sexual debut that is rapidly manifested following caregiver change and persists for a short period. The association is strongest at early ages, and there is a cumulative effect of multiple caregiver changes. The results highlight the importance of studying family stability in sub-Saharan Africa, as distinct from family structure, and for attention to dimensions such as age and recency.

  2. Cell Cycle Synchronization of HeLa Cells to Assay EGFR Pathway Activation.

    PubMed

    Wee, Ping; Wang, Zhixiang

    2017-01-01

    Progression through the cell cycle causes changes in the cell's signaling pathways that can alter EGFR signal transduction. Here, we describe drug-derived protocols to synchronize HeLa cells in various phases of the cell cycle, including G1 phase, S phase, G2 phase, and mitosis, specifically in the mitotic stages of prometaphase, metaphase, and anaphase/telophase. The synchronization procedures are designed to allow synchronized cells to be treated for EGF and collected for the purpose of Western blotting for EGFR signal transduction components.S phase synchronization is performed by thymidine block, G2 phase with roscovitine, prometaphase with nocodazole, metaphase with MG132, and anaphase/telophase with blebbistatin. G1 phase synchronization is performed by culturing synchronized mitotic cells obtained by mitotic shake-off. We also provide methods to validate the synchronization methods. For validation by Western blotting, we provide the temporal expression of various cell cycle markers that are used to check the quality of the synchronization. For validation of mitotic synchronization by microscopy, we provide a guide that describes the physical properties of each mitotic stage, using their cellular morphology and DNA appearance. For validation by flow cytometry, we describe the use of imaging flow cytometry to distinguish between the phases of the cell cycle, including between each stage of mitosis.

  3. Genetic and physical analyses of Methylobacterium organophilum XX genes encoding methanol oxidation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Machlin, S.M.; Tam, P.E.; Bastien, C.A.

    When allyl alcohol was used as a suicide substrate, spontaneous mutants and UV light- and nitrous acid-generated mutants of Methylobacterium organophilum XX were selected which grew on methylamine but not on methanol. There was no detectable methanol dehydrogenase (MDH) activity in crude extracts of these mutants, yet Western blots revealed that some mutants still produced MDH protein. Complementation of 50 mutants by a cosmid gene bank of M. organophilum XX demonstrated that three major regions of the genome, each of which was separated by a minimum of 40 kilobases, were required for expression of active MDH. By subcloning and Tn5more » insertion mutagenesis of subcloned fragments, at least 11 genes clustered within these three regions were subsequently identified. The identity of the MDH structural gene, which was initially determined by hybridization to the structural gene of Methylobacterium sp. strain AM1, was confirmed by Western blot analysis of an MDH-..beta..-galactosidase fusion protein.« less

  4. Effects of selexipag and its active metabolite in contrasting the profibrotic myofibroblast activity in cultured scleroderma skin fibroblasts.

    PubMed

    Cutolo, Maurizio; Ruaro, Barbara; Montagna, Paola; Brizzolara, Renata; Stratta, Emanuela; Trombetta, Amelia Chiara; Scabini, Stefano; Tavilla, Pier Paolo; Parodi, Aurora; Corallo, Claudio; Giordano, Nicola; Paolino, Sabrina; Pizzorni, Carmen; Sulli, Alberto; Smith, Vanessa; Soldano, Stefano

    2018-05-02

    Myofibroblasts contribute to fibrosis through the overproduction of extracellular matrix (ECM) proteins, primarily type I collagen (COL-1) and fibronectin (FN), a process which is mediated in systemic sclerosis (SSc) by the activation of fibrogenic intracellular signaling transduction molecules, including extracellular signal-regulated kinases 1 and 2 (Erk1/2) and protein kinase B (Akt). Selexipag is a prostacyclin receptor agonist synthesized for the treatment of pulmonary arterial hypertension. The study investigated the possibility for selexipag and its active metabolite (ACT-333679) to downregulate the profibrotic activity in primary cultures of SSc fibroblasts/myofibroblasts and the fibrogenic signaling molecules involved. Fibroblasts from skin biopsies obtained with Ethics Committee (EC) approval from patients with SSc, after giving signed informed consent, were cultured until the 3 rd culture passage and then either maintained in normal growth medium (untreated cells) or independently treated with different concentrations of selexipag (from 30 μM to 0.3 μM) or ACT-333679 (from 10 μM to 0.1 μM) for 48 h. Protein and gene expressions of α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (S100A4), COL-1, and FN were investigated by western blotting and quantitative real-time PCR. Erk1/2 and Akt phosphorylation was investigated in untreated and ACT-333679-treated cells by western botting. Selexipag and ACT-333679 significantly reduced protein synthesis and gene expression of α-SMA, S100A4, and COL-1 in cultured SSc fibroblasts/myofibroblasts compared to untreated cells, whereas FN was significantly downregulated at the protein level. Interestingly, ACT-333679 significantly reduced the phosphorylation of Erk1/2 and Akt in cultured SSc fibroblasts/myofibroblasts. Selexipag and mainly its active metabolite ACT-333679 were found for the first time to potentially interfere with the profibrotic activity of cultured SSc fibroblasts

  5. Inactivation of Phaeodactylum tricornutum urease gene using transcription activator-like effector nuclease-based targeted mutagenesis.

    PubMed

    Weyman, Philip D; Beeri, Karen; Lefebvre, Stephane C; Rivera, Josefa; McCarthy, James K; Heuberger, Adam L; Peers, Graham; Allen, Andrew E; Dupont, Christopher L

    2015-05-01

    Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle. © 2014 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  6. East-Asian Teaching Practices through the Eyes of Western Learners

    ERIC Educational Resources Information Center

    Chen, Rainbow Tsai-Hung

    2014-01-01

    Many East-Asian countries are actively positioning themselves as receiving countries of international students. Consequently, the number of international students in these countries is steadily growing. Given the differences between Eastern and Western conceptions of teaching and learning, it could be expected that Western learners studying in the…

  7. Western States and National Energy Policy: The New States' Rights.

    ERIC Educational Resources Information Center

    Hall, Timothy A.; And Others

    1978-01-01

    Energy development in western states has contributed to demands for new states' rights. Western states want a more active part in federal energy policy formulation. Article discusses recent intergovernmental relations, federal-state relations, and the effects of national energy, environmental, and related policies on energy resource development in…

  8. Nuclear factor-kappaB activation correlates with better prognosis and Akt activation in human gastric cancer.

    PubMed

    Lee, Byung Lan; Lee, Hye Seung; Jung, Jieun; Cho, Sung Jin; Chung, Hee-Yong; Kim, Woo Ho; Jin, Young-Woo; Kim, Chong Soon; Nam, Seon Young

    2005-04-01

    Because the biological significance of constitutive nuclear factor-kappaB (NF-kappaB) activation in human gastric cancer is unclear, we undertook this study to clarify the regulatory mechanism of NF-kappaB activation and its clinical significance. Immunohistochemistry for NF-kappaB/RelA was done on 290 human gastric carcinoma specimens placed on tissue array slides. The correlations between NF-kappaB activation and clinicopathologic features, prognosis, Akt activation, tumor suppressor gene expression, or Bcl-2 expression were analyzed. We also did luciferase reporter assay, Western blot analysis, and reverse transcription-PCR using the SNU-216 human gastric cancer cell line transduced with retroviral vectors containing constitutively active Akt or the NF-kappaB repressor mutant of IkappaBalpha. Nuclear expression of RelA was found in 18% of the gastric carcinomas and was higher in early-stage pathologic tumor-node-metastasis (P = 0.019). A negative correlation was observed between NF-kappaB activation and lymphatic invasion (P = 0.034) and a positive correlation between NF-kappaB activation and overall survival rate of gastric cancer patients (P = 0.0228). In addition, NF-kappaB activation was positively correlated with pAkt (P = 0.047), p16 (P = 0.004), adenomatous polyposis coli (P < 0.001), Smad4 (P = 0.002), and kangai 1 (P < 0.001) expression. An in vitro study showed that NF-kappaB activity in gastric cancer cells is controlled by and controls Akt. NF-kappaB activation was frequently observed in early-stage gastric carcinoma and was significantly correlated with better prognosis and Akt activation. These findings suggest that NF-kappaB activation is a valuable prognostic variable in gastric carcinoma.

  9. Effects of TGF-β1 on plasminogen activation in human dental pulp cells: Role of ALK5/Smad2, TAK1 and MEK/ERK signalling.

    PubMed

    Chang, Mei-Chi; Chang, Hsiao-Hua; Lin, Po-Shuan; Huang, Yu-An; Chan, Chiu-Po; Tsai, Yi-Ling; Lee, Shen-Yang; Jeng, Po-Yuan; Kuo, Han-Yueh; Yeung, Sin-Yuet; Jeng, Jiiang-Huei

    2018-04-01

    Transforming growth factor-β1 (TGF-β1) plays an important role in the pulpal repair and dentinogenesis. Plasminogen activation (PA) system regulates extracellular matrix turnover. In this study, we investigated the effects of TGF-β1 on PA system of dental pulp cells and its signalling pathways. Dental pulp cells were treated with different concentrations of TGF-β1. MTT assay, reverse transcription-polymerase chain reaction, Western blotting and enzyme-linked immunosorbant assay (ELISA) were used to detect the effect of TGF-β1 on cell viability, mRNA and protein expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) as well as their secretion. The phosphorylation of Smad2 and TAK1 was analysed by Pathscan ELISA or Western blotting. Cells were pretreated with SB431542 (ALK5/Smad2/3 inhibitor), 5z-7-oxozeaenol (TAK1 inhibitor) and U0126 (MEK/ERK inhibitor) for examining the related signalling. TGF-β1 slightly inhibited cell growth that was reversed by SB431542. TGF-β1 upregulated both RNA and protein expression of PAI-1 and uPAR, whereas it downregulated uPA expression. Accordingly, TGF-β1 stimulated PAI-1 and soluble uPAR (suPAR) secretion of pulp cells, whereas uPA secretion was inhibited. TGF-β1 induced the phosphorylation of Smad2 and TAK1. In addition, SB431542, 5z-7-oxozeaenol and U0126 attenuated the TGF-β1-induced secretion of PAI-1 and suPAR. These results indicate that TGF-β1 is possibly involved in the repair/regeneration and inflammatory processes of dental pulp via regulation of PAI-1, uPA and uPAR. These effects of TGF-β1 are related to activation of ALK5/Smad2, TAK1 and MEK/ERK signalling pathways. Clarifying the signal transduction for the effects of TGF-β1 is helpful for pulpo-dentin regeneration and tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  10. Betaine supplement enhances skeletal muscle differentiation in murine myoblasts via IGF-1 signaling activation

    PubMed Central

    2013-01-01

    Background Betaine (BET) is a component of many foods, including spinach and wheat. It is an essential osmolyte and a source of methyl groups. Recent studies have hypothesized that BET might play a role in athletic performance. However, BET effects on skeletal muscle differentiation and hypertrophy are still poorly understood. Methods We examined BET action on neo myotubes maturation and on differentiation process, using C2C12 murine myoblastic cells. We used RT2-PCR array, Western blot and immunofluorescence analysis to study the BET effects on morphological features of C2C12 and on signaling pathways involved in muscle differentiation and hypertrophy. Results We performed a dose–response study, establishing that 10 mM BET was the dose able to stimulate morphological changes and hypertrophic process in neo myotubes. RT2-PCR array methodology was used to identify the expression profile of genes encoding proteins involved in IGF-1 pathway. A dose of 10 mM BET was found to promote IGF-1 receptor (IGF-1 R) expression. Western blot and immunofluorescence analysis, performed in neo myotubes, pointed out that 10 mM BET improved IGF-1 signaling, synthesis of Myosin Heavy Chain (MyHC) and neo myotubes length. In addition, we investigated BET role on myoblasts proliferation and differentiation. During proliferation, BET did not modify C2C12 proliferative rate, but promoted myogenic induction, enhancing MyoD protein content and cellular elongation. During differentiation, BET caused an increase of muscle-specific markers and IGF-1 R protein levels. Conclusions Our findings provide the first evidence that BET could promote muscle fibers differentiation and increase myotubes size by IGF-1 pathway activation, suggesting that BET might represent a possible new drug/integrator strategy, not only in sport performance but also in clinical conditions characterized by muscle function impairment. PMID:23870626

  11. The Modulation of Tropical Storm Activity in the Western North Pacific by the Madden-Julian Oscillation in GEOS-5 AGCM Experiments

    NASA Technical Reports Server (NTRS)

    Kim, Dongmin; Lee, Myong-In; Kim, Hye-Mi; Schubert, Siegfried D.; Yoo, Jin Ho

    2014-01-01

    This study examines the influence of the Madden-Julian Oscillation (MJO) on tropical storm (TS) activity in the western North Pacific, using observations and GEOS-5 simulations at 50-km horizontal resolution. While GEOS-5 produces an MJO of faster propagation and weaker amplitude, it nevertheless reproduces the observed modulation of TS activity by the MJO with the highest TS genesis and increased track density in the active phases of MJO. The study suggests that the simulation of the sub-seasonal variability of TS activity could be improved by improving the simulations of the MJO in climate models.

  12. Cross-Sectional Serological Survey of Human Fascioliasis in Canutama Municipality in Western Amazon, Brazil

    PubMed Central

    Lima, Walter dos Santos; de Almeida, Francisco Lazaro Moreira; Coelho, Leila Inês Aguiar Raposo Câmara; Araújo, Guilherme Alfredo Novelino; Lima, Mariana Gomes; Maciel, Luiz Henrique Gonçalves; Pereira, Cíntia Aparecida de Jesus; Maciel, Thaís Costa da Silva; Guerra, Jorge Augusto de Oliveira; Santana, Rosa Amélia Gonçalves; Guerra, Maria das Graças Vale Barbosa

    2018-01-01

    Background Fascioliasis is an important parasitic disease. In the northern region of Brazil, a human parasite infection has been reported through a coprological survey. Eggs of Fasciola hepatica were found in fecal samples of 11 individuals. Knowledge of the infection in animals or the presence of snails is necessary to address the possibility of the parasite cycle occurrence in that region. The aim of this study was to describe the transmission of human fascioliasis in Canutama, Amazonas, in Western Amazonia, Brazil. Methods Serological (ELISA and Western Blot, WB) and parasitological analyses were carried out in humans. In addition, the presence of the intermediate snail host within the community was examined. Results A total of 434 human samples were included in the study, of which 36 (8.3%) were reactive by ELISA and 8 (1.8%) were reactive by WB. Fasciola hepatica eggs were found in one human sample. The occurrence of the intermediated host was recorded and 31/43 specimens were identified as Lymnaea columella. Conclusion. Canutama constitutes a focus of transmission of human fascioliasis. This study describes the first serological survey for human fascioliasis, as well as its simultaneous occurrence in human hosts and possible intermediates performed in northern Brazil. PMID:29593895

  13. Interleukin 31 mediates MAP kinase and STAT1/3 activation in intestinal epithelial cells and its expression is upregulated in inflammatory bowel disease

    PubMed Central

    Dambacher, Julia; Beigel, Florian; Seiderer, Julia; Haller, Dirk; Göke, Burkhard; Auernhammer, Christoph J; Brand, Stephan

    2007-01-01

    Background/aim Interleukin 31 (IL31), primarily expressed in activated lymphocytes, signals through a heterodimeric receptor complex consisting of the IL31 receptor alpha (IL31Rα) and the oncostatin M receptor (OSMR). The aim of this study was to analyse IL31 receptor expression, signal transduction, and specific biological functions of this cytokine system in intestinal inflammation. Methods Expression studies were performed by RT‐PCR, quantitative PCR, western blotting, and immunohistochemistry. Signal transduction was analysed by western blotting. Cell proliferation was measured by MTS assays, cell migration by restitution assays. Results Colorectal cancer derived intestinal epithelial cell (IEC) lines express both IL31 receptor subunits, while their expression in unstimulated primary murine IEC was low. LPS and the proinflammatory cytokines TNF‐α, IL1β, IFN‐γ, and sodium butyrate stimulation increased IL31, IL31Rα, and OSMR mRNA expression, while IL31 itself enhanced IL8 expression in IEC. IL31 mediates ERK‐1/2, Akt, STAT1, and STAT3 activation in IEC resulting in enhanced IEC migration. However, at low cell density, IL31 had significant antiproliferative capacities (p<0.005). IL31 mRNA expression was not increased in the TNFΔARE mouse model of ileitis but in inflamed colonic lesions compared to non‐inflamed tissue in patients with Crohn's disease (CD; average 2.4‐fold increase) and in patients with ulcerative colitis (UC; average 2.6‐fold increase) and correlated with the IL‐8 expression in these lesions (r = 0.564 for CD; r = 0.650 for UC; total number of biopsies analysed: n = 88). Conclusion IEC express the functional IL31 receptor complex. IL31 modulates cell proliferation and migration suggesting a role in the regulation of intestinal barrier function particularly in intestinal inflammation. PMID:17449633

  14. Response of western mountain ecosystems to climatic variability and change: The Western Mountain Initiative

    USGS Publications Warehouse

    Stephenson, Nathan L.; Peterson, Dave; Fagre, Daniel B.; Allen, Craig D.; McKenzie, Donald; Baron, Jill S.; O'Brian, Kelly

    2007-01-01

    Mountain ecosystems within our national parks and other protected areas provide valuable goods and services such as clean water, biodiversity conservation, and recreational opportunities, but their potential responses to expected climatic changes are inadequately understood. The Western Mountain Initiative (WMI) is a collaboration of scientists whose research focuses on understanding and predicting responses of western mountain ecosystems to climatic variability and change. It is a legacy of the Global Change Research Program initiated by the National Park Service (NPS) in 1991 and continued by the U.S. Geological Survey (USGS) to this day as part of the U.S. Climate Change Science Program (http://www.climatescience.gov/). All WMI scientists are active participants in CIRMOUNT, and seek to further its goals.

  15. Marine actinomycete crude extracts with potent TRAIL-resistance overcoming activity against breast cancer cells.

    PubMed

    Elmallah, Mohammed I Y; Micheau, Olivier; Eid, Mennat Allah G; Hebishy, Ali M S; Abdelfattah, Mohamed S

    2017-06-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent, as it can kill tumor cells selectively. In our search of bioactive natural products to overcome TRAIL-resistance, we isolated 47 actinomycete strains from different sediments and seawater samples collected from the Red Sea coast in Egypt and found four crude extracts (EGY1, EGY3, EGY24 and EGY34) displaying TRAIL sensitizing activity in the resistant breast cancer cell line MDA-MB-231. None of these crude extracts exhibited cytotoxic effect on normal mouse embryonic fibroblasts (MEF), with the exception of EGY34. Analysis of the signaling pathways underlying the sensitization of MDA-MB-231 cells to TRAIL-induced apoptosis, by western blotting, revealed that all crude extracts facilitated initiator caspase‑8/-10 activation upon TRAIL stimulation, but that in addition, EGY3 and EGY34, alone, induced strong ER-stress activation, with the appearance of BiP in the cytosolic extracts. Our results pave the way to the discovery and the development of marine-derived drugs for cancer therapy.

  16. Aberrant AKT activation drives well-differentiated liposarcoma

    PubMed Central

    Gutierrez, Alejandro; Snyder, Eric L.; Marino-Enriquez, Adrian; Zhang, Yi-Xiang; Sioletic, Stefano; Kozakewich, Elena; Grebliunaite, Ruta; Ou, Wen-bin; Sicinska, Ewa; Raut, Chandrajit P.; Demetri, George D.; Perez-Atayde, Antonio R.; Wagner, Andrew J.; Fletcher, Jonathan A.; Fletcher, Christopher D. M.; Look, A. Thomas

    2011-01-01

    Well-differentiated liposarcoma (WDLPS), one of the most common human sarcomas, is poorly responsive to radiation and chemotherapy, and the lack of animal models suitable for experimental analysis has seriously impeded functional investigation of its pathobiology and development of effective targeted therapies. Here, we show that zebrafish expressing constitutively active Akt2 in mesenchymal progenitors develop WDLPS that closely resembles the human disease. Tumor incidence rates were 8% in p53 wild-type zebrafish, 6% in p53 heterozygotes, and 29% in p53-homozygous mutant zebrafish (P = 0.013), indicating that aberrant Akt activation collaborates with p53 mutation in WDLPS pathogenesis. Analysis of primary clinical specimens of WDLPS, and of the closely related dedifferentiated liposarcoma (DDLPS) subtype, revealed immunohistochemical evidence of AKT activation in 27% of cases. Western blot analysis of a panel of cell lines derived from patients with WDLPS or DDLPS revealed robust AKT phosphorylation in all cell lines examined, even when these cells were cultured in serum-free media. Moreover, BEZ235, a small molecule inhibitor of PI3K and mammalian target of rapamycin that effectively inhibits AKT activation in these cells, impaired viability at nanomolar concentrations. Our findings are unique in providing an animal model to decipher the molecular pathogenesis of WDLPS, and implicate AKT as a previously unexplored therapeutic target in this chemoresistant sarcoma. PMID:21930930

  17. Production of Hybrids between Western Gray Wolves and Western Coyotes

    PubMed Central

    Mech, L. David; Christensen, Bruce W.; Asa, Cheryl S.; Callahan, Margaret; Young, Julie K.

    2014-01-01

    Using artificial insemination we attempted to produce hybrids between captive, male, western, gray wolves (Canis lupus) and female, western coyotes (Canis latrans) to determine whether their gametes would be compatible and the coyotes could produce and nurture offspring. The results contribute new information to an ongoing controversy over whether the eastern wolf (Canis lycaon) is a valid unique species that could be subject to the U. S. Endangered Species Act. Attempts with transcervically deposited wolf semen into nine coyotes over two breeding seasons yielded three coyote pregnancies. One coyote ate her pups, another produced a resorbed fetus and a dead fetus by C-section, and the third produced seven hybrids, six of which survived. These results show that, although it might be unlikely for male western wolves to successfully produce offspring with female western coyotes under natural conditions, western-gray-wolf sperm are compatible with western-coyote ova and that at least one coyote could produce and nurture hybrid offspring. This finding in turn demonstrates that gamete incompatibility would not have prevented western, gray wolves from inseminating western coyotes and thus producing hybrids with coyote mtDNA, a claim that counters the view that the eastern wolf is a separate species. However, some of the difficulties experienced by the other inseminated coyotes tend to temper that finding and suggest that more experimentation is needed, including determining the behavioral and physical compatibility of western gray wolves copulating with western coyotes. Thus although our study adds new information to the controversy, it does not settle it. Further study is needed to determine whether the putative Canis lycaon is indeed a unique species. PMID:24586418

  18. Production of hybrids between western gray wolves and western coyotes.

    PubMed

    Mech, L David; Christensen, Bruce W; Asa, Cheryl S; Callahan, Margaret; Young, Julie K

    2014-01-01

    Using artificial insemination we attempted to produce hybrids between captive, male, western, gray wolves (Canis lupus) and female, western coyotes (Canis latrans) to determine whether their gametes would be compatible and the coyotes could produce and nurture offspring. The results contribute new information to an ongoing controversy over whether the eastern wolf (Canis lycaon) is a valid unique species that could be subject to the U. S. Endangered Species Act. Attempts with transcervically deposited wolf semen into nine coyotes over two breeding seasons yielded three coyote pregnancies. One coyote ate her pups, another produced a resorbed fetus and a dead fetus by C-section, and the third produced seven hybrids, six of which survived. These results show that, although it might be unlikely for male western wolves to successfully produce offspring with female western coyotes under natural conditions, western-gray-wolf sperm are compatible with western-coyote ova and that at least one coyote could produce and nurture hybrid offspring. This finding in turn demonstrates that gamete incompatibility would not have prevented western, gray wolves from inseminating western coyotes and thus producing hybrids with coyote mtDNA, a claim that counters the view that the eastern wolf is a separate species. However, some of the difficulties experienced by the other inseminated coyotes tend to temper that finding and suggest that more experimentation is needed, including determining the behavioral and physical compatibility of western gray wolves copulating with western coyotes. Thus although our study adds new information to the controversy, it does not settle it. Further study is needed to determine whether the putative Canis lycaon is indeed a unique species.

  19. Production of hybrids between western gray wolves and western coyotes

    USGS Publications Warehouse

    Mech, L. David; Christensen, Bruce W.; Asa, Cheryl S.; Callahan, Margaret; Young, Julie K.

    2014-01-01

    Using artificial insemination we attempted to produce hybrids between captive, male, western, gray wolves (Canis lupus) and female, western coyotes (Canis latrans) to determine whether their gametes would be compatible and the coyotes could produce and nurture offspring. The results contribute new information to an ongoing controversy over whether the eastern wolf (Canis lycaon) is a valid unique species that could be subject to the U. S. Endangered Species Act. Attempts with transcervically deposited wolf semen into nine coyotes over two breeding seasons yielded three coyote pregnancies. One coyote ate her pups, another produced a resorbed fetus and a dead fetus by C-section, and the third produced seven hybrids, six of which survived. These results show that, although it might be unlikely for male western wolves to successfully produce offspring with female western coyotes under natural conditions, western-gray-wolf sperm are compatible with western-coyote ova and that at least one coyote could produce and nurture hybrid offspring. This finding in turn demonstrates that gamete incompatibility would not have prevented western, gray wolves from inseminating western coyotes and thus producing hybrids with coyote mtDNA, a claim that counters the view that the eastern wolf is a separate species. However, some of the difficulties experienced by the other inseminated coyotes tend to temper that finding and suggest that more experimentation is needed, including determining the behavioral and physical compatibility of western gray wolves copulating with western coyotes. Thus although our study adds new information to the controversy, it does not settle it. Further study is needed to determine whether the putative Canis lycaon is indeed a unique species.

  20. [Analytic study of dot blotting for the detection of anti-Jo-1, anti-M2, anti-ribosomes and anti-LKM].

    PubMed

    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.

  1. Cross-sectional study of serum antibodies against Sarcocystis neurona in cats tested for antibodies against Toxoplasma gondii.

    PubMed

    Rossano, Mary G; Murphy, Alice J; Vrable, Ruth A; Vanzo, Nicole E; Lewis, Stacy K; Sheline, Katherine D; Kaneene, John B; Mansfield, Linda S

    2002-08-15

    To determine apparent seroprevalence of antibodies against Sarcocystis neurona in a population of domestic cats previously tested for antibodies against Toxoplasma gondii. Cross-sectional study. Serum from 196 domestic cats. Banked serum samples submitted to the Michigan State University Animal Health Diagnostic Laboratory for T. gondii diagnostic testing were tested for antibodies against S. neurona by use of an indirect fluorescent antibody (IFA) test and a western blot test. Submission records were analyzed to determine descriptive statistics and test for associations between positive results of a test for S. neurona and other variables in the data set. 10 of 196 (5%) samples yielded positive results for antibodies against S. neurona by use of western blot analysis, whereas 27 samples yielded positive results by use of the IFA. No association was found between S. neurona western blot test results and T. gondii test results, age, sex, or the reason for T. gondii testing. The S. neurona IFA titer was positively and significantly associated with positive results of western blot analysis. Domestic cats are not likely to play a substantial role as intermediate hosts in the natural life cycle of S. neurona. Results indicate that natural infection of domestic cats may occur, and small animal practitioners should be aware of this fact when evaluating cats with neurologic disease. The S. neurona IFA test had lower specificity than western blot analysis.

  2. Dasatinib is preclinically active against Src-overexpressing human transitional cell carcinoma of the urothelium with activated Src signaling.

    PubMed

    Levitt, Jonathan M; Yamashita, Hideyuki; Jian, Weiguo; Lerner, Seth P; Sonpavde, Guru

    2010-05-01

    Dasatinib is an orally administered multitargeted kinase inhibitor that targets Src family tyrosine kinases, Abl, c-Kit, and PDGFR. A preclinical study was conducted to evaluate dasatinib alone or combined with cisplatin for human transitional cell carcinoma (TCC). Expression of Src in a human TCC tissue microarray was evaluated by immunohistochemistry. The activity of dasatinib and/or cisplatin was evaluated in six human TCC cell lines. Western blot was done to assess Src and phosphorylated-Src (p-Src) expression. The activity of dasatinib alone and in combination with cisplatin was determined in murine subcutaneous xenografts. Sixty-two percent to 75% of human TCC expressed Src. Dasatinib displayed significant antiproliferative activity at nanomolar concentrations against two human TCC cell lines (RT4 and Hu456) that exhibited high Src and p-Src expression and were cisplatin-resistant. RT4 cells were the most sensitive and displayed the highest level of Src pathway activation (p-Src/Src ratio). Dasatinib downregulated p-Src in either sensitive or resistant cells. TCC cells that were sensitive to cisplatin (5637 and TCC-SUP) were highly resistant to dasatinib and exhibited low Src expression. Dasatinib showed antitumor activity in RT4 murine xenografts, and the combination of dasatinib and cisplatin was significantly more active than placebo. Combination dasatinib plus cisplatin significantly inhibited proliferation and promoted apoptosis in vivo. In conclusion, dasatinib displayed significant preclinical antitumor activity against Src-overexpressing human TCC with active Src signaling and was highly active in combination with cisplatin in vivo. Further clinical development might be warranted in selected human subjects.

  3. Morus alba and active compound oxyresveratrol exert anti-inflammatory activity via inhibition of leukocyte migration involving MEK/ERK signaling.

    PubMed

    Chen, Yi-Ching; Tien, Yin-Jing; Chen, Chun-Houh; Beltran, Francesca N; Amor, Evangeline C; Wang, Ran-Juh; Wu, Den-Jen; Mettling, Clément; Lin, Yea-Lih; Yang, Wen-Chin

    2013-02-23

    Morus alba has long been used in traditional Chinese medicine to treat inflammatory diseases; however, the scientific basis for such usage and the mechanism of action are not well understood. This study investigated the action of M. alba on leukocyte migration, one key step in inflammation. Gas chromatography-mass spectrometry (GC-MS) and cluster analyses of supercritical CO2 extracts of three Morus species were performed for chemotaxonomy-aided plant authentication. Phytochemistry and CXCR4-mediated chemotaxis assays were used to characterize the chemical and biological properties of M. alba and its active compound, oxyresveratrol. fluorescence-activated cell sorting (FACS) and Western blot analyses were conducted to determine the mode of action of oxyresveratrol. Chemotaxonomy was used to help authenticate M. alba. Chemotaxis-based isolation identified oxyresveratrol as an active component in M. alba. Phytochemical and chemotaxis assays showed that the crude extract, ethyl acetate fraction and oxyresveratrol from M. alba suppressed cell migration of Jurkat T cells in response to SDF-1. Mechanistic study indicated that oxyresveratrol diminished CXCR4-mediated T-cell migration via inhibition of the MEK/ERK signaling cascade. A combination of GC-MS and cluster analysis techniques are applicable for authentication of the Morus species. Anti-inflammatory benefits of M. alba and its active compound, oxyresveratrol, may involve the inhibition of CXCR-4-mediated chemotaxis and MEK/ERK pathway in T and other immune cells.

  4. Screening of Organophosphorus Acid Anhydrases from different Sources by Western Blot Analysis

    DTIC Science & Technology

    1991-01-01

    agents) have been found3 in both procaryotic and eukaryotic organisms. These enzymes, referred to as organophosphorous acid anhydrases (OPA anhydrases...of organisms, both procaryotic and eukaryotic. Previously, these enzymes were known as DFPases, somanases, paraoxonases or parathion hydrolases, based...cell (SLM Aminco) at 16,000 psi. Cellular debris was removed by centrifugation (46,000xg) for 30 minutes at 40C. The crude cell supernatant which

  5. Periostin activates pathways involved in epithelial-mesenchymal transition in adamantinomatous craniopharyngioma.

    PubMed

    Chen, Ming; Zheng, Shi-hao; Liu, Yi; Shi, Jin; Qi, Song-tao

    2016-01-15

    Periostin (POSTN) is an extracellular matrix protein (ECM) critical for epithelial-mesenchymal transitions (EMT) in several kinds of tumor cells. Previous studies have indicated that EMT exists in craniopharyngioma (CP), and expression of POSTN is a significant factor in the prognosis of CP. However, it has never been explored whether POSTN exists in CP, or how it activates CP's EMT. The expression of POSTN was examined in adamantinomatous craniopharyngioma (ACP) primary cells and tissues by immunohistochemistry, PCR and Western blot, respectively. The effects and mechanisms of POSTN on ACP cells' EMT were also analyzed. It was found that POSTN expression increased in ACP-associated fibroblasts. Overexpressed POSTN significantly elevated the EMT of ACP cells by regulating the expression of associated genes. More importantly, our further study revealed that the upregulated POSTN activated Akt signaling pathway to regulate the EMT. This study showed that POSTN is responsible for the EMT of ACP cells, and POSTN might be a potential molecular therapeutic target for ACP treatment in future. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Nucleoporin 62 and Ca(2+)/calmodulin dependent kinase kinase 2 regulate androgen receptor activity in castrate resistant prostate cancer cells.

    PubMed

    Karacosta, Loukia G; Kuroski, Laura A; Hofmann, Wilma A; Azabdaftari, Gissou; Mastri, Michalis; Gocher, Angela M; Dai, Shuhang; Hoste, Allen J; Edelman, Arthur M

    2016-02-15

    Re-activation of the transcriptional activity of the androgen receptor (AR) is an important factor mediating progression from androgen-responsive to castrate-resistant prostate cancer (CRPC). However, the mechanisms regulating AR activity in CRPC remain incompletely understood. Ca(2+) /calmodulin-dependent kinase kinase (CaMKK) 2 was previously shown to regulate AR activity in androgen-responsive prostate cancer cells. Our objective was to further explore the basis of this regulation in CRPC cells. The abundance of CaMKK2 in nuclear fractions of androgen-responsive prostate cancer and CRPC, cells were determined by subcellular fractionation and Western blotting. CaMKK2 association with nuclear pore complexes (NPCs) and nucleoporins (Nups) including Nup62, were imaged by structured illumination and super-resolution fluorescence microscopy and co-immunoprecipitation, respectively. The abundance and subcellular localization of CaMKK2 and Nup62 in human clinical specimens of prostate cancer was visualized by immunohistochemistry. The role of Nups in the growth and viability of CRPC cells was assessed by RNA interference and cell counting. The involvement of CaMKK2 and Nup62 in regulating AR transcriptional activity was addressed by RNA interference, chromatin immunoprecipitation, androgen response element reporter assay, and Western blotting. CaMKK2 was expressed at higher levels in the nuclear fraction of CPRC C4-2 cells, than in that of androgen-responsive LNCaP cells. In C4-2 cells, CaMKK2 associated with NPCs of the nuclear envelope and physically interacted with Nup62. CaMKK2 and Nup62 demonstrated pronounced, and similar increases in both expression and perinuclear/nuclear localization in human clinical specimens of advanced prostate cancer relative to normal prostate. Knockdown of Nup62, but not of Nups, 98 or 88, reduced growth and viability of C4-2 cells. Knockdown of Nup62 produced a greater reduction of the growth and viability of C4-2 cells than of non

  7. Eicosapentaenoic acid-enriched phosphatidylcholine isolated from Cucumaria frondosa exhibits anti-hyperglycemic effects via activating phosphoinositide 3-kinase/protein kinase B signal pathway.

    PubMed

    Hu, Shiwei; Xu, Leilei; Shi, Di; Wang, Jingfeng; Wang, Yuming; Lou, Qiaoming; Xue, Changhu

    2014-04-01

    Eicosapentaenoic acid-enriched phosphatidylcholine was isolated from the sea cucumber Cucumaria frondosa (Cucumaria-PC) and its effects on streptozotocin (STZ)-induced hyperglycemic rats were investigated. Male Sprague-Dawley rats were randomly divided into normal control, model control (STZ), low- and high-dose Cucumaria-PC groups (STZ + Cucumaria-PC at 25 and 75 mg/Kg·b·wt, intragastrically, respectively). Blood glucose, insulin, glycogen in liver and gastrocnemius were determined over 60 days. Insulin signaling in the rats' gastrocnemius was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The results showed that Cucumaria-PC significantly decreased blood glucose level, increased insulin secretion and glycogen synthesis in diabetic rats. RT-PCR analysis revealed that Cucumaria-PC significantly promoted the expressions of glycometabolism-related genes of insulin receptor (IR), insulin receptor substrate-1 (IRS-1), phosphoinositide 3-kinase (PI3K), protein kinase B (PKB), and glucose transporter 4 (GLUT4) in gastrocnemius. Western blotting assay demonstrated that Cucumaria-PC remarkably enhanced the proteins abundance of IR-β, PI3K, PKB, GLUT4, as well as phosphorylation of Tyr-IR-β, p85-PI3K, Ser473-PKB (P < 0.05 and P < 0.01). These findings suggested that Cucumaria-PC exhibited significant anti-hyperglycemic activities through up-regulating PI3K/PKB signal pathway mediated by insulin. Nutritional supplementation with Cucumaria-PC, if validated for human studies, may offer an adjunctive therapy for diabetes mellitus. Copyright © 2013 The Society for Biotechnology, Japan. All rights reserved.

  8. Myo-inositol reduces β-catenin activation in colitis

    PubMed Central

    Bradford, Emily M; Thompson, Corey A; Goretsky, Tatiana; Yang, Guang-Yu; Rodriguez, Luz M; Li, Linheng; Barrett, Terrence A

    2017-01-01

    AIM To assess dietary myo-inositol in reducing stem cell activation in colitis, and validate pβ-cateninS552 as a biomarker of recurrent dysplasia. METHODS We examined the effects of dietary myo-inositol treatment on inflammation, pβ-cateninS552 and pAkt levels by histology and western blot in IL-10-/- and dextran sodium sulfate-treated colitic mice. Additionally, we assessed nuclear pβ-cateninS552 in patients treated with myo-inositol in a clinical trial, and in patients with and without a history of colitis-induced dysplasia. RESULTS In mice, pβ-cateninS552 staining faithfully reported the effects of myo-inositol in reducing inflammation and intestinal stem cell activation. In a pilot clinical trial of myo-inositol administration in patients with a history of low grade dysplasia (LGD), two patients had reduced numbers of intestinal stem cell activation compared to the placebo control patient. In humans, pβ-cateninS552 staining discriminated ulcerative colitis patients with a history of LGD from those with benign disease. CONCLUSION Enumerating crypts with increased numbers of pβ-cateninS552 - positive cells can be utilized as a biomarker in colitis-associated cancer chemoprevention trials. PMID:28811707

  9. Analysis of creatine kinase activity with evaluation of protein expression under the effect of heat and hydrogen peroxide.

    PubMed

    Rakhmetov, A D; Pil, Lee Sang; Ostapchenko, L I; Zoon, Chae Ho

    2015-01-01

    Protein oxidation has detrimental effects on the brain functioning, which involves inhibition of the crucial enzyme, brain type creatine kinase (CKBB), responsible for the CK/phosphocreatine shuttle system. Here we demonstrate a susceptibility of CKBB to several ordinary stressors. In our study enzymatic activity of purified recombinant brain-type creatine kinase was evaluated. We assayed 30 nMconcentration of CKBB under normal and stress conditions. In the direction of phosphocreatine formation hydrogen peroxide and heat treatments altered CKBB activity down to 26 and 14%, respectively. Also, examination of immunoblotted membrane patterns by SDS-PAGE electrophoresis and western blot analysis showed a decrease in expression levels of intrinsic CKBB enzyme in HeLa andA549 cells. Hence, our results clearly show that cytosolic CKBB is extremely sensitive to oxidative stress and heat induced inactivation. Therefore, due to its susceptibility, this enzyme may be defined as a potential target in brain damage.

  10. Effects of Antioxidant Components of AREDS Vitamins and Zinc Ions on Endothelial Cell Activation: Implications for Macular Degeneration

    PubMed Central

    Zeng, Shemin; Hernández, Jasmine

    2012-01-01

    Purpose. To investigate whether the benefit of Age-Related Eye Disease Study (AREDS) formula multivitamins and zinc in the progression of age-related macular degeneration (AMD) may occur through inhibiting inflammatory events in the choroid. Methods. Mouse C166 endothelial cells (ECs) and, for some experiments, human retinal pigment epithelium (RPE)–choroid organ cultures were treated with AREDS multivitamin solution (MVS) or ZnCl2. The cytotoxicity of MVS was evaluated using a lactate dehydrogenase colorimetric assay. Cell motility was assessed using a scratch assay. Macrophage adhesion to EC monolayers or ICAM-1 protein was determined after MVS and zinc treatment and with or without lipopolysaccharide (LPS). Quantitative reverse transcription PCR and Western blot analysis were used to determine the effects of MVS on the expression of proinflammatory molecules in treated and untreated cells. Results. AREDS MVS and zinc did not affect C166 EC viability until the 56th hour after treatment. Scratch assays showed partial inhibition of MVS and zinc on EC migration. In cell adhesion assays, MVS and zinc decreased the number of macrophages bound to EC and to ICAM-1 protein. Quantitative PCR showed that LPS increased the expression of ICAM-1 in both C166 and human RPE-choroid cultures, which was partially offset by MVS and zinc. MVS and zinc also mitigated LPS-induced ICAM-1 protein expression on Western blot analysis. Conclusions. Treatment with AREDS MVS and zinc may affect both angiogenesis and endothelial-macrophage interactions. These results suggest that AREDS vitamins and zinc ions may slow the progression of AMD, in part through the attenuation of EC activation. PMID:22247465

  11. Activation of microglial cells triggers a release of brain-derived neurotrophic factor (BDNF) inducing their proliferation in an adenosine A2A receptor-dependent manner: A2A receptor blockade prevents BDNF release and proliferation of microglia

    PubMed Central

    2013-01-01

    Background Brain-derived neurotrophic factor (BDNF) has been shown to control microglial responses in neuropathic pain. Since adenosine A2A receptors (A2ARs) control neuroinflammation, as well as the production and function of BDNF, we tested to see if A2AR controls the microglia-dependent secretion of BDNF and the proliferation of microglial cells, a crucial event in neuroinflammation. Methods Murine N9 microglial cells were challenged with lipopolysaccharide (LPS, 100 ng/mL) in the absence or in the presence of the A2AR antagonist, SCH58261 (50 nM), as well as other modulators of A2AR signaling. The BDNF cellular content and secretion were quantified by Western blotting and ELISA, A2AR density was probed by Western blotting and immunocytochemistry and cell proliferation was assessed by BrdU incorporation. Additionally, the A2AR modulation of LPS-driven cell proliferation was also tested in primary cultures of mouse microglia. Results LPS induced time-dependent changes of the intra- and extracellular levels of BDNF and increased microglial proliferation. The maximal LPS-induced BDNF release was time-coincident with an LPS-induced increase of the A2AR density. Notably, removing endogenous extracellular adenosine or blocking A2AR prevented the LPS-mediated increase of both BDNF secretion and proliferation, as well as exogenous BDNF-induced proliferation. Conclusions We conclude that A2AR activation plays a mandatory role controlling the release of BDNF from activated microglia, as well as the autocrine/paracrine proliferative role of BDNF. PMID:23363775

  12. Autophagic activity in BC3H1 cells exposed to yessotoxin.

    PubMed

    Korsnes, Mónica Suárez; Kolstad, Hilde; Kleiveland, Charlotte Ramstad; Korsnes, Reinert; Ørmen, Elin

    2016-04-01

    The marine toxin yessotoxin (YTX) can induce programmed cell death through both caspase-dependent and -independent pathways in various cellular systems. It appears to stimulate different forms of cellular stress causing instability among cell death mechanisms and making them overlap and cross-talk. Autophagy is one of the key pathways that can be stimulated by multiple forms of cellular stress which may determine cell survival or death. The present work evaluates a plausible link between ribotoxic stress and autophagic activity in BC3H1 cells treated with YTX. Such treatment produces massive cytoplasmic compartments as well as double-membrane vesicles termed autophagosomes which are typically observed in cells undergoing autophagy. The observed autophagosomes contain a large amount of ribosomes associated with the endoplasmic reticulum (ER). Western blotting analysis of Atg proteins and detection of the autophagic markers LC3-II and SQSTM1/p62 by flow cytometry and immunofluorescence verified autophagic activity during YTX-treatment. The present work supports the idea that autophagic activity upon YTX exposure may represent a response to ribotoxic stress. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Ergosterol peroxide from Chaga mushroom (Inonotus obliquus) exhibits anti-cancer activity by down-regulation of the β-catenin pathway in colorectal cancer.

    PubMed

    Kang, Ju-Hee; Jang, Jeong-Eun; Mishra, Siddhartha Kumar; Lee, Hee-Ju; Nho, Chu Won; Shin, Dongyun; Jin, Mirim; Kim, Mi Kyung; Choi, Changsun; Oh, Seung Hyun

    2015-09-15

    In this study, we examined the effect of different fractions and components of Chaga mushroom (Inonotus Obliquus) on viability and apoptosis of colon cancer cells. Among them, one component showed the most effective growth inhibition and was identified as ergosterol peroxide by NMR analysis. We investigated the anti-proliferative and apoptosis mechanisms of ergosterol peroxide associated with its anti-cancer activities in human colorectal cancer (CRC) cell lines and tested its anti-tumor effect on colitis-induced CRC developed by Azoxymethane (AOM)/Dextran sulfate sodium (DSS) in a mouse model. We used MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, flow cytometry assays, Western blot analysis, colony formation assays, reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and AOM/DSS mouse models to study the molecular mechanism of metastatic activities in CRC cells. Ergosterol peroxide inhibited cell proliferation and also suppressed clonogenic colony formation in HCT116, HT-29, SW620 and DLD-1 CRC cell lines. The growth inhibition observed in these CRC cell lines was the result of apoptosis, which was confirmed by FACS analysis and Western blotting. Ergosterol peroxide inhibited the nuclear levels of β-catenin, which ultimately resulted in reduced transcription of c-Myc, cyclin D1, and CDK-8. Ergosterol peroxide administration showed a tendency to suppress tumor growth in the colon of AOM/DSS-treated mice, and quantification of the IHC staining showed a dramatic decrease in the Ki67-positive staining and an increase in the TUNEL staining of colonic epithelial cells in AOM/DSS-treated mice by ergosterol peroxide for both prevention and therapy. Our data suggest that ergosterol peroxide suppresses the proliferation of CRC cell lines and effectively inhibits colitis-associated colon cancer in AOM/DSS-treated mice. Ergosterol peroxide down-regulated β-catenin signaling, which exerted anti-proliferative and

  14. Persistence of RSV promotes proliferation and epithelial-mesenchymal transition of bronchial epithelial cells through Nodal signaling.

    PubMed

    Xiang, Zhao; Liang, Zhang; Yanfeng, Huang; Leitao, Kang

    2017-10-01

    Nodal may play an important role in the development of cancers. The present study was designed to determine the effects of Nodal induced by respiratory syncytial virus (RSV) infection on the occurrence and development of lung cancer and the underlying mechanisms. After verification of RSV infection by observation of cytopathic effect and indirect immunofluorescence, real-time PCR, Western blot and methylation assays were used to verify the influence of RSV on Nodal expression. Then, a Nodal overexpressed vector was constructed and the effects of Nodal on the proliferation and apoptosis of bronchial epithelial cells (BECs) and epithelial-mesenchymal transition (EMT) were assayed by flow cytometry and Western blot, respectively. Moreover, Lefty and pSmad2/3 were assayed by Western blot and Cyclin D1, CDK4, c-myc and Bcl-2 induced by Nodal overepression or RSV infection were also assayed by real-time PCR. The results showed that Nodal over expression and demethylation of the promoter were observed in BECs after RSV infection. Activation of Nodal promoted proliferation, colony formation and EMT and inhibited apoptosis of BECs. Nodal also promoted malignant change by promoting expression of cyclin D1 and related-dependent kinase and inhibiting apoptosis. Besides, RSV infection inhibited Lefty expression and promoted the activation of pSmad2/3. RSV also promoted Cyclin D1, CDK4, c-myc and Bcl-2 expression through the activation of pSmad2/3. Our data showed that persistence of RSV promoted the proliferation, epithelial-mesenchymal transition and expression of oncogenes through Nodal signaling, which may be associated with the occurrence and development of lung cancers.

  15. Magmatism evolution on the last Neoproterozoic development stage of the western Siberian active continental margin

    NASA Astrophysics Data System (ADS)

    Vernikovskaya, Antonina E.; Vernikovsky, Valery A.; Matushkin, Nikolay Yu.; Kadilnikov, Pavel I.; Romanova, Irina V.

    2017-04-01

    Rocks from active continental margin complexes are characterized by a wide variety of chemical compositions from depleted in alkali to alkali differentiates. When addressing issues of geodynamic settings in which such rocks form, it is important to understand the evolution of the host tectonic structure, as well as the chemical affiliation of the various rocks composing it. The Yenisey Ridge orogen located in the south-western framing of Siberia is one of the more studied regions with a long history of Neoproterozoic magmatic events. This orogen was formed during the collision of the Central Angara terrane with Siberia, which took place 761-718 Ma. Subsequent subduction-related events in the orogen have been recorded in the coeval magmatism (711-629 Ma) of two complexes: one is the active continental margin complex (Nb enriched igneous rocks - gabbroids, trachybasalts, A-type granites and carbonatites, including contact metasomatites zones with Nb mineralization), and the other one is an island arc complex (differentiated series volcanics, gabbroids and plagiogranites). The rocks of these complexes are respectively located in two suture zones: the Tatarka-Ishimba zone that formed due to the collision mentioned above, and the Yenisei suture marking the subduction zone [Vernikovsky et al., 2003; 2008]. The final Neoproterozoic stage in the evolution of the active margin of Siberia is manifested as adakite-gabbro-anorthosite magmatism in the 576-546 Ma interval. Our results indicate a genetic relationship between the adakites and their host NEB-type metabasites of the Zimovey massif. These Neoproterozoic adakites could have formed in a setting of transform-strike-slip drift of lithospheric plates after the subduction stopped, both from a crustal and mantle-crustal source, similarly to the Cenozoic magmatic complexes of the transform margin in the eastern framing of Eurasia [Khanchuk et al., 2016]. Vernikovsky V.A., Vernikovskaya A.E., Kotov A.B., Sal'nikova E

  16. Silica nanoparticle-induced oxidative stress and mitochondrial damage is followed by activation of intrinsic apoptosis pathway in glioblastoma cells

    PubMed Central

    Kusaczuk, Magdalena; Krętowski, Rafał; Naumowicz, Monika; Stypułkowska, Anna; Cechowska-Pasko, Marzanna

    2018-01-01

    Introduction Recently, the focus of oncological research has been on the optimization of therapeutic strategies targeted at malignant diseases. Nanomedicine utilizing silicon dioxide nanoparticles (SiNPs) is one such strategy and is rapidly developing as a promising tool for cancer diagnosis, imaging, and treatment. Nevertheless, little is known about the mechanisms of action of SiNPs in brain tumors. Materials and methods Here, we explored the effects of 5–15 nm SiNPs in the human glioblastoma cell line LN229. In this respect, MTT assays, microscopic observations, flow cytometry analyses, and luminescent assays were performed. Moreover, RT-qPCR and Western blot analyses were done to determine gene and protein expressions. Results We demonstrated that SiNPs triggered evident cytotoxicity, with microscopic observations of the nuclei, annexin V–fluorescein isothiocyanate/propidium iodide staining, and elevated caspase 3/7 activity, suggesting that SiNPs predominantly induced apoptotic death in LN229 cells. We further showed the occurrence of oxidative stress induced by enhanced reactive oxygen-species generation. This effect was followed by deregulated expression of genes encoding the antioxidant enzymes SOD1, SOD2, and CAT, and impaired mitochondria function. SiNP- induced mitochondrial dysfunction was characterized by membrane-potential collapse, ATP depletion, elevated expression of BAX, PUMA, and NOXA with simultaneous downregulation of BCL2/BCL2L1, and activation of caspase 9. Moreover, RT-qPCR and Western blot analyses demonstrated increased levels of the endoplasmic reticulum stress markers GRP78, GRP94, and DDIT3, as well as strongly increased expressions of the IL1B and COX2 genes, suggesting activation of endoplasmic reticulum stress and a proinflammatory response. Conclusions Altogether, our data indicate that in LN229 cells, SiNPs evoke cell death via activation of the intrinsic apoptosis pathway and suggest that other aspects of cellular function may

  17. Combined use of leptin and mechanical stress has osteogenic effects on ossification of the posterior longitudinal ligament.

    PubMed

    Chen, Shuai; Zhu, Haifeng; Wang, Gangliang; Xie, Ziang; Wang, Jiying; Chen, Jian

    2018-06-16

    To evaluate the effects of leptin/leptin receptor (LepR) combined with mechanical stress on the development of ossification of the posterior longitudinal ligament (OPLL), which is a disease characterized by ectopic bone formation of the posterior longitudinal ligament (PLL) and can lead to radiculopathy and myelopathy. Six human samples of the PLL were analyzed for the expression of leptin and LepR by RT-PCR and western blotting. PLL cells were stimulated with leptin and mechanical stress delivered via a Flexcell tension system, and osteogenic differentiation was evaluated by RT-PCR and western blotting analysis of osteogenic marker expression as well as by alkaline phosphatase (ALP) staining and alizarin red S staining. Activation of mitogen-activated protein kinase (MAPK), Janus kinase (JAK) 2-signal transducer, activator of transcription (STAT) 3 and phosphatidylinositol 3-kinase (PI3K)-Akt was evaluated by western blotting. Samples from the OPLL group had higher LepR mRNA and protein levels and lower leptin levels than those from healthy controls. Exposure to leptin and Flexcell increased the number of ALP-positive cells and calcium nodules in a dose-dependent manner; this effect was accompanied by upregulation of the osteogenic markers osteocalcin, runt-related transcription factor 2 (RUNX2) and osteopontin. Extracellular signal-regulated kinase, P38 MAPK, JAK2, STAT3, PI3K and Akt signaling, was also activated by the combined effects of leptin and mechanical stress. Leptin and LepR are differentially expressed in OPLL tissues, and the combined use of leptin/LepR and mechanical stress promotes osteogenic differentiation of PLL cells via MAPK, JAK2-STAT3 and PI3K/Akt signaling. These slides can be retrieved under Electronic Supplementary Material.

  18. Forest fire weather in western Oregon and western Washington in 1957.

    Treesearch

    Owen P. Cramer

    1957-01-01

    Severity of 1957 fire weather west of the Cascade Range summit in Oregon and Washington was near the average of the previous 10 years. The season (April 1 through October 31) was slightly more severe than 1956 in western Oregon and about the same as 1956 in western Washington. Spring fire weather was near average severity in both western Washington and western Oregon....

  19. Cardiac progenitor cell‑derived exosomes promote H9C2 cell growth via Akt/mTOR activation.

    PubMed

    Li, Shentang; Jiang, Jie; Yang, Zuocheng; Li, Zhuoying; Ma, Xing; Li, Xin

    2018-05-21

    Exosomes are cell‑derived vesicles released from a variety of mammalian cells that are involved in cell‑to‑cell signalling. It has been reported that cardiac progenitor cells (CPCs) derived from an adult heart are one of the most promising stem cell types for cardioprotection and repair. The mammalian target of rapamycin (mTOR) signalling pathway is a pivotal regulator in CPCs, therefore, CPC‑derived exosomes were used in the present study to investigate whether it can promote H9C2 cell growth through the protein kinase B (PKB, or Akt)/mTOR signalling pathway. The CPCs were isolated from Sprague‑Dawley hearts. Following treatment with a specific medium, the exosomes were purified and identified by electron micrograph and western blot assays, using CD63 and CD81 as markers. The methyl‑thiazolyl‑tetrazolium and 5‑ethynyl‑2'‑deoxyuridine methods were used to detect H9C2 cell growth. The expression of Akt and mTOR were detected by western blot analysis following treatment with 200 or 400 µg/ml of exosomes for 24 or 48 h, respectively. It was found that, compared with higher concentrations of exosomes, prolonging the duration of exposure promoted cell growth. Accordingly, CPC‑derived exosomes stimulated the expression of Akt to a marked degree; groups treated with exosomes for 48 h showed higher expression of Akt than those treated for 24 h at the same concentration. mTOR was also stimulated by CPC‑derived exosomes. The activation of mTOR increased in accordance with the treatment time at an exosome concentration of 200 µg/ml and decreased with treatment time at an exosome concentration of 400 µg/ml. In conclusion, the present study demonstrated that CPC‑derived exosomes promoted H9C2 cell growth via the activation of Akt/mTOR in a time‑dependent manner at a relatively low exosome concentration, which may provide a novel therapy for cardiovascular disease.

  20. Antiviral activity of polymethoxylated flavones from "Guangchenpi", the edible and medicinal pericarps of citrus reticulata 'Chachi'.

    PubMed

    Xu, Jiao-Jiao; Wu, Xia; Li, Man-Mei; Li, Guo-Qiang; Yang, Yi-Ting; Luo, Hu-Jie; Huang, Wei-Huang; Chung, Hau Yin; Ye, Wen-Cai; Wang, Guo-Cai; Li, Yao-Lan

    2014-03-12

    The present study found that the supercritical fluid extract of "Guangchenpi" possessed in vitro antiviral activity against respiratory syncytial virus (RSV). Bioassay-guided isolation and identification of this extract led to obtain five active polymethoxylated flavones (1-5). Cytopathic effect (CPE) reduction assay exhibited that tangeretin (2) and nobiletin (3), two major polymethoxylated flavones in the extract, possessed better anti-RSV effect comparable to the positive control ribavirin. Plaque reduction assay revealed that tangeretin dose-dependently inhibited RSV-induced plaque formation on the HEp-2 cells. This polymethoxylated flavone mainly affected the intracellular replication of RSV, and it also could inhibit RSV entry into the HEp-2 cells. Further investigations with quantitative real-time PCR and confocal and Western blot assays indicated that tangeretin downregulated the expression of RSV phosphoprotein (P protein). Results suggest the potential application of the supercritical fluid extract of "Guangchenpi" and tangeretin in the treatment and the prevention of RSV infection.

  1. In vitro and in vivo effect of PD-1/PD-L1 blockade on microglia/macrophage activation and T cell subset balance in cryptococcal meningitis.

    PubMed

    Che, Yuan-Mei; Zhang, Yi; Li, Ming; Li, Xiao-Peng; Zhang, Lun-Li

    2018-04-01

    This study aimed to investigate the PD-1/ PD-L1 signaling pathway and its effects the activation of microglia/macrophage and balancing T cell subsets in cryptococcal meningitis (CM). A total of 126 CM patients and 126 healthy individuals were recruited for the study. The CM patients were treated with amphotericin B (AmB). Seventy five C57BL/6 mice were grouped into the normal control, CM model, CM + AmB, sham, and CM + PD-1 antibodies (Ab) groups. CD4 + and CD8 + T cells as well as microglia/macrophages were analyzed by means of flow cytometry. Ionized calcium-binding adaptor molecule 1 (Ibal) expression was detected using western blotting and immunohistochemistry techniques. And the expression of Rab5 and Rab11 were detected using an immunofluorescence assay. Both PD-1 and PD-L1 mRNA and protein expression among the mice in the study were evaluated by qRT-PCR and western blotting methods. Compared to the CM model group, the CM + AmB and CM + PD-1 Ab groups exhibited increased levels of Th1 cytokines and chemokines expression, and reduced levels of Th2 cytokines expressions. Elevated cell purity and viability of CD4 + T cell were recorded as well as increases in microglia, however, there were reductions in the number of CD8 + T cells. Depleted expressions of Ibal, Rab5, and Rab11 as well as reduced mRNA expressions of PD-1 and PD-L1 in CD4 + , microglia, and macrophage cells. The findings suggested that suppression of the PD-1/PD-L1 signaling pathway restricts the proliferation of CM by down-regulating the expressions of Th2 cells and suppressing microglia and macrophage activation. © 2017 Wiley Periodicals, Inc.

  2. Western European Art Foundations and Publishing

    ERIC Educational Resources Information Center

    King, Lindsay M.; Clement, Russell T.

    2011-01-01

    Western European art foundations create invaluable opportunities for research and exhibition by artists, curators, and scholars. These activities are often documented and disseminated via high-quality publications. This article highlights an important but under-recognized collecting resource for academic and museum libraries by profiling several…

  3. Measles Elimination Activities in the Western Pacific Region: Experience from the Republic of Korea

    PubMed Central

    Choe, Young June; Jee, Youngmee; Oh, Myoung-don

    2015-01-01

    We describe the global status of measles control and elimination, including surveillance and vaccination coverage data provided by the World Health Organization (WHO). Since 2000, two doses of measles vaccine (MCV2) became recommended globally and the achievement of high vaccination coverage has led to dramatic decrease in the measles incidence. Our finding indicates that, in the Western Pacific Region (WPR), substantial progress has been made to control measles transmission in some countries; however, the measles virus continues to circulate, causing outbreaks. The Republic of Korea (ROK) experienced a series of resurgence of measles due to the importation and healthcare-associated transmission in infants, however overall incidence and surveillance indicators met the WHO criteria for measles elimination. The ROK was verified to be measles-free along with Australia, Mongolia, and Macau, China in 2014. One of the effective elimination activities was the establishment of solid keep-up vaccination system in school settings. The lessons learnt from the measles elimination activities in Korea may contribute to enhancing the surveillance schemes and strengthening of vaccination programs in member countries and areas of WPR. PMID:26617443

  4. Measles Elimination Activities in the Western Pacific Region: Experience from the Republic of Korea.

    PubMed

    Choe, Young June; Jee, Youngmee; Oh, Myoung-don; Lee, Jong-Koo

    2015-11-01

    We describe the global status of measles control and elimination, including surveillance and vaccination coverage data provided by the World Health Organization (WHO). Since 2000, two doses of measles vaccine (MCV2) became recommended globally and the achievement of high vaccination coverage has led to dramatic decrease in the measles incidence. Our finding indicates that, in the Western Pacific Region (WPR), substantial progress has been made to control measles transmission in some countries; however, the measles virus continues to circulate, causing outbreaks. The Republic of Korea (ROK) experienced a series of resurgence of measles due to the importation and healthcare-associated transmission in infants, however overall incidence and surveillance indicators met the WHO criteria for measles elimination. The ROK was verified to be measles-free along with Australia, Mongolia, and Macau, China in 2014. One of the effective elimination activities was the establishment of solid keep-up vaccination system in school settings. The lessons learnt from the measles elimination activities in Korea may contribute to enhancing the surveillance schemes and strengthening of vaccination programs in member countries and areas of WPR.

  5. [Curcumin inhibited rat colorectal carcinogenesis by activating PPAR-γ: an experimental study].

    PubMed

    Liu, Liu-bin; Duan, Chang-nong; Ma, Zeng-yi; Xu, Gang

    2015-04-01

    To explore the chemopreventive effect of curcumin on DMH induced colorectal carcinogenesis and the underlining mechanism. Totally 40 Wistar rats were divided into the model group and the curcumin group by random digit table, 20 in each group. Meanwhile, a normal control group was set up (n =10). A colorectal cancer model was induced by subcutaneously injecting 20 mg/kg DMH. The tumor incidence and the inhibition rate were calculated. The effect of curcumin on the expression of peroxisome proliferator-activated receptor gamma (PPARγ) in rat colon mucosal tissues was observed using immunohistochemistry and Western blot. HT 29 cell line were cultured and divided into a control group, the curcumin + GW9662 (2-chloro-5-nitro-N-4-phenylbenzamide) intervention group, and the curcumin group. The inhibition of different concentrations curcumin on HT29 cell line was detected using MTT. The expression of curcumin on PPARy was also detected using Western blot. The tumor incidence was 80. 00% (12/15 cases) in the model group, obviously higher than that of the curcumin group (58. 82%, 10/17 cases, P <0. 05). The inhibition rate of curcumin on DMH induced colorected carcinoma reached 26. 46%. Compared with the normal control group, the expression of PPARγ protein was significantly increased in the curcumin group and the model group (P <0. 01). Compared with the model group at the same time point, the expression of PPARy protein was significantly enhanced in the curcumin group (P <0. 05). MTT analysis showed that curcumin could inhibit the proliferation of in vitro HT 29 cells in dose and time dependent manners. The expression of PPARy protein was significantly increased in the GW9662 group and the curcumin group, showing statistical difference when compared with the normal control group (P <0. 01). Compared with the GW9662 group, the expression of PPARγ protein was significantly increased in the curcumin group (P <0. 01). Curcumin could inhibit DMH-induced rat colorectal

  6. Acceleration of thaw slump activity in glaciated landscapes of the Western Canadian Arctic

    NASA Astrophysics Data System (ADS)

    Segal, Rebecca A.; Lantz, Trevor C.; Kokelj, Steven V.

    2016-03-01

    Climate change is increasing the frequency and intensity of thermokarst, but the influences of regional climate and physiography remain poorly understood. Retrogressive thaw slumping is one of the most dynamic forms of thermokarst and affects many areas of glaciated terrain across northwestern Canada. In this study, we used airphotos and satellite imagery to investigate the influence of climate and landscape factors on thaw slump dynamics. We assessed slump size, density, and growth rates in four regions of ice-rich terrain with contrasting climate and physiographic conditions: the Jesse Moraine, the Tuktoyaktuk Coastlands, the Bluenose Moraine, and the Peel Plateau. Observed increases in: (1) the area impacted by slumps (+2 to +407%), (2) average slump sizes (+0.31 to +1.82 ha), and (3) slump growth rates (+169 to +465 m2 yr-1) showed that thermokarst activity is rapidly accelerating in ice-rich morainal landscapes in the western Canadian Arctic, where slumping has become a dominant driver of geomorphic change. Differences in slump characteristics among regions indicate that slump development is strongly influenced by topography, ground ice conditions, and Quaternary history. Observed increases in slump activity occurred in conjunction with increases in air temperature and precipitation, but variation in slump activity among the four regions suggests that increased precipitation has been an important driver of change. Our observation that the most rapid intensification of slump activity occurred in the coldest environment (the Jesse Moraine on Banks Island) indicates that ice-cored landscapes in cold permafrost environments are highly vulnerable to climate change.

  7. The Role of IQGAP1 in Breast Carcinoma

    DTIC Science & Technology

    2011-10-01

    study! of! the! pathogenesis! of! breast! cancer.! These! include! analysis ! of! intracellular! signaling!by!Western!blotting,! determination!of! cell...proliferation!by! sulforhodamine!B! staining,! fluorescence: activated!cell!sorting!(FACS)! analysis ,!stable!cell!line!generation,!production!of!and...transduction!using!retroviral! and!lentiviral!supernatants,! immunocytochemistry!and!confocal! laser!microscopy,! immunohistochemistry,!and! analysis

  8. Curcumin decreases the expression of Pokemon by suppressing the binding activity of the Sp1 protein in human lung cancer cells.

    PubMed

    Cui, Jiajun; Meng, Xianfeng; Gao, Xudong; Tan, Guangxuan

    2010-03-01

    Pokemon, which stands for POK erythroid myeloid ontogenic factor, can regulate expression of many genes and plays an important role in tumorigenesis. Curcumin, a natural and non-toxic yellow compound, has capacity for antioxidant, free radical scavenger, anti-inflammatory properties. Recent studies shows it is a potential inhibitor of cell proliferation in a variety of tumour cells. To investigate whether curcumin can regulate the expression of Pokemon, a series of experiments were carried out. Transient transfection experiments demonstrated that curcumin could decrease the activity of the Pokemon promoter. Western blot analysis suggested that curcumin could significantly decrease the expression of the Pokemon. Overexpression of Sp1 could enhance the activity of the Pokemon promoter, whereas knockdown of Sp1 could decrease its activity. More important, we also found that curcumin could decrease the expression of the Pokemon by suppressing the stimulation of the Sp1 protein. Therefore, curcumin is a potential reagent for tumour therapy which may target Pokemon.

  9. An advanced dual labeled gold nanoparticles probe to detect Cryptosporidium parvum using rapid immuno-dot blot assay.

    PubMed

    Thiruppathiraja, Chinnasamy; Kamatchiammal, Senthilkumar; Adaikkappan, Periyakaruppan; Alagar, Muthukaruppan

    2011-07-15

    The zoonotic protozoan parasite Cryptosporidium parvum poses a significant risk to public health. Due to the low infectious dose of C. parvum, remarkably sensitive detection methods are required for water and food industries analysis. However PCR affirmed sensing method of the causative nucleic acid has numerous advantages, still criterion demands for simple techniques and expertise understanding to extinguish its routine use. In contrast, protein based immuno detecting techniques are simpler to perform by a commoner, but lack of sensitivity due to inadequate signal amplification. In this paper, we focused on the development of a mere sensitive immuno detection method by coupling anti-cyst antibody and alkaline phosphatase on gold nanoparticle for C. parvum is described. Outcome of the sensitivity in an immuno-dot blot assay detection is enhanced by 500 fold (using conventional method) and visually be able to detect up to 10 oocysts/mL with minimal processing period. Techniques reported in this paper substantiate the convenience of immuno-dot blot assay for the routine screening of C. parvum in water/environmental examines and most importantly, demonstrates the potential of a prototype development of simple and inexpensive diagnostic technique. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. The expression of xenobiotic-metabolizing enzymes in human prostate and in prostate epithelial cells (PECs) derived from primary cultures.

    PubMed

    Al-Buheissi, S Z; Cole, K J; Hewer, A; Kumar, V; Bryan, R L; Hudson, D L; Patel, H R; Nathan, S; Miller, R A; Phillips, D H

    2006-06-01

    Dietary heterocyclic amines (HCAs) are carcinogenic in rodent prostate requiring activation by enzymes such as cytochrome P450 (CYP) and N-acetyltransferase (NAT). We investigated by Western blotting and immunohistochemistry the expression of CYP1A1, CYP1A2, and NAT1 in human prostate and in prostate epithelial cells (PECs) derived from primary cultures and tested their ability to activate the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and its N-hydroxy metabolite (N-OH-IQ) to DNA-damaging moieties. Western blotting identified CYP1A1, CYP1A2, and NAT1. Immunohistochemistry localized NAT1 to the cytoplasm of PECs. Inter-individual variation was observed in the expression levels of CYP1A1, 1A2, and NAT1 (11, 75, and 35-fold, respectively). PECs expressed CYP1A1 and NAT1 but not CYP1A2. When incubated with IQ or N-OH-IQ, PECs formed DNA adducts indicating their ability to metabolically activate these compounds. Prostate cells possess the capacity to activate dietary carcinogens. PECs may provide a useful model system to study their role in prostate carcinogenesis.

  11. Is Oxidative Stress in Mice Brain Regions Diminished by 2-[(2,6-Dichlorobenzylidene)amino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile?

    PubMed Central

    Fortes, A. C.; Almeida, A. A. C.; Oliveira, G. A. L.; Santos, P. S.; De Lucca Junior, W.; Mendonça Junior, F. J. B.; Freitas, R. M.; Soares-Sobrinho, J. L.; Soares, M. F. R.

    2013-01-01

    2-[(2,6-Dichlorobenzylidene)amino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile, 5TIO1, is a new 2-aminothiophene derivative with promising pharmacological activities. The aim of this study was to evaluate its antioxidant activity in different areas of mice central nervous system. Male Swiss adult mice were intraperitoneally treated with Tween 80 dissolved in 0.9% saline (control group) and 5TIO1 (0.1, 1, and 10 mg kg−1). Brain homogenates—hippocampus, striatum, frontal cortex, and cerebellum—were obtained after 24 h of observation. Superoxide dismutase and catalase activities, lipid peroxidation and nitrite content were measured using spectrophotometrical methods. To clarify the 5TIO1's mechanism on oxidative stress, western blot analysis of superoxide dismutase and catalase was also performed. 5TIO1 decreased lipid peroxidation and nitrite content in all brain areas and increased the antioxidant enzymatic activities, specially, in cerebellum. The data of Western blot analysis did not demonstrate evidence of the upregulation of these enzymes after the administration of this compound. Our findings strongly support that 5TIO1 can protect the brain against neuronal damages regularly observed during neuropathologies. PMID:23577220

  12. Inhibition of calpain on oxygen glucose deprivation-induced RGC-5 necroptosis.

    PubMed

    Chen, Shuang; Yan, Jie; Deng, Hai-Xiao; Long, Ling-Ling; Hu, Yong-Jun; Wang, Mi; Shang, Lei; Chen, Dan; Huang, Ju-Fang; Xiong, Kun

    2016-10-01

    The purpose of this study was to investigate the effect of inhibition of calpain on retinal ganglion cell-5 (RGC-5) necroptosis following oxygen glucose deprivation (OGD). RGC-5 cells were cultured in Dulbecco's-modified essential medium and necroptosis was induced by 8-h OGD. PI staining and flow cytometry were performed to detect RGC-5 necrosis. The calpain expression was detected by Western blotting and immunofluorescence staining. The calpain activity was tested by activity detection kit. Flow cytometry was used to detect the effect of calpain on RGC-5 necroptosis following OGD with or without N-acetyl-leucyl-leucyl-norleucinal (ALLN) pre-treatment. Western blot was used to detect the protein level of truncated apoptosis inducing factor (tAIF) in RGC-5 cells following OGD. The results showed that there was an up-regulation of the calpain expression and activity following OGD. Upon adding ALLN, the calpain activity was inhibited and tAIF was reduced following OGD along with the decreased number of RGC-5 necroptosis. In conclusion, calpain was involved in OGD-induced RGC-5 necroptosis with the increased expression of its downstream molecule tAIF.

  13. Withaferin A activates stress signalling proteins in high risk acute lymphoblastic leukemia

    PubMed Central

    Shi, Li-Huan; Wu, Xi-Jun; Liu, Jun-Shan; Gao, Yin-Bo

    2015-01-01

    Withaferin A, the principal bio-active component isolated from the Withaniasomnifera, has shown promising anti-leukemic activity in addition to anti-invasive and anti-metastatic activity. The present study demonstrates the effect of withaferin A on the cell cycle status and the phosphorylation/activation of proteins involved in signal transduction in t(4;11) and non-t(4;11) acute lymphoblastic leukemia (ALL) cell lines after treatment with withaferin A. The cells after treatment with the vehicle or 25 μM withaferin A for 1, 2, 4 and 8 h were examined using flow cytometric analysis. The results revealed that withaferin A treatment induced cell growth arrest at the S to G2/M phase transition of the cell cycle. Withaferin A treatment also induced the phosphorylation of stress signalling proteins, including the p38 mitogen-activated protein kinase, the c-Jun N-terminal kinase, c-Jun, the heat shock protein 27 and protein kinase B within 0 to 16 h. These results were observed using multiplex technology and Western blotting analysis. Thus withaferin A induces stress response leading to cell death. Therefore, withaferin A can be a potent therapeutic agent for the treatment of high risk ALL with chromosomal translocation t(4;11). PMID:26884834

  14. Withaferin A activates stress signalling proteins in high risk acute lymphoblastic leukemia.

    PubMed

    Shi, Li-Huan; Wu, Xi-Jun; Liu, Jun-Shan; Gao, Yin-Bo

    2015-01-01

    Withaferin A, the principal bio-active component isolated from the Withaniasomnifera, has shown promising anti-leukemic activity in addition to anti-invasive and anti-metastatic activity. The present study demonstrates the effect of withaferin A on the cell cycle status and the phosphorylation/activation of proteins involved in signal transduction in t(4;11) and non-t(4;11) acute lymphoblastic leukemia (ALL) cell lines after treatment with withaferin A. The cells after treatment with the vehicle or 25 μM withaferin A for 1, 2, 4 and 8 h were examined using flow cytometric analysis. The results revealed that withaferin A treatment induced cell growth arrest at the S to G2/M phase transition of the cell cycle. Withaferin A treatment also induced the phosphorylation of stress signalling proteins, including the p38 mitogen-activated protein kinase, the c-Jun N-terminal kinase, c-Jun, the heat shock protein 27 and protein kinase B within 0 to 16 h. These results were observed using multiplex technology and Western blotting analysis. Thus withaferin A induces stress response leading to cell death. Therefore, withaferin A can be a potent therapeutic agent for the treatment of high risk ALL with chromosomal translocation t(4;11).

  15. Antitumor activity of ginseng sapogenins, 25-OH-PPD and 25-OCH3-PPD, on gastric cancer cells.

    PubMed

    Zhao, Chen; Su, Guangyue; Wang, Xude; Zhang, Xiaoshu; Guo, Shuang; Zhao, Yuqing

    2016-01-01

    25-Hydroxyprotopanaxadiol (25-OH-PPD) and 25-methoxylprotopanaxadiol (25-OCH3-PPD), two ginseng sapogenins, have potent antitumor activity and their effects on gastric cancer (BGC-823, SGC-7901, MKN-28) cells and a gastric mucosa (GES-1) cell line are reported. Both compounds significantly inhibited the growth of gastric cancer cells, while having lesser inhibitory effects on GES-1 cells by MTT assay. A mechanistic study revealed that the two ginseng sapogenins could induce apoptosis in BGC-823 cells by morphological observation, DNA fragmentation, flow cytometry and western blot analysis. Besides, the apoptosis was inhibited by Ac-DEVD-CHO, a caspase 3 inhibitor, which was confirmed by cell viability analysis. These results indicate that 25-OH-PPD and 25-OCH3-PPD have potential to be promising agents for the treatment of gastric cancer.

  16. Adiponectin improves insulin sensitivity via activation of autophagic flux.

    PubMed

    Ahlstrom, Penny; Rai, Esther; Chakma, Suharto; Cho, Hee Ho; Rengasamy, Palanivel; Sweeney, Gary

    2017-11-01

    Skeletal muscle insulin resistance is known to play an important role in the pathogenesis of diabetes, and one potential causative cellular mechanism is endoplasmic reticulum (ER) stress. Adiponectin mediates anti-diabetic effects via direct metabolic actions and by improving insulin sensitivity, and we recently demonstrated an important role in stimulation of autophagy by adiponectin. However, there is limited knowledge on crosstalk between autophagy and ER stress in skeletal muscle and in particular how they are regulated by adiponectin. Here, we utilized the model of high insulin/glucose (HIHG)-induced insulin resistance, determined by measuring Akt phosphorylation (T308 and S473) and glucose uptake in L6 skeletal muscle cells. HIHG reduced autophagic flux measured by LC3 and p62 Western blotting and tandem fluorescent RFP/GFP-LC3 immunofluorescence (IF). HIHG also induced ER stress assessed by thioflavin T/KDEL IF, pIRE1, pPERK, peIF2α and ATF6 Western blotting and induction of a GRP78-mCherry reporter. Induction of autophagy by adiponectin or rapamycin attenuated HIHG-induced ER stress and improved insulin sensitivity. The functional significance of enhanced autophagy was validated by demonstrating a lack of improved insulin sensitivity in response to adiponectin in autophagy-deficient cells generated by overexpression of dominant negative mutant of Atg5. In summary, adiponectin-induced autophagy in skeletal muscle cells alleviated HIHG-induced ER stress and insulin resistance. © 2017 Society for Endocrinology.

  17. Western bats as a reservoir of novel Streptomyces species with antifungal activity

    USGS Publications Warehouse

    Hamm, Paris S.; Caimi, Nicole A.; Northup, Diana E.; Valdez, Ernest W.; Buecher, Debbie C.; Dunlap, Christopher A.; Labeda, David P.; Lueschow, Shiloh; Porras-Alfaro, Andrea

    2017-01-01

    At least two-thirds of commercial antibiotics today are derived from Actinobacteria, more specifically from the genus Streptomyces. Antibiotic resistance and new emerging diseases pose great challenges in the field of microbiology. Cave systems, in which actinobacteria are ubiquitous and abundant, represent new opportunities for the discovery of novel bacterial species and the study of their interactions with emergent pathogens. White-nose syndrome is an invasive bat disease caused by the fungus Pseudogymnoascus destructans, which has killed more than six million bats in the last 7 years. In this study, we isolated naturally occurring actinobacteria from white-nose syndrome (WNS)-free bats from five cave systems and surface locations in the vicinity in New Mexico and Arizona, USA. We sequenced the 16S rRNA region and tested 632 isolates from 12 different bat species using a bilayer plate method to evaluate antifungal activity. Thirty-six actinobacteria inhibited or stopped the growth of P. destructans, with 32 (88.9%) actinobacteria belonging to the genus Streptomyces. Isolates in the genera Rhodococcus, Streptosporangium, Luteipulveratus, and Nocardiopsis also showed inhibition. Twenty-five of the isolates with antifungal activity against P. destructans represent 15 novel Streptomyces spp. based on multilocus sequence analysis. Our results suggest that bats in western North America caves possess novel bacterial microbiota with the potential to inhibit P. destructans.

  18. Lactoferrin Expression in Human and Murine Ocular Tissue.

    PubMed

    Rageh, Abrar A; Ferrington, Deborah A; Roehrich, Heidi; Yuan, Ching; Terluk, Marcia R; Nelson, Elizabeth F; Montezuma, Sandra R

    2016-07-01

    Lactoferrin (LF) is a multifunctional protein known to provide innate defense due to its antimicrobial and anti-inflammatory properties. In the eye, LF has been identified in the tears and vitreous humor. Its presence in other ocular tissues has not been determined. Our aim is to assess the presence of LF in the cornea, iris, retina and retinal pigment epithelium (RPE) of humans and mice. To test for the endogenous production of LF, reverse transcription polymerase chain reaction was performed in cultured human cells from the cornea and RPE and in murine tissues. To confirm LF localization in specific ocular tissue, immunohistochemistry was performed on flat mounts of cornea, retina and RPE in human donor eyes. The presence of LF was assessed by western blotting in human and mouse ocular tissue and human culture cells (cornea and RPE). To verify antibody specificity, purified human LF and transferrin (TF) were used on 1D and 2D western blots. LF gene expression was confirmed in the cornea and RPE cell cultures from humans, suggesting that LF is an endogenously produced protein. PCR results from mouse ocular tissue showed LF expression in cornea, iris, RPE, but not in retina. These results were also consistent with immunohistochemical localization of LF in human donor tissue. Antibody reaction for human LF was specific and western blotting showed its presence in the cornea, iris and RPE tissues. A faint reaction for the retina was observed but was likely due to contamination from other ocular tissues. Multiple commercially available antibodies for murine LF cross-reacted with TF, so no reliable results were obtained for murine western blot. LF is expressed in multiple eye tissues of humans and mice. This widespread expression and multifunctional activity of LF suggests that it may play an important role in protecting eye tissues from inflammation-associated diseases.

  19. Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS.

    PubMed

    Zuidervaart, W; van Nieuwpoort, F; Stark, M; Dijkman, R; Packer, L; Borgstein, A-M; Pavey, S; van der Velden, P; Out, C; Jager, M J; Hayward, N K; Gruis, N A

    2005-06-06

    In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma.

  20. Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS

    PubMed Central

    Zuidervaart, W; van Nieuwpoort, F; Stark, M; Dijkman, R; Packer, L; Borgstein, A-M; Pavey, S; van der Velden, P; Out, C; Jager, M J; Hayward, N K; Gruis, N A

    2005-01-01

    In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma. PMID:15928660