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Sample records for time western blot

  1. Western blotting using capillary electrophoresis.

    Science.gov (United States)

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

    2011-02-15

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

  2. Western blotting.

    Science.gov (United States)

    Kurien, Biji T; Scofield, R Hal

    2006-04-01

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

  3. Multiplexed Western Blotting Using Microchip Electrophoresis.

    Science.gov (United States)

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

    2016-07-05

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

  4. Western Blotting using Capillary Electrophoresis

    OpenAIRE

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

    2011-01-01

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

  5. Western Blotting of the Endocannabinoid System.

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    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

    Directory of Open Access Journals (Sweden)

    Sean C. Taylor

    2014-01-01

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

  7. Western blotting: an introduction.

    Science.gov (United States)

    Kurien, Biji T; Scofield, R Hal

    2015-01-01

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

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

    OpenAIRE

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

    2013-01-01

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

  9. Multistrip western blotting to increase quantitative data output.

    Science.gov (United States)

    Kiyatkin, Anatoly; Aksamitiene, Edita

    2009-01-01

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

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

    OpenAIRE

    Silva, Jillian M.; McMahon, Martin

    2014-01-01

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

  11. Multistrip Western blotting to increase quantitative data output

    OpenAIRE

    Kiyatkin, Anatoly; Aksamitiene, Edita

    2009-01-01

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

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

    Science.gov (United States)

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

    2008-09-01

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

  13. Western blotting using chemiluminescent substrates.

    Science.gov (United States)

    Alegria-Schaffer, Alice

    2014-01-01

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

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

    Science.gov (United States)

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

    2016-09-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2008-01-01

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

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

    Science.gov (United States)

    García-Solaesa, Virginia; Abad, Sara Ciria

    2016-01-01

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

  18. Western Blot of Stained Proteins from Dried Polyacrylamide Gels

    Science.gov (United States)

    Gruber, Claudia; Stan-Lotter, Helga

    1996-01-01

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

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

    Science.gov (United States)

    Chang, Ming-Mei; Lovett, Janice

    2011-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

  2. Positive IgG Western Blot for Borrelia burgdorferi in Colombia

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    Palacios Ricardo

    1999-01-01

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

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

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    de Souza Marcilia MA

    2011-01-01

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

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

    Science.gov (United States)

    Abeyrathne, Priyanka D; Lam, Joseph S

    2007-04-01

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

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

    Science.gov (United States)

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

    2000-01-01

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

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

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    Tappe D

    2009-09-01

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

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

    NARCIS (Netherlands)

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

    2015-01-01

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

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

    Science.gov (United States)

    Ołdak, Elzbieta; Rozkiewicz, Doroto; Sulik, Artur

    2008-01-01

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

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

    OpenAIRE

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

    2007-01-01

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

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

    Science.gov (United States)

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

  11. Protein blotting protocol for beginners.

    Science.gov (United States)

    Petrasovits, Lars A

    2014-01-01

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

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

    Science.gov (United States)

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

    2008-07-01

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

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

    Directory of Open Access Journals (Sweden)

    A Halajian

    2009-05-01

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

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

    Science.gov (United States)

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

    2017-11-01

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

  15. The early days of blotting.

    Science.gov (United States)

    Southern, Edwin

    2015-01-01

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

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

    Science.gov (United States)

    Hazzalin, Catherine A; Mahadevan, Louis C

    2017-01-01

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

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

    OpenAIRE

    Penna, Aubin; Cahalan, Michael

    2007-01-01

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

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

    Directory of Open Access Journals (Sweden)

    D.V. Pilonetto

    2009-03-01

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

  19. Proteínas inmunodominantes de Brucella Melitensis evaluadas por Western Blot

    Directory of Open Access Journals (Sweden)

    Elizabeth Anaya

    1997-01-01

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

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

    OpenAIRE

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

    2017-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-03-15

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

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

    Directory of Open Access Journals (Sweden)

    R. Rosario Diéguez Castro

    2009-12-01

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

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

    Science.gov (United States)

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

    2009-01-01

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

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

    Science.gov (United States)

    Flisiak, R; Wierzbicka, I; Prokopowicz, D

    1998-01-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

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

    2000-01-01

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

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

    DEFF Research Database (Denmark)

    Uhlén, Mathias

    2017-01-01

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

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

    Science.gov (United States)

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

    2017-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Nilton Thaumaturgo

    2002-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Samantha L Eaton

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

  11. Detection of proteins on blot transfer membranes.

    Science.gov (United States)

    Sasse, Joachim; Gallagher, Sean R

    2003-11-01

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

  12. Cy5 total protein normalization in Western blot analysis.

    Science.gov (United States)

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

    2015-10-01

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

  13. Delineation of pulmonary airway fluid protein fractions with HRPO binding-avidity by far-Western ligand blot and mass spectrometry analyses: a model methodology for detecting mannose-binding protein expression profiles.

    Science.gov (United States)

    Coyne, Cody P; Rashmir-Raven, Ann; Jones, Toni; Mochal, Cathleen; Linford, Robert L; Brashier, Michael; Eddy, Alison

    2009-01-01

    Limited research to date has characterized the potential for HRPO to function as a primary molecular probe. Pulmonary airway fluid was developed by non-reducing far-Western (ligand) blot analyses utilizing conjugated HRPO-strepavidin or non-conjugated HRPO without the presence of primary immunoglobulin. Endogenous esterase-like biochemical activity of fractions within pulmonary airway fluid was inactivated to determine if they were capable of biochemically converting HRPO chemiluminescent substrate. Complementary analyses modified pulmonary fluid and HRPO with beta-galactosidase and alpha-mannosidase respectively, in addition to determining the influence of mannose and maltose competitive binding on HRPO far-Western (ligand) blot analyses. Identification of pulmonary fluid fractions detected by HRPO far-Western blot analyses was determined by mass spectrometry. Modification of pulmonary fluid with beta-galactosidase, and HRPO with alpha-mannosidase in concert with maltose and mannose competitive binding analyses altered the intensity and spectrum of pulmonary fluid fractions detected by HRPO far-Western blot analysis. Identity of pulmonary airway fluid fractions detected by HRPO far-Western (ligand) blot analysis were transferrin, dynein, albumin precursor, and two 156 kDa equine peptide fragments. HRPO can function as a partially-selective primary molecular probe when applied in either a conjugated or non-conjugated form. Some protein fractions can form complexes with HRPO through molecular mechanisms that involve physical interactions at the terminal alpha-mannose-rich regions of HRPO glycan side-chains. Based on its known molecular composition and structure, HRPO provides an opportunity for the development of diagnostics methodologies relevant to disease biomarkers that possess mannose-binding avidity.

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

    Science.gov (United States)

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

    2015-10-01

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

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

    Science.gov (United States)

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

    2012-06-01

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

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

    Science.gov (United States)

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

    2005-05-01

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

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

    Science.gov (United States)

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

    2001-01-01

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

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

    Science.gov (United States)

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

    2011-12-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Taki, Takao

    2015-01-01

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

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

    Science.gov (United States)

    Kurien, Biji T; Scofield, R Hal

    2015-01-01

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

  2. A comparison of the immune parameters of dogs infected with visceral leishmaniasis using Western blot and neutralization techniques Comparação dos parâmetros imunológicos de cães infectados com leishmaniose visceral usando as técnicas de Western blot e neutralização

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    Yeda L. Nogueira

    2007-12-01

    Full Text Available The Western blot technique was used to demonstrate the presence of antibodies in the blood of dogs that presented canine visceral leishmaniasis. This technique was used against some specific molecules present in the lysate of the promastigote form of Leshmania chagasi.Through the association of the results of the Western blot technique with the morphological alterations seen as a result of the serum neutralization technique performed in McCoy cells (which mimetizes the macrophage it was possible to observe the role of some molecules of great relevance in determining the disease in symptomatic dogs as well as that of some other molecules associated with asymptomatic infected dogs that may become transmitters as well as differentiating them as asymptomatic resistant dogs. In the sera analyses carried out during the immunobloting a variation of 9 to 27 immunoreacting bands was observed, which were then compared using Dice's similarity coefficient. In the dendrogram constructed on the basis of the coefficient, 50% similarity was observed among the total number of reagent bands with the promastigote lysate, thus creating five groups. The main difference observed related to the clinical condition of the dogs: symptomatic and asymptomatic dogs were found in separate groups. The asymptomatic group of dogs was distributed in two different places in the dendrogram because they presented two different behavior patterns regarding the cellular morphology in the serum neutralization reaction: the presence or absence of cellular lysis. According to this analysis it is possible to evaluate the immune status and associate it with specific markers observed in the reaction found in the Western blot strips.A técnica de Western blot foi utilizada para demonstrar a presença de anticorpos do soro de cães, que apresentavam leishmaniose visceral canina, contra algumas moléculas específicas no lisado da forma promastigota de Leshmania chagasi.Através da associa

  3. 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.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Garth L. Nicolson

    2012-03-01

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

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

    Science.gov (United States)

    Davelois, Kelly; Escalante, Hermes; Jara, César

    2016-01-01

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

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

    Science.gov (United States)

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

    1996-05-01

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

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

    Science.gov (United States)

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

    1992-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Luiz Claudio Pereira Ribeiro

    2013-09-01

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

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

    Science.gov (United States)

    Kurien, Biji T; Scofield, R Hal

    2009-01-01

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

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

    Directory of Open Access Journals (Sweden)

    MORALES Olga Lucía

    2002-01-01

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

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

    Science.gov (United States)

    Lin, Wen-Wei; Chen, I-Ju; Cheng, Ta-Chun; Tung, Yi-Ching; Chu, Pei-Yu; Chuang, Chih-Hung; Hsieh, Yuan-Chin; Huang, Chien-Chiao; Wang, Yeng-Tseng; Kao, Chien-Han; Roffler, Steve R; Cheng, Tian-Lu

    2016-01-01

    Molecular weight markers that can tolerate denaturing conditions and be auto-detected by secondary antibodies offer great efficacy and convenience for Western Blotting. Here, we describe M&R LE protein markers which contain linear epitopes derived from the heavy chain constant regions of mouse and rabbit immunoglobulin G (IgG Fc LE). These markers can be directly recognized and stained by a wide range of anti-mouse and anti-rabbit secondary antibodies. We selected three mouse (M1, M2 and M3) linear IgG1 and three rabbit (R1, R2 and R3) linear IgG heavy chain epitope candidates based on their respective crystal structures. Western blot analysis indicated that M2 and R2 linear epitopes are effectively recognized by anti-mouse and anti-rabbit secondary antibodies, respectively. We fused the M2 and R2 epitopes (M&R LE) and incorporated the polypeptide in a range of 15-120 kDa auto-detecting markers (M&R LE protein marker). The M&R LE protein marker can be auto-detected by anti-mouse and anti-rabbit IgG secondary antibodies in standard immunoblots. Linear regression analysis of the M&R LE protein marker plotted as gel mobility versus the log of the marker molecular weights revealed good linearity with a correlation coefficient R2 value of 0.9965, indicating that the M&R LE protein marker displays high accuracy for determining protein molecular weights. This accurate, regular and auto-detected M&R LE protein marker may provide a simple, efficient and economical tool for protein analysis.

  12. Estandarización de la técnica de Western blot para el diagnóstico de la fasciolosis humana utilizando antígenos de excreción-secreción de Fasciola hepática Western blot technique standardization of the diagnosis of human fasciolosis using Fasciola hepatica excreted-secreted antigens

    Directory of Open Access Journals (Sweden)

    Hermes Escalante

    2011-09-01

    Full Text Available Objetivos. Evaluar la eficacia de la técnica de electroinmunotransferencia (EITB o Western blot utilizando antígenos de excreción-secreción de las formas adultas de Fasciola hepatica (Fh E/S Ag para el diagnóstico de la fasciolosis humana. Materiales y métodos. Los antígenos fueron obtenidos a las 18 horas de incubación en medio Minimum Essential Eagle y preparados a la concentración proteica de 0,15 ug/uL; los cuales, al ser enfrentados con un pool de sueros de pacientes con fasciolosis confirmada por el hallazgo de huevos del parásito en las heces, se detectaron los antígenos de 10, 12, 17, 23, 27, 30, 36, 43, 66 y 136 KDa, con los cuales se desarrolló la técnica de Western blot. La sensibilidad se evaluó empleando sueros de 67 pacientes con fasciolosis, y la especificidad con sueros de 57 pacientes con otras parasitosis y diez sueros de personas no parasitadas. Resultados. De los 67 sueros, 64 reaccionaron con la banda de 23 KDa y 61 con la banda de 17KDa. Estas dos bandas no fueron detectadas por ninguno de los sueros de pacientes con otras parasitosis, ni de personas no parasitadas, siendo por ello consideradas como específicas y diagnósticas. Conclusiones. La sensibilidad de la prueba, utilizando las bandas de 17 y 23 KDa, fue de 95,5 % cuando se presenta reacción positiva en una o en las dos bandas, siendo la especificidad para estos dos antígenos de 100 % con un valor predictivo positivo de 100 % y un valor predictivo negativo de 95,71 %.Objectives. To evaluate the performance of the enzyme-linked immunoelectrotransfer blot assay (EITB, Western blot using excretory/secretory antigens from adult forms of Fasciola hepatica (Fh E/S Ag for the diagnosis of human fasciolosis. Materials and methods. Antigens were obtained after 18 hours of incubation in culture medium Minimum Essential Eagle, prepared at a protein concentration of 0.15 ug/uL and run against a pool of sera of patients with proven fasciolosis (confirmed by the

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

    Science.gov (United States)

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

    1998-02-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Nicholas H Ogden

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

  16. Direct visualization of epidermal growth factor receptors (EGFR) in A431 and placental cell membrane by western blot with 125I-EGF

    International Nuclear Information System (INIS)

    Lin, P.H.; Selinfreund, R.; Wharton, W.

    1986-01-01

    Using the western blot technique, they have devised a new procedure that allowed the direct visualization of both the 150KD and the 170KD forms of EGFR by its natural ligand, 125 I-EGF. A431, and placental plasmalemma were purified and solubilized in either SDS-PAGE buffer (without DTT, EDTA) or Triton X-100 (0.5%), resolved on PAGE and electrophoretically transferred onto nitrocellulose (NC) paper. In the absence of boiling, SDS did not denature the EGFR. Although EGER band can be detected after hybridization with 125 I-EGF, the receptor signal was considerably improved with the addition of 0.1% Tween-20. The binding of 125 I-EGF to the both the 150KD and the 170KD bands of the EGFR was specific, reversible and increased with the amount of membrane protein present. The direct visualization of the EGFR using its natural ligand eliminated the necessity for the time consuming antibody preparation. Presently, they are using this technique to identify specific receptors for other ligands

  17. ANALYSIS OF Treponema pallidum RECOMBINANT ANTIGENS FOR DIAGNOSIS OF SYPHILIS BY WESTERN BLOTTING TECHNIQUE Análise de antígenos recombinantes de Treponema pallidum no diagnóstico da sífilis utilizando a técnica de Western Blotting

    Directory of Open Access Journals (Sweden)

    Neuza Satomi SATO

    1999-03-01

    Full Text Available Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rTp17 antigen. Some samples were non-reactive or showed a weak reaction with GST-rTp47 and/or GST-rTp15, and apparently there was no correlation with the stage of disease. There was no seropositivity among blood donors. No sample reacted with purified GST. We concluded that due to their specificity these recombinant antigens can be used as GST fusion protein for development of syphilis diagnostic assays.Os antígenos recombinantes de Treponema pallidum GST-rTp47, GST-rTp17 e GST-rTp15, produzidos em fusão com glutationa S-transferase (GST em E. coli, foram analisados quanto ao potencial diagnóstico da sífilis pela técnica de Western blotting. Foram testadas 53 amostras, sendo 25 de pacientes em diferentes estágios clínicos da sífilis, com resultados positivos no teste treponêmico clássico; 25 amostras procedentes de doadores de banco de sangue, com sorologia negativa e 3 de pacientes com doença sexualmente transmissível não relacionado à sífilis. Todas as amostras de pacientes com sífilis apresentaram alta reatividade com o antígeno GST-rTp17. Quanto aos antígenos GST-rTp47 e GST-Tp15 verificou-se uma variação na presença ou na intensidade da reação em diferentes amostras de pacientes com sífilis, sem mostrar correlação com o estágio da doença. Nenhuma reatividade contra quaisquer desses antígenos foi observada com as amostras do grupo controle. Nenhuma das amostras testadas apresentaram reatividade com a GST purificada. A

  18. Northern blotting analysis

    DEFF Research Database (Denmark)

    Josefsen, Knud; Nielsen, Henrik

    2011-01-01

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

  19. Rendimiento diagnóstico del Western Blot para detectar simultáneamente anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana

    Directory of Open Access Journals (Sweden)

    Kelly Davelois

    Full Text Available Objetivo. Determinar el rendimiento diagnóstico de la técnica de Western Blot para detectar simultáneamente anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana. Materiales y métodos. Estudio transversal de evaluación de prueba diagnóstica. Se obtuvieron los antígenos de excreción-secreción de las larvas de Taenia solium, quistes de Echinococcus granulosus; y la forma adulta de Fasciola hepática; que luego fueron separados electroforéticamente en geles de poliacrilamida individuales, transferidos y fijados a una membrana de nitrocelulosa para ser enfrentados con sueros de pacientes con las tres parasitosis. La sensibilidad de la técnica se evaluó empleando 300 sueros individuales, 60 pools de dos parasitosis y 20 pools de tres parasitosis y la especificidad con 75 sueros de pacientes con otras parasitosis, 10 de pacientes con otras enfermedades y 15 sueros de personas no parasitadas. Resultados. La técnica reconoció trece glicoproteínas (GP: GP 35, 31, 24, 23, 18, 17, 14 y 13 kDa para cisticercosis, GP 8,16 y 21 kDa para hidatidosis y GP: 17 y 23 kDa para fascioliasis. La prueba detectó la presencia de anticuerpos alcanzando una sensibilidad de 96% (IC95%: 94,62-98,54% en la detección de una o las trece bandas, una especificidad de 100% (IC95%: 99,50 - 100,00%; individualmente, se tuvo una sensibilidad para cisticercosis de 97% (IC95%: 93,16-100%, para hidatidosis de 94% (IC95%: 88,85-99,15% y para fascioliasis de 96% (IC95%: 91,66-100%. Conclusiones. La prueba de Western blot es eficaz en la detección, simultanea de anticuerpos en pacientes con cisticercosis, hidatidosis y fascioliasis humana, y puede ser utilizada como prueba de descarte o confirmatoria en zonas endémicas.

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

    Science.gov (United States)

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

    2013-10-01

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

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

    Science.gov (United States)

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

    2016-08-01

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

  2. Southern blotting.

    Science.gov (United States)

    Brown, T

    2001-05-01

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

  3. Far Western: probing membranes.

    Science.gov (United States)

    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.

    Science.gov (United States)

    Ravera, Ivan; Ferreira, Diana; Gallego, Laia Solano; Bardagí, Mar; Ferrer, Lluís

    2015-08-01

    The aim of this study was to investigate the presence of canine immunoglobulins (Ig) G against Demodex proteins in the sera of healthy dogs and of dogs with juvenile generalized demodicosis (CanJGD) with or without secondary pyoderma. Demodex mites were collected from dogs with CanJGD. Protein concentration was measured and a western blot technique was performed. Pooled sera from healthy dogs reacted mainly with antigen bands ranging from 55 to 72 kDa. Pooled sera from dogs with CanJGD without secondary pyoderma reacted either with 10 kDa antigen band or 55 to 72 kDa bands. Pooled sera from dogs with CanJGD with secondary pyoderma reacted only with a 10 kDa antigen band. The results of this study suggest that both healthy dogs and dogs with CanJGD develop a humoral response against different proteins of Demodex canis. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    Hood-Degrenier, Jennifer K

    2008-01-01

    The movement of newly synthesized proteins through the endomembrane system of eukaryotic cells, often referred to generally as the secretory pathway, is a topic covered in most intermediate-level undergraduate cell biology courses. An article previously published in this journal described a laboratory exercise in which yeast mutants defective in two distinct steps of protein secretion were differentiated using a genetic reporter designed specifically to identify defects in the first step of the pathway, the insertion of proteins into the endoplasmic reticulum (Vallen, 2002). We have developed two versions of a Western blotting assay that serves as a second way of distinguishing the two secretory mutants, which we pair with the genetic assay in a 3-wk laboratory module. A quiz administered before and after students participated in the lab activities revealed significant postlab gains in their understanding of the secretory pathway and experimental techniques used to study it. A second survey administered at the end of the lab module assessed student perceptions of the efficacy of the lab activities; the results of this survey indicated that the experiments were successful in meeting a set of educational goals defined by the instructor.

  6. Introduction to protein blotting.

    Science.gov (United States)

    Kurien, Biji T; Scofield, R Hal

    2009-01-01

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

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  8. Evaluación de la técnica Western blot para la detección de antígenos de Hymenolepis nana

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    Flora Chávez-Salas

    2013-04-01

    Full Text Available Este trabajo tuvo como objetivo evaluar la técnica de inmunoelectrotransferencia (Western Blot para detectar los antígenos específicos de excreción/secreción de Hymenolepis nana en sueros de pacientes con himenolepiosis y con otras helmintiosis confirmadas. Se utilizó a Mesocricetus auratus “hamster” para obtener ejemplares adultos de H. nana. Los antígenos de excreción/secreción fueron obtenidos en el medio MEM (Minimum Essential Medium Eagle, y enfrentados con un grupo de sueros de pacientes con himenolepiosis confirmada para evaluar su calidad inmunológica y con sueros individuales de pacientes con himenolepiosis y con otras helmintiosis confirmadas para detectar mediante la técnica de “Western Blot”, los antígenos específicos de este cestode. El grupo de sueros de pacientes con himenolepiosis confirmada reconoció las bandas antigénicas de 50,1; 42,6; 38,9; 32,9; 26,3; 22,4 y 18,6 kDa; sin embargo, los sueros individuales reconocieron diferente número de bandas, siendo la de 50,1 KDa la que fue reconocida por todos ellos. Los sueros de pacientes con helmintiosis confirmadas no reconocieron la banda de 50,1 kDa; sin embargo, dieron reacción cruzada con algunas de las demás bandas, a excepción de los sueros de pacientes con cisticercosis que no reconocieron a ninguna de las bandas de estos antígenos. Se concluye que el antígeno de excreción/secreción de H. nana de 50,1 kDa es específico de este cestode por ser reconocido por todos los sueros de pacientes con himenolepiosis confirmada y no por sueros de pacientes con otras helmintiosis utilizando la técnica de “Western Blot”.

  9. Detection of Tumor Markers in Prostate Cancer and Comparison of Sensitivity between Real Time and Nested PCR

    OpenAIRE

    Matsuoka, Takayuki; Shigemura, Katsumi; Yamamichi, Fukashi; Fujisawa, Masato; Kawabata, Masato; Shirakawa, Toshiro

    2012-01-01

    The objective of this study is to investigate and compare the sensitivity in conventional PCR, quantitative real time PCR, nested PCR and western blots for detection of prostate cancer tumor markers using prostate cancer (PCa) cells. We performed conventional PCR, quantitative real time PCR, nested PCR, and western blots using 5 kinds of PCa cells. Prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and androgen receptor (AR) were compared for their detection sensitivi...

  10. A bead-based western for high-throughput cellular signal transduction analyses

    Science.gov (United States)

    Treindl, Fridolin; Ruprecht, Benjamin; Beiter, Yvonne; Schultz, Silke; Döttinger, Anette; Staebler, Annette; Joos, Thomas O.; Kling, Simon; Poetz, Oliver; Fehm, Tanja; Neubauer, Hans; Kuster, Bernhard; Templin, Markus F.

    2016-01-01

    Dissecting cellular signalling requires the analysis of large number of proteins. The DigiWest approach we describe here transfers the western blot to a bead-based microarray platform. By combining gel-based protein separation with immobilization on microspheres, hundreds of replicas of the initial blot are created, thus enabling the comprehensive analysis of limited material, such as cells collected by laser capture microdissection, and extending traditional western blotting to reach proteomic scales. The combination of molecular weight resolution, sensitivity and signal linearity on an automated platform enables the rapid quantification of hundreds of specific proteins and protein modifications in complex samples. This high-throughput western blot approach allowed us to identify and characterize alterations in cellular signal transduction that occur during the development of resistance to the kinase inhibitor Lapatinib, revealing major changes in the activation state of Ephrin-mediated signalling and a central role for p53-controlled processes. PMID:27659302

  11. Northern blotting analysis

    DEFF Research Database (Denmark)

    Josefsen, Knud; Nielsen, Henrik

    2011-01-01

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

  12. HIV-1/2 indeterminate Western blot results: follow-up of asymptomatic blood donors in Belo Horizonte, Minas Gerais, Brazil

    Directory of Open Access Journals (Sweden)

    CARNEIRO-PROIETTI A.B.F.

    1999-01-01

    Full Text Available The clinical and public health importance of indeterminate results in HIV-1/2 testing is still difficult to evaluate in volunteer blood donors. At Fundação Hemominas, HIV-1/2 ELISA is used as the screening test and, if reactive, is followed by Western blot (WB. We have evaluated 84 blood donors who had repeatedly reactive ELISA tests for HIV-1/2, but indeterminate WB results. Sixteen of the 84 donors (19.0% had history of sexually transmitted diseases; 18/84 (21.4% informed receiving or paying for sex; 3/84 (3.6% had homosexual contact; 2/26 women (7.6% had past history of multiple illegal abortions and 3/84 (3.6% had been previously transfused. Four out of 62 donors (6.5% had positive anti-nuclear factor (Hep2, with titles up to 1:640. Parasitological examination of the stool revealed eggs of S. mansoni in 4/62 (6.4% donors and other parasites in 8/62 (12.9%. Five (5.9% of the subjects presented overt seroconversion for HIV-1/2, 43/84 (51.2% had negative results on the last visit, while 36/84 (42.9% remained WB indeterminate. Although some conditions could be found associated with the HIV-1/2 indeterminate WB results and many donors had past of risky behavior, the significance of the majority of the results remains to be determined.

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

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    NUNES Cáris Maroni

    1997-01-01

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

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

    Science.gov (United States)

    Walpurgis, Katja; Thomas, Andreas; Schänzer, Wilhelm; Thevis, Mario

    2016-02-01

    Myostatin is a key regulator of skeletal muscle growth and inhibition of its signaling pathway results in an increased muscle mass and function. The aim of this study was to develop a qualitative detection assay for myostatin-neutralizing antibodies for doping control purposes by using immunological approaches. To detect different types of myostatin-neutralizing antibodies irrespective of their amino acid sequence, an immunological assay specific for antibodies directed against myostatin and having a human Fc domain was established. Affinity purification and Western blotting strategies were combined to allow extracting and identifying relevant analytes from 200 μL of plasma/serum in a non-targeted approach. The assay was characterized regarding specificity, linearity, precision, robustness, and recovery. The assay was found to be highly specific, robust, and linear from 0.1 to 1 μg/mL. The precision was successfully specified at three different concentrations and the recovery of the affinity purification was 58%. Within this study, an immunological detection assay for myostatin-neutralizing antibodies present in plasma/serum specimens was developed and successfully characterized. The presented approach can easily be modified to include other therapeutic antibodies and serves as proof-of-concept for the detection of antibody-based myostatin inhibitors in doping control samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

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

    2013-08-01

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

  16. Automated design of genomic Southern blot probes

    Directory of Open Access Journals (Sweden)

    Komiyama Noboru H

    2010-01-01

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

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

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    Seyyed Javad SEYYEDTABAEI

    2017-06-01

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

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

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

    Science.gov (United States)

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

    2012-04-30

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

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

    Science.gov (United States)

    Gilboa-Garber, Nechama; Lerrer, Batya; Lesman-Movshovich, Efrat; Dgani, Orly

    2005-12-01

    Human milk, serum, saliva, and seminal fluid glycoproteins (gps) nourish and protect newborn and adult tissues. Their saccharides, which resemble cell membrane components, may block pathogen adhesion and infection. In the present study, they were examined by a battery of lectins from plants, animals, and bacteria, using hemagglutination inhibition and Western blot analyses. The lectins included galactophilic ones from Aplysia gonad, Erythrina corallodendron, Maclura pomifera (MPL), peanut, and Pseudomonas aeruginosa (PA-IL); fucose-binding lectins from Pseudomonas aeruginosa (PA-IIL), Ralstonia solanacearum (RSL), and Ulex europaeus (UEA-I), and mannose/glucose-binding Con A. The results demonstrated the chosen lectin efficiency for differential analysis of human secreted gps as compared to CBB staining. They unveiled the diversity of these body fluid gp glycans (those of the milk and seminal fluid being highest): the milk gps interacted most strongly with PA-IIL, followed by RSL; the saliva gps with RSL, followed by PA-IIL and MPL; the serum gps with Con A and MPL, followed by PA-IIL and RSL, and the seminal plasma gps with RSL and MPL, followed by UEA-I and PA-IIL. The potential usage of these lectins as probes for scientific, industrial, and medical purposes, and for quality control of the desired gps is clearly indicated.

  1. Low Proviral Load is Associated with Indeterminate Western Blot Patterns in Human T-Cell Lymphotropic Virus Type 1 Infected Individuals: Could Punctual Mutations be Related?

    Directory of Open Access Journals (Sweden)

    Camila Cánepa

    2015-10-01

    Full Text Available Background: indeterminate Western blot (WB patterns are a major concern for diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1 infection, even in non-endemic areas. Objectives: (a to define the prevalence of indeterminate WB among different populations from Argentina; (b to evaluate if low proviral load (PVL is associated with indeterminate WB profiles; and (c to describe mutations in LTR and tax sequence of these cases. Results: Among 2031 samples, 294 were reactive by screening. Of them, 48 (16.3% were WB indeterminate and of those 15 (31.3% were PCR+. Quantitative real-time PCR (qPCR was performed to 52 HTLV-1+ samples, classified as Group 1 (G1: 25 WB+ samples from individuals with pathologies; Group 2 (G2: 18 WB+ samples from asymptomatic carriers (AC; and Group 3 (G3: 9 seroindeterminate samples from AC. Median PVL was 4.78, 2.38, and 0.15 HTLV-1 copies/100 PBMCs, respectively; a significant difference (p=0.003 was observed. Age and sex were associated with PVL in G1 and G2, respectively. Mutations in the distal and central regions of Tax Responsive Elements (TRE 1 and 2 of G3 were observed, though not associated with PVL.The 8403A>G mutation of the distal region, previously related to high PVL, was absent in G3 but present in 50% of WB+ samples (p = 0.03. Conclusions: indeterminateWBresults confirmed later as HTLV-1 positive may be associated with low PVL levels. Mutations in LTR and tax are described; their functional relevance remains to be determined.

  2. Western blot seroindeterminate individuals for Human T-lymphotropic Virus 1/2 (HTLV-1/2 in Fortaleza (Brazil: a serological and molecular diagnostic and epidemiological approach

    Directory of Open Access Journals (Sweden)

    Santos Terezinha de Jesus Teixeira

    2003-01-01

    Full Text Available How to handle Western blot (WB seroindeterminate individuals for Human T-lymphotropic Virus 1/2 (HTLV-1/2 constitutes a challenge for blood banks and fam ilies. We made a cross-sectional study of 191 enzyme linked immunoassay (EIA reactive individuals from the hematological center (HEMOCE of Fortaleza (Brazil, examining their serological (WB and molecular (PCR diagnosis, and demographic profiles, as well as a possible association of their condition with other infectious pathologies and risk factors. Ethical institutional approval and personal consent were obtained. Out of 191 EIA reactive individuals, 118 were WB seroindeterminate and 73 were seropositive for HTLV-1/2. In the PCR analysis of 41 WB seroindeterminate individuals, 9 (22% were positive and 32 (78% were negative for HTLV-1/2. The demographic analysis indicated a trend towards a predominance of males among the seroindeterminate individuals and females in the seropositive ones. The seroindeterminate individuals were younger than the seropositive ones. We did not find any association of these conditions with syphilis, Chagas disease or HIV or hepatitis, and with risk factors such as breast-feeding, blood transfusion, STD (syphilis and IDU.

  3. Western blot seroindeterminate individuals for Human T-lymphotropic Virus 1/2 (HTLV-1/2 in Fortaleza (Brazil: a serological and molecular diagnostic and epidemiological approach

    Directory of Open Access Journals (Sweden)

    Terezinha de Jesus Teixeira Santos

    Full Text Available How to handle Western blot (WB seroindeterminate individuals for Human T-lymphotropic Virus 1/2 (HTLV-1/2 constitutes a challenge for blood banks and fam ilies. We made a cross-sectional study of 191 enzyme linked immunoassay (EIA reactive individuals from the hematological center (HEMOCE of Fortaleza (Brazil, examining their serological (WB and molecular (PCR diagnosis, and demographic profiles, as well as a possible association of their condition with other infectious pathologies and risk factors. Ethical institutional approval and personal consent were obtained. Out of 191 EIA reactive individuals, 118 were WB seroindeterminate and 73 were seropositive for HTLV-1/2. In the PCR analysis of 41 WB seroindeterminate individuals, 9 (22% were positive and 32 (78% were negative for HTLV-1/2. The demographic analysis indicated a trend towards a predominance of males among the seroindeterminate individuals and females in the seropositive ones. The seroindeterminate individuals were younger than the seropositive ones. We did not find any association of these conditions with syphilis, Chagas disease or HIV or hepatitis, and with risk factors such as breast-feeding, blood transfusion, STD (syphilis and IDU.

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

    Science.gov (United States)

    Stramer, Susan L; Townsend, Rebecca L; Foster, Gregory A; Johnson, Ramona; Weixlmann, Barbara; Dodd, Roger Y

    2018-03-01

    Human T-lymphotropic virus (HTLV) blood donation screening has used a dual-testing algorithm beginning with either a chemiluminescent immunoassay or enzyme-linked immunosorbent screening assay (ELISA). Before the availability of a licensed HTLV supplemental assay, repeat-reactive (RR) samples on a first assay (Assay 1) were retested with a second screening assay (Assay 2). Donors with RR results by Assay 2 were deferred from blood donation and further tested using an unlicensed supplemental test to confirm reactivity while nonreactive (NR) donors remained eligible for donation until RR on a subsequent donation. This "dual-test" algorithm was replaced in May 2016 with the requirement that all RRs by Assay 1 be further tested by a licensed HTLV supplemental test (Western blot [WB]). In this study, we have requalified the dual-test algorithm using the available licensed HTLV WB. We tested 100 randomly selected HTLV RRs on screening Assay 1 (Abbott PRISM chemiluminescent immunoassay) but NR on screening Assay 2 (Avioq ELISA) by a Food and Drug Administration-licensed WB (MP Biomedicals) to ensure that no confirmed positives were among those that were RR by Assay 1 but NR by Assay 2. Of the 100 samples evaluated, 79 of 100 were WB seronegative, 21 of 100 indeterminate, and 0 of 100 seropositive. Of the 79 of 100 seronegative specimens, 73 of 79 did not express any bands on WB. We demonstrated that none of the 100 samples RR on Assay 1 but NR on Assay 2 were confirmed positive. This algorithm prevents such donors from requiring further testing and from being deferred. © 2018 AABB.

  5. Molecular evidence for the occurrence of beet western yellows virus on chickpea in Morocco.

    NARCIS (Netherlands)

    Fortass, M.; Wilk, van der F.; Heuvel, van de J.F.J.M.; Goldbach, R.W.

    1997-01-01

    A luteovirus isolate infecting chickpea in Morocco was experimentally transmitted by Myzus persicae to Physalis floridana, on which it produced mild symptoms. When tested in western blots against antisera to known legume luteoviruses, this isolate reacted strongly to beet western yellows virus

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

    Science.gov (United States)

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

    2008-01-01

    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

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

    Science.gov (United States)

    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…

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

    Science.gov (United States)

    Lee, Der-Yen; Chang, Geen-Dong

    2015-01-01

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

  9. Evaluación de las pruebas dot blot y aglutinación de látex para el diagnóstico de cisticercosis en Perú

    Directory of Open Access Journals (Sweden)

    Eduardo Miranda-Ulloa

    Full Text Available Con el objetivo de evaluar las pruebas dot blot y aglutinación de látex para la detección de cisticercosis humana con antígeno de líquido de cisticerco de Taenia solium, se usaron 125 sueros humanos, de los cuales 60 procedían de personas con cisticercosis confirmada por Western Blot, 45 de personas con otras enfermedades parasitarias y 20 de personas aparentemente sanas. La concentración óptima del antígeno para impregnar las tiras dot blot fue de 0,01 ug/uL, y para impregnar las partículas de látex fue de 0,092 ug/uL. Para la prueba dot blot se encontró una sensibilidad del 100% y especificidad del 87,7%; para la aglutinación de látex una sensibilidad del 93,3% y especificidad del 89,2%. Ambas pruebas podrían ser de utilidad y factibles de implementar como alternativas de diagnóstico serológico en laboratorios de áreas endémicas del Perú

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

    International Nuclear Information System (INIS)

    Bodkin, D.K.

    1985-01-01

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

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

    NARCIS (Netherlands)

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

    1993-01-01

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

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

    Science.gov (United States)

    Nilsen, Timothy W

    2015-03-02

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

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

    International Nuclear Information System (INIS)

    Kroczek, R.A.; Siebert, E.

    1990-01-01

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

  14. Western blot data using two distinct anti-O-GlcNAc monoclonal antibodies showing unique glycosylation status on cellular proteins under 2-deoxy-d-glucose treatment

    Directory of Open Access Journals (Sweden)

    Tetsuya Okuda

    2017-02-01

    Full Text Available Protein modification by O-linked N-acetylglucosamine (O-GlcNAcylation is one of the post transcriptional modifications occurring on cellular proteins. This paper provides a data set relating to the O-GlcNAcylation of cellular proteins detected by RL2 and CTD110.6 antibodies, which are commonly used for detection of protein O-GlcNAcylation, in 2-deoxy-d-glucose (2DG-treated human teratocarcinoma NCCIT cells in support of the research article entitled “A novel, promoter-based, target-specific assay identifies 2-deoxy-d-glucose as an inhibitor of globotriaosylceramide biosynthesis” (Okuda et al., 2009 [1]. The main article described a suppressive effect of 2DG on an Sp1 target gene in NCCIT cells and discussed the relationship between the effect of 2DG and O-GlcNAcylation status of Sp1. The data in this paper complements this relationship by Western blotting and clearly showed that the 2DG treatment increased O-GlcNAcylation of cellular proteins in NCCIT cells, whereas the RL2 and CTD110.6 epitopes were detected in a different manner. The RL2 epitope was detected on Sp1 during 2DG treatment, and the level was transiently increased at 24 h. In contrast, the CTD110.6 epitope became detectable on Sp1 over 72 h after 2DG treatment, and then the other proteins containing CTD110.6 epitopes also appeared in the cell lysates and the anti-Sp1 antibody precipitates.

  15. Detection of tumor markers in prostate cancer and comparison of sensitivity between real time and nested PCR.

    Science.gov (United States)

    Matsuoka, Takayuki; Shigemura, Katsumi; Yamamichi, Fukashi; Fujisawa, Masato; Kawabata, Masato; Shirakawa, Toshiro

    2012-06-27

    The objective of this study is to investigate and compare the sensitivity in conventional PCR, quantitative real time PCR, nested PCR and western blots for detection of prostate cancer tumor markers using prostate cancer (PCa) cells. We performed conventional PCR, quantitative real time PCR, nested PCR, and western blots using 5 kinds of PCa cells. Prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), and androgen receptor (AR) were compared for their detection sensitivity by real time PCR and nested PCR. In real time PCR, there was a significant correlation between cell number and the RNA concentration obtained (R(2)=0.9944) for PSA, PSMA, and AR. We found it possible to detect these markers from a single LNCaP cell in both real time and nested PCR. By comparison, nested PCR reached a linear curve in fewer PCR cycles than real time PCR, suggesting that nested PCR may offer PCR results more quickly than real time PCR. In conclusion, nested PCR may offer tumor maker detection in PCa cells more quickly (with fewer PCR cycles) with the same high sensitivity as real time PCR. Further study is necessary to establish and evaluate the best tool for PCa tumor marker detection.

  16. Western aeronautical test range real-time graphics software package MAGIC

    Science.gov (United States)

    Malone, Jacqueline C.; Moore, Archie L.

    1988-01-01

    The master graphics interactive console (MAGIC) software package used on the Western Aeronautical Test Range (WATR) of the NASA Ames Research Center is described. MAGIC is a resident real-time research tool available to flight researchers-scientists in the NASA mission control centers of the WATR at the Dryden Flight Research Facility at Edwards, California. The hardware configuration and capabilities of the real-time software package are also discussed.

  17. Individual Case Analysis of Postmortem Interval Time on Brain Tissue Preservation.

    Directory of Open Access Journals (Sweden)

    Jeffrey A Blair

    Full Text Available At autopsy, the time that has elapsed since the time of death is routinely documented and noted as the postmortem interval (PMI. The PMI of human tissue samples is a parameter often reported in research studies and comparable PMI is preferred when comparing different populations, i.e., disease versus control patients. In theory, a short PMI may alleviate non-experimental protein denaturation, enzyme activity, and other chemical changes such as the pH, which could affect protein and nucleic acid integrity. Previous studies have compared PMI en masse by looking at many different individual cases each with one unique PMI, which may be affected by individual variance. To overcome this obstacle, in this study human hippocampal segments from the same individuals were sampled at different time points after autopsy creating a series of PMIs for each case. Frozen and fixed tissue was then examined by Western blot, RT-PCR, and immunohistochemistry to evaluate the effect of extended PMI on proteins, nucleic acids, and tissue morphology. In our results, immunostaining profiles for most proteins remained unchanged even after PMI of over 50 h, yet by Western blot distinctive degradation patterns were observed in different protein species. Finally, RNA integrity was lower after extended PMI; however, RNA preservation was variable among cases suggesting antemortem factors may play a larger role than PMI in protein and nucleic acid integrity.

  18. Investigation of the effects of experimental autolysis on the detection of abnormal prion protein in lymphoid and central nervous system tissues from elk and sheep using the Western blotting method.

    Science.gov (United States)

    Huang, Hongsheng; Soutyrine, Andrei; Rendulich, Jasmine; O'Rourke, Katherine; Balachandran, Aru

    2011-01-01

    Tissues unsuitable for standard immunohistochemical and histopathological examinations for chronic wasting disease (CWD) in cervids and for scrapie in sheep are frequently submitted for testing. This study investigated the effects of experimental autolysis on the detection of abnormal prion protein (PrPsc) in lymphoid and central nervous system (CNS) tissues from elk and sheep. The PrPsc was detected using a Western blotting (WB) test following PrPsc enrichment using sodium phosphotungstic acid (PTA) precipitation (PTA-WB). A commercial enzyme-linked immunosorbent assay (ELISA) was used as a reference test for quantitative measurement. This study showed that the amount of PrPsc in lymphoid and CNS tssues from elk and sheep decreased gradually as a result of autolysis, but PrPsc was still detectable after 5 and 15 d incubation at 37°C by PTA-WB for all lymphoid and CNS samples. The results of the ELISA supported those of PTA-WB, particularly for CNS tissues. In conclusion, autolysis at 37°C for 15 d would not significantly affect the detection of PrPsc in lymphoid and CNS tissues by WB and ELISA and, particularly, PTA-WB is a valuable and alternative confirmatory test to detect PrPsc in autolyzed lymphoid and CNS samples.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

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

    NARCIS (Netherlands)

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

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

  1. Timing is everything : western Canada natural gas supply

    International Nuclear Information System (INIS)

    George, R.

    1998-01-01

    The issue of the energy market's ability to support Western Canadian pipeline expansion projects was re-visited. Related topics discussed included the ex-basin transportation bottleneck, the large 'basis' differential, estimates of the magnitude of impact on 'basis', trends in drilling and the decline in well productivity. The conclusions drawn from the analysis were that drilling activity must remain high, that more gas-directed drilling was needed, along with a reduction in drilling time. A shift towards more exploratory drilling and higher quality reserves further north and west was recommended to 'fill the pipes'. 2 tabs., 5 figs

  2. Lectin-Array Blotting.

    Science.gov (United States)

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

    2017-09-01

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

  3. Detection and attribution of streamflow timing changes to climate change in the Western United States

    Science.gov (United States)

    Hidalgo, H.G.; Das, T.; Dettinger, M.D.; Cayan, D.R.; Pierce, D.W.; Barnett, T.P.; Bala, G.; Mirin, A.; Wood, A.W.; Bonfils, Celine; Santer, B.D.; Nozawa, T.

    2009-01-01

    This article applies formal detection and attribution techniques to investigate the nature of observed shifts in the timing of streamflow in the western United States. Previous studies have shown that the snow hydrology of the western United States has changed in the second half of the twentieth century. Such changes manifest themselves in the form of more rain and less snow, in reductions in the snow water contents, and in earlier snowmelt and associated advances in streamflow "center" timing (the day in the "water-year" on average when half the water-year flow at a point has passed). However, with one exception over a more limited domain, no other study has attempted to formally attribute these changes to anthropogenic increases of greenhouse gases in the atmosphere. Using the observations together with a set of global climate model simulations and a hydrologic model (applied to three major hydrological regions of the western United States_the California region, the upper Colorado River basin, and the Columbia River basin), it is found that the observed trends toward earlier "center" timing of snowmelt-driven streamflows in the western United States since 1950 are detectably different from natural variability (significant at the p analysis, and it is the only basin that showed a detectable signal when the analysis was performed on individual basins. It should be noted that although climate change is an important signal, other climatic processes have also contributed to the hydrologic variability of large basins in the western United States. ?? 2009 American Meteorological Society.

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

    Science.gov (United States)

    Rey, María-Dolores; Prieto, Pilar

    2017-01-01

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

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

    Science.gov (United States)

    Methogo, Ruth Menque; Dansette, Patrick M.; Klarskov, Klaus

    2007-12-01

    A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.

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

    Science.gov (United States)

    Hubbard, Katherine; Hegarty, Peter

    2016-01-01

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

  7. Blotting from PhastGel to Membranes by Ultrasound.

    Science.gov (United States)

    Kost, Joseph; Azagury, Aharon

    2015-01-01

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

  8. Immunocytochemical electron microscopic study and western blot analysis of paramyosin in different invertebrate muscle cell types of the fruit fly Drosophila melanogaster, the earthworm Eisenia foetida, and the snail Helix aspersa.

    Science.gov (United States)

    Royuela, M; García-Anchuelo, R; Arenas, M I; Cervera, M; Fraile, B; Paniagua, R

    1996-04-01

    The presence and distribution pattern of paramyosin have been examined in different invertebrate muscle cell types by means of Western blot analysis and electron microscopy immunogold labelling. The muscles studied were: transversely striated muscle with continuous Z lines (flight muscle from Drosophila melanogaster), transversely striated muscle with discontinuous Z lines (heart muscle from the snail Helix aspersa), obliquely striated body wall muscle from the earthworm Eisenia foetida, and smooth muscles (retractor muscle from the snail and pseudoheart outer muscular layer from the earthworm). Paramyosin-like immunoreactivity was localized in thick filaments of all muscles studied. Immunogold particle density was similar along the whole thick filament length in insect flight muscle but it predominated in filament tips of fusiform thick filaments in both snail heart and earthworm body wall musculature when these filaments were observed in longitudinal sections. In obliquely sectioned thick filaments, immunolabelling was more abundant at the sites where filaments disappeared from the section. These results agree with the notion that paramyosin extended along the whole filament length, but that it can only be immunolabelled when it is not covered by myosin. In all muscles examined, immunolabelling density was lower in cross-sectioned myofilaments than in longitudinally sectioned myofilaments. This suggests that paramyosin does not form a continuous filament. The results of a semiquantitative analysis of paramyosin-like immunoreactivity indicated that it was more abundant in striated than in smooth muscles, and that, within striated muscles, transversely striated muscles contain more paramyosin than obliquely striated muscles.

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

    International Nuclear Information System (INIS)

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

    1991-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yukihiro Shoyama

    2013-01-01

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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

  12. Western blotting of high and low molecular weight proteins using heat.

    Science.gov (United States)

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

    DEFF Research Database (Denmark)

    Christensen, C B; Theander, T G

    1997-01-01

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

  14. Changes in snowmelt runoff timing in western North America under a 'business as usual' climate change scenario

    Science.gov (United States)

    Stewart, I.T.; Cayan, D.R.; Dettinger, M.D.

    2004-01-01

    Spring snowmelt is the most important contribution of many rivers in western North America. If climate changes, this contribution may change. A shift in the timing of springtime snowmelt towards earlier in the year already is observed during 1948-2000 in many western rivers. Streamflow timing changes for the 1995-2099 period are projected using regression relations between observed streamflow-timing responses in each river, measured by the temporal centroid of streamflow (CT) each year, and local temperature (TI) and precipitation (PI) indices. Under 21st century warming trends predicted by the Parallel Climate Model (PCM) under business-as-usual greenhouse-gas emissions, streamflow timing trends across much of western North America suggest even earlier springtime snowmelt than observed to date. Projected CT changes are consistent with observed rates and directions of change during the past five decades, and are strongest in the Pacific Northwest, Sierra Nevada, and Rocky Mountains, where many rivers eventually run 30-40 days earlier. The modest PI changes projected by PCM yield minimal CT changes. The responses of CT to the simultaneous effects of projected TI and PI trends are dominated by the TI changes. Regression-based CT projections agree with those from physically-based simulations of rivers in the Pacific Northwest and Sierra Nevada.

  15. Seroprevalence of antibodies to Borrelia burgdorferi sensu lato in healthy adults from western Norway: risk factors and methodological aspects.

    Science.gov (United States)

    Hjetland, Reidar; Nilsen, Roy M; Grude, Nils; Ulvestad, Elling

    2014-11-01

    The aim of this study was to assess the seroprevalence of antibodies to Borrelia burgdorferi sensu lato in a healthy adult population from Sogn and Fjordane county in western Norway by different assays. Sera from 1213 blood donors at four different blood banks were analysed in Enzygnost Lyme link VlsE/IgG (IgG), Enzygnost Borreliosis IgM (IgM), and Immunetics C6 Lyme ELISA kit (C6). Sera showing positive or grey-zone reactivities were further examined with Borrelia-EUROLine-RN-AT IgG blot and Borrelia-EUROLine-RN-AT IgM blot. The seroprevalences were 9.6%, 8.2%, 8.4%, 6.4% and 5.7%, respectively. The seroprevalence for IgG was lower in the eastern part of the county and in owners of pet animals. It was higher in men, and increased with age and number of tick bites. C6 and IgG gave comparable results. IgM only was found in 4.5%, more often in women, did not increase with age, and showed no relationship with geography, and 56.4% were positive in IgM blot. In conclusion, antibodies to B. burgdorferi s.l. are common in blood donors in western Norway. The results may be used for evaluation of predictive values of test results in patients, as well as a basis for test algorithms in the laboratory. © 2014 APMIS. Published by John Wiley & Sons Ltd.

  16. Screening for simian foamy virus infection by using a combined antigen Western blot assay: evidence for a wide distribution among Old World primates and identification of four new divergent viruses

    International Nuclear Information System (INIS)

    Hussain, Althaf I.; Shanmugam, Vedapuri; Bhullar, Vinod B.; Beer, Brigitte E.; Vallet, Dominique; Gautier-Hion, Annie; Wolfe, Nathan D.; Karesh, William B.; Kilbourn, Annelisa M.; Tooze, Zeena; Heneine, Walid; Switzer, William M.

    2003-01-01

    Simian foamy viruses (SFVs) belong to a genetically and antigenically diverse class of retroviruses that naturally infect a wide range of nonhuman primates (NHPs) and can also be transmitted to humans occupationally exposed to NHPs. Current serologic detection of SFV infection requires separate Western blot (WB) testing by using two different SFV antigens [SFV AGM (African green monkey) and SFV CPZ (chimpanzee)]. However, this method is labor intensive and validation is limited to only small numbers of NHPs. To facilitate serologic SFV testing, we developed a WB assay that combines antigens from both SFV AGM and SFV CPZ . The combined-antigen WB (CA-WB) assay was validated with 145 serum samples from 129 NHPs (32 African and Asian species) and 16 humans, all with known SFV infection status determined by PCR. Concordant CA-WB results were obtained for all 145 PCR-positive or -negative primate and human specimens, giving the assay a 100% sensitivity and specificity. In addition, no reactivity was observed in sera from persons positive for human immunodeficiency virus or human T cell lymphotropic virus (HIV/HTLV) (n = 25) or HIV/HTLV-negative U.S. blood donors (n = 100). Using the CA-WB assay, we screened 360 sera from 43 Old World primate species and found an SFV prevalence of about 68% in both African and Asian primates. We also isolated SFV from the blood of four seropositive primates (Allenopithecus nigroviridis, Trachypithecus francoisi, Hylobates pileatus, and H. leucogenys) not previously known to be infected with SFV. Phylogenetic analysis of integrase sequences from these isolates confirmed that all four SFVs represent new, distinct, and highly divergent lineages. These results demonstrate the ability of the CA-WB assay to detect infection in a large number of NHP species, including previously uncharacterized infections with divergent SFVs

  17. Immunological diagnosis of human hydatid cyst using Western immunoblotting technique

    Directory of Open Access Journals (Sweden)

    Mahboubeh Hadipour

    2016-01-01

    Full Text Available Background: Echinococcosis is a parasitic disease with worldwide distribution which is caused by the tapeworms Echinococcus granulosus. Diagnosis of the disease relies on imaging techniques, but the techniques are not able to differentiate the cyst from benign or malignant tumors; hence, appropriate serologic methods are required for the differential diagnosis of the infection. Materials and Methods: In this investigation, different sheep hydatid cyst antigens probed with thirty sera of patients with hydatid cyst and also thirty human normal sera using Western immunoblotting technique. Considering results of surgery as gold standard, sensitivity and specificity of Western blotting was estimated. Results: Sera of 29, 26, and 16 patients with hydatid cyst reacted with specific bands of hydatid cyst fluid (HCF, protoscolex crude antigen, and cyst wall crude antigen, respectively. However, none of the normal human sera reacted with those specific bands. Conclusion: A 20 kDa band of sheep HCF is an appropriate antigen for serodiagnosis of hydatid cyst infection.

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

    Science.gov (United States)

    Shiokai, Sachiko; Kitashiba, Hiroyasu; Nishio, Takeshi

    2010-08-01

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

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

    International Nuclear Information System (INIS)

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

    1986-01-01

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

  20. Characterization of serum amyloid A (SAA) in rainbow trout using a new monoclonal antibody

    DEFF Research Database (Denmark)

    Kania, Per Walter; Chettri, Jiwan Kumar; Buchmann, Kurt

    2014-01-01

    Serum amyloid A (SAA) is an integral part of the innate immune response in mammals and considered to be important during the acute phase response. The present study was undertaken to elucidate the role of SAA protein in the innate immune response of rainbow trout. A monoclonal antibody raised...... against a recombinant peptide of rainbow trout SAA was characterized using Western blot, dot blot, ELISA and immunohistochemistry. SAA association with high density lipoprotein (HDL) complicated band identification in Western blot, but delipidization of the SAA-HDL isolate highly increased the quality...... of reaction in the western blot. Rainbow trout fry (87 days post hatch) infected with Yersinia ruckeri showed a significant up-regulation of the SAA gene at 72 h post infection with an increase until 96 h post infection. Non-significant up-regulations were seen at earlier time points i.e. 4 and 24 h...

  1. Timing of susceptibility to post-fire debris flows in the western USA

    Science.gov (United States)

    DeGraff, Jerome V.; Cannon, Susan H.; Gartner, Joseph E.

    2015-01-01

    Watersheds recently burned by wildfires can have an increased susceptibility to debris flow, although little is known about how long this susceptibility persists, and how it changes over time. We here use a compilation of 75 debris-flow response and fire-ignition dates, vegetation and bedrock class, rainfall regime, and initiation process from throughout the western U.S. to address these issues. The great majority (85 percent) of debris flows occurred within the first 12 months following wildfire, with 71 percent within the first six months. Seven percent of the debris flows occurred between 1 and 1.5 years after a fire, or during the second rainy season to impact an area. Within the first 1.5 years following fires, all but one of the debris flows initiated through runoff-dominated processes, and debris flows occurred in similar proportions in forested and non-forested landscapes. Geologic materials affected how long debris-flow activity persisted, and the timing of debris flows varied within different rainfall regimes. A second, later period of increased debris flow susceptibility between 2.2 and 10 years after fires is indicated by the remaining 8 percent of events, which occurred primarily in forested terrains and initiated largely through landslide processes. The short time period between fire and debris-flow response within the first 1.5 years after ignition, and the longer-term response between 2.2 and 10 years after fire, demonstrate the necessity of both rapid and long-term reactions by land managers and emergency-response agencies to mitigate hazards from debris flows from recently burned areas in the western U.S.

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  3. Phenylglyoxal-Based Visualization of Citrullinated Proteins on Western Blots

    Directory of Open Access Journals (Sweden)

    Sanne M. M. Hensen

    2015-04-01

    Full Text Available Citrullination is the conversion of peptidylarginine to peptidylcitrulline, which is catalyzed by peptidylarginine deiminases. This conversion is involved in different physiological processes and is associated with several diseases, including cancer and rheumatoid arthritis. A common method to detect citrullinated proteins relies on anti-modified citrulline antibodies directed to a specific chemical modification of the citrulline side chain. Here, we describe a versatile, antibody-independent method for the detection of citrullinated proteins on a membrane, based on the selective reaction of phenylglyoxal with the ureido group of citrulline under highly acidic conditions. The method makes use of 4-azidophenylglyoxal, which, after reaction with citrullinated proteins, can be visualized with alkyne-conjugated probes. The sensitivity of this procedure, using an alkyne-biotin probe, appeared to be comparable to the antibody-based detection method and independent of the sequence surrounding the citrulline.

  4. Insecticide Efficacy and Timing for Control of Western Bean Cutworm (Lepidoptera: Noctuidae) in Dry and Snap Beans.

    Science.gov (United States)

    Goudis, L A; Trueman, C L; Baute, T S; Hallett, R H; Gillard, C L

    2016-02-01

    The western bean cutworm, Striacosta albicosta (Smith) (Lepidoptera: Noctuidae), is a recent pest of corn, dry,and snap beans, in the Great Lakes region, and best practices for its management in beans need to be established.Insecticide efficacy and application timing field studies, conducted in 2011–2013, determined that lambda-cyhalothrin and chlorantraniliprole were capable of reducing western bean cutworm feeding damage in dry beans from 2.3 to 0.4% in preharvest samples, and in snap beans from 4.8 to 0.1% of marketable pods, respectively. The best application timing in dry beans was determined to be 4–18 d after 50% egg hatch. No economic benefit was found when products were applied to dry beans, and despite high artificial inoculation rates, damage to marketable yield was relatively low. Thiamethoxam, methoxyfenozide, and spinetoram were also found to be effective at reducing western bean cutworm damage in dry bean to as low as 0.3% compared to an untreated control with 2.5% damaged pods. In snap beans, increased return on investment between CAD$400 and CAD$600 was seen with multiple applications of lambda-cyhalothrin, and with chlorantraniliprole applied 4 d after egg mass infestation.

  5. Modifying a standard method allows simultaneous extraction of RNA and protein, enabling detection of enzymes in the rat retina with low expressions and protein levels.

    Science.gov (United States)

    Agardh, Elisabet; Gustavsson, Carin; Hagert, Per; Nilsson, Marie; Agardh, Carl-David

    2006-02-01

    The aim of the study was to evaluate messenger RNA and protein expression in limited amounts of tissue with low protein content. The Chomczynski method was used for simultaneous extraction of RNA, and protein was modified in the protein isolation step. Template mass and cycling time for the complementary DNA synthesis step of real-time reverse transcription-polymerase chain reaction (RT-PCR) for analysis of catalase, copper/zinc superoxide dismutase, manganese superoxide dismutase, the catalytic subunit of glutamylcysteine ligase, glutathione peroxidase 1, and the endogenous control cyclophilin B (CypB) were optimized before PCR. Polymerase chain reaction accuracy and efficacy were demonstrated by calculating the regression (R2) values of the separate amplification curves. Appropriate antibodies, blocking buffers, and running conditions were established for Western blot, and protein detection and multiplex assays with CypB were performed for each target. During the extraction procedure, the protein phase was dissolved in a modified washing buffer containing 0.1% sodium dodecyl sulfate, followed by ultrafiltration. Enzyme expression on real-time RT-PCR was accomplished with high reliability and reproducibility (R2, 0.990-0.999), and all enzymes except for glutathione peroxidase 1 were detectable in individual retinas on Western blot. Western blot multiplexing with CypB was possible for all targets. In conclusion, connecting gene expression directly to protein levels in the individual rat retina was possible by simultaneous extraction of RNA and protein. Real-time RT-PCR and Western blot allowed accurate detection of retinal protein expressions and levels.

  6. Western scrub-jays allocate longer observation time to more valuable information.

    Science.gov (United States)

    Watanabe, Arii; Grodzinski, Uri; Clayton, Nicola S

    2014-07-01

    When humans mentally reconstruct past events and imagine future scenarios, their subjective experience of mentally time travelling is accompanied by the awareness of doing so. Despite recent popularity of studying episodic memory in animals, such phenomenological consciousness has been extremely difficult to demonstrate without agreed behavioural markers of consciousness in non-linguistic subjects. We presented western scrub-jays (Aphelocoma californica) with a task requiring them to allocate observing time between two peepholes to see food being hidden in either of two compartments, one where observing the hiding location was necessary to later relocate the food, and another where food could easily be found without watching. Jays first separately experienced these consequences of possessing information in each compartment and subsequently, once given a choice, made more looks and spent more time looking into the compartment where information was necessary than into the compartment where it was unnecessary. Thus, the jays can collect information to solve a future problem. Moreover, they can differentiate sources of information according to their potential value and modify behaviour to efficiently collect important, usable information. This is the first evidence of metacognition in a species that passes the behavioural criteria for both retrospective and prospective mental time travel.

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

    DEFF Research Database (Denmark)

    Larsen, S; Rygaard, K; Asnaes, S

    1992-01-01

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

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

    Directory of Open Access Journals (Sweden)

    N.S. Sato

    2004-07-01

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

  9. Repeated and time-correlated morphological convergence in cave-dwelling harvestmen (Opiliones, Laniatores from Montane Western North America.

    Directory of Open Access Journals (Sweden)

    Shahan Derkarabetian

    Full Text Available BACKGROUND: Many cave-dwelling animal species display similar morphologies (troglomorphism that have evolved convergent within and among lineages under the similar selective pressures imposed by cave habitats. Here we study such ecomorphological evolution in cave-dwelling Sclerobuninae harvestmen (Opiliones from the western United States, providing general insights into morphological homoplasy, rates of morphological change, and the temporal context of cave evolution. METHODOLOGY/PRINCIPAL FINDINGS: We gathered DNA sequence data from three independent gene regions, and combined these data with Bayesian hypothesis testing, morphometrics analysis, study of penis morphology, and relaxed molecular clock analyses. Using multivariate morphometric analysis, we find that phylogenetically unrelated taxa have convergently evolved troglomorphism; alternative phylogenetic hypotheses involving less morphological convergence are not supported by Bayesian hypothesis testing. In one instance, this morphology is found in specimens from a high-elevation stony debris habitat, suggesting that troglomorphism can evolve in non-cave habitats. We discovered a strong positive relationship between troglomorphy index and relative divergence time, making it possible to predict taxon age from morphology. Most of our time estimates for the origin of highly-troglomorphic cave forms predate the Pleistocene. CONCLUSIONS/SIGNIFICANCE: While several regions in the eastern and central United States are well-known hotspots for cave evolution, few modern phylogenetic studies have addressed the evolution of cave-obligate species in the western United States. Our integrative studies reveal the recurrent evolution of troglomorphism in a perhaps unexpected geographic region, at surprisingly deep time depths, and in sometimes surprising habitats. Because some newly discovered troglomorphic populations represent undescribed species, our findings stress the need for further biological

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

    Science.gov (United States)

    Maccari, Francesca; Volpi, Nicola

    2002-09-01

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

  11. Non-linear modelling of monthly mean vorticity time changes: an application to the western Mediterranean

    Directory of Open Access Journals (Sweden)

    M. Finizio

    Full Text Available Starting from a number of observables in the form of time-series of meteorological elements in various areas of the northern hemisphere, a model capable of fitting past records and predicting monthly vorticity time changes in the western Mediterranean is implemented. A new powerful statistical methodology is introduced (MARS in order to capture the non-linear dynamics of time-series representing the available 40-year history of the hemispheric circulation. The developed model is tested on a suitable independent data set. An ensemble forecast exercise is also carried out to check model stability in reference to the uncertainty of input quantities.

    Key words. Meteorology and atmospheric dynamics · General circulation ocean-atmosphere interactions · Synoptic-scale meteorology

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

    Directory of Open Access Journals (Sweden)

    Gusti Ngurah Permana

    2014-08-01

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

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

    International Nuclear Information System (INIS)

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

    1985-01-01

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

  14. Introduction: Imams in Western Europe

    DEFF Research Database (Denmark)

    Vinding, Niels Valdemar; Hashas, Mohammed; de Ruiter, Jan Jaap

    2018-01-01

    by this source of religious authority in the context of the secular-liberal societies of Western Europe since the Second World War and the subsequent migration and refugee flows. At the same time, this research also serves to highlight secular-liberal institutions and their adaptation, or lack thereof......, to the multiculturalism that characterizes Western European states. The social facts of globalization, transnational migration, and various interpretations of secularism have challenged the visibility of religion in the public sphere in Western societies. This has most importantly and urgently required religious...

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

    Science.gov (United States)

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

    2013-04-01

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

  16. Novel Autoantibody Serum and Cerebrospinal Fluid Biomarkers in Veterans with Gulf War Illness

    Science.gov (United States)

    2017-10-01

    using Western blot and ELISA assays. PURPOSE: Development of peripheral biomarkers for GWI. Scope of the Research: Serum and plasma from 250 Gulf War...basic protein (MBP), Myelin Associated Glycoprotein (MAG), CaMKII, alpha-synuclein, GFAP, S100B, Western Blot, ELISA , chronic fatigue syndrome (CFS...Milestone(s) Achieved: Site 1, 4 and 5 serum and CSF data collected and set up for laboratory assays ( ELISA , western blot). Autoantibody data shipped

  17. Dietary isothiocyanate sulforaphene induces reactive oxygen ...

    African Journals Online (AJOL)

    intracellular oxygen species (ROS) measurement, mitochondrial membrane depolarization and western blot analysis were performed in four time-intervals to explore sulforaphene activity. ..... proteins were transferred to PVDF membranes.

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

    Science.gov (United States)

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

    2016-01-01

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

  19. Inmunofluorescencia indirecta como prueba alternativa para la confirmación diagnóstica de infección por VIH en el Perú

    Directory of Open Access Journals (Sweden)

    Ada Valverde

    1997-01-01

    Full Text Available Desde 1990 en el INS-Perú se viene utilizando la técnica de Western Blot (WB para la confirmación del Diagnóstico de VIH. En este estudio se evalúa la prueba de Inmunofluorescencia indirecta (IFI como una alternativa de confirmación al Western Blot. Se utilizaron 132 sueros de la seroteca de la División de Virología del INS-Perú con diagnóstico previo de VIH por WB. Para el diagnóstico de IFI se usó un Kit producido en el Centro Nacional de Referencia de SIDA(Chile-Argentina. De los 132 sueros procesados 56 (42,4% correspondieron a Western Blot positivo, 52 (39,4% a Western Blot negativos y 24 (18,2 con Western Blot indeterminado. La sensibilidad y especificidad de la técnica IFI en comparación con la de Western Blot fue de 98,2% y 98% respectivamente. Los valores predictivo positivo y negativo fueron 98,2% y 100% respectivamente. Estos resultados permiten incorporar a la técnica de IFI como una prueba alternativa para la confirmación del diagnóstico de infección por VIH.

  20. Comparison of the performance in detection of HPV infections between the high-risk HPV genotyping real time PCR and the PCR-reverse dot blot assays.

    Science.gov (United States)

    Zhang, Lahong; Dai, Yibei; Chen, Jiahuan; Hong, Liquan; Liu, Yuhua; Ke, Qiang; Chen, Yiwen; Cai, Chengsong; Liu, Xia; Chen, Zhaojun

    2018-01-01

    A new multiplex real-time PCR assay, the high-risk HPV genotyping real time PCR assay (HR HPV RT-PCR), has been developed to detect 15 high-risk HPV types with respective viral loads. In this report, a total of 684 cervical specimens from women diagnosed with vaginitis were assessed by the HR HPV RT-PCR and the PCR reaction and reverse dot blot (PCR-RDB) assays, using a PCR-sequencing method as a reference standard. A total coincidence of 97.7% between the HR HPV RT PCR and the PCR-RDB assays was determined with a Kappa value of 0.953. The HR HPV RT PCR assay had sensitivity, specificity, and concordance rates (accuracy) of 99.7%, 99.7%, and 99.7%, respectively, as confirmed by PCR-sequencing, while the PCR-RDB assay had respective rates of 98.8%, 97.1%, and 98.0%. The overall rate of HPV infection, determined by PCR-sequencing, in women diagnosed with vaginitis was 49.85%, including 36.26% of single infection and 13.6% of multiple infections. The most common infections among the 15 high-risk HPV types in women diagnosed with vaginitis were HPV-52, HPV-16, and HPV-58, with a total detection rate of 10.23%, 7.75%, and 5.85%, respectively. We conclude that the HR HPV RT PCR assay exhibits better clinical performance than the PCR-RDB assay, and is an ideal alternative method for HPV genotyping. In addition, the HR HPV RT PCR assay provides HPV DNA viral loads, and could serve as a quantitative marker in the diagnosis and treatment of single and multiple HPV infections. © 2017 Wiley Periodicals, Inc.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  2. Extent, causes and timing of moose calves mortality in western interior Alaska

    Science.gov (United States)

    Osbourne, Timothy O.; Paragi, Thomas F.; Bodkin, James L.; Loranger, Andre J.; Johnson, W.N.

    1991-01-01

    We examined the causes and timing of moose (Alces alces) calf mortality during 1988­ 90 in 2 locations in western interior Alaska. Annual survival rates of all calves in 1988 (0.34, n = 42) and 1989 (0.29, n =47) on the Nowitna National Wildlife Refuge (NWR) and in 1990 (0.25, n = 62) on the Koyukuk NWR were not significantly different. Survival rates of male and female calves were not significantly different during any year. Annual survival of single calves was significantly higher than that for twins in 1989 (0.56 vs. 0.20) and 1990 (0.37 vs. 0.21), but not in 1988. Black bears (Ursus americanus) killed 40% of all calves. Wolves (Canis lupus) killed 9% of the calves, unknown predators killed 8%, grizzly bears (Ursus arctos) killed 3%, and 5% died from other causes.

  3. Nuremberg Counting Jetons of XVI–XIX centuries: from Western Europe to Western Siberia

    Directory of Open Access Journals (Sweden)

    Andrey A. Pushkarev

    2015-12-01

    Full Text Available The article is devoted to the history of Western counting jetons, which have spread over a vast area from Western Europe to Siberia. The history of jetons is very dynamic. For more than three centuries in Western Europe tokens were used as a tool for calculation in trading shops, government offices, etc. In the second half of the XVI century because the spread of the written account the functions of jetons fundamentally changed. They are being used as chips in card games, they were presented as souvenirs, scattered among the crowd at weddings and festivals, etc. At the same time, jetons became the translation tool for public information about the government, in the form of images of the reigning monarch, as well as key political events. Through trade exchange jetons penetrate into the territory of Western Siberia, where their function changed again. All counting tokens from burial graves in Western Siberia, have holes for hanging or sewing, which indicate their use as ornaments. However, in the culture of the indigenous population decorations had not only aesthetic but also a sacred, religious meaning.

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

    Science.gov (United States)

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

    2013-06-01

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

  5. Utilization Pattern and Drug Use of Traditional Chinese Medicine, Western Medicine, and Integrated Chinese-Western Medicine Treatments for Allergic Rhinitis Under the National Health Insurance Program in Taiwan.

    Science.gov (United States)

    Huang, Sheng-Kang; Lai, Chih-Sung; Chang, Yuan-Shiun; Ho, Yu-Ling

    2016-10-01

    Patients in Taiwan with allergic rhinitis seek not only Western medicine treatment but also Traditional Chinese Medicine treatment or integrated Chinese-Western medicine treatment. Various studies have conducted pairwise comparison on Traditional Chinese Medicine, Western medicine, and integrated Chinese-Western medicine treatments. However, none conducted simultaneous analysis of the three treatments. This study analyzed patients with allergic rhinitis receiving the three treatments to identify differences in demographic characteristic and medical use and thereby to determine drug use patterns of different treatments. The National Health Insurance Research Database was the data source, and included patients were those diagnosed with allergic rhinitis (International Classification of Diseases, Ninth Revision, Clinical Modification codes 470-478). Chi-square test and Tukey studentized range (honest significant difference) test were conducted to investigate the differences among the three treatments. Visit frequency for allergic rhinitis treatment was higher in female than male patients, regardless of treatment with Traditional Chinese Medicine, Western medicine, or integrated Chinese-Western medicine. Persons aged 0-19 years ranked the highest in proportion of visits for allergic rhinitis. Traditional Chinese Medicine treatment had more medical items per person-time and daily drug cost per person-time and had the lowest total expenditure per person-time. In contrast, Western medicine had the lowest daily drug cost per person-time and the highest total expenditure per person-time. The total expenditure per person-time, daily drug cost per person-time, and medical items per person-time of integrated Chinese-Western medicine treatment lay between those seen with Traditional Chinese Medicine and Western medicine treatments. Although only 6.82 % of patients with allergic rhinitis chose integrated Chinese-Western medicine treatment, the visit frequency per person-year of

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

    Science.gov (United States)

    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.

  7. Expression of the tumor suppressor genes NF2, 4.1B, and TSLC1 in canine meningiomas.

    Science.gov (United States)

    Dickinson, P J; Surace, E I; Cambell, M; Higgins, R J; Leutenegger, C M; Bollen, A W; LeCouteur, R A; Gutmann, D H

    2009-09-01

    Meningiomas are common primary brain tumors in dogs; however, little is known about the molecular genetic mechanisms involved in their tumorigenesis. Several tumor suppressor genes have been implicated in meningioma pathogenesis in humans, including the neurofibromatosis 2 (NF2), protein 4.1B (4.1 B), and tumor suppressor in lung cancer-1 (TSLC1) genes. We investigated the expression of these tumor suppressor genes in a series of spontaneous canine meningiomas using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) (NF2; n = 25) and western blotting (NF2/merlin, 4.1B, TSLC1; n = 30). Decreased expression of 4.1B and TSLC1 expression on western blotting was seen in 6/30 (20%) and in 15/30 (50%) tumors, respectively, with 18/30 (60%) of meningiomas having decreased or absent expression of one or both proteins. NF2 gene expression assessed by western blotting and RT-PCR varied considerably between individual tumors. Complete loss of NF2 protein on western blotting was not seen, unlike 4.1B and TSLC1. Incidence of TSLC1 abnormalities was similar to that seen in human meningiomas, while perturbation of NF2 and 4.1B appeared to be less common than reported for human tumors. No association was observed between tumor grade, subtype, or location and tumor suppressor gene expression based on western blot or RT-PCR. These results suggest that loss of these tumor suppressor genes is a frequent occurrence in canine meningiomas and may be an early event in tumorigenesis in some cases. In addition, it is likely that other, as yet unidentified, genes play an important role in canine meningioma formation and growth.

  8. [The beginning of western medical education].

    Science.gov (United States)

    Kee, C D

    1992-01-01

    Our country had quite an advanced system of medical education during the era of the Koryo Kingdom, and during the Choson Dynasty, the Kyong Guk Dae Jon, in which a systematized medical education was clearly described, was compiled in the era of King Sejong. However, the educational system was not for Western medicine. Western medicine was first introduced to our country in the 9th year of King Injo (1631) when Chong Du Won, Yi Yong Jun, etc. returned from Yon Gyong (Beiuin) with Chik Bang Oe Gi. Knowledge of Western medicine was disseminated by Shil Hak (practical learning) scholars who read a translation in Chinese characters, of Chik Bang Oe Gi. Yi Ik (Song Ho), Yi Gyu Gyong (O ju), Choe Han Gi (Hye Gang), Chong Yak Yong (Ta San), etc., read books of Western medicine and introduced in writing the excellent theory of Western medicine. In addition, Yu Hyong Won (Pan Gye), Pak Ji Won (Yon Am), Pak Je Ga (Cho Jong), etc., showed much interest in Western medicine, but no writings by them about western medicine can be found. With the establishment of a treaty of amity with Japan in the 13th year of King Kojong (1876), followed by the succession of amity treaties with Western powers, foreigners including medical doctors were permitted to flow into this country. At that time, doctors Horace N. Allen, W. B. Scranton, John W. Heron, Rosetta Sherwood (Rosetta S. Hall), etc., came to Korea and inaugurated hospitals, where they taught Western medicine to Korean students. Dr. Horace N. Allen, with the permission of king Kojong, established Che Jung Won in April 1885, and in March 1886, he began at the hospital to provide education of Western medicine to Korean students who were recrutied by the Korean Government. However, the education was not conduted on a regular basis, only training them for work as assistants. This is considered to be the pioneer case of Western medical education in this country. Before that time, Japanese medical doctors came to Korea, but there are no

  9. Apatite fission track dating of the Northern Western Shield, Western Australia

    International Nuclear Information System (INIS)

    Weber, U.D.; Kohn, B.P.; Gleadow, A.J.W.

    1999-01-01

    Full text:The investigation of the thermotectonic evolution of the northern part of the Precambrian Western Shield of Western Australia using apatite fission track (AFT) thermochronology is the main focus of this study. The study area encompasses Precambrian rocks of the Pilbara Craton and the northern part of the Yilgarn Craton including the Narryer Gneiss Complex. AFT data, mostly from the Archaean cratons reveal cooling ages ranging between 260±8 Ma and 400±20 Ma. Mean confined horizontal track lengths fall between ∼12 and 13 μm with standard deviations ranging from 1.1-2.2 μm. Forward modelling of time-temperature history paths for representative samples reveals a period of regional cooling of at least ∼50 deg C in the late Palaeozoic. Most paths also show a second period of cooling of 25 deg C from temperatures of ∼80-85 deg C. This later cooling episode occurred in the Mesozoic but its timing is less well constrained. Assuming that the average present day geothermal gradient of ∼18±2 deg C per km - 1 was prevalent since the late Palaeozoic, then the minimum of ∼75 deg C of cooling predicted by the fission track modelling suggests overall denudation of at least ∼ 3.7-4.6 km of section since that time. Phanerozoic basins (Perth, Carnarvon and Canning) adjacent to the north and west of the northern Western Shield mostly continue offshore and form complex structures containing up to ∼ 15 km of predominantly clastic sediments of early Ordovician to late Cretaceous age. The basins are likely to have been depocentres for much of the detritus derived from the denudation inferred from the cooling recorded by the AFT. Possible causative events which could be linked to the observed late Palaeozoic cooling are tectonism related to the collision of Gondwanaland with Laurussia in Carboniferous time forming the supercontinent Pangea or a possible far-field effect related to the Alice Springs Orogeny. Further low temperature thermochronological studies

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

    International Nuclear Information System (INIS)

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

    2002-01-01

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

  11. Human transmissible spongiform encephalopathy: Case report

    Directory of Open Access Journals (Sweden)

    Duque Velásquez, Camilo

    2014-07-01

    Full Text Available We report the case of a 64 year-old woman with motor and cognitive deterioration that progressed rapidly during eight months. She was unsuccessfully treated with quinacrine, and died in a terminal status, by septic shock secondary to bronchopneumonia by broncho-aspiration. The brain was donated for research and the histopathological analysis showed spongiform changes, astrogliosis and prion protein (PrPRes deposits, confirmed by Western blot (WB. These features are considered characteristic of prion diseases, which are uncommon in Colombia. We highlight that its diagnosis was made for the first time in this country by the simultaneous use of immunohistochemistry and Western blot.

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

    International Nuclear Information System (INIS)

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

    1982-01-01

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

  13. GPCR Interaction: 59 [GRIPDB[Archive

    Lifescience Database Archive (English)

    Full Text Available required for their anti-HIV-1 activity. 11854425, 14716309, 10725362, 12089144 Point mutation, bioinforma...tics approach BRET Western blot Cross-linking in the presence of agonist, immunoprecipitation, western blot NP_000570.1 ...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1985-09-01

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

  15. Utility of Western Blot Analysis for the Diagnosis of Cutaneous Leishmaniasis

    Directory of Open Access Journals (Sweden)

    Marzieh ASHRAFMANSOURI

    2015-12-01

    Full Text Available Background: Cutaneous leishmaniasis (CL is a parasitic disease with a relatively wide distribution in different areas of the world, including Iran. The parasite is mainly diagnosed microscopically, but serological approaches might be useful for diagnosis as well.  This study aimed to assess the efficacy of an immunoblotting system for serodiagnosis of cutaneous leishmaniasis in Iran.Methods: Sixty-one sera samples from parasitologically confirmed CL patients and 50 sera samples from healthy controls along with 50 sera sample from non-CL patients were collected. Native strain of Leishmania major was cultured in Schnei­der medium and soluble Leishmania antigens were prepared from amastigotes-like parasites. All of sera samples were evaluated by an immunoblot­ting system.Results: Components of 14 to 135 kDa were detectable by the sera of CL pa­tients. From 61 sera of CL patients, 59 cases (96.7% detected a 63 kDa subunit and 51 cases (83.6% recognized a 32-35 kDa component. Among all subunits, the 63 kDa band showed the highest sensitivity (96.7% and a 75 kDa band had the highest (98% specificity.Conclusion: Immunoblotting has a satisfactory performance in diagnosis of CL and this test can be used, as an aid, for proper diagnosis of CL.

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

    Science.gov (United States)

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

    2016-06-14

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

  17. Indeterminate HIV-1 Western Blots: Etiology, Natural History, and Psychological Reactions

    Science.gov (United States)

    1992-09-16

    William Lafferty, Departments of Medicine. Laboratori’ Medicine. Epidenrioloý k. .nd Thomas S. Inui, Pamela H. Louie, Carol A. Gates, Biostatistics ...and dentistry . Finally, the psychosoclal onsequences of lWB are considerable.16 1655;LBISOTO2 JAB 13:33 03-17-92 P0003 U3 C9439 0021 11655 JGLM Nov

  18. Functional Characterization and Expression of Molluscan Detoxification Enzymes and Transporters Involved in Dietary Allelochemical Resistance

    Science.gov (United States)

    2008-06-01

    melanogasrer (43), and Homo sapiens (40) (Ding et al. 2003). To date, seven soluble cytosolic GST classes, encoding proteins of approximately 200 amino acids...Fasciola hepatica (P56598), Haliotis discus discus (ABF67506, ABF67507), Haemaphysalis longicornis (AAQ74441), Homo sapiens (NP_665683, AAV38750, NP_000840...MorciTa 2003 Mtilus Mdulis Western blot Jonsson ct al. 2004 CYP2H Mtilus go/lh,-ro/ii Western blot Peters el al. 19 9 0a (Y P2 Mvti/us ’dlis Western blot

  19. Reduction of the nitro group during sample preparation may cause underestimation of the nitration level in 3-nitrotyrosine immunoblotting

    DEFF Research Database (Denmark)

    Söderling, Ann-Sofi; Hultman, Lena; Delbro, Dick

    2007-01-01

    We noted differences in the antibody response to 3-nitrotyrosine (NO(2)Tyr) in fixed and non-fixed tissues, and studied therefore potential problems associated with non-fixed tissues in Western blot analyses. Three different monoclonal anti-nitrotyrosine antibodies in Western blot analysis of inf...... is not detected by anti-NO(2)Tyr antibodies. Western blot analysis may therefore underestimate the level of tissue nitration, and factors causing a reduction of NO(2)Tyr during sample preparation might conceal the actual nitration of proteins....

  20. Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases.

    Science.gov (United States)

    Jadwin, Joshua A; Oh, Dongmyung; Curran, Timothy G; Ogiue-Ikeda, Mari; Jia, Lin; White, Forest M; Machida, Kazuya; Yu, Ji; Mayer, Bruce J

    2016-04-12

    While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding.

  1. Molecular studies of BKCa channels in intracranial arteries: presence and localization

    DEFF Research Database (Denmark)

    Johansson, Helle Wulf; Hay-Schmidt, Anders; Poulsen, Asser Nyander

    2008-01-01

    of the BK(Ca) channel in rat basilar, middle cerebral, and middle meningeal arteries by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, and Western blotting. Distribution patterns were investigated using in situ hybridization and immunofluorescence studies. RT......Large conductance calcium-activated potassium channels (BK(ca)) are crucial for the regulation of cerebral vascular basal tone and might be involved in cerebral vasodilation relevant to migraine and stroke. We studied the differential gene expression of mRNA transcript levels and protein expression......-PCR and quantitative real-time PCR detected the expression of the BK(Ca) channel mRNA transcript in rat basilar, middle cerebral, and middle meningeal arteries, with the transcript being expressed more abundantly in rat basilar arteries than in middle cerebral and middle meningeal arteries. Western blotting detected...

  2. Molecular studies of BKCa channels in intracranial arteries

    DEFF Research Database (Denmark)

    Wulf, Helle; Hay-Schmidt, Anders; Poulsen, Asser Nyander

    2008-01-01

    expression of the BK(Ca) channel in rat basilar, middle cerebral, and middle meningeal arteries by reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, and Western blotting. Distribution patterns were investigated using in situ hybridization and immunofluorescence studies. RT......  Large conductance calcium-activated potassium channels (BK(ca)) are crucial for the regulation of cerebral vascular basal tone and might be involved in cerebral vasodilation relevant to migraine and stroke. We studied the differential gene expression of mRNA transcript levels and protein......-PCR and quantitative real-time PCR detected the expression of the BK(Ca) channel mRNA transcript in rat basilar, middle cerebral, and middle meningeal arteries, with the transcript being expressed more abundantly in rat basilar arteries than in middle cerebral and middle meningeal arteries. Western blotting detected...

  3. [Effect of down-regulation of HE4 gene expression on biologic behavior of ovarian cancer cells].

    Science.gov (United States)

    Zhou, Lei; Xiao, Ran; Chen, Ying; Zhang, Jing; Lu, Ren-quan; Guo, Lin

    2013-10-01

    To investigate the effects of HE4 gene knockdown on the proliferation, adhesion and invasion of the ovarian cancer cells SKOV3. The knockdown of HE4 gene was performed by RNAi technology. The recombinant plasmids (pSUPER-HE4 shDNAs) were constructed and transfected into human ovarian cancer cells SKOV3. HE4 expression was then identified by real-time PCR and Western blot analysis. The invasion and adhesion ability of transduced cells were determined. In addition, cell proliferation and growth were analyzed by colonies formation assay. Knockdown of HE4 was achieved, and further confirmed by real-time PCR and Western blot. The proliferation of HE4-down-regulated cells was not affected, but the invasion ability of the transfected cells was reduced (P cells.

  4. Effect of corilagin on cerebral ischaemia/reperfusioninduced ...

    African Journals Online (AJOL)

    At the end of the treatments, neurological deficit, brain oedema, and volume of ... tissues was evaluated by immunofluorescence, real-time PCR, and western blotting. Results: There was a significant decrease in neurological deficit, brain ...

  5. Up-regulation of ALG-2 in hepatomas and lung cancer tissue

    DEFF Research Database (Denmark)

    la Cour, Jonas Marstrand; Mollerup, Jens; Winding, Pernille

    2003-01-01

    , a result confirmed by immunohistochemical analysis. Staining of four different lung cancer tissue microarrays including specimens of 263 patients showed that ALG-2 is mainly localized to epithelial cells and significantly up-regulated in small-cell lung cancers and in non-small-cell lung cancers. Our...... using Western blot analysis and immunohistochemistry. Western blot analysis of 15 different adult mouse tissues demonstrated that ALG-2 is ubiquitously expressed. We found that ALG-2 was more than threefold overexpressed in rat liver hepatoma compared to normal rat liver using Western blot analysis...

  6. Molecular analysis of mutant and wild type alcohol dehydrogenase alleles from Drosophila

    International Nuclear Information System (INIS)

    Batzer, M.A.

    1988-01-01

    Wild type alcohol dehydrogenase polypeptides (ADH) from Drosophila melanogaster transformants were examined using western blots and polyclonal antiserum specific for Drosophila melanogaster ADH. Mutants induced in Drosophila spermatozoa at the alcohol dehydrogenase (Adh) locus using X-rays, 1-ethyl-1-nitrosourea (ENU) or ethyl methanesulfonate (EMS) were characterized using genetic complementation tests, western blots, Southern blots, northern blots and enzymatic amplification of the Adh locus. Genetic complementation tests showed that 22/30 X-ray-induced mutants, and 3/13 ENU and EMS induced mutants were multi-locus deficiencies. Western blot analysis of the intragenic mutations showed that 4/7 X-ray-induced mutants produced detectable polypeptides, one of which was normal in molecular weight and charge. In contrast 8/10 intragenic ENU and EMS induced mutants produced normal polypeptides. Southern blot analysis showed that 5/7 intragenic X-ray induced mutants and all 10 of the intragenic ENU and EMS induced mutants were normal with respect to the alleles they were derived from

  7. 2012 Annual Report [WESTERN AREA POWER ADMINISTRATION

    Energy Technology Data Exchange (ETDEWEB)

    None

    2012-01-01

    Fiscal Year 2012 brought some tumultuous and uncertain times to Western. The utility industry and technology continued to evolve, and the demand for constant flow of power and transmission system reliability continued to increase. Western kept pace by continuing to deliver reliable, cost-based hydropower while reviewing and updating business practices that took into account how the energy industry is evolving. During this time of exponential change, Western tackled many challenges, including: Reviewing the Transmission Infrastructure Program processes and procedures; Responding to Secretary of Energy Steven Chu’s memorandum to create a modern, efficient and reliable transmission grid; Weathering record-breaking natural disasters in our service territory; Completing our role in TIP’s flagship project—the Montana Alberta Tie Ltd. transmission line; Incorporating new, far-reaching regulations and industry trends.

  8. Western Sufism

    DEFF Research Database (Denmark)

    Sedgwick, Mark

    Western Sufism is sometimes dismissed as a relatively recent "new age" phenomenon, but in this book, Mark Sedgwick argues that it actually has very deep roots, both in the Muslim world and in the West. In fact, although the first significant Western Sufi organization was not established until 1915......, the first Western discussion of Sufism was printed in 1480, and Western interest in some of the ideas that are central to Sufi thought goes back to the thirteenth century. Sedgwick starts with the earliest origins of Western Sufism in late antique Neoplatonism and early Arab philosophy, and traces later......, the year in which the first Western Sufi order based not on the heritage of the European Middle Ages, Renaissance and Enlightenment, but rather on purely Islamic models, was founded. Later developments in this and other orders are also covered. Western Sufism shows the influence of these origins...

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

    Science.gov (United States)

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

    2017-07-01

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

  10. Time- and cell-type specific changes in iron, ferritin, and transferrin in the gerbil hippocampal CA1 region after transient forebrain ischemia

    Directory of Open Access Journals (Sweden)

    Dae Young Yoo

    2016-01-01

    Full Text Available In the present study, we used immunohistochemistry and western blot analysis to examine changes in the levels and cellular localization of iron, heavy chain ferritin (ferritin-H, and transferrin in the gerbil hippocampal CA1 region from 30 minutes to 7 days following transient forebrain ischemia. Relative to sham controls, iron reactivity increased significantly in the stratum pyramidale and stratum oriens at 12 hours following ischemic insult, transiently decreased at 1-2 days and then increased once again within the CA1 region at 4-7 days after ischemia. One day after ischemia, ferritin-H immunoreactivity increased significantly in the stratum pyramidale and decreased at 2 days. At 4-7 days after ischemia, ferritin-H immunoreactivity in the glial components in the CA1 region was significantly increased. Transferrin immunoreactivity was increased significantly in the stratum pyramidale at 12 hours, peaked at 1 day, and then decreased significantly at 2 days after ischemia. Seven days after ischemia, Transferrin immunoreactivity in the glial cells of the stratum oriens and radiatum was significantly increased. Western blot analyses supported these results, demonstrating that compared to sham controls, ferritin H and transferrin protein levels in hippocampal homogenates significantly increased at 1 day after ischemia, peaked at 4 days and then decreased. These results suggest that iron overload-induced oxidative stress is most prominent at 12 hours after ischemia in the stratum pyramidale, suggesting that this time window may be the optimal period for therapeutic intervention to protect neurons from ischemia-induced death.

  11. Growth factors regulate glutamine synthetase activity in ...

    African Journals Online (AJOL)

    Khaled

    2012-07-10

    Jul 10, 2012 ... glutamate and ammonia, which in turn, cells are supplied with ammonia ... out to determine the maximum growth time at which cells will be .... Western blot technique for detection the glutamine synthetase enzyme. Lane 1;.

  12. Oral and Fecal Campylobacter concisus Strains induce Barrier dysfunction by Apoptosis in HT-29/B6 Intestinal Epithelial Cells

    DEFF Research Database (Denmark)

    Nielsen, Hans Linde; Nielsen, Henrik Ib; Ejlertsen, Tove

    in Ussing chambers. Tight junction (TJ) protein expression was determined by Western blotting, and subcellular TJ distribution was analyzed by confocal laser-scanning microscopy. Apoptosis induction was examined by TUNEL-staining and Western blot of caspase-3 activation. All strains invaded confluent HT-29...

  13. Appraising timing response of paleoenvironmental proxies to the Bond cycle in the western Mediterranean over the last 20 kyr

    Science.gov (United States)

    Rodrigo-Gámiz, Marta; Martínez-Ruiz, Francisca; Rodríguez-Tovar, Francisco J.; Pardo-Igúzquiza, Eulogio; Ortega-Huertas, Miguel

    2018-04-01

    The timing of climate responses to the Bond cycle is investigated in the western Mediterranean. Periodicities had been previously reported in a marine sediment record from this region spanning the last 20 kyr, and registered by diverse paleoenvironmental proxies, in particular those associated with terrigenous input, redox conditions, productivity, sea surface temperature (SST) and salinity. Further cross-spectral analyses on these time series reveal leads-lags in the 1400 year climate cycle. Considering as reference a terrigenous input proxy (the K/Al ratio), all the paleoenvironmental proxies displayed time shifts varying from ca. 700 year to ca. 350 year. SST and salinity variations show a first leaded response with the inflow of cold and less salty Atlantic waters. Followed by a time lead of 525 year, progresively arid conditions with an increase of eolian dust transport to the area, given by the Zr/Al signal, are observed. The intensification of dust transport could have triggered a latest biological response, lead by 350 year, with an increase of productivity, as suggested by the Ba/Al ratio. Lastly changes in the Mediterranean thermohaline circulation, indicated by a selected redox proxy (the U/Th ratio), are observed. These results support that the oceanic response triggered the atmospheric response to the Bond cycle in the western Mediterranean. Changes in the North Atlantic Oscillation mode and in the Inter-Tropical Convergence Zone migrations with variations in the monsoon activity or Saharan winds system, are considered as main forcing mechanisms, with a complex relationship of the involved phenomena.

  14. Radiation dose determines the method for quantification of DNA double strand breaks

    International Nuclear Information System (INIS)

    Bulat, Tanja; Keta, Olitija; Korićanac, Lela; Žakula, Jelena; Petrović, Ivan; Ristić-Fira, Aleksandra; Todorović, Danijela

    2016-01-01

    Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci. (author)

  15. Radiation dose determines the method for quantification of DNA double strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Bulat, Tanja; Keta, Olitija; Korićanac, Lela; Žakula, Jelena; Petrović, Ivan; Ristić-Fira, Aleksandra [University of Belgrade, Vinča Institute of Nuclear Sciences, Belgrade (Serbia); Todorović, Danijela, E-mail: dtodorovic@medf.kg.ac.rs [University of Kragujevac, Faculty of Medical Sciences, Kragujevac (Serbia)

    2016-03-15

    Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci. (author)

  16. Time-related variation of volatile contents of Western Ghats volcanic formations, Deccan, India

    Science.gov (United States)

    Marzoli, Andrea; Callegaro, Sara; Baker, Don R.; De Min, Angelo; Renne, Paul R.

    2016-04-01

    Deccan volcanism in India covered more than 1 million square km and reached a maximum thickness of about 3 km, as presently preserved in the Western Ghats volcanic lava piles. Volcanic activity started at about 66.4 Ma (Jawhar formation) and ended at about 65.5 Ma (Mahabaleshwar unit; Renne et al., 2015). Deccan volcanism straddled the Cretaceous-Paleogene boundary (ca. 66.0 Ma) and possibly contributed to the end-Cretaceous mass extinction event through emission of gases such as SO2, CO2, Cl, F that may have triggered global climate changes. Severe pollution by volcanic gases is supported by the high S and Cl contents (up to 1400 and up to 900 ppm, respectively; Self et al., 2008) measured in a few olivine- and plagioclase-hosted melt inclusions from the Jawhar, Neral, and Thakurvadi Formations (early lava flows, ca. 66.3-66.4 ± 0.1 Ma; Renne et al., 2015) and by magmatic S contents (up to 1800 ppm; Callegaro et al., 2014) calculated from S measurements in clinopyroxenes from the Mahabaleshwar unit (ca. 65.5 ± 0.1; Schoene et al., 2015). Here, we present new analyses of S, Cl, and F, obtained by ion-probe and synchrotron light micro-fluorescence analyses on clinopyroxenes and plagioclase phenocrysts from ?al? lava flow units of the Western Ghats. The volatile contents of the host magmas have been calculated from recently published clinopyroxene/basalt partition coefficients. These new data will describe the time-related variation of volatile elements hosted and eventually emitted by Deccan lavas and shed light on their environmental impact. References: Callegaro S. et al. (2014). Geology 42, 895-898. Renne P.R. et al. (2015). Science 350, 76-78. Schoene B. et al. (2015). Science 347, 192-184. Self S. et al. (2008). Science 319, 1654-1657.

  17. Philosophy and Ethics in Western Australian Secondary Schools

    Science.gov (United States)

    Millett, Stephan; Tapper, Alan

    2014-01-01

    The introduction of Philosophy and Ethics to the Western Australian Certificate of Education courses in 2008 brought philosophy into the Western Australian secondary school curriculum for the first time. How philosophy came to be included is part of a larger story about the commitment and perseverance of a relatively small number of Australian…

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

    DEFF Research Database (Denmark)

    Bundgaard, Karen Marie

    2005-01-01

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

  19. [Expression of DKKL1 in spermatozoa of men with asthenospermia].

    Science.gov (United States)

    Yan, Qiu-Xia; Ma, Yi; Chen, Run-Qiang; Zhou, Xiu-Qin; Qiao, Jing; Xian, Ying-Jie; Feng, Ling; Chen, Cai-Rong

    2018-03-20

    To compare the expression of DKKL1 in ejaculated spermatozoa of normal fertile men and men with asthenospermia and investigate the role of DKKL1 in the pathogenesis of asthenospermia. The characteristics of semen samples collected from normal fertile men and men with asthenospermia were analyzed using computer-assisted sperm analysis according to WHO criteria. The ejaculated sperms were isolated by Percoll discontinuous density gradients to detect the expression of DKKL1 mRNA and protein using real-time PCR and Western blotting. The expression of DKKL1 mRNA was significantly down-regulated by 11.1 times in asthenospermic men as compared with that in normal fertile men (P<0.01). Western blotting showed that the expression of DKKL1 protein was down-regulated by 2.4 times in asthenospermic men compared to normal fertile men. The expression of DKKL1, which may play an important role in sperm motility,is significantly decreased in ejaculated spermatozoa of men with asthenospermia.

  20. Westerns fra hele verden

    DEFF Research Database (Denmark)

    Bjerre, Thomas Ærvold

    2014-01-01

    Om den amerikanske western, spaghettiwesterns, kommunistiske westerns og danske westerns - i forbindelse med Kristian Levrings The Salvation (2014).......Om den amerikanske western, spaghettiwesterns, kommunistiske westerns og danske westerns - i forbindelse med Kristian Levrings The Salvation (2014)....

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

    NARCIS (Netherlands)

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

    1996-01-01

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

  2. Cross-Sectional Serological Survey of Human Fascioliasis in Canutama Municipality in Western Amazon, Brazil

    Directory of Open Access Journals (Sweden)

    Marcel Gonçalves Maciel

    2018-01-01

    Full Text Available 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.

  3. Production of hybrids between western gray wolves and western coyotes.

    Directory of Open Access Journals (Sweden)

    L David Mech

    Full Text Available 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.

  4. [Analytic study of dot blotting for the detection of anti-Jo-1, anti-M2, anti-ribosomes and anti-LKM].

    Science.gov (United States)

    Huguet, S; Sghiri, R; Ballot, E; Johanet, C

    2004-01-01

    The Cyto-Dot 4 HM043 kit commercialised by BMD, has replaced the Cyto-Dot HM010 kit that allowed three auto-antibodies detection (anti-Jo-1, anti-M2 and anti-ribosomal protein). Detection of anti-LKM1 auto-antibody was added. These four auto-antibodies have in common only the intracytoplasmic localisation of their respective antigen. The aim of our study was to evaluate this new kit using 104 sera and to compare our results with reference techniques (indirect immunofluorescence IF for anti-M2, anti-ribosomal protein and anti-LKM1, double immunodiffusion ID for anti-Jo-1 and anti-LKM1, western blotting WB for anti-M2) and with Cyto-Dot HM010. The one hundred and four sera were divided into five groups: Group I (n = 12) with anti-Jo-1 detected by ID; Group II (n = 28) with 26 anti-M2 positive by IF and WB, 2 anti-M2 positive only by WB; Group III (n = 10) with anti-ribosomal protein detected by IF 5 of which precipitated by ID; Group IV (n = 32) with anti-LKM1 by IF and ID divided into 18 AIH2 and 14 HCV; Group V (n = 22) consisting of 14 healthy individuals and 8 patients with hypergammaglobulinemia. Results of this study are similar to those of Cyto-Dot HM010 for the three auto-antibodies already in use. Cyto-Dot 4 is a very good anti-LKM1 confirmation method as it is ID. Copyright John Libbey Eurotext 2003.

  5. Cross-sectional study of serum antibodies against Sarcocystis neurona in cats tested for antibodies against Toxoplasma gondii.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2005-04-01

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

  7. Secular trends in Cherokee cranial morphology: Eastern vs Western bands.

    Science.gov (United States)

    Sutphin, Rebecca; Ross, Ann H; Jantz, Richard L

    2014-01-01

    The research objective was to examine if secular trends can be identified for cranial data commissioned by Boas in 1892, specifically for cranial breadth and cranial length of the Eastern and Western band Cherokee who experienced environmental hardships. Multiple regression analysis was used to test the degree of relationship between each of the cranial measures: cranial length, cranial breadth and cephalic index, along with predictor variables (year-of-birth, location, sex, admixture); the model revealed a significant difference for all craniometric variables. Additional regression analysis was performed with smoothing Loess plots to observe cranial length and cranial breadth change over time (year-of-birth) separately for Eastern and Western Cherokee band females and males born between 1783-1874. This revealed the Western and Eastern bands show a decrease in cranial length over time. Eastern band individuals maintain a relatively constant head breadth, while Western Band individuals show a sharp decline beginning around 1860. These findings support negative secular trend occurring for both Cherokee bands where the environment made a detrimental impact; this is especially marked with the Western Cherokee band.

  8. Anti-Taenia solium metacestode IgG antibodies in serum samples from inhabitants of a central-western region of Brazil

    Directory of Open Access Journals (Sweden)

    Oliveira Heliana B. de

    2006-01-01

    Full Text Available A total of 354 serum samples from inhabitants who frequent the Clinical Laboratory in Catalão, Goiás, in the central-western region of Brazil, were collected from June to August, 2002. The samples were evaluated by indirect immunofluorescence antibody tests and an enzyme linked immunosorbent assay in order to detect anti-Taenia solium metacestode IgG antibodies. Reactive and inconclusive samples were tested by Western blotting (WB. Considering WB as a confirmation, the frequency of antibodies in the serum samples of the above population was 11.3% (CI 5.09 - 17.51. The immunodominant bands most frequently recognized in WB were 64-68 kDa (97.5% and 47-52 kDa (80%. The percentage of seropositivity to cysticercosis was significantly higher for individuals residing in areas without sewage systems (p < 0.0001. In conclusion, the results indicate a probable endemic situation of cysticercosis in this population. These results reinforce the urgent need for control and prevention measures to be taken by the local public health services.

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

    NARCIS (Netherlands)

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

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

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

    Science.gov (United States)

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

    2010-10-01

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

  11. [The transition of acupuncture and moxibustion in Japan in modern times after western medicine spreading to the East].

    Science.gov (United States)

    Li, Su-Yun

    2014-04-01

    The research methods, such as philology of medicine history and comparison between tradition and modern and so on were adopted in this article to study the acupuncture-moxibustion development after western medicine spreading to the East in Japan and its main transition under the impact of western medicine. The results showed that from Meiji to Showa period, under the influence of western medicine, the transition of Japanese acupuncture-moxibustion mainly embodied in following three aspects, incuinng acupuncture works absorbing western medicine knowledge, applying experiment measures to explore acupuncture principle and launching acupuncture teaching in accordance with Europe and America academy educational pattern. The changes on acupuncture works, teaching materials and methods of researching and teaching have triggered the transition and transformation of Japanese acupuncture-moxibustion from tradition to modern.

  12. miR-497 suppresses epithelial–mesenchymal transition and metastasis in colorectal cancer cells by targeting fos-related antigen-1

    Directory of Open Access Journals (Sweden)

    Zhang N

    2016-10-01

    Full Text Available Nan Zhang,1 Quan Shen,2 Pingping Zhang3 1Department of General Surgery, First Affiliated Hospital of Henan University of Chinese Medicine, 2Department of Hepatobiliary Surgery, Henan Provincial People’s Hospital, Zhengzhou, 3Department of Integrated Chinese and Western Medicine, Tianjin First Central Hospital, Tianjin, People’s Republic of China Objective: MicroRNAs have key roles in tumor metastasis. The acquisition of metastatic capability by cancer cells is associated with epithelial–mesenchymal transition (EMT. Here, we describe the role and molecular mechanism of miR-497 in colorectal cancer (CRC cell EMT, migration, and invasion.Methods: Quantitative real-time polymerase chain reaction and Western blot assays were performed to detect the expression levels of miR-497 and Fos-related antigen-1 (Fra-1 in the CRC cells. HCT116 and SW480 cells with miR-497 overexpression or Fra-1 low expression were constructed by lipofection. Target prediction and luciferase reporter assays were performed to investigate whether Fra-1 is one of the targets of miR-497. Western blot and Transwell assays were performed to detect the effects of miR-497 and Fra-1 on CRC cell EMT, migration and invasion.Results: We searched the miRanda, TargetScan, and PicTar databases and found that Fra-1, a key driver of CRC metastasis, is a potential target of miR-497. Quantitative real-time polymerase chain reaction and Western blot analysis verified downregulation of miR-497 and upregulation of Fra-1 in CRC cells. Western blot and Transwell assays showed that overexpression of miR-497 suppresses CRC cell EMT, migration, and invasion. Luciferase gene reporter assay revealed that Fra-1 is a downstream target of miR-497 as miR-497 bound directly to the 3' untranslated region of Fra-1 messenger RNA. An inverse correlation was also found between miR-497 and Fra-1 in HCT116 and SW480 cells. Furthermore, knockdown of Fra-1 recuperated the effects of miR-497 overexpression

  13. A 15-year-long Southern blotting analysis of FMR1 to detect female carriers and for prenatal diagnosis of fragile X syndrome in Taiwan.

    Science.gov (United States)

    Tzeng, C-C; Tsai, L-P; Chang, Y-K; Hung, Y-J; Chang, Y-Y; Su, Y-P; Jiang, J-J; Liang, H-M

    2017-08-01

    Here, we review the results of Southern blotting analyses of the FMR1 gene performed in our reference laboratory in Taiwan over a 15-year period. In total, 725 high-risk women with a family history of fragile X syndrome (FXS) or idiopathic intellectual disability, 3911 low-risk pregnant women without such family history, and prenatal diagnosis data for 32 foetuses from 24 carrier mothers were included. Only 2 carriers were in the low-risk group, which indicated a prevalence of 1 of 1955 women (95% confidence interval: 1/7156-1/539). A total of 100 carriers were found to be in the high-risk group, thus revealing a significantly higher frequency than the low-risk group (100/725 vs 2/3911, P<0.0001). Eight of the 14 foetuses that inherited the maternal mutant allele were verified to have a full mutation, with the smallest maternal pre-mutation allele carrying 56 CGG repeats. The overall findings confirmed that the carrier prevalence among low-risk women in Taiwan is significantly lower than that reported in western countries. Therefore, the most important step for preventing FXS in Taiwan would be to focus on high-risk women by promoting general awareness of this disease and spreading knowledge regarding the benefits of carrier screening and prenatal testing. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. New Nordic Diet induced weight loss is accompanied by changes in metabolism and AMPK signalling in adipose tissue

    DEFF Research Database (Denmark)

    Fritzen, Andreas Mæchel; Lundsgaard, Annemarie; Jordy, Andreas Børsting

    2015-01-01

    adipose tissue (SCAT) were obtained at week 0 and 26. OUTCOME: Gene and protein expressions were analysed by real time PCR and western blotting. RESULTS: Improved HOMA-IR index and lowered plasma triacylglycerol concentration after NND coincided with molecular adaptations in SCAT, but not in skeletal...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-08-26

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

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

    International Nuclear Information System (INIS)

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

    2016-01-01

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

  17. Production and characterization of polyclonal antibody against a synthetic peptide from β-actin protein

    Directory of Open Access Journals (Sweden)

    Nazila Amini

    2014-06-01

    Full Text Available Objective(s:Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. Materials and Methods: A synthetic peptide derived from β-actin protein was designed and conjugated to Keyhole limpet hemocyanin (KLH (and used to immunize a white New Zealand rabbit. The antibody was purified from serum by affinity chromatography column. The purity of the antibody was determined by SDS-PAGE and its ability to recognize the immunizing peptide was measured by ELISA. The reactivity of the antibody with β-actin protein in a panel of different cell lysates was then evaluated by western blot. In addition, the reactivity of the antibody with the corresponding protein was also evaluated by Immunocytochemistry and Immunohistochemistry in different samples. Results: The antibody could recognize the immunizing peptide in ELISA. It could also recognize            β-actin protein in western blot as well as in immunocytochemistry and immunohistochemistry. Conclusion: Our data suggest that this antibody may be used as an internal control in western blot analyses as well as in other immunological applications such as ELISA,immunocytochemistry and immunohistochemistry.

  18. Expression of Recombinant Human Coagulation Factor VII by the Lizard Leishmania Expression System

    Directory of Open Access Journals (Sweden)

    Sina Mirzaahmadi

    2011-01-01

    Full Text Available The variety of recombinant protein expression systems have been developed as a resource of FVII gene expression. In the current study, the authors used a novel protein expression system based on the Iranian Lizard Leishmania, a trypanosomatid protozoan as a host for expression of FVII. Plasmid containing cDNA encoding full-length human FVII was introduced into Lizard Leishmania and positive transfectants were analyzed by SDS-PAGE and Western blot analysis. Furthermore, biological activity of purified protein was detected by PT assay. The recombinant strain harboring a construct was analyzed for expression of FVII at the mRNA and protein level. Purified rFVII was obtained and in order to confirm the purified compound was in fact rFVII. Western blot analysis was carried out. Clotting time in PT assay was reduced about 30 seconds with the purified rFVII. In Conclusion, this study has demonstrated, for the first time, that Leishmania cells can be used as an expression system for producing recombinant FVII.

  19. ET-1 Promotes Differentiation of Periodontal Ligament Stem Cells into Osteoblasts through ETR, MAPK, and Wnt/β-Catenin Signaling Pathways under Inflammatory Microenvironment

    Science.gov (United States)

    Liang, Li; Zhou, Wei; Yang, Nan; Yu, Jifeng; Liu, Hongchen

    2016-01-01

    Periodontitis is a kind of chronic inflammatory disease that affects the tooth-supporting tissues. ET-1 is related to periodontitis and involved in the regulation of cytokines, but the mechanisms remain unclear. The aim of this study is to investigate how ET-1 affects proinflammatory cytokine expression and differentiation in human periodontal ligament stem cells (PDLSCs). PDLSCs were isolated from the periodontal ligament tissues of periodontitis patients and then treated with ET-1 (1, 10, or 100 nM) for 12 h, 24 h, or 72 h. The osteogenic potential of PDLSCs was tested using ALP staining. TNF-α, IL-1β, and IL-6 levels were evaluated by ELISA and western blot. Runx2, OCN, and COL1 mRNA and western levels were detected by RT-PCR and western blot, respectively. To examine the signaling pathways and molecular mechanisms involved in ET-1-mediated cytokine expression and osteogenic differentiation, ETR pathway, MAPKs pathway, Wnt/β-catenin pathway, and Wnt/Ca2+ pathway were detected by RT-PCR and western blot, respectively. ET-1 promoted differentiation of PDLSCs into osteoblasts by increasing secretion of TNF-α, IL-1β, and IL-6 in a dose- and time-dependent manner. ET-1 also increased expression of Runx2, OCN, and COL1. ET-1 promotes differentiation of PDLSCs into osteoblasts through ETR, MAPK, and Wnt/β-catenin signaling pathways under inflammatory microenvironment. PMID:26884650

  20. ET-1 Promotes Differentiation of Periodontal Ligament Stem Cells into Osteoblasts through ETR, MAPK, and Wnt/β-Catenin Signaling Pathways under Inflammatory Microenvironment

    Directory of Open Access Journals (Sweden)

    Li Liang

    2016-01-01

    Full Text Available Periodontitis is a kind of chronic inflammatory disease that affects the tooth-supporting tissues. ET-1 is related to periodontitis and involved in the regulation of cytokines, but the mechanisms remain unclear. The aim of this study is to investigate how ET-1 affects proinflammatory cytokine expression and differentiation in human periodontal ligament stem cells (PDLSCs. PDLSCs were isolated from the periodontal ligament tissues of periodontitis patients and then treated with ET-1 (1, 10, or 100 nM for 12 h, 24 h, or 72 h. The osteogenic potential of PDLSCs was tested using ALP staining. TNF-α, IL-1β, and IL-6 levels were evaluated by ELISA and western blot. Runx2, OCN, and COL1 mRNA and western levels were detected by RT-PCR and western blot, respectively. To examine the signaling pathways and molecular mechanisms involved in ET-1-mediated cytokine expression and osteogenic differentiation, ETR pathway, MAPKs pathway, Wnt/β-catenin pathway, and Wnt/Ca2+ pathway were detected by RT-PCR and western blot, respectively. ET-1 promoted differentiation of PDLSCs into osteoblasts by increasing secretion of TNF-α, IL-1β, and IL-6 in a dose- and time-dependent manner. ET-1 also increased expression of Runx2, OCN, and COL1. ET-1 promotes differentiation of PDLSCs into osteoblasts through ETR, MAPK, and Wnt/β-catenin signaling pathways under inflammatory microenvironment.

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

    International Nuclear Information System (INIS)

    Maria Lina R; Budiman Bela; Andi Yasmon

    2009-01-01

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

  2. Dietary intake, leisure time activities and obesity among adolescents in Western Sweden: a cross-sectional study.

    Science.gov (United States)

    Winkvist, Anna; Hultén, Bodil; Kim, Jeong-Lim; Johansson, Ingegerd; Torén, Kjell; Brisman, Jonas; Bertéus Forslund, Heléne

    2016-04-21

    Overweight and obesity among adolescents are increasing worldwide. Risk factors include dietary intake characteristics and high levels of physical inactivity. In Sweden, few large comprehensive population-based surveys of dietary intake and lifestyle among adolescents have been carried out. Thus, the purpose of the current study was to describe dietary intake and food choices as well as leisure time activities in relation to overweight and obesity in a total sample of all schoolchildren aged 15 years in Western Sweden. In 2008, a questionnaire was sent to all 21,651 adolescents born in 1992 in Västra Götaland Region, Sweden. Participation rate was 54.3 % (50.7 % girls/49.3 % boys). The questionnaire included a 73-item semi-quantitative food frequency questionnaire and questions on lifestyle. Results were evaluated against the Nordic Nutrition Recommendations and Swedish indicators of healthy diet and exercise habits. Associations with concurrent overweight and obesity were evaluated in multiple linear regression analysis. Among girls, 49.5 % reached the goal of consuming fruit and vegetables at least daily, whereas for boys the figure was 34.4 %. Among both sexes, 15 % reached the goal of consuming fish at least twice weekly. Two-thirds of both sexes reached the goal of regular moderate or vigorous physical activity weekly. In total, 12.4 % were overweight and 2.4 % were obese. More girls than boys were underweight, whereas more boys than girls were overweight or obese (p bread and potatoes and fast food (p < 0.001). Frequent intake of candies and chocolate was reported by both sexes. Among girls and boys, living in rural areas, living in apartments and reporting no frequent leisure time physical activity were significant risk factors for being overweight or obese, also when adjusted for other risk factors. Dietary habits of adolescents in Western Sweden warrant improvements. Public health actions should be taken to increase consumption of fruit, vegetables and

  3. Note sur la présence de lames aménagées par technique de Kostienki dans les couches gravettiennes du Blot (Cerzat,Haute-Loire).

    OpenAIRE

    Klaric , Laurent

    2000-01-01

    International audience; The unprecedented presence of Kostienki-technique prepared blades (also called Kostienki knives) in the Gravettian layers at Le Blot leads to a new analysis of these artefacts. Thorough technological study has pointed to the possible role of these items as cores, in association with or complementary to burin-forms, in particular context of backed-bladelet production. Le Blot is the second French site yielding such artefacts, the other being Corbiac (Dordogne). The aim ...

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

    Science.gov (United States)

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

    2009-04-01

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

  5. Journal of Biosciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    The expression patterns of CHOP and glucose-regulated protein (GRP) 78, a good marker of ER stress, were assessed by Western blotting, real-time PCR, Hoechst and immunohistochemistry in the hippocampus, cortex and striatum on a status epilepticus (SE) model. Double-fluorescent staining of CHOP and the terminal ...

  6. Isolation and characterization of a PUF-domain of pumilio gene from ...

    African Journals Online (AJOL)

    Drosophila pumilio antibody all could specifically detect BmPUM expressed in prokaryotic cells. The tissue expression pattern performed by real-time PCR and western blotting demonstrated that BmPUM expressed in various tissues, especially in testis and ovary. Those data collectively indicated that BmPUM belongs to ...

  7. Communication and relationship satisfaction in Chinese, Western, and intercultural Chinese-Western couples.

    Science.gov (United States)

    Hiew, Danika N; Halford, W Kim; van de Vijver, Fons J R; Liu, Shuang

    2016-03-01

    The current study compared Chinese, Western, and intercultural Chinese-Western couples' communication and examined how culture moderates the association of communication with relationship satisfaction. We coded the communication of 33 Western couples, 36 Chinese couples, and 54 intercultural Chinese-Western couples when discussing a relationship problem and when reminiscing about positive relationship events. Couples with Chinese female partners showed fewer positive behaviors and more negative behaviors (as classified in existing Western coding systems) than couples with Western female partners. The male partner's culture had few associations with couples' rates of communication behavior. Relationship satisfaction was associated with low rates of negative behaviors and high rates of most of the positive behaviors across cultural groups, and these associations were more evident in problem discussions than positive reminiscences. (c) 2016 APA, all rights reserved).

  8. Similar diagnostic performance for neurocysticercosis of three glycoprotein preparations from Taenia solium metacestodes.

    Science.gov (United States)

    Villota, Guido E; Gomez, Diana I; Volcy, Michel; Franco, Andrés F; Cardona, Edgar A; Isaza, Rodrigo; Sanzón, Fernando; Teale, Judy M; Restrepo, Blanca I

    2003-03-01

    The detection of antibodies to Taenia solium metacestodes is very important in the differential diagnosis of neurocysticercosis (NCC). In this study, an electroimmunotransfer blot (EITB) assay that uses an elaborate protocol with metacestode glycoproteins as antigens was compared with two other Western blots that use glycoproteins obtained using simpler methods, including an eluate from a lectin column, or the vesicular fluid (VF) of the parasite. The concordance between the three assays was 91% in patients with active NCC and 100% in patients with suspected NCC and previous documentation of negative serology. The specificities for the Western blots and the EITB assay were 98% and 100%, respectively (98% concordance). These data suggest that the simplest of these immunoassays, the one that uses the VF of T. solium metacestodes in a Western blot format, can be reliably used for the serologic diagnosis of NCC in developing countries where access to the EITB assay is difficult.

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

    Science.gov (United States)

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

    2012-05-01

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

  10. Western lowland gorillas (Gorilla gorilla gorilla) change their activity patterns in response to frugivory.

    Science.gov (United States)

    Masi, Shelly; Cipolletta, Chloé; Robbins, Martha M

    2009-02-01

    The most important environmental factor explaining interspecies variation in ecology and sociality of the great apes is likely to be variation in resource availability. Relatively little is known about the activity patterns of western lowland gorillas (Gorilla gorilla gorilla), which inhabit a dramatically different environment from the well-studied mountain gorillas (G. beringei beringei). This study aims to provide a detailed quantification of western lowland gorillas' activity budgets using direct observations on one habituated group in Bai Hokou, Central African Republic. We examined how activity patterns of both sexes are shaped by seasonal frugivory. Activity was recorded with 5-min instantaneous sampling between December 2004 and December 2005. During the high-frugivory period the gorillas spent less time feeding and more time traveling than during the low-frugivory period. The silverback spent less time feeding but more time resting than both females and immatures, which likely results from a combination of social and physiological factors. When compared with mountain gorillas, western lowland gorillas spend more time feeding (67 vs. 55%) and traveling (12 vs. 6.5%), but less time resting (21 vs. 34%) and engaging in social/other activities (0.5 vs. 3.6%). This disparity in activity budgets of western lowland gorillas and mountain gorillas may be explained by the more frugivorous diet and the greater dispersion of food resources experienced by western lowland gorillas. Like other apes, western lowland gorillas change their activity patterns in response to changes in the diet. (c) 2008 Wiley-Liss, Inc.

  11. Brief communication: timing of spheno-occipital closure in modern Western Australians.

    Science.gov (United States)

    Franklin, Daniel; Flavel, Ambika

    2014-01-01

    The spheno-occipital synchondrosis is a craniofacial growth centre between the occipital and sphenoid bones-its ossification persists into adolescence, which for the skeletal biologist, means it has potential application for estimating subadult age. Based on previous research the timing of spheno-occipital fusion is widely variable between and within populations, with reports of complete fusion in individuals as young as 11 years of age and nonfusion in adults. The aim of this study is, therefore, to examine this structure in a mixed sex sample of Western Australian individuals that developmentally span late childhood to adulthood. The objective is to develop statistically quantified age estimation standards based on scoring the degree of spheno-occipital fusion. The sample comprises multidetector computed tomography (MDCT) scans of 312 individuals (169 male; 143 female) between 5 and 25 years of age. Each MDCT scan is visualized in a standardized sagittal plane using three-dimensional oblique multiplanar reformatting. Fusion status is scored according to a four-stage system. Transition analysis is used to calculate age ranges for each defined stage and determine the mean age for transition between an unfused, fusing and fused status. The maximum likelihood estimates for the transition from open to fusing in the endocranial half is 14.44 years (male) and 11.42 years (female); transition from fusion in the ectocranial half to complete fusion is 16.16 years (male) and 13.62 years (female). This study affirms the potential value of assessing the degree of fusion in the spheno-occipital synchondrosis as an indicator of skeletal age. Copyright © 2013 Wiley Periodicals, Inc.

  12. Apatite fission track and (U-Th)/He dating of teschenite intrusions gives time constraints on accretionary processes and development of planation surfaces in the Outer Western Carpathians

    Energy Technology Data Exchange (ETDEWEB)

    Danisik, Martin; Frisch, Wolfgang [Tuebingen Univ. (Germany). Inst. of Geosciences; Panek, Tomas [Ostrava Univ. (Czech Republic). Dept. of Physical Geography and Geoecology; Matysek, Dalibor [Technical Univ. of Ostrava (Czech Republic). Dept. of Geological Engineering; Dunkl, Istvan [Geoscience Center Goettingen (Germany). Sedimentology and Environmental Geology

    2008-09-15

    The age of planation surfaces in the Podbeskydska pahorkatina Upland in the Outer Western Carpathians (OWC, Czech Republic) is constrained by low-temperature thermochronological dating methods for the first time. Our apatite fission track and apatite (U-Th)/He data measured on teschenite intrusions show that planation surfaces in the study area formed in post-Pannonian time (>7.1 Ma) and are therefore younger than traditionally believed. This contradicts the classical concepts, which stipulate that a large regional planation surface of Pannonian age (the so-called ''midmountain level'') developed in the whole Western Carpathians. Geodynamic implications of our data are the following: (i) the investigated Tesin and Godula nappes of the OWC were buried and thermally overprinted in the accretionary wedge in different ways, and consequently experienced different cooling histories. This indicates a dynamic basin setting with an active accretionary process in a subduction zone; (ii) accretionary processes in the OWC were active already during Late Eocene times. (orig.)

  13. High CpG island methylation of p16 gene and loss of p16 protein

    Indian Academy of Sciences (India)

    Methylation-specific polymerase chain reaction (MSP) was employed to detect CpG island methylation in p16 promoter region andWestern blotting was used to detect p16 expression of all subjects. Real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) was performed to test p16 mRNA expression.

  14. Hath1 inhibits proliferation of colon cancer cells probably through up-regulating expression of Muc2 and p27 and down-regulating expression of cyclin D1.

    Science.gov (United States)

    Zhu, Dai-Hua; Niu, Bai-Lin; Du, Hui-Min; Ren, Ke; Sun, Jian-Ming; Gong, Jian-Ping

    2012-01-01

    Previous studies showed that Math1 homologous to human Hath1 can cause mouse goblet cells to differentiate. In this context it is important that the majority of colon cancers have few goblet cells. In the present study, the potential role of Hath1 in colon carcinogenesis was investigated. Sections of paraffin-embedded tissues were used to investigate the goblet cell population of normal colon mucosa, mucosa adjacent colon cancer and colon cancer samples from 48 patients. Hath1 and Muc2 expression in these samples were tested by immunohistochemistry, quantitative real-time reverse transcription -PCR and Western blotting. After the recombinant plasmid, pcDNA3.1(+)-Hath1 had been transfected into HT29 colon cancer cells, three clones were selected randomly to test the levels of Hath1 mRNA, Muc2 mRNA, Hath1, Muc2, cyclin D1 and p27 by quantitative real-time reverse transcription-PCR and Western blotting. Moreover, the proliferative ability of HT29 cells introduced with Hath1 was assessed by means of colony formation assay and xenografting. Expression of Hath1, Muc2, cyclin D1 and p27 in the xenograft tumors was also detected by Western blotting. No goblet cells were to be found in colon cancer and levels of Hath1 mRNA and Hath1, Muc2 mRNA and Muc2 were significantly down-regulated. Hath1 could decrease cyclin D1, increase p27 and Muc2 in HT29 cells and inhibit their proliferation. Hath1 may be an anti-oncogene in colon carcinogenesis.

  15. Identification of Causes and Treatments for Chronic Pain in a Model of Gulf War Illness

    Science.gov (United States)

    2017-10-01

    saline delivered. Euthanasia and necropsy at days 5 and 20 post acidic/normal saline, and tissue analysis performed ( ELISA , Western Blot, LC-MS...performed ( ELISA , Western Blot, LC-MS). Milestone 3: the association between GWI-induced musculoskeletal pain and neuroinflammation, as well as the...thresholds. Serum samples analysed by ELISA . Major Task 7: Acidic/normal saline administered 180 days after final DFP injection; pharmacological reversal

  16. 'Western Balkans': Political context and the media usage

    Directory of Open Access Journals (Sweden)

    Svilar Predrag

    2010-01-01

    Full Text Available By accessing the content analysis of media archives, available both on the Internet presentations of the most significant and most influential media, press agencies, web search engines, and official public institutions, international government and non-government organizations, we will make an attempt to point to political conditioning, appearances and uses, as well as political etymology of the term Western Balkans. The attention will also be called to the fact that the term has its origin in Anglo-Saxon political and historiographical tradition, through the examples of its use, as well as to the similarities of political relations which bring to its applicability. By analyzing the content of media inscriptions, reports, documents, official announcements, authorial reviews and analyses, it came to a conclusion that Western Balkans occurs as a regional reference in particular historical and political circumstances and that geographical frames it is being used within, could be located outside the time frames and there are specified the particularities of its use. We will demonstrate that through the analysis of the political context and media usage, Western Balkans could be interpreted on more than one level. Western Balkans can at the same time represent both a terminological construction and a regional reference which expresses the indecisiveness of the West on social and cultural properties of the Balkans and the founding of Balkan cultures as immanently European, but at the same time the means of measurement, which is not only the determination of regionalism, but the determination of historical and political moment likewise. Besides, Western Balkans could also be interpreted as a mean of practical methodology and classification of naming the societies on the Balkans, which are the only outside the European Union frames, but also stand for the synchronization of the Balkanistic discourse with current political and historical circumstances and

  17. Influences of spawning timing, water temperature, and climatic warming on early life history phenology in western Alaska sockeye salmon

    Science.gov (United States)

    Sparks, Morgan M.; Falke, Jeffrey A.; Quinn, Thomas P.; Adkison, Milo D.; Schindler, Daniel E.; Bartz, Krista K.; Young, Daniel B.; Westley, Peter A. H.

    2018-01-01

    We applied an empirical model to predict hatching and emergence timing for 25 western Alaska sockeye salmon (Oncorhynchus nerka) populations in four lake-nursery systems to explore current patterns and potential responses of early life history phenology to warming water temperatures. Given experienced temperature regimes during development, we predicted hatching to occur in as few as 58 d to as many as 260 d depending on spawning timing and temperature. For a focal lake spawning population, our climate-lake temperature model predicted a water temperature increase of 0.7 to 1.4 °C from 2015 to 2099 during the incubation period, which translated to a 16 d to 30 d earlier hatching timing. The most extreme scenarios of warming advanced development by approximately a week earlier than historical minima and thus climatic warming may lead to only modest shifts in phenology during the early life history stage of this population. The marked variation in the predicted timing of hatching and emergence among populations in close proximity on the landscape may serve to buffer this metapopulation from climate change.

  18. Impact of the zero-markup drug policy on hospitalisation expenditure in western rural China: an interrupted time series analysis.

    Science.gov (United States)

    Yang, Caijun; Shen, Qian; Cai, Wenfang; Zhu, Wenwen; Li, Zongjie; Wu, Lina; Fang, Yu

    2017-02-01

    To assess the long-term effects of the introduction of China's zero-markup drug policy on hospitalisation expenditure and hospitalisation expenditures after reimbursement. An interrupted time series was used to evaluate the impact of the zero-markup drug policy on hospitalisation expenditure and hospitalisation expenditure after reimbursement at primary health institutions in Fufeng County of Shaanxi Province, western China. Two regression models were developed. Monthly average hospitalisation expenditure and monthly average hospitalisation expenditure after reimbursement in primary health institutions were analysed covering the period 2009 through to 2013. For the monthly average hospitalisation expenditure, the increasing trend was slowed down after the introduction of the zero-markup drug policy (coefficient = -16.49, P = 0.009). For the monthly average hospitalisation expenditure after reimbursement, the increasing trend was slowed down after the introduction of the zero-markup drug policy (coefficient = -10.84, P = 0.064), and a significant decrease in the intercept was noted after the second intervention of changes in reimbursement schemes of the new rural cooperative medical insurance (coefficient = -220.64, P markup drug policy in western China. However, hospitalisation expenditure and hospitalisation expenditure after reimbursement were still increasing. More effective policies are needed to prevent these costs from continuing to rise. © 2016 John Wiley & Sons Ltd.

  19. Detection of serologic responses to GB virus C/hepatitis G virus infection.

    Science.gov (United States)

    Lo, Shih-Yen; Ku, Chia-Wen; Ma, Hsin-Chieh; Li, Yi-Hwei; Yu, Jui-Hung; Lin, Hsien-Hong; Lua, Ahai C; Lee, Ming-Liang

    2002-09-01

    To investigate the prevalence of GB virus C/hepatitis G virus (GBV-C/HGV) and compare the serologic responses to various GBV-C/HGV markers in eastern Taiwan aborigines. We used RT-PCR and anti-HGenv u-plate to investigate the prevalence of GBV-C/HGV in eastern Taiwan aborigines. We also used ELISA, dot blot assay, and Western blot to detect the serologic responses to various GBV-C/HGV markers. The prevalence of GBV-C/HGV RNA in the general population of eastern Taiwan aborigines is about 5% (17/317), while 14% (43/317) have anti-E2 antibodies. There were no significant differences in antibody titer against one consensus core peptide (PPSSAAACSRGSPR) between GBV-C/HGV RNA-positive and -negative sera. Only 23 of 42 serum samples positive in the anti-HGenv u-plate EIA assay were positive (55%) in the dot blot assay. No positive signal was detected by Western blot using either recombinant NS3 or commercial E2 proteins. Antibodies against one consensus core peptide (PPSSAAACSRGSPR) may not constitute a good marker for the detection of GBV-C/HGV viremia. For the detection of anti-E2 antibodies, the anti-HGenv u-plate assay is more sensitive than the dot blot assay. Western blot assay is not a sensitive method for detecting GBV-C/HGV infection.

  20. Impact of ecological and social factors on ranging in western gorillas.

    Science.gov (United States)

    Doran-Sheehy, Diane M; Greer, David; Mongo, Patrice; Schwindt, Dylan

    2004-10-01

    We examined the influence of ecological (diet, swamp use, and rainfall) and social (intergroup interaction rate) factors on ranging behavior in one group of western gorillas (Gorilla gorilla gorilla) during a 16-month study. Relative to mountain gorillas, western gorillas live in habitats with reduced herb densities, more readily available fruit (from seasonal and rare fruit trees), and, at some sites, localized large open clearings (swamps and "bais"). Ranging behavior reflects these ecological differences. The daily path length (DPL) of western gorillas was longer (mean=2,014 m) than that of mountain gorillas, and was largely related to fruit acquisition. Swamp use occurred frequently (27% of days) and incurred a 50% increase in DPL, and 77% of the variation in monthly frequency of swamp use was explained by ripe fruit availability within the swamp, and not by the absence of resources outside the swamp. The annual home-range size was 15.4 km2. The western gorilla group foraged in larger areas each month, and reused them more frequently and consistently through time compared to mountain gorillas. In contrast to mountain gorillas, intergroup encounters occurred at least four times more frequently, were usually calm rather than aggressive, and had no consistent effect on DPL or monthly range size for one group of western gorillas. High genetic relatedness among at least some neighboring males [Bradley et al., Current Biology, in press] may help to explain these results, and raises intriguing questions about western gorilla social relationships.

  1. Interpretation of biological and mechanical variations between the Lowry versus Bradford method for protein quantification.

    Science.gov (United States)

    Lu, Tzong-Shi; Yiao, Szu-Yu; Lim, Kenneth; Jensen, Roderick V; Hsiao, Li-Li

    2010-07-01

    The identification of differences in protein expression resulting from methodical variations is an essential component to the interpretation of true, biologically significant results. We used the Lowry and Bradford methods- two most commonly used methods for protein quantification, to assess whether differential protein expressions are a result of true biological or methodical variations. MATERIAL #ENTITYSTARTX00026; Differential protein expression patterns was assessed by western blot following protein quantification by the Lowry and Bradford methods. We have observed significant variations in protein concentrations following assessment with the Lowry versus Bradford methods, using identical samples. Greater variations in protein concentration readings were observed over time and in samples with higher concentrations, with the Bradford method. Identical samples quantified using both methods yielded significantly different expression patterns on Western blot. We show for the first time that methodical variations observed in these protein assay techniques, can potentially translate into differential protein expression patterns, that can be falsely taken to be biologically significant. Our study therefore highlights the pivotal need to carefully consider methodical approaches to protein quantification in techniques that report quantitative differences.

  2. The life and times of a western refinery

    International Nuclear Information System (INIS)

    Thomas, W.

    1998-01-01

    An historical account is given of the discovery of oil by Imperial Oil Ltd., at Leduc in 1947, and the opening of a refinery in Edmonton in 1948. The discovery at Leduc was the first major strike to be made in western Canada, but Imperial Oil did not have a refinery at the site to process the new found crude. Given the economic constraints of the postwar era, Imperial Oil decided to dismantle and move an idle and virtually brand-new refinery located at Whitehorse, Yukon. It took 10 months to reassemble the refinery at its newly prepared site at Edmonton. Regular expansions and improvements helped to ensure that the refinery kept up with the advances in technology and market demand. By the early 1970s, small refineries were becoming uneconomical as distribution of products by pipelines became possible. In 1976 Imperial Oil spent more than $300 million to consolidate its four Prairie refineries, those in Calgary, Winnipeg, Regina and Edmonton, into one large facility at the Edmonton site. The role that the refinery plays in today's community and the effort that Imperial Oil makes to comply with stringent environmental regulations were described. Today, the refinery is producing 165,000 barrels a day of fuels and lubricants and another 20,000 barrels of asphalt. The refinery supplies more than 200 different products to customers across Canada, with unit costs which are amongst the lowest of North American refineries. figs

  3. Periostin: a promising target of therapeutical intervention for prostate cancer

    Directory of Open Access Journals (Sweden)

    Ding Weihong

    2011-06-01

    Full Text Available Abstract Background In our recent study, Periostin was up-regulated in prostate cancer(PCa compared with benign prostate hyperplasia (BPH by proteomics analysis of prostate biopsies. We investigated the effect of sliencing Periostin by RNA interference (RNAi on the proliferation and migration of PCa LNCap cell line. Methods All the prostate biopsies from PCa, BPH and BPH with local prostatic intraepithelial neoplasm(PIN were analyzed by iTRAQ(Isobaric tags for relative and absolute quantification technology. Western blotting and immunohistochemical staining were used to verify Periostin expression in the tissues of PCa. Periostin expression in different PCa cell lines was determined by immunofluorescence staining, western blotting and reverse transcription PCR(RT-PCR. The LNCap cells with Periostin expression were used for transfecting shRNA-Periostin lentiviral particles. The efficancy of transfecting shRNA lentiviral particles was evaluated by immunofluorescence, western blotting and Real-time PCR. The effect of silencing Periostin expression by RNAi on proliferation of LNCap cells was determined by MTT assay and tumor xenografts. The tissue slices from theses xenografts were analyzed by hematoxylin and eosin(HE staining. The expression of Periostin in the xenografts was deteminned by Immunohistochemical staining and western blotting. The migration of LNCap cells after silencing Periostin gene expression were analyzed in vitro. Results Periostin as the protein of interest was shown 9.12 fold up-regulation in PCa compared with BPH. The overexpression of Periostin in the stroma of PCa was confirmed by western blotting and immunohistochemical staining. Periostin was only expressed in PCa LNCap cell line. Our results indicated that the transfection ratio was more than 90%. As was expected, both the protein level and mRNA level of Periostin in the stably expressing shRNA-Periostin LNCap cells were significantly reduced. The stably expressing sh

  4. Mosquito densonucleosis virus non-structural protein NS2 is necessary for a productive infection

    International Nuclear Information System (INIS)

    Azarkh, Eugene; Robinson, Erin; Hirunkanokpun, Supanee; Afanasiev, Boris; Kittayapong, Pattamaporn; Carlson, Jonathan; Corsini, Joe

    2008-01-01

    Mosquito densonucleosis viruses synthesize two non-structural proteins, NS1 and NS2. While NS1 has been studied relatively well, little is known about NS2. Antiserum was raised against a peptide near the N-terminus of NS2, and used to conduct Western blot analysis and immuno-fluorescence assays. Western blots revealed a prominent band near the expected size (41 kDa). Immuno-fluorescence studies of mosquito cells transfected with AeDNV indicate that NS2 has a wider distribution pattern than does NS1, and the distribution pattern appears to be a function of time post-infection. Nuclear localization of NS2 requires intact C-terminus but does not require additional viral proteins. Mutations ranging from complete NS2 knock-out to a single missense amino acid substitution in NS2 can significantly reduce viral replication and production of viable progeny

  5. VEGF-mediated angiogenesis stimulates neural stem cell proliferation and differentiation in the premature brain

    International Nuclear Information System (INIS)

    Sun, Jinqiao; Sha, Bin; Zhou, Wenhao; Yang, Yi

    2010-01-01

    This study investigated the effects of angiogenesis on the proliferation and differentiation of neural stem cells in the premature brain. We observed the changes in neurogenesis that followed the stimulation and inhibition of angiogenesis by altering vascular endothelial growth factor (VEGF) expression in a 3-day-old rat model. VEGF expression was overexpressed by adenovirus transfection and down-regulated by siRNA interference. Using immunofluorescence assays, Western blot analysis, and real-time PCR methods, we observed angiogenesis and the proliferation and differentiation of neural stem cells. Immunofluorescence assays showed that the number of vWF-positive areas peaked at day 7, and they were highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at every time point. The number of neural stem cells, neurons, astrocytes, and oligodendrocytes in the subventricular zone gradually increased over time in the VEGF up-regulation group. Among the three groups, the number of these cells was highest in the VEGF up-regulation group and lowest in the VEGF down-regulation group at the same time point. Western blot analysis and real-time PCR confirmed these results. These data suggest that angiogenesis may stimulate the proliferation of neural stem cells and differentiation into neurons, astrocytes, and oligodendrocytes in the premature brain.

  6. CEPF Western Ghats Special Series: Fish fauna of Indrayani River, northern Western Ghats, India

    Directory of Open Access Journals (Sweden)

    Neelesh Dahanukar

    2012-01-01

    Full Text Available The freshwater fish fauna of the Indrayani River, a northern tributary of the Krishna River system in the Western Ghats of India was studied. A total of 57 species of freshwater fish belonging to 18 families and 39 genera were recorded. However, based on the previous literature it is possible that the Indrayani River harbours around 67 species. Out of the 57 species in the present collection, 12 are endemic to the Western Ghats while six are endemic to the Krishna River system. Neotropius khavalchor, an endemic fish of the Krishna River system, was recorded for the first time from the northern tributaries. The fish fauna of the Indrayani River is threatened due to seven introduced species and anthropogenic activities such as deforestation leading to siltation, tourism, sand mining, over fishing and organic and inorganic pollution. Since the Indrayani River hosts endemic and threatened species, including Glyptothorax poonaensis, conservation measures to ensure habitat protection in the river are essential.

  7. Spatial δ18Osw-SSS relationship across the western tropical Pacific Ocean

    Science.gov (United States)

    Thompson, D. M.; Conroy, J. L.; Wyman, A.; Read, D.

    2017-12-01

    Dynamic hydroclimate processes across the western tropical Pacific lead to strong spatial and temporal variability in δ18Osw and sea-surface salinity (SSS) across the western Pacific. Corals in this region have therefore provided key information about past SSS variability, as δ18Osw contributes strongly to coral δ18O across this region. However, uncertainties in the δ18Osw-SSS relationship across space and time often limit quantitative SSS reconstructions from such coral records. Recent work demonstrates considerable variability in the δ18Osw-SSS relationship across the Pacific, which may lead to over- or under-estimation of the contribution of SSS to coral δ18O, particularly across the western tropical Pacific (Conroy et al. 2017). Here we assess the spatial δ18Osw-SSS relationship across the dynamic western tropical Pacific, capitalizing on a transit between Subic Bay, Philippines and Townsville, Australia aboard the International Ocean Discovery program's JOIDES Resolution. Water samples and weather conditions were collected 3 times daily (6:00, 12:00, 18:00) en route, resulting in a network of 47 samples spaced at semi-regular 130-260 km intervals across the western Pacific from 14°N to 18°S. The route also crossed near long-term δ18Osw monitoring sites at Papua New Guinea and Palau (Conroy et al. 2017), allowing us to compare the spatial and temporal δ18Osw-SSS relationships at these sites and test the space-for-time assumption. We present the δ18Osw-SSS relationship across this region, compare the relationship across space and time, and discuss the implications of our results for SSS reconstructions from coral δ18O.

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

    Science.gov (United States)

    Ng, Khong Y; Machida, Kazuya

    2017-01-01

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

  9. [Overexpression of liver kinase B1 inhibits the proliferation of lung cancer cells].

    Science.gov (United States)

    Li, Yang; Zhang, Libin; Wang, Ping

    2017-01-01

    Objective To explore the effect of overexpressed liver kinase B1(LKB1) on the proliferation of lung cancer cell lines. Methods The expression levels of LKB1 and PTEN in A549, NCI-H23, NCI-H157, XWLC-05, NCI-H446 lung cancer cells were detected by immunocytochemistry (ICC) and Western blotting. Plasmid pcDNA3.1 + -LKB1 and empty vector pcDNA3.1 + -null were separately transfected into the above five cell lines, and then the expression of LKB1 mRNA and protein were determined by quantitative real-time PCR and Western blotting, respectively. Finally, CCK-8 assay was used to analyze the proliferation ability of the transfected cells. Results LKB1 and PTEN were positive in NCI-H23 cells; LKB1 was negative while PTEN was positive in A549 and NCI-H446 cells; both LKB1 and PTEN were negative in NCI-H157 and XWLC-05 cells. Quantitative real-time PCR and Western blotting showed that the expression level of LKB1 significantly increased in the above cell lines transfected with plasmid pcDNA3.1 + -LKB1 compared with the ones with empty vector pcDNA3.1 + -null. Besides, CCK-8 assay showed that the overexpression of LKB1 in the lung cancer cells transfected with pcDNA3.1 + -LKB1 had an obvious inhibitory effect on cell proliferation. Conclusion The expression of LKB1 is down-regulated in most of the lung cell lines to different extent and the over-expression of LKB1 can remarkably inhibit the proliferation ability of lung cancer cell lines.

  10. Cattle Rustling and Banditry in North-Western Kenya

    African Journals Online (AJOL)

    rejecting all forms of Western influence for a long time. ..... children are spared. ... Nearly all the Pokot adopted in one way or another these short-term survival ..... raids in living memory against their neighbours, the Marakwet (Magut inter-.

  11. Precipitation-snowmelt timing and snowmelt augmentation of large peak flow events, western Cascades, Oregon

    Science.gov (United States)

    Keith Jennings; Julia A. Jones

    2015-01-01

    This study tested multiple hydrologic mechanisms to explain snowpack dynamics in extreme rain-on-snow floods, which occur widely in the temperate and polar regions. We examined 26, 10 day large storm events over the period 1992–2012 in the H.J. Andrews Experimental Forest in western Oregon, using statistical analyses (regression, ANOVA, and wavelet coherence) of hourly...

  12. Water avoidance stress induces frequency through cyclooxygenase-2 expression: a bladder rat model.

    Science.gov (United States)

    Yamamoto, Keisuke; Takao, Tetsuya; Nakayama, Jiro; Kiuchi, Hiroshi; Okuda, Hidenobu; Fukuhara, Shinichiro; Yoshioka, Iwao; Matsuoka, Yasuhiro; Miyagawa, Yasushi; Tsujimura, Akira; Nonomura, Norio

    2012-02-01

    Water avoidance stress is a potent psychological stressor and it is associated with visceral hyperalgesia, which shows degeneration of the urothelial layer mimicking interstitial cystitis. Cyclooxygenase-2 inhibitors have been recognized to ameliorate frequency both in clinical and experimental settings. We investigated the voiding pattern and cyclooxygenase-2 expression in a rat bladder model of water avoidance stress. After being subjected to water avoidance stress or a sham procedure, rats underwent metabolic cage analysis and cystometrography. Real time reverse transcription polymerase chain reaction was carried out to examine cyclooxygenase-2 messenger ribonucleic acid in bladders of rats. Protein expression of cyclooxygenase-2 was analyzed with immunohistochemistry and western blotting. Furthermore, the effects of the cyclooxygenase-2 inhibitor, etodolac, were investigated by carrying out cystometrography, immunohistochemistry and western blotting. Metabolic cage analysis and cystometrography showed significantly shorter intervals and less volume of voiding in water avoidance stress rats. Significantly higher expression of cyclooxygenase-2 messenger ribonucleic acid was verified by reverse transcription polymerase chain reaction. Immunohistochemistry and western blotting showed significantly higher cyclooxygenase-2 protein levels in water avoidance stress bladders. Furthermore, immunohistochemistry showed high cyclooxygenase-2 expression exclusively in smooth muscle cells. All water avoidance stress-induced changes were reduced by cyclooxygenase-2 inhibitor pretreatment. Chronic stress might cause frequency through cyclooxygenase-2 gene upregulation in bladder smooth muscle cells. Further study of cyclooxygenase-2 in the water avoidance stress bladder might provide novel therapeutic modalities for interstitial cystitis. © 2011 The Japanese Urological Association.

  13. Methods to uncover an antibody epitope in the KPI domain of Alzheimer's amyloid precursor protein for immunohistochemistry in human brain.

    Science.gov (United States)

    Campbell, E; Pearson, R C; Parkinson, D

    1999-11-15

    A novel polyclonal antibody (Ab993), specific for a KPI domain epitope of APP, was characterised for use in immunoprecipitation, Western blotting and immunohistochemistry. Conditioned medium from NTera2/D1 cells was used for immunoprecipitation and Western blots. Paraffin-embedded human brain sections were used for immunohistochemistry. The antibody recognised KPI-containing APP on Western blots after standard solubilisation but immunoprecipitation of soluble APP required reduction with 2-mercaptoethanol followed by alkylation of reduced sulphydryl bonds with sodium iodoacetate. Immunohistochemical staining of human brain sections was significantly enhanced by this pre-treatment. Microwaving of sections also increased immunolabelling, by a mechanism that was additive to reduction and alkylation. Incubation in 80% formic acid did not confer any enhancement of immunoreactivity. Ab993, applied with the methods reported here, is expected to be valuable in investigations of the pathogenesis of Alzheimer's disease to determine the source of the beta-amyloid peptide.

  14. Attenuation of everolimus-induced cytotoxicity by a protective autophagic pathway involving ERK activation in renal cell carcinoma cells

    Science.gov (United States)

    Zeng, Yizhou; Tian, Xiaofang; Wang, Quan; He, Weiyang; Fan, Jing; Gou, Xin

    2018-01-01

    Aim The mammalian target of rapamycin (mTOR) pathway is a critical target for cancer treatment and the mTOR inhibitor everolimus (RAD001) has been approved for treatment of renal cell carcinoma (RCC). However, the limited efficacy of RAD001 has led to the development of drug resistance. Autophagy is closely related to cell survival and death, which may be activated under RAD001 stimulation. The aim of the present study was to identify the underlying mechanisms of RAD001 resistance in RCC cells through cytoprotective autophagy involving activation of the extracellular signal-regulated kinase (ERK) pathway. Methods and results: RAD001 strongly induced autophagy of RCC cells in a dose- and time-dependent manner, as confirmed by Western blot analysis. Importantly, suppression of autophagy by the pharmacological inhibitor chloroquine effectively enhanced RAD001-induced apoptotic cytotoxicity, as demonstrated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Western blot analysis, indicating a cytoprotective role for RAD001-induced autophagy. In addition, as was shown by the MTT assay, flow cytometry, and Western blot analysis, RAD001 robustly activated ERK, but not c-Jun N-terminal kinase and p38. Activation of ERK was inhibited by the pharmacological inhibitor selumetinib (AZD6244), which effectively promoted RAD001-induced cell death. Moreover, employing AZD6244 markedly attenuated RAD001-induced autophagy and enhanced RAD001-induced apoptosis, which play a central role in RAD001-induced cell death. Furthermore, RAD001-induced autophagy is regulated by ERK-mediated phosphorylation of Beclin-1 and B-cell lymphoma 2, as confirmed by Western blot analysis. Conclusion These results suggest that RAD001-induced autophagy involves activation of the ERK, which may impair cytotoxicity of RAD001 in RCC cells. Thus, inhibition of the activation of ERK pathway-mediated autophagy may be useful to overcome chemoresistance to RAD001. PMID:29719377

  15. Expression of MICA, MICB and NKG2D in human leukemic myelomonocytic and cervical cancer cells

    Directory of Open Access Journals (Sweden)

    Mendoza-Rincon Jorge

    2011-04-01

    Full Text Available Abstract Background Cancer cells are known to secrete the stress molecules MICA and MICB that activate cytotoxicity by lymphocytes and NK cells through their NKG2D receptor as a mechanism of immunological defense. This work was undertaken to evaluate if cancer cells can also express this receptor as a possible mechanisms of depletion of MIC molecules and thus interfere with their immune recognition. Methods Myelomonocytic leukemic (TPH-1 and U-937 and cervical cancer (CALO and INBL cell lines were evaluated by Western Blot, ELISA, flow cytometry and immunocytochemistry to evaluate their capacity to express and secrete MICA and MICB and to be induced to proliferate by these molecules as well as to express their receptor NKG2D. Statistical analysis was performed by two-way ANOVA for time course analysis and Student's t-test for comparison between groups. Values were considered significantly different if p Results THP-1 and U-937 produce and secrete the stress MICA and MICB as shown by Western Blot of lysed cells and by ELISA of their conditioned media. By Western Blot and flow cytometry we found that these cells also express the receptor NKG2D. When THP-1 and U-937 were cultured with recombinant MICA and MICB they exhibited a dose dependent induction for their proliferation. CALO and INBL also produce MICA and MICB and were induced to proliferate by these stress molecules. By Western Blot, flow cytometry and immunocytochemistry we also found that these cells express NKG2D. Conclusions Our novel results that tumor cells can simultaneously secrete MIC molecules and express their receptor, and to be induced for proliferation by these stress molecules, and that tumor epithelial cells can also express the NKG2D receptor that was thought to be exclusive of NK and cytotoxic lymphocytes is discussed as a possible mechanism of immunological escape and of tumor growth induction.

  16. Nuclear energy in Western Europe

    International Nuclear Information System (INIS)

    Loennroth, M.; Walker, W.

    1984-01-01

    This is an overview of nuclear energy in Western Europe, as seen by two Western Europeans, attempting to place the topic into the context not only of energy supply but also of industrial relations, institutional structure, and sociocultural factors. Although its main focus is Western Europe, it is sometimes necessary to glance at the wider context, in particular the industrial relations with the United States and Japan. Export markets are also considered, in particular, in the Pacific. The paper does not, however, deal with nonproliferation policies and the possible difference of opinion within Western Europe and between Western Europe and other regions over this topic. (author)

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

    International Nuclear Information System (INIS)

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

    1985-01-01

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

  18. Internal globalization of Western Balkan

    Directory of Open Access Journals (Sweden)

    Vukotić Veselin

    2006-01-01

    Full Text Available What are potential and real effects of the globalization process on the economic connection between Western Balkan countries? What is the crucial change in relations between Western Balkan countries and its economies inexorably brought by globalization? What are the elements of political economy of Western Balkan globalization? What are reflections of the conflict between political and economic areas of Western Balkan? These are some of the issues discuses in this paper.

  19. Peritoneal Dialysis in Western Countries.

    Science.gov (United States)

    Struijk, Dirk G

    2015-12-01

    Peritoneal dialysis (PD) for the treatment of end-stage renal failure was introduced in the 1960s. Nowadays it has evolved to an established therapy that is complementary to hemodialysis (HD), representing 11% of all patients treated worldwide with dialysis. Despite good clinical outcomes and similar results in patient survival between PD and HD, the penetration of PD is decreasing in the Western world. First the major events in the history of the development of PD are described. Then important insights into the physiology of peritoneal transport are discussed and linked to the changes in time observed in biopsies of the peritoneal membrane. Furthermore, the developments in peritoneal access, more biocompatible dialysate solutions, automated PD at home, the establishment of parameters for dialysis adequacy and strategies to prevent infectious complications are mentioned. Finally non-medical issues responsible for the declining penetration in the Western world are analyzed. Only after introduction of the concept of continuous ambulatory PD by Moncrief and Popovich has this treatment evolved in time to a renal replacement therapy. Of all structures present in the peritoneal membrane, the capillary endothelium offers the rate-limiting hindrance for solute and water transport for the diffusive and convective transport of solutes and osmosis. The functional and anatomical changes in the peritoneal membrane in time can be monitored by the peritoneal equilibrium test. Peritonitis incidence decreased by introduction of the Y-set and prophylaxis using mupirocin on the exit site. The decrease in the proportion of patients treated with PD in the Western world can be explained by non-medical issues such as inadequate predialysis patient education, physician experience and training, ease of HD initiation, overcapacity of in-center HD, lack of adequate infrastructure for PD treatment, costs and reimbursement issues of the treatment. (1) PD is cheaper than HD and provides a

  20. Use of capillary Western immunoassay (Wes) for quantification of dystrophin levels in skeletal muscle of healthy controls and individuals with Becker and Duchenne muscular dystrophy.

    Science.gov (United States)

    Beekman, Chantal; Janson, Anneke A; Baghat, Aabed; van Deutekom, Judith C; Datson, Nicole A

    2018-01-01

    Duchenne muscular dystrophy (DMD) is a neuromuscular disease characterized by progressive weakness of the skeletal and cardiac muscles. This X-linked disorder is caused by open reading frame disrupting mutations in the DMD gene, resulting in strong reduction or complete absence of dystrophin protein. In order to use dystrophin as a supportive or even surrogate biomarker in clinical studies on investigational drugs aiming at correcting the primary cause of the disease, the ability to reliably quantify dystrophin expression in muscle biopsies of DMD patients pre- and post-treatment is essential. Here we demonstrate the application of the ProteinSimple capillary immunoassay (Wes) method, a gel- and blot-free method requiring less sample, antibody and time to run than conventional Western blot assay. We optimized dystrophin quantification by Wes using 2 different antibodies and found it to be highly sensitive, reproducible and quantitative over a large dynamic range. Using a healthy control muscle sample as a reference and α-actinin as a protein loading/muscle content control, a panel of skeletal muscle samples consisting of 31 healthy controls, 25 Becker Muscle dystrophy (BMD) and 17 DMD samples was subjected to Wes analysis. In healthy controls dystrophin levels varied 3 to 5-fold between the highest and lowest muscle samples, with the reference sample representing the average of all 31 samples. In BMD muscle samples dystrophin levels ranged from 10% to 90%, with an average of 33% of the healthy muscle average, while for the DMD samples the average dystrophin level was 1.3%, ranging from 0.7% to 7% of the healthy muscle average. In conclusion, Wes is a suitable, efficient and reliable method for quantification of dystrophin expression as a biomarker in DMD clinical drug development.

  1. Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

    International Nuclear Information System (INIS)

    Stolf, A.M.S.; Umezawa, E.S.; Zingales, B.

    1990-01-01

    A radioactive Western blotting technique was developed by which the reactivity of Immunoglobulins (IGs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse. T.cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgC specific antibodies. A different pattern of reactivity with acute Chagas disease patients sera was thus obtained. (author)

  2. A Premature Termination of Human Epidermal Growth Factor Receptor Transcription in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jihene Elloumi-Mseddi

    2014-01-01

    Full Text Available Our success in producing an active epidermal growth factor receptor (EGFR tyrosine kinase in Escherichia coli encouraged us to express the full-length receptor in the same host. Despite its large size, we were successful at producing the full-length EGFR protein fused to glutathione S-transferase (GST that was detected by Western blot analysis. Moreover, we obtained a majoritarian truncated GST-EGFR form detectable by gel electrophoresis and Western blot. This truncated protein was purified and confirmed by MALDI-TOF/TOF analysis to belong to the N-terminal extracellular region of the EGFR fused to GST. Northern blot analysis showed two transcripts suggesting the occurrence of a transcriptional arrest.

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

    Science.gov (United States)

    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.

  4. Extraordinary Diversity of Immune Response Proteins among Sea Urchins: Nickel-Isolated Sp185/333 Proteins Show Broad Variations in Size and Charge

    Science.gov (United States)

    Sherman, Lauren S.; Schrankel, Catherine S.; Brown, Kristy J.; Smith, L. Courtney

    2015-01-01

    Effective protection against pathogens requires the host to produce a wide range of immune effector proteins. The Sp185/333 gene family, which is expressed by the California purple sea urchin Strongylocentrotus purpuratus in response to bacterial infection, encodes a highly diverse repertoire of anti-pathogen proteins. A subset of these proteins can be isolated by affinity to metal ions based on multiple histidines, resulting in one to four bands of unique molecular weight on standard Western blots, which vary depending on the individual sea urchin. Two dimensional gel electrophoresis (2DE) of nickel-isolated protein samples followed by Western blot was employed to detect nickel-isolated Sp185/333 (Ni-Sp185/333) proteins and to evaluate protein diversity in animals before and after immune challenge with marine bacteria. Ni-Sp185/333 proteins of the same molecular weight on standard Western blots appear as a broad complex of variants that differ in pI on 2DE Western blots. The Ni-Sp185/333 protein repertoire is variable among animals, and shows a variety of changes among individual sea urchins in response to immune challenges with both the same and different species of bacteria. The extraordinary diversity of the Ni-Sp185/333 proteins may provide significant anti-pathogen capabilities for sea urchins that survive solely on innate immunity. PMID:26406912

  5. Multifractals in Western Major STOCK Markets Historical Volatilities in Times of Financial Crisis

    Science.gov (United States)

    Lahmiri, Salim

    In this paper, the generalized Hurst exponent is used to investigate multifractal properties of historical volatility (CHV) in stock market price and return series before, during and after 2008 financial crisis. Empirical results from NASDAQ, S&P500, TSE, CAC40, DAX, and FTSE stock market data show that there is strong evidence of multifractal patterns in HV of both price and return series. In addition, financial crisis deeply affected the behavior and degree of multifractality in volatility of Western financial markets at price and return levels.

  6. Identification of antigenic domains in the non-structural protein of Muscovy duck parvovirus.

    Science.gov (United States)

    Yu, Tian-Fei; Li, Ming; Yan, Bing; Shao, Shu-Li; Fan, Xing-Dong; Wang, Jia; Wang, Dan-Na

    2016-08-01

    Muscovy duck parvovirus (MDPV) infection is widespread in many Muscovy-duck-farming countries, leading to a huge economic loss. By means of overlapping peptides expressed in Escherichia coli in combination with Western blot, antigenic domains on the non-structural protein (NSP) of MDPV were identified for the first time. On the Western blot, the fragments NS(481-510), NS (501-530), NS (521-550), NS (541-570), NS (561-590), NS (581-610) and NS (601-627) were positive (the numbers in parentheses indicate the location of amino acids), and other fragments were negative. These seven fragments were also reactive in an indirect enzyme-linked immunosorbent assay (i-ELISA). We therefore conclude that a linear antigenic domain of the NSP is located at its C-terminal end (amino acid residues 481-627). These results may facilitate future investigations into the function of NSP of MDPV and the development of immunoassays for the diagnosis of MDPV infection.

  7. Effects of Lead on the Morphology and Structure of the Nucleolus in the Root Tip Meristematic Cells of Allium cepa L.

    Directory of Open Access Journals (Sweden)

    Ze Jiang

    2014-07-01

    Full Text Available To study the toxic mechanisms of lead (Pb in plants, the effects of Pb on the morphology and structure of the nucleolus in root tip meristematic cells of Allium cepa var. agrogarum L. were investigated. Fluorescence labeling, silver-stained indirect immunofluorescent microscopy and western blotting were used. Fluorescence labeling showed that Pb ions were localized in the meristematic cells and the uptake and accumulation of Pb increased with treatment time. At low concentrations of Pb (1–10 μM there were persistent nucleoli in some cells during mitosis, and at high concentration (100 μM many of the nucleolar organizing regions were localized on sticky chromosomes in metaphase and anaphase cells. Pb induced the release of particles containing argyrophilic proteins to be released from the nucleus into the cytoplasm. These proteins contained nucleophosmin and nucleolin. Pb also caused the extrusion of fibrillarin from the nucleus into the cytoplasm. Western blotting demonstrated the increased expression of these three major nucleolar proteins under Pb stress.

  8. CEPF Western Ghats Special Series : Parasitoids (Hymenoptera of xylophagous beetles (Coleoptera attacking dead wood in southern Western Ghats, Kerala, India, with descriptions of two new species

    Directory of Open Access Journals (Sweden)

    P.M. Sureshan

    2013-05-01

    Full Text Available An account is given of four species of Hymenoptera parasitoids probably of the wood boring beetle Clytocera chinospila Gahan (Coleoptera: Cerambycidae from Chinnar Wildlife Sanctuary, southern Western Ghats, Kerala. Two new hymenopteran species, Eurytoma chinnarensis (Eurytomidae and Foenatopus idukkiensis (Stephanidae are described. Solenura ania Walker (Pteromalidae is reported for the first time from Kerala and Western Ghats with a new host record, and Doryctus sp. (Braconidae is reported here.

  9. Teaching the Western.

    Science.gov (United States)

    Lenihan, John H.

    2002-01-01

    Discusses the content of a course on the genre of western films that was utilized as a film study and a U.S. cultural history credit. Describes in detail the film, "Winchester '73," and addresses other films utilized in the course. States that the course also focuses on the development of the western genre. (CMK)

  10. Population models of burrowing mayfly recolonization in Western Lake Erie

    Science.gov (United States)

    Madenjian, C.P.; Schloesser, D.W.; Krieger, K.A.

    1998-01-01

    Burrowing mayflies, Hexagenia spp. (H. limbata and H. rigida), began recolonizing western Lake Erie during the 1990s. Survey data for mayfly nymph densities indicated that the population experienced exponential growth between 1991 and 1997. To predict the time to full recovery of the mayfly population, we fitted logistic models, ranging in carrying capacity from 600 to 2000 nymphs/m2, to these survey data. Based on the fitted logistic curves, we forecast that the mayfly population in western Lake Erie would achieve full recovery between years 1998 and 2000, depending on the carrying capacity of the western basin. Additionally, we estimated the mortality rate of nymphs in western Lake Erie during 1994 and then applied an age-based matrix model to the mayfly population. The results of the matrix population modeling corroborated the exponential growth model application in that both methods yielded an estimate of the population growth rate, r, in excess of 0.8 yr-1. This was the first evidence that mayfly populations are capable of recolonizing large aquatic ecosystems at rates comparable with those observed in much smaller lentic ecosystems. Our model predictions should prove valuable to managers of power plant facilities along the western basin in planning for mayfly emergences and to managers of the yellow perch (Perca flavescens) fishery in western Lake Erie.

  11. Communication Patterns in Adult-Infant Interactions in Western and Non-Western Cultures.

    Science.gov (United States)

    Keller, Heidi; And Others

    1988-01-01

    Analyzes the early communication structure in adult-child interactions with two- to six-month old babies in Western (West Germany, Greece) and non-Western (Yanomami, Trobriand) societies. Discusses universal international verbal and non-verbal structures reflecting intuitive parenting programs. (FMW)

  12. Analytical assessment of the osteoinductive material COLLOSSE

    DEFF Research Database (Denmark)

    Nienhuijs, M E L; Poulsen, K; van der Zande, M

    2009-01-01

    In this study, the growth factors in COLLOSSE were analyzed, using ELISA tests, mass spectrometry, western blotting, and a 24-day cell culture experiment using osteoblast-like cells. The results of the ELISA testing, mass spectrometry, and western blotting all confirmed that TGF-beta1 was the mai...... of osteoinductive factors in COLLOSSE causes a synergistic effect, explaining the new bone formation found in previously described in vivo studies, with much lower growth factor concentrations when compared with recombinant BMPs....

  13. Role of Obesity in Prostate Cancer Development

    Science.gov (United States)

    2011-04-01

    Western blot analysis . Representative staining from the immunohistochemistry is shown in Figure 6. Expression of AdipoR1 was found in all prostate tumor...with goat serum instead of primary antibody was negative (Fig 6D). Western blot analysis of frozen tissue from the same mice was also performed and...TRAMP) model. Cancer Res., 57, 3325-3330. 41. Williams,T.M., Hassan,G.S., Li,J., Cohen,A.W., Medina,F., Frank,P.G., Pestell ,R.G., Di Vizio,D., and

  14. Breastfeeding Duration and Residential Isolation amid Aboriginal Children in Western Australia

    Directory of Open Access Journals (Sweden)

    Stephen R. Zubrick

    2012-12-01

    Full Text Available Objectives: To examine factors that impact on breastfeeding duration among Western Australian Aboriginal children. We hypothesised that Aboriginal children living in remote locations in Western Australia were breastfed for longer than those living in metropolitan locations. Methods: A population-based cross-sectional survey was conducted from 2000 to 2002 in urban, rural and remote settings across Western Australia. Cross-tabulations and multivariate logistic regression analyses were performed, using survey weights to produce unbiased estimates for the population of Aboriginal children. Data on demographic, maternal and infant characteristics were collected from 3932 Aboriginal birth mothers about their children aged 0–17 years (representing 22,100 Aboriginal children in Western Australia. Results: 71% of Aboriginal children were breastfed for three months or more. Accounting for other factors, there was a strong gradient for breastfeeding duration by remoteness, with Aboriginal children living in areas of moderate isolation being 3.2 times more likely to be breastfed for three months or more (p < 0.001 compared to children in metropolitan Perth. Those in areas of extreme isolation were 8.6 times more likely to be breastfed for three months or longer (p < 0.001. Conclusions: Greater residential isolation a protective factor linked to longer breastfeeding duration for Aboriginal children in our West Australian cohort.

  15. Characteristics of seroconversion and implications for diagnosis of post-treatment Lyme disease syndrome: acute and convalescent serology among a prospective cohort of early Lyme disease patients.

    Science.gov (United States)

    Rebman, Alison W; Crowder, Lauren A; Kirkpatrick, Allison; Aucott, John N

    2015-03-01

    Two-tier serology is often used to confirm a diagnosis of Lyme disease. One hundred and four patients with physician diagnosed erythema migrans rashes had blood samples taken before and after 3 weeks of doxycycline treatment for early Lyme disease. Acute and convalescent serologies for Borrelia burgdorferi were interpreted according to the 2-tier antibody testing criteria proposed by the Centers for Disease Control and Prevention. Serostatus was compared across several clinical and demographic variables both pre- and post-treatment. Forty-one patients (39.4%) were seronegative both before and after treatment. The majority of seropositive individuals on both acute and convalescent serology had a positive IgM western blot and a negative IgG western blot. IgG seroconversion on western blot was infrequent. Among the baseline variables included in the analysis, disseminated lesions (p Lyme disease. Furthermore, these findings underline the difficulty for rheumatologists in identifying a prior exposure to Lyme disease in caring for patients with medically unexplained symptoms or fibromyalgia-like syndromes.

  16. Western Slope Colorado

    International Nuclear Information System (INIS)

    Epis, R.C.; Callender, J.F.

    1981-01-01

    A conference on the geology and geologic resources of the Western Slope of western Colorado and eastern Utah is presented. Fourteen papers from the conference have been abstracted and indexed for the Department of Energy's Energy Data Base. These papers covered such topics as uranium resources, oil shale deposits, coal resources, oil and gas resources, and geothermal resources of the area

  17. Milk--the promoter of chronic Western diseases.

    Science.gov (United States)

    Melnik, Bodo C

    2009-06-01

    Common chronic diseases of Western societies, such as coronary heart disease, diabetes mellitus, cancer, hypertension, obesity, dementia, and allergic diseases are significantly influenced by dietary habits. Cow's milk and dairy products are nutritional staples in most Western societies. Milk and dairy product consumption is recommended by most nutritional societies because of their beneficial effects for calcium uptake and bone mineralization and as a source of valuable protein. However, the adverse long-term effects of milk and milk protein consumption on human health have been neglected. A hypothesis is presented, showing for the first time that milk protein consumption is an essential adverse environmental factor promoting most chronic diseases of Western societies. Milk protein consumption induces postprandial hyperinsulinaemia and shifts the growth hormone/insulin-like growth factor-1 (IGF-1) axis to permanently increased IGF-1 serum levels. Insulin/IGF-1 signalling is involved in the regulation of fetal growth, T-cell maturation in the thymus, linear growth, pathogenesis of acne, atherosclerosis, diabetes mellitus, obesity, cancer and neurodegenerative diseases, thus affecting most chronic diseases of Western societies. Of special concern is the possibility that milk intake during pregnancy adversely affects the early fetal programming of the IGF-1 axis which will influence health risks later in life. An accumulated body of evidence for the adverse effects of cow's milk consumption from fetal life to childhood, adolescence, adulthood and senescence will be provided which strengthens the presented hypothesis.

  18. Industrial aspects of radioactive waste management in Western Europe

    International Nuclear Information System (INIS)

    Marcus, F.R.

    1976-01-01

    Various aspects of waste management are discussed from the viewpoint of the nuclear industry. Future amounts of waste generated in the 15 Foratom countries in Western Europe are estimated. Industrial waste questions--as seen by electricity producers, reprocessors, and waste operators--are discussed; questions concerning decommissioning are also dealt with. A number of recommendations for further action, primarily on the part of national authorities and international organizations, are put forward. One conclusion of the study is that there is no reason for waste-management problems to impede the timely development of nuclear energy as a large-scale industrial activity in Western Europe

  19. Non-Western International Relations Theory: Myth or Reality?

    Directory of Open Access Journals (Sweden)

    Marina Mikhailovna Lebedeva

    2017-12-01

    Full Text Available In recent years, Russian and foreign literature increasingly raises the question on national theories of international relations. A special interest is manifested towards non-Western theories of international relations. The article analyzes the reasons for such interest. It is noted that the main motive for scholars to search for national schools is the transformation of the political organization of the world that emerged in the West and was developing largely on the Western model. This transformation encompasses three levels of political organization of the modern world: the Westphalian system, the system of international (interstate relations and the political systems of a state. Three levels of political organization of the world changing at the same time today reinforce each other and generate synergies. With such a large-scale transformation, when all three levels are “moving”, the world is facing for the first time, although the change of the second and especially the third levels were before. As far as the system of political organization of the world undergoes major changes, IR theories, which appeared in the West, are in crisis. Researchers’ attention to non-Western, primarily Asian TMO to find answers due to the following reasons: 1 the rapid economic growth of the region; 2 the development of scientific research in Asia; 3 the crisis of the Western model of political organization in the world that encourages the search for solutions in other civilizational structures. The article substantiates the necessity and possibility of “project activities” for reforming the political organization of the world and include practices that exist in different regions of the world. In order to implement such activities, the work of specialists from different brunches of social sciences is required.

  20. Glacigenic landforms and sediments of the Western Irish Shelf

    Science.gov (United States)

    McCarron, Stephen; Monteys, Xavier; Toms, Lee

    2013-04-01

    Vibrocoring of possible glacigenic landforms identified from high resolution bathymetric coverage of the Irish Shelf by the Irish National Seabed Survey (INSS) has provided several clusters of short (<3m) cores that, due to a regional post-glacial erosional event, comprise last glacial age stratigraphies. In addition, new shallow seismic data and sedimentological information from across the Western Irish Shelf provide new insights into aspects of the nature, timing and pattern of shelf occupation by grounded lobate extensions of the last Irish Ice Sheet. Restricted chronological control of deglacial sequences in several cores indicates that northern parts of the western mid-shelf (south of a prominent outer Donegal Bay ridge) were ice free by ~24 ka B.P., and that ice had also probably retreated from outer shelf positions (as far west as the Porcupine Bank) at or before this time.

  1. Western United States Dams Challenges Faced, Options, and Opportunities

    Science.gov (United States)

    Raff, D.

    2017-12-01

    Water management in the Western United States relies significantly upon a fleet of small to very large engineered dams to store water during times of runoff and distribute that water during times of need. Much of this infrastructure is Federally owned and/or operated, and was designed and funded during the first half of the twentieth century through a complex set of repayment contracts for Federally authorized purposes addressing water supply, recreation, and hydropower, and other water management objectives. With environmental laws, namely the Endangered Species Act, and other environmental concerns taking a more active role in water resources in the mid to latter half of the twentieth century, this infrastructure is being stressed even greater than anticipated to provide authorized purposes. Additionally, weather and climate norms being experienced are certainly near the edges, if not outside, of anticipated variability in the climate and hydrology scenarios for which the infrastructure was designed. And, finally, these dams, economically designed for a lifespan of 50 - 100 years, are experiencing maintenance challenges from routine to significant. This presentation will focus on identifying some of the history and challenges facing the water infrastructure in the Western United States. Additionally, some perspectives on future paths to meet the needs of western irrigation and hydropower production will be provided.

  2. Time to delivery: Transfers for threatened preterm labour and prelabour rupture of membranes in Western Australia.

    Science.gov (United States)

    Hollingworth, Jade; Pietsch, Rachel; Epee-Bekima, Mathias; Nathan, Elizabeth

    2018-02-01

    To describe the outcomes of patients transferred to King Edward Memorial Hospital (KEMH) with signs of labour at preterm gestations. A retrospective observational study of the 69 cases transferred to KEMH during 2015. Patient transfers from all locations across Western Australia (WA) to the sole tertiary perinatal centre in Perth. Pregnant women within WA with threatened or actual preterm labour (PTL) or preterm prelabour rupture of membranes (PPROM) between 23 and 32 weeks gestation. The occurrence of delivery during the admission and time-to-delivery as well as length of admission and association between clinical factors and time-to-delivery. The percentage of the study population delivered during the admission following transfer was 72.5%. Eighty-six per cent of those who delivered did so within 72 hours of transfer. The median time from transfer to delivery was 1 day. Sixty-three per cent of those who did not deliver during the admission progressed to 36 weeks gestation. Patients transferred with PPROM were less likely to deliver during the admission compared to those with uterine activity (50% versus 19.6%, P = 0.007) and nulliparas were more likely to deliver (93.5% versus 55.3%, P < 0.001). The majority of women transferred with signs of PTL progress to delivery during the same admission with the highest risk of delivery being the first 72 hours following transfer. If the pregnancy is ongoing at 72 hours, there is a reasonable chance of progression to late preterm gestation supporting the return of woman to their place of origin for antenatal care following discharge. © 2017 National Rural Health Alliance Inc.

  3. Insect-resistance and high-yield transgenic tobacco obtained by ...

    African Journals Online (AJOL)

    The modified synthesized VHb gene and insectidal gene (GFMcryIA) were transferred to tobacco plants by Agrobacterium-mediated transformation. The bivalent genes were inserted successfully into the tobacco genome and detected by PCR amplification. Southern blot and Western blot analyses showed that VHb gene ...

  4. Computer Simulation Western

    International Nuclear Information System (INIS)

    Rasmussen, H.

    1992-01-01

    Computer Simulation Western is a unit within the Department of Applied Mathematics at the University of Western Ontario. Its purpose is the development of computational and mathematical methods for practical problems in industry and engineering and the application and marketing of such methods. We describe the unit and our efforts at obtaining research and development grants. Some representative projects will be presented and future plans discussed. (author)

  5. Antibody reaction of human anti-Toxoplasma gondii positive and negative sera with Neospora caninum antigens

    OpenAIRE

    Nam, Ho-Woo; Kang, Seung-Won; Choi, Won-Young

    1998-01-01

    Anti-Neospora caninum antibody was detected in anti-Toxoplasma gondii positive and negative human sera by ELISA, western blot and immunofluorescence assay (IFA). Twelve cases out of 172 (6.7%) Toxoplasma-positive sera cross-reacted with both T. gondii and N. caninum antigens, and one out of 110 Toxoplasma-negative sera reacted with N. caninum antigen by ELISA. By western blot, all 12 sera reacted with T. gondii antigens with various banding patterns but specifically at 30 kDa (SAG1) and 22 kD...

  6. Globalisation and western music historiography

    Directory of Open Access Journals (Sweden)

    Romanou Katy

    2015-01-01

    Full Text Available Globalisation of musicology and music history aims to fuse the divisions created during Western music’s acme, and is referred to as “post-European historical thinking”. Therefore, “post” and “pre” European historical thinking have much in common. One aspect of this process of fragmentation was that music history was separated from theory and that Western Music Histories succeeded General Music Histories (a development described in some detail in the article. Connecting global music history with “post-European” historical thinking is one among numerous indications of Western awareness that European culture has reached some sort of a terminal phase. Concurrently, countries that have been developing by following Western Europe as a prototype, are leading today some past phase of Western development, which, with the ideas of cultural relativism prevailing, are not considered inferior.

  7. The genome-wide expression profile of Curcuma longa-treated cisplatin-stimulated HEK293 cells

    Science.gov (United States)

    Sohn, Sung-Hwa; Ko, Eunjung; Chung, Hwan-Suck; Lee, Eun-Young; Kim, Sung-Hoon; Shin, Minkyu; Hong, Moochang; Bae, Hyunsu

    2010-01-01

    AIM The rhizome of turmeric, Curcuma longa (CL), is a herbal medicine used in many traditional prescriptions. It has previously been shown that CL treatment showed greater than 47% recovery from cisplatin-induced cell damage in human kidney HEK 293 cells. This study was conducted to evaluate the recovery mechanisms of CL that occur during cisplatin induced nephrotoxicity by examining the genome wide mRNA expression profiles of HEK 293 -cells. METHOD Recovery mechanisms of CL that occur during cisplatin-induced nephrotoxicity were determined by microarray, real-time PCR, immunofluorescent confocal microscopy and Western blot analysis. RESULTS The results of microarray analysis and real-time PCR revealed that NFκB pathway-related genes and apoptosis-related genes were down-regulated in CL-treated HEK 293 cells. In addition, immunofluorescent confocal microscopy and Western blot analysis revealed that NFκB p65 nuclear translocation was inhibited in CL-treated HEK 293 cells. Therefore, the mechanism responsible for the effects of CL on HEK 293 cells is closely associated with regulation of the NFκB pathway. CONCLUSION CL possesses novel therapeutic agents that can be used for the prevention or treatment of cisplatin-induced renal disorders. PMID:20840446

  8. Expression of Toll-like receptor 4 in hippocampus of rat model with temporal lobe epilepsy

    Directory of Open Access Journals (Sweden)

    PAN Li-ping

    2013-12-01

    Full Text Available Objective To investigate the expression of Toll-like receptor 4 (TLR4 protein in hippocampus of rat model with temporal lobe epilepsy after status epilepticus (SE and explore its function in the pathogenesis of temporal lobe epilepsy. Methods Rat model with temporal lobe epilepsy was induced by lithium chloride (LiCl-pilocarpine. Total protein was extracted from hippocampus and rat brain slices were obtained at different time points (0, 1, 6, 12, 24, 48 h and 7, 10, 30, 50 d after SE. Western blotting and immunohistochemical staining were used for detection of the expression of TLR4 in the hippocampus. Results The results of Western blotting showed the TLR4 protein expression at 0, 1, 6, 12, 24, 48 h and 7, 10, 30 d after SE was higher than that in the control group (P 0.05. Conclusion TLR4 protein was mainly expressed in cytoplasm of pyramidal cells in CA3 area of hippocampus. TLR4 protein expression in the hippocampus was increased in varying degrees at different observation time points after SE, indicating that TLR4 may play an important role in the development of epilepsy.

  9. On the Eastern and Western Cultures as Reflected in Differences in Food Culture

    Institute of Scientific and Technical Information of China (English)

    黄卓; 张海南

    2015-01-01

    When talking about differences between Eastern and Western culture,we should first think of the eating cultural differences.There are many differences in Eastern and Western food cultures,in this paper it will introduce the different food concepts,the different eating goals,the different eating habits,etc.A comparison study of Chinese and Western food culture still makes sense through the analysis of cultural differences between Chinese and Western food,we can understand their own cultural traditions in China and the West.At the same time it is able to carry out improvement and innovation of Chinese culture.Throughout the comparisons,coupled with the differences of the concept of Western food culture,objects,methods,ownership and nature,it studies these differences,identifies areas for mastery of the place,promotes cultural exchange.Thus it enables China to the world,and to make the world know China better.

  10. On the Eastern and Western Cultures as Reflected in Differences in Food Culture

    Institute of Scientific and Technical Information of China (English)

    黄卓; 张海南

    2015-01-01

    When talking about differences between Eastern and Western culture,we should first think of the eating cultural differences.There are many differences in Eastern and Western food cultures,in this paper it will introduce the different food concepts,the different eating goals,the different eating habits,etc. A comparison study of Chinese and Western food culture still makes sense through the analysis of cultural differences between Chinese and Western food,we can understand their own cultural traditions in China and the West.At the same time it is able to carry out improvement and innovation of Chinese culture. Throughout the comparisons,coupled with the differences of the concept of Western food culture,objects,methods,ownership and nature,it studies these differences,identifies areas for mastery of the place,promotes cultural exchange.Thus it enables China to the world,and to make the world know China better.

  11. Systematic analysis of phosphotyrosine antibodies recognizing single phosphorylated EPIYA-motifs in CagA of Western-type Helicobacter pylori strains.

    Directory of Open Access Journals (Sweden)

    Judith Lind

    Full Text Available The clinical outcome of Helicobacter pylori infections is determined by multiple host-pathogen interactions that may develop to chronic gastritis, and sometimes peptic ulcers or gastric cancer. Highly virulent strains encode a type IV secretion system (T4SS that delivers the effector protein CagA into gastric epithelial cells. Translocated CagA undergoes tyrosine phosphorylation at EPIYA-sequence motifs, called A, B and C in Western-type strains, by members of the oncogenic Src and Abl host kinases. Phosphorylated EPIYA-motifs mediate interactions of CagA with host signaling factors--in particular various SH2-domain containing human proteins--thereby hijacking multiple downstream signaling cascades. Observations of tyrosine-phosphorylated CagA are mainly based on the use of commercial phosphotyrosine antibodies, which originally were selected to detect phosphotyrosines in mammalian proteins. Systematic studies of phosphorylated EPIYA-motif detection by the different antibodies would be very useful, but are not yet available. To address this issue, we synthesized phospho- and non-phosphopeptides representing each predominant Western CagA EPIYA-motif, and determined the recognition patterns of seven different phosphotyrosine antibodies in Western blots, and also performed infection studies with diverse representative Western H. pylori strains. Our results show that a total of 9-11 amino acids containing the phosphorylated EPIYA-motifs are necessary and sufficient for specific detection by these antibodies, but revealed great variability in sequence recognition. Three of the antibodies recognized phosphorylated EPIYA-motifs A, B and C similarly well; whereas preferential binding to phosphorylated motif A and motifs A and C was found with two and one antibodies, respectively, and the seventh anti-phosphotyrosine antibody did not recognize any phosphorylated EPIYA-motif. Controls showed that none of the antibodies recognized the corresponding non

  12. Solar energy futures in a Western European context

    Energy Technology Data Exchange (ETDEWEB)

    Nakicenovic, N; Messner, S

    1983-02-01

    The study considers three limiting scenarios that specify possible but not necessarily likely transitions to sustainable energy futures for Western Europe. Two scenarios consider exclusively solar futures--one based on centralized solar technologies (Hard scenario) and the other on decentralized, user-oriented technologies (Soft scenario). The third scenario, based on nuclear technologies, incorporates an intermediate degree of centralization in the energy system and serves as a comparison to the two exclusively solar scenarios. All three scenarios lead to sustainable energy futures before the year 2100, which is the time horizon of the study. While all three scenarios eliminate Western Europe's dependence on domestic and foreign fossil energy sources, the Hard Solar scenario requires substantial imports of solar produced hydrogen. The scenarios are based on dynamic balances of energy demand and supply using detailed models to achieve consistency. The overall implications of each scenario are that fundamental but different changes of the whole energy system, economic structure and life-styles are necessary in order to achieve sustainable energy futures in Western Europe.

  13. Solar energy futures in a Western European context

    Energy Technology Data Exchange (ETDEWEB)

    Nakicenovic, N; Messner, S

    1983-02-01

    The study considers three limiting scenarios that specify possible but not necessarily likely transitions to sustainable energy future for Western Europe. Two scenarios consider exclusively solar futures - one based on centralized solar technologies (hard scenario) and the other on decentralized, user-oriented technologies (soft scenario). The third scenario, based on nuclear technologies, incorporates an intermediate degree of centralization in the energy system and serves as a comparison to the two exclusively solar scenarios. All three scenarios lead to sustainable energy futures before the year 2100, which is the time horizon of the study. While all three scenarios eliminate Western Europe's dependence on domestic and foreign fossil energy sources, the Hard Solar scenario requires substantial imports of solar produced hydrogen. The scenarios are based on dynamic balances of energy demand and supply using detailed models to achieve consistency. The overall implications of each scenario are that fundamental but different changes of the whole energy system, economic structure and life-styles are necessary in order to achieve sustainable energy futures in Western Europe.

  14. Identification of proteins in a human pleural exudate using two-dimensional preparative liquid-phase electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry.

    Science.gov (United States)

    Nilsson, C L; Puchades, M; Westman, A; Blennow, K; Davidsson, P

    1999-01-01

    Pleural effusion may occur in patients suffering from physical trauma or systemic disorders such as infection, inflammation, or cancer. In order to investigate proteins in a pleural exudate from a patient with severe pneumonia, we used a strategy that combined preparative two-dimensional liquid-phase electrophoresis (2-D LPE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Western blotting. Preparative 2-D LPE is based on the same principles as analytical 2-D gel electrophoresis, except that the proteins remain in liquid phase during the entire procedure. In the first dimension, liquid-phase isoelectric focusing allows for the enrichment of proteins in liquid fractions. In the Rotofor cell, large volumes (up to 55 mL) and protein amounts (up to 1-2 g) can be loaded. Several low abundance proteins, cystatin C, haptoglobin, transthyretin, beta2-microglobulin, and transferrin, were detected after liquid-phase isoelectric focusing, through Western blotting analysis, in a pleural exudate (by definition, >25 g/L total protein). Direct MALDI-TOF-MS analysis of proteins in a Rotofor fraction is demonstrated as well. MALDI-TOF-MS analysis of a tryptic digest of a continuous elution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fraction confirmed the presence of cystatin C. By applying 2-D LPE, MALDI-TOF-MS, and Western blotting to the analysis of this pleural exudate, we were able to confirm the identity of proteins of potential diagnostic value. Our findings serve to illustrate the usefulness of this combination of methods in the analysis of pathological fluids.

  15. [Knockdown of DNA-PKcs inhibits cell cycle and its mechanism of drug-resistant Bel7402/5-Fu hepatocellular carcinoma cells].

    Science.gov (United States)

    Li, Dayu; Liu, Yun; Yu, Chunbo; Liu, Xiping; Fan, Fang

    2017-12-01

    Objective To study the effect of the knock-down of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) on the cell cycle of the multidrug-resistant (MDR) Bel7402/5-Fu hepatocellular carcinoma cells and its MDR mechanism. Methods After cationic liposome-mediated siDNA-PKcs oligonucleotide transfection, the drug sensitivity of Bel7402/5-Fu cells to 5-fluorouracil (5-Fu) and adriamycin (ADM) was determined by MTT assay; the cell cycle were detected by flow cytometry; meanwhile, the protein expressions of cell cycle-related proteins P21, cell cycle protein B1 (cyclin B1), cell cycle division protein 2 (CDC2) were tested by Western blotting; the expressions of ataxia telangiectasia mutated (ATM) and p53 at both mRNA and protein levels were detected by real-time PCR and Western blot analysis. Results The MTT results showed siDNA-PKcs increased the chemotherapeutic sensitivity of Bel7402/5-Fu cells to 5-Fu and ADM. The flow cytometric analysis showed siDNA-PKcs decreased the percentage of S-phase cells but increased the percentage of G2/M phase cells. Western blotting showed siDNA-PKcs increased the protein expression of P21 but decreased cyclinB1 and CDC2 proteins. In addition, siDNA-PKcs also increased the expressions of ATM and p53. Conclusion DNA-PKcs silencing increases P21 while decreases cyclin B1 and CDC2 expressions, and finally induces G2/M phase arrest in Bel7402/5-Fu cells, which may be related to ATM-p53 signaling pathway.

  16. Cloning, Expression and Purification of the Recombinant HIV-1 Tat-Nef Fusion Protein in Prokaryotic Expression System

    Directory of Open Access Journals (Sweden)

    Somayeh Kadkhodayan

    2016-07-01

    Full Text Available Abstract Background: Nef is one of the HIV-1 critical proteins, because it is essential for viral replication and AIDS disease progression and induction of immune response against it can partially inhibit viral infection. Moreover, a domain of the HIV-1 Trans-Activator of Transcription (Tat, 48-60 aa could act as a cell penetrating peptide (CPP. In current study, cloning and expression of Tat-Nef fusion protein was performed in E. coli for the first time. The protein expression was confirmed by western blot analysis and was purified using reverse staining method. Materials and Methods: In this experimental study, primarily, cloning of Tat-Nef fusion gene was done in pGEX6p2 expression vector. Then, the expression of Tat-Nef recombinat protein in E.coli BL21 (DE3 strain was performed by using IPTG inducer. The protein expression was confirmed by SDS-PAGE and western blotting using anti-Nef monoclonal antibody. Then, the recombinant fusion protein was purified from gel using reverse staining method. Results: The results of PCR analysis and enzyme digestion showed a clear band of ~ 726 bp in agarose gel indicating the correct Tat-Nef fusion cloning in pGEX6p2 prokaryotic expression vector. In addition, a 54 kDa band of Tat-Nef on SDS-PAGE revealed Tat-Nef protein expression that western blot analysis using anti-Nef monoclonal antibody confirmed it. Conclusion: The purified Tat-Nef recombinant fusion protein will be used as an antigen for protein vaccine design against HIV infection.

  17. Antigenic Cross-Reactivity Anti-Birtoxin Antibody against Androctonus crassicauda Venom

    Directory of Open Access Journals (Sweden)

    SuhandanAdigüzel Van-Zoelen

    2015-10-01

    Full Text Available Background: Antivenom is still widely used in the treatment of envenomation as there are no vaccines or other effective agents available against animal venoms. Recently, neurotoxins named birtoxin family have been described from Parabuthus transvaalicus and Androctonus crassicauda. The aim of the present study was to test the antibirtoxinantibodies for their ability to neutralize the lethal effects of A. crassicauda scorpion venom.Methods: SDS-PAGE and Western blotting used the presence of components from A. crassicauda and P.transvaalicus scorpion venoms and to determine the degree of cross-reactivity. The Minimum Lethal Dose (MLD of venom was assessed by subcutaneously (sc injections in mice.Results: The MLD of the A. crassicauda venom was 35 μg/ 20g mouse by sc injection route. Western blotting showed the presence of components from A. crassicauda and P. transvaalicus scorpion venoms strongly cross react with the A. crassicauda antivenom. However, Western blotting of the A. crassicauda scorpion venom using the Refik Saydam Public Health Agency (RSPHA generated antibody showed that not all the venom components cross reacted with the anti-birtoxin antibody. The antibodies only cross reacted with components falling under the 19 kDa protein size of A. crassicauda venom.Conclusion: The bioassays and Western blotting of A. crassicauda venom with the anti-birtoxin antibodies produced against a synthetic peptide showed that these antibodies cross reacted but did not neutralize the venom of A. crassicauda.

  18. Formulation, Characterization, and Antitumor Properties of Trans- and Cis-Citral in the 4T1 Breast Cancer Xenograft Mouse Model.

    Science.gov (United States)

    Zeng, San; Kapur, Arvinder; Patankar, Manish S; Xiong, May P

    2015-08-01

    Citral is composed of a random mixture of two geometric stereoisomers geranial (trans-citral) and neral (cis-citral) yet few studies have directly compared their in vivo antitumor properties. A micelle formulation was therefore developed. Geranial and neral were synthesized. Commercially-purchased citral, geranial, and neral were formulated in PEG-b-PCL (block sizes of 5000:10,000, Mw/Mn 1.26) micelles. In vitro degradation, drug release, cytotoxicity, flow cytometry, and western blot studies were conducted. The antitumor properties of drug formulations (40 and 80 mg/kg based on MTD studies) were evaluated on the 4T1 xenograft mouse model and tumor tissues were analyzed by western blot. Micelles encapsulated drugs with >50% LE at 5-40% drug to polymer (w/w), displayed sustained release (t1/2 of 8-9 h), and improved drug stability at pH 5.0. The IC50 of drug formulations against 4T1 cells ranged from 1.4 to 9.9 μM. Western blot revealed that autophagy was the main cause of cytotoxicity. Geranial at 80 mg/kg was most effective at inhibiting tumor growth. Geranial is significantly more potent than neral and citral at 80 mg/kg (p < 0.001) and western blot of tumor tissues confirms that autophagy and not apoptosis is the major mechanism of tumor growth inhibition in p53-null 4T1 cells.

  19. The P2X7 receptor regulates cell survival, migration and invasion of pancreatic ductal adenocarcinoma cells

    DEFF Research Database (Denmark)

    Giannuzzo, Andrea; Pedersen, Stine Helene Falsig; Novak, Ivana

    2015-01-01

    of the ATP receptors, the P2X7 receptor (P2X7R) could be an important player in PDAC behaviour. METHODS: We determined the expression (real time PCR and Western blot) and localization (immunofluorescence) of P2X7R in human PDAC cell lines (AsPC-1, BxPC-3, Capan-1, MiaPaCa-2, Panc-1) and a "normal" human...

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

    Science.gov (United States)

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

    2018-02-01

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

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

    Science.gov (United States)

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

    2013-03-07

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

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

    Science.gov (United States)

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

    1993-01-01

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

  3. Western values and the Russian energy weapon

    Science.gov (United States)

    Domingues, Bennett K.

    become heavily involved in oil and gas extraction projects in the region, in particular in Kazakhstan. Yet, efforts to transport that energy to western markets without Russian involvement have met with strong resistance. This thesis demonstrates that part of the reason western firms have been less successful than they hoped to be is because western firms, in particular American firms, are so bound by western ethical norms and the statues that codify them, that they have lost their competitive advantage. The thesis concludes with a discussion of the current day balance of energy resources in Europe, noting that Europe is rapidly growing heavily dependent on Russian oil and gas resources. Given the precedents shown in Georgia and Ukraine, it is only a matter of time before Europe grows so dependent on Russian energy that the individual countries will lose their political and economic independence and in turn, their ability to project western values and values-based ideas throughout the world with impunity.

  4. Review of western Baltic cod (Gadus morhua) recruitment dynamics

    DEFF Research Database (Denmark)

    Hüssy, Karin

    2011-01-01

    Important processes in the recruitment dynamics of western Baltic cod (Gadus morhua) are identified. Spawning areas are in the deep, saline waters below 20–40 m, depending on area topography. Spatial distribution remains relatively stable over time. Peak spawning shows an area-specific pattern...

  5. Violence the Western way.

    Science.gov (United States)

    Roth, B E

    1997-10-01

    Despite the quiet revolution in response to changing conceptualizations of gender in psychoanalysis, the Western has remained the domain of aggressive phallic masculinity. The iconic imagery of the Western, when combined with its narrative trajectory, is used to tell stories of violent encounters between men. The acceptance of the genre, and its duplication by other cultures and film makers, indicates that the Westerns' imagery and moral solutions tap into some basic deep structures of anxiety and pleasure in violence between men. As long as societies require subtle sublimations of aggressive and violent drives, it is likely that men will seek imaginary regressive experiences to discharge frustrations.

  6. Paparan zat besi pada ekspresi protein spesifik extracellular polymeric substance biofilm Aggregatibacter actinomycetemcomitans

    Directory of Open Access Journals (Sweden)

    Marchella Hendrayanti W

    2014-06-01

    Full Text Available Background: The study of biofilms bacteria could be an alternative of preventive treatment in reducing prevalence of aggressive periodontitis in the community, because biofilm protects the bacteria from environmental conditions, including the attack of immune system and antimicrobial. Aggregatibacter actinomycetemcomitans is a major cause of bacterial aggressive periodontitis. Purpose: This study aims to examine the iron exposure to specific protein expression of extracellular polymeric substance (EPS of Aggregatibacter actinomycetemcomitans biofilm. Methods: Protein containing EPS biofilm was isolated from cultures of A.actinomycetemcomitans. The protein was processed through several procedures: electrophoresis , electroelution , immunization of rabbits , serum isolation , and purification of antibodies. After the Western blotting procedure the antibody was used. Protein containing EPS biofilms exposed to iron, then once again isolated from cultures of A. actinomycetemcomitans. The electrophoresis and Western blotting were done on the isolated protein. Results: The result showed that the the expression of specific proteins in EPS biofilm decreased in response to iron exposure. Conclusions: Iron exposure could influenced the specific protein expression in EPS biofilm of Aggregatibacter actinomycetemcomitans.Latar belakang: Penelitian terhadap bakteri biofilm dapat menjadi alternatif perawatan preventif dalam menurunkan prevalensi periodontitis agresif di masyarakat, karena biofilm melindungi bakteri terhadap kondisi lingkungan, termasuk serangan sistem imun dan antimikroba. Aggregatibacter actinomycetemcomitans merupakan bakteri penyebab utama periodontitis agresif. Tujuan: Studi ini bertujuan meneliti paparan zat besi terhadap ekspresi protein spesifik extracellular polymeric substance (EPS Aggregatibacter actinomycetemcomitans. Metode: Protein yang mengandung EPS biofilm diisolasi dari kultur A. actinomycetemcomitans. Protein yang diisolasi

  7. Life time fatality risk assessment due to variation of indoor radon concentration in dwellings in western Haryana, India

    International Nuclear Information System (INIS)

    Kansal, Sandeep; Mehra, Rohit; Singh, N.P.

    2012-01-01

    Indoor radon measurements in 60 dwellings belonging to 12 villages of Sirsa, Fatehbad and Hisar districts of western Haryana, India, have been carried out, using LR-115 type II cellulose nitrate films in the bare mode. The annual average indoor radon value in the studied area varies from 76.00 to 115.46 Bq m −3 , which is well within the recommended action level 200–300 Bq m −3 (). The winter/summer ratio of indoor radon ranges from 0.78 to 2.99 with an average of 1.52. The values of annual average dose received by the residents and Life time fatality risk assessment due to variation of indoor radon concentration in dwellings of studied area suggests that there is no significance threat to the human beings due to the presence of natural radon in the dwellings. - Highlights: ► The radon concentration values in the dwellings are 2–3 times more than the world average of 40 Bq m −3 . ► These values are lower than the recommended action level of 200–300 Bq m −3 (). ► The annual effective dose is less than the recommended action level of 3–10 mSv per year (). ► The values of life time fatality risk determined for the studied area are within safe standards. ► There is no significant threat to the human beings due to the presence of natural radon in the dwellings.

  8. Yersinia enterocolitica in the Western Cape | Finlayson | South ...

    African Journals Online (AJOL)

    Yersinia enterocolitica, serotype 3, phage type 9a, has been isolated for the first time in the Western Cape. Sera from 59 abattoir workers were investigated for the presence of 0 and H agglutinins. These were present in one sample, suggesting a past infection. Sera from 115 Nama-speaking adults of the Kuboes area ...

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    1999-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Khaled DJELOUAH

    2015-01-01

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

  12. Phenotypic and genetic characterization of western prairie clover collections from the western USA

    Science.gov (United States)

    Kishor Bhattarai; B. Shaun Bushman; Douglas A. Johnson; John G. Carman

    2010-01-01

    Few North American legumes are available for rangeland revegetation in the semiarid western United States. Western prairie clover (Dalea ornata [Douglas ex Hook.] Eaton & J. Wright) is a perennial legume with desirable forage characteristics and is distributed in the northern Great Basin, Snake River Basin, and southern Columbia Plateau. Understanding the...

  13. True Time API Link (real time arrival info)

    Data.gov (United States)

    Allegheny County / City of Pittsburgh / Western PA Regional Data Center — This link will take you to the site where you can create an account to access Port Authority's real time arrival information. To request access to Port Authority's...

  14. Language Ability and Adjustment: Western Expatriates in China

    DEFF Research Database (Denmark)

    Selmer, Jan

    2006-01-01

    was directed to Western business expatriates assigned to China. Controlling for the time expatriates had spent in China, results showed that their language ability had a positive association with their sociocultural adjustment. Not surprisingly, this positive relationship was strongest for interaction...... adjustment and weakest for work adjustment. The straightforward implications of these clear findings are discussed in detail....

  15. Harvesting systems for western stand health improvement cuttings

    Science.gov (United States)

    Bruce R. Hartsough; Bryce J. Stokes; Joseph F. McNeel; William F. Watson

    1995-01-01

    A significant percentage of the forested area in the western United States is comprised of stands that have been altered over time by human activities, especially fire suppression, and are now being damaged by droughts, insect attacks, and wildfires. These stands should be returned to a condition where "biotic and abiotic influences do not threaten resource...

  16. 78 FR 62350 - FFP Qualified Hydro 14, LLC; Western Minnesota Municipal Power Agency; Notice Announcing...

    Science.gov (United States)

    2013-10-18

    .... 14491-000] FFP Qualified Hydro 14, LLC; Western Minnesota Municipal Power Agency; Notice Announcing... applications were filed by FFP Qualified Hydro 14, LLC for Project No. 13579-002 and Western Minnesota... regular business day. See 18 CFR 385.2001(a)(2) (2013). On October 21, 2013, at 10:00 a.m. (Eastern Time...

  17. Lipocalin 2 Suppresses Ocular Inflammation by Inhibiting the Activation of NF-κβ Pathway in Endotoxin-Induced Uveitis

    Directory of Open Access Journals (Sweden)

    Wenyi Tang

    2018-03-01

    Full Text Available Background/Aims: Lipocalin 2 (LCN2, an important mediator of a variety of cellular processes, is involved in regulating the inflammatory response, but its roles in different inflammatory diseases are controversial. Because the role of LCN2 in ocular inflammation has been unclear until now, we explored the function of LCN2 in lipopolysaccharide (LPS-induced ocular inflammation in vivo and in vitro. Methods: Endotoxin-induced uveitis (EIU was induced in male Sprague Dawley rats by the intravitreal injection of LPS. The expression and location of LCN2 in the retina were detected with western blotting and immunohistochemistry, respectively. We determined the clinical scores for anterior inflammation, quantified the infiltrated inflammatory cells, and measured the pro-inflammatory factors to determine the anti-inflammatory effects of LCN2 in EIU eyes. Cultured primary rat Müller cells were stimulated with LPS and the expression and secretion of LCN2 were measured with real-time PCR, western blotting, and an ELISA. After Müller cells were cotreated with LPS and LCN2 or PBS, the expression and secretion of TNF-α, IL-6, and MCP-1 were examined with realtime PCR, western blotting, and ELISAs. Western blotting and immunofluorescence were used to detect the phosphorylation and cellular distribution of nuclear factor kappaB (NF-κB subunit p65. Results: In EIU, the expression of LCN2 was significantly upregulated in the retina, especially in the outer nuclear layer (mainly composed of Müller cells. LPS stimulation of cultured Müller cells also markedly elevated LCN2 expression. Intravitreal injection of LCN2 significantly reduced the clinical scores, inflammatory infiltration, and protein leakage in EIU, which correlated with the reduced levels of proinflammatory factors in the aqueous humor and retina. LCN2 treatment also reduced the expression and secretion of TNF-α, IL-6, and MCP-1 in LPS-stimulated Müller cells. LCN2 inhibited the inflammatory

  18. 137Cs in the western South Pacific Ocean

    International Nuclear Information System (INIS)

    Yamada, Masatoshi; Wang Zhongliang

    2007-01-01

    The 137 Cs activities were determined for seawater samples from the East Caroline, Coral Sea, New Hebrides, South Fiji and Tasman Sea (two stations) Basins of the western South Pacific Ocean by γ spectrometry using a low background Ge detector. The 137 Cs activities ranged from 1.4 to 2.3 Bq m -3 over the depth interval 0-250 m and decreased exponentially from the subsurface to 1000 m depth. The distribution profiles of 137 Cs activity at these six western South Pacific Ocean stations did not differ from each other significantly. There was a remarkable difference for the vertical profiles of 137 Cs activity between the East Caroline Basin station in this study and the GEOSECS (Geochemical Ocean Sections Study) station at the same latitude in the Equatorial Pacific Ocean; the 137 Cs inventory over the depth interval 100-1000 m increased from 400 ± 30 Bq m -2 to 560 ± 30 Bq m -2 during the period from 1973 to 1992. The total 137 Cs inventories in the western South Pacific Ocean ranged from 850 ± 70 Bq m -2 in the Coral Sea Basin to 1270 ± 90 Bq m -2 in the South Fiji Basin. Higher 137 Cs inventories were observed at middle latitude stations in the subtropical gyre than at low latitude stations. The 137 Cs inventories were 1.9-4.5 times (2.9 ± 0.7 on average) and 1.7-4.3 times (3.1 ± 0.7 on average) higher than that of the expected deposition density of atmospheric global fallout at the same latitude and that of the estimated 137 Cs deposition density in 10 o latitude by 10 deg. longitude grid data obtained by Aoyama et al. [Aoyama M, Hirose K, Igarashi Y. Re-construction and updating our understanding on the global weapons tests 137 Cs fallout. J Environ Monit 2006;8:431-438], respectively. The possible processes for higher 137 Cs inventories in the western South Pacific Ocean than that of the expected deposition density of atmospheric global fallout may be attributable to the inter-hemisphere dispersion of the atmospheric nuclear weapons testing 137 Cs from

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

    International Nuclear Information System (INIS)

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

    1988-01-01

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

  20. Use of social media by Western European hospitals: longitudinal study.

    Science.gov (United States)

    Van de Belt, Tom H; Berben, Sivera A A; Samsom, Melvin; Engelen, Lucien J L P G; Schoonhoven, Lisette

    2012-05-01

    Patients increasingly use social media to communicate. Their stories could support quality improvements in participatory health care and could support patient-centered care. Active use of social media by health care institutions could also speed up communication and information provision to patients and their families, thus increasing quality even more. Hospitals seem to be becoming aware of the benefits social media could offer. Data from the United States show that hospitals increasingly use social media, but it is unknown whether and how Western European hospitals use social media. To identify to what extent Western European hospitals use social media. In this longitudinal study, we explored the use of social media by hospitals in 12 Western European countries through an Internet search. We collected data for each country during the following three time periods: April to August 2009, August to December 2010, and April to July 2011. We included 873 hospitals from 12 Western European countries, of which 732 were general hospitals and 141 were university hospitals. The number of included hospitals per country ranged from 6 in Luxembourg to 347 in Germany. We found hospitals using social media in all countries. The use of social media increased significantly over time, especially for YouTube (n = 19, 2% to n = 172, 19.7%), LinkedIn (n =179, 20.5% to n = 278, 31.8%), and Facebook (n = 85, 10% to n = 585, 67.0%). Differences in social media usage between the included countries were significant. Social media awareness in Western European hospitals is growing, as well as its use. Social media usage differs significantly between countries. Except for the Netherlands and the United Kingdom, the group of hospitals that is using social media remains small. Usage of LinkedIn for recruitment shows the awareness of the potential of social media. Future research is needed to investigate how social media lead to improved health care.

  1. Developing Biomass Equations for Western Hemlock and Red Alder Trees in Western Oregon Forests

    Science.gov (United States)

    Krishna Poudel; Hailemariam Temesgen

    2016-01-01

    Biomass estimates are required for reporting carbon, assessing feedstock availability, and assessing forest fire threat. We developed diameter- and height-based biomass equations for Western hemlock (Tsuga heterophylla (Raf.) Sarg.) and red alder (Alnus rubra Bong.) trees in Western Oregon. A system of component biomass...

  2. Airborne measurements of western U.S. wildfire emissions: Comparison with prescribed burning and air quality implications: Western U.S. Wildfire Emissions

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xiaoxi [School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta Georgia USA; Now at Cooperative Institute for Research in Environmental Sciences, University of Colorado Boulder, Boulder Colorado USA; Now at Department of Chemistry, University of Colorado Boulder, Boulder Colorado USA; Huey, L. Gregory [School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta Georgia USA; Yokelson, Robert J. [Department of Chemistry, University of Montana, Missoula Montana USA; Selimovic, Vanessa [Department of Chemistry, University of Montana, Missoula Montana USA; Simpson, Isobel J. [Department of Chemistry, University of California, Irvine California USA; Müller, Markus [Department of Chemistry, University of Montana, Missoula Montana USA; Institute for Ion Physics and Applied Physics, University of Innsbruck, Innsbruck Austria; Jimenez, Jose L. [Cooperative Institute for Research in Environmental Sciences, University of Colorado Boulder, Boulder Colorado USA; Department of Chemistry, University of Colorado Boulder, Boulder Colorado USA; Campuzano-Jost, Pedro [Cooperative Institute for Research in Environmental Sciences, University of Colorado Boulder, Boulder Colorado USA; Department of Chemistry, University of Colorado Boulder, Boulder Colorado USA; Beyersdorf, Andreas J. [NASA Langley Research Center, Hampton Virginia USA; Now at Department of Chemistry, California State University, San Bernardino California USA; Blake, Donald R. [Department of Chemistry, University of California, Irvine California USA; Butterfield, Zachary [Earth and Environmental Sciences Division, Los Alamos National Laboratory, Los Alamos New Mexico USA; Now at Department of Atmospheric, Oceanic, and Space Sciences, University of Michigan, Ann Arbor Michigan USA; Choi, Yonghoon [NASA Langley Research Center, Hampton Virginia USA; Science Systems and Applications, Inc., Hampton Virginia USA; Crounse, John D. [Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena California USA; Day, Douglas A. [Cooperative Institute for Research in Environmental Sciences, University of Colorado Boulder, Boulder Colorado USA; Department of Chemistry, University of Colorado Boulder, Boulder Colorado USA; Diskin, Glenn S. [NASA Langley Research Center, Hampton Virginia USA; Dubey, Manvendra K. [Earth and Environmental Sciences Division, Los Alamos National Laboratory, Los Alamos New Mexico USA; Fortner, Edward [Center for Aerosol and Cloud Chemistry, Aerodyne Research Inc., Billerica Massachusetts USA; Hanisco, Thomas F. [Atmospheric Chemistry and Dynamics Laboratory, NASA Goddard Space Flight Center, Greenbelt Maryland USA; Hu, Weiwei [Cooperative Institute for Research in Environmental Sciences, University of Colorado Boulder, Boulder Colorado USA; Department of Chemistry, University of Colorado Boulder, Boulder Colorado USA; King, Laura E. [School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta Georgia USA; Kleinman, Lawrence [Environmental and Climate Sciences Department, Brookhaven National Laboratory, Upton New York USA; Meinardi, Simone [Department of Chemistry, University of California, Irvine California USA; Mikoviny, Tomas [Department of Chemistry, University of Oslo, Oslo Norway; Onasch, Timothy B. [Center for Aerosol and Cloud Chemistry, Aerodyne Research Inc., Billerica Massachusetts USA; Palm, Brett B. [Cooperative Institute for Research in Environmental Sciences, University of Colorado Boulder, Boulder Colorado USA; Department of Chemistry, University of Colorado Boulder, Boulder Colorado USA; Peischl, Jeff [Cooperative Institute for Research in Environmental Sciences, University of Colorado Boulder, Boulder Colorado USA; Earth System Research Laboratory, National Oceanic and Atmospheric Administration, Boulder Colorado USA; Pollack, Ilana B. [Cooperative Institute for Research in Environmental Sciences, University of Colorado Boulder, Boulder Colorado USA; Earth System Research Laboratory, National Oceanic and Atmospheric Administration, Boulder Colorado USA; Now at Department of Atmospheric Science, Colorado State University, Fort Collins Colorado USA; Ryerson, Thomas B. [Earth System Research Laboratory, National Oceanic and Atmospheric Administration, Boulder Colorado USA; Sachse, Glen W. [NASA Langley Research Center, Hampton Virginia USA; Sedlacek, Arthur J. [Environmental and Climate Sciences Department, Brookhaven National Laboratory, Upton New York USA; Shilling, John E. [Atmospheric Sciences and Global Change Division, Pacific Northwest National Laboratory, Richland Washington USA; Springston, Stephen [Environmental and Climate Sciences Department, Brookhaven National Laboratory, Upton New York USA; St. Clair, Jason M. [Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena California USA; Now at Atmospheric Chemistry and Dynamics Laboratory, NASA Goddard Space Flight Center, Greenbelt Maryland USA; Now at Joint Center for Earth Systems Technology, University of Maryland, Baltimore County, Baltimore Maryland USA; Tanner, David J. [School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta Georgia USA; Teng, Alexander P. [Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena California USA; Wennberg, Paul O. [Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena California USA; Division of Engineering and Applied Science, California Institute of Technology, Pasadena California USA; Wisthaler, Armin [Institute for Ion Physics and Applied Physics, University of Innsbruck, Innsbruck Austria; Department of Chemistry, University of Oslo, Oslo Norway; Wolfe, Glenn M. [Atmospheric Chemistry and Dynamics Laboratory, NASA Goddard Space Flight Center, Greenbelt Maryland USA; Joint Center for Earth Systems Technology, University of Maryland, Baltimore County, Baltimore Maryland USA

    2017-06-14

    Wildfires emit significant amounts of pollutants that degrade air quality. Plumes from three wildfires in the western U.S. were measured from aircraft during the Studies of Emissions and Atmospheric Composition, Clouds, and Climate Coupling by Regional Surveys (SEAC4RS) and the Biomass Burning Observation Project (BBOP), both in summer 2013. This study reports an extensive set of emission factors (EFs) for over 80 gases and 5 components of submicron particulate matter (PM1) from these temperate wildfires. These include rarely, or never before, measured oxygenated volatile organic compounds and multifunctional organic nitrates. The observed EFs are compared with previous measurements of temperate wildfires, boreal forest fires, and temperate prescribed fires. The wildfires emitted high amounts of PM1 (with organic aerosol (OA) dominating the mass) with an average EF that is more than two times the EFs for prescribed fires. The measured EFs were used to estimate the annual wildfire emissions of carbon monoxide, nitrogen oxides, total nonmethane organic compounds, and PM1 from 11 western U.S. states. The estimated gas emissions are generally comparable with the 2011 National Emissions Inventory (NEI). However, our PM1 emission estimate (1530 ± 570 Gg yr-1) is over three times that of the NEI PM2.5 estimate and is also higher than the PM2.5 emitted from all other sources in these states in the NEI. This study indicates that the source of OA from BB in the western states is significantly underestimated. In addition, our results indicate prescribed burning may be an effective method to reduce fine particle emissions.

  3. Naringenin modulates the metastasis of human prostate cancer cells by down regulating the matrix metalloproteinases -2/-9 via ROS/ERK1/2 pathways

    Directory of Open Access Journals (Sweden)

    Er-Jiang Lin

    2014-08-01

    Full Text Available Metastasis is a multifactorial condition that complicates cancer treatment options and widens the target of treatment. Matrix mettalopriteinases (MMPs of the extracellular matrix (ECM are involved in metastasis, thus they present as potential targets in halting cancer metastasis. The study was undertaken to investigate the influence of naringenin, a naturally occurring flavonoid on the metastasis of human prostate cancer cells (PC-3 and DU145. Naringenin was observed to be effective in reducing the viability and migratory percentage of PC-3 and DU145 cells. Naringenin significantly reduced the expression and activities of the chief MMPs (MMP-2 and MMP-9 as assessed by western blotting, real-time PCR and gelatin zymography analysis. The influence of naringenin on extracellular signal-regulated kinase (ERK -ERK1/2 was analysed by western blotting. The results indicated that naringenin was able to effectively inhibit ERK1/2. Naringenin exposure also significantly suppressed the levels of reactive oxygen species (ROS. Naringenin thus stands as an effective chemotherapeutic agent for prostate cancer treatment that could be further explored.

  4. Gemcitabine inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells.

    Science.gov (United States)

    Yong-Xian, Gui; Xiao-Huan, Li; Fan, Zhang; Guo-Fang, Tian

    2016-10-01

    The aim of the study is to investigate the underlying molecular mechanisms by which gemcitabine (gem) inhibits proliferation and induces apoptosis in human pancreatic cancer PANC-1 cells in vitro. After PANC-1 cells had been treated by indicated concentration (0, 5, and 25 mg/L) of gem for 48 h, cell proliferation was evaluated by 3'-(4, 5 dimethyl-thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay; cell morphology was observed by transmission electron microscopy; Expression of c-IAP2 and Bcl-2 proteins was analyzed by Western blot; the activity of caspase-3 and -9 was detected by spectrophotometry. Gem significantly inhibited cell proliferation and could induce apoptosis of human pancreatic cancer PANC-1 cells, with a dose-dependent manner. Western blot analysis showed that gem significantly reduced c-IAP2 and Bcl-2 proteins expression level (P PANC-1 cells. Gem could induce apoptosis of human pancreatic cancer PANC-1 cells, probably through downregulating c-IAP2 and Bcl-2 expression levels, and at the same time activating caspase-3 and -9.

  5. Identification of the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available Immunoproteomics was used to screen the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R. The spore and vegetative proteins were separated by 2D gel electrophoresis and transferred to polyvinylidene difluoride membranes, and then western blotting was performed with rabbit immune serum against B.anthracis live spores. Immunogenic spots were cut and digested by trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was performed to identify the proteins. As a result, 11 and 45 immunogenic proteins were identified in the spores and vegetative cells, respectively; 26 of which have not been reported previously. To verify their immunogenicity, 12 of the identified proteins were selected to be expressed, and the immune sera from the mice vaccinated by the 12 expressed proteins, except BA0887, had a specific western blot band with the A16R whole cellular lytic proteins. Some of these immunogenic proteins might be used as novel vaccine candidates themselves or for enhancing the protective efficacy of a protective-antigen-based vaccine.

  6. [Preparation and preliminary application of rabbit anti-human PON2 antibodies(paraoxonase-2)].

    Science.gov (United States)

    Chen, Miao; Yang, Jin-Ju; Li, Shu-Zhen; Liu, Xiao-Lan; Liu, Ying; Zhang, Lin-Jie; Gao, Jian-En; Sun, Qi-Hong

    2008-07-01

    To preparation and characterize the rabbit polyclonal antibodies against human PON2 (paraoxonase-2). A fragment of human PON2 gene which was of low homology with rabbits but of higher hydrophilicity and immunogenicity was selected for recombinant expression in prokaryotic expression system. The rabbits were immunized with the purified GST fusion protein 3 times. The specificity and sensitivity of the anti-human PON2 polyclonal antibodies were detected by Western blot and indirect immunofluorescence. The GST-PON2 fusion protein was highly expressed in Ecoli with a molecular weight of 46 kDa. Western blot analysis proved the rabbit polyclonal antibodies could specifically recognize 39 kDa native PON2 protein expressed in several cells and tissues, such as HeLa cells, U937 cells, and human liver tissue. Indirect immunofluorescence analysis confirmed that PON2 protein was located in the cytoplasm of SY5Y cells. The rabbit polyclonal antibodies against human PON2 can specifically recognize natural protein expressed in human cells and tissues, Which can be used for further study and clinical detection of human PON2.

  7. Factors affecting the use of prenatal care by non-western women in industrialized western countries: a systematic review.

    OpenAIRE

    Boerleider, A.W.; Wiegers, T.A.; Manniën, J.; Francke, A.L.; Devillé, W.L.J.M.

    2013-01-01

    Background: Despite the potential of prenatal care for addressing many pregnancy complications and concurrent health problems, non-western women in industrialized western countries more often make inadequate use of prenatal care than women from the majority population do. This study aimed to give a systematic review of factors affecting non-western women's use of prenatal care (both medical care and prenatal classes) in industrialized western countries.Methods: Eleven databases (PubMed, Embas...

  8. Rare inborn errors associated with chronic hepatitis B virus infection

    DEFF Research Database (Denmark)

    Zhao, Qiang; Peng, Liang; Huang, Weijun

    2012-01-01

    further studied its expression by immunohistochemistry, real-time polymerase chain reaction, and western blotting. Our results showed that it was strongly expressed by healthy hepatocytes, but its expression was reduced in liver tissues with CHB, hepatitis B viral (HBV) genome-containing HepG2.2.15 cells......, as compared with healthy liver tissues and non-HBV genome-containing HepG2 cells (P = 0.022 and 0.0036, respectively)....

  9. Evidence that spinal astrocytes but not microglia contribute to the pathogenesis of paclitaxel-induced painful neuropathy

    OpenAIRE

    Zhang, Haijun; Yoon, Seo-Yeon; Zhang, Hongmei; Dougherty, Patrick M.

    2012-01-01

    Paclitaxel often induces persistent painful neuropathy as its most common treatment limiting side effect. Little is known concerning the underlying mechanisms. Given the prominent role of glial cells in many types of neuropathic pain, we investigated here the morphological and functional changes of spinal astrocytes and microglia in a rat model of paclitaxel-induced neuropathy. Immunohistochemistry, western blotting and real-time polymerase chain reaction (rt-PCR) were performed with samples ...

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

    Directory of Open Access Journals (Sweden)

    Bowlin Gary L

    2007-10-01

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

  11. 77 FR 6786 - Western Pacific Fishery Management Council; Public Meetings

    Science.gov (United States)

    2012-02-09

    ... Pacific Fishery Management Council; Public Meetings AGENCY: National Marine Fisheries Service (NMFS... meeting dates and times. SUMMARY: The Western Pacific Fishery Management Council (Council) announces... Merizo Community Meeting 6 p.m.-9 p.m., Wednesday, February 29, 2012 1. Introduction 2. Community Based...

  12. Effects of increased low-level diode laser irradiation time on extraction socket healing in rats.

    Science.gov (United States)

    Park, Joon Bong; Ahn, Su-Jin; Kang, Yoon-Goo; Kim, Eun-Cheol; Heo, Jung Sun; Kang, Kyung Lhi

    2015-02-01

    In our previous studies, we confirmed that low-level laser therapy (LLLT) with a 980-nm gallium-aluminum-arsenide diode laser was beneficial for the healing of the alveolar bone in rats with systemic disease. However, many factors can affect the biostimulatory effects of LLLT. Thus, we attempted to investigate the effects of irradiation time on the healing of extraction sockets by evaluating the expressions of genes and proteins related to bone healing. The left and right first maxillary molars of 24 rats were extracted. Rats were randomly divided into four groups in which extraction sockets were irradiated for 0, 1, 2, or 5 min each day for 3 or 7 days. Specimens containing the sockets were examined using quantitative real-time reverse transcription polymerase chain reaction and western blotting. LLLT increased the expressions of all tested genes, Runx2, collagen type 1, osteocalcin, platelet-derived growth factor-B, and vascular endothelial growth factor, in a time-dependent manner. The highest levels of gene expressions were in the 5-min group after 7 days. Five minutes of irradiation caused prominent increases of the expression of all tested proteins after both 3 and 7 days. The expression level of each protein in group 4 was higher by almost twofold compared with group 1 after 7 days. Laser irradiation for 5 min caused the highest expressions of genes and proteins related to bone healing. In conclusion, LLLT had positive effects on the early stages of bone healing of extraction sockets in rats, which were irradiation time-dependent.

  13. Western Military Culture and Counterinsurgency:

    African Journals Online (AJOL)

    francois

    with a threat both abroad and within their homeland societies. Civilians fulfil a ..... we have now with the use of force and forces is their persistent structuring ... advanced equipment remains the main feature of Western military culture. Western.

  14. Crocodilian faunal renewal in the Upper Oligocene of Western Europe

    Science.gov (United States)

    Antunes, Miguel Telles; Cahuzac, Bruno

    1999-01-01

    The presence of Tomistomine Crocodilians ( Tomistoma sp.) is first recorded in the Upper Oligocene of Western Europe (Chattian, Saint-Geours-de-Maremne, Aquitaine basin). Immigration, probably of Asiatic origin, occurred ca. 26 Ma, prior to the previously earliest records for Lowermost Miocene. Tomistoma points to warm, at least subtropical environments, in agreement with data on invertebrates and fish fauna. Immigrations to Western Europe of Tomistoma (and of Gavialis, in Miocene times) during Upper Oligocene and Lower-Middle Miocene seem related to climatic warming and eustatic rise events. There was a renewal of the European Crocodilian fauna, much impoverished in Late Eocene and reduced to Diplocynodon.

  15. Duff mound consumption and cambium injury for centuries-old western larch from prescribed burning in western Montana

    Science.gov (United States)

    Michael G. Harrington

    2012-01-01

    Western larch is one of the most fire-adapted conifers in western North America. Its historical perpetuation depended upon regular fire disturbances, which creates open stand conditions and mineral seedbeds. A stand of 200- to 500-year-old larch in western Montana with deep duff mounds resulting from an unusually long 150-year fire-free period was mechanically thinned...

  16. Chronic consumption of a western diet modifies the DNA methylation profile in the frontal cortex of mice.

    Science.gov (United States)

    Yokoyama, Amy S; Dunaway, Keith; Rutkowsky, Jennifer; Rutledge, John C; Milenkovic, Dragan

    2018-02-21

    In our previous work in mice, we have shown that chronic consumption of a Western diet (WD; 42% kcal fat, 0.2% total cholesterol and 34% sucrose) is correlated with impaired cognitive function. Cognitive decline has also been associated with alterations in DNA methylation. Additionally, although there have been many studies analyzing the effect of maternal consumption of a WD on DNA methylation in the offspring, few studies have analyzed how an individual's consumption of a WD can impact his/her DNA methylation. Since the frontal cortex is involved in the regulation of cognitive function and is often affected in cases of cognitive decline, this study aimed to examine how chronic consumption of a WD affects DNA methylation in the frontal cortex of mice. Eight-week-old male mice were fed either a control diet (CD) or a WD for 12 weeks, after which time alterations in DNA methylation were analyzed. Assessment of global DNA methylation in the frontal cortex using dot blot analysis revealed that there was a decrease in global DNA methylation in the WD-fed mice compared with the CD-fed mice. Bioinformatic analysis identified several networks and pathways containing genes displaying differential methylation, particularly those involved in metabolism, cell adhesion and cytoskeleton integrity, inflammation and neurological function. In conclusion, the results from this study suggest that consumption of a WD alters DNA methylation in the frontal cortex of mice and could provide one of the mechanisms by which consumption of a WD impairs cognitive function.

  17. Regulation of the Na+/Ca2+ exchanger in rat pancreatic ducts

    DEFF Research Database (Denmark)

    Ankorina-Stark, I; Amstrup, J; Novak, I

    2002-01-01

    by hormones/agonists affecting pancreatic secretion. Whole pancreas, pure pancreatic acini and ducts were obtained from rats and used for RT-PCR and Western blot analysis, immunohistochemistry and intracellular Ca2+ measurements using Fura-2. RT-PCR analysis indicated Na+/Ca2+-exchanger isoforms NCX1.......3 and NCX1.7 in acini and pancreas. Western blot with NCX1 antibody identified bands of 70, 120 and 150 kDa in isolated ducts, acini and pancreas. Immunofluorescence experiments showed the Na+/Ca2+ exchanger on the basolateral membrane of acini and small intercalated/intralobular ducts, but in larger...

  18. Calcitonin gene-related peptide and its receptor components in the human sphenopalatine ganglion -- interaction with the sensory system

    DEFF Research Database (Denmark)

    Csati, Anett; Tajti, Janos; Tuka, Bernadett

    2012-01-01

    , Western blot technique was used to demonstrate the existence of CGRP receptor components in rat SPG. CGRP immunoreactive fibers were frequently found intraganglionic in the SPG in the vicinity of neurons. CLR immunoreactivity was observed in satellite glial cells (SGCs) as well as in nerve fibers......, but not in neurons. RAMP1 immunoreactivity was localized in many neurons and SGCs. Thus, the two CGRP receptor components together were found in the SGCs. In addition, Western blot revealed the presence of RAMP1 and CLR in rat SPG. Our results suggest a possible sensory influence in the parasympathetic cranial...

  19. The impact of FCN2 polymorphisms and haplotypes on the Ficolin-2 serum levels

    DEFF Research Database (Denmark)

    Munthe-Fog, L; Hummelshøj, T; Hansen, B E

    2007-01-01

    Ficolin-2 in serum samples and identified FCN2 genotypes with a Taq Man-based minor groove binder assay and by sequencing. Serum samples were applied to gel-permeation chromatography and fractions were analysed by an ELISA, SDS-PAGE and subsequently Western blotting. In 214 Danish blood donors, the median...... Ficolin-2 serum concentration was determined to 5.4 microg/ml (range: 1.0-12.2 microg/ml). An ELISA, SDS-PAGE and Western blot analysis of gel-permeation chromatography fractions showed that Ficolin-2 comprises a mixture of covalently and non-covalently linked Ficolin-2 oligomers independent...

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

    Science.gov (United States)

    2014-01-01

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

  1. Time from HIV infection to virological suppression: dramatic fall from 2007 to 2016.

    Science.gov (United States)

    Medland, Nicholas A; Nicholson, Suellen; Chow, Eric P F; Read, Timothy R H; Bradshaw, Catriona S; Denham, Ian; Fairley, Christopher K

    2017-11-13

    Time from HIV infection to virological suppression: dramatic fall from 2007 to 2016. We examined the time from HIV infection to virological suppression in MSM who were first diagnosed at Melbourne Sexual Health Centre between 2007 and 2016. Retrospective cohort. Date of infection was imputed from the testing history or serological evidence of recent infection (negative or indeterminate western blot) or baseline CD4 cell count. Date of virological suppression was determined using clinical viral load data. We analysed predictors of diagnosis with serological evidence of recent infection (logistic regression) and time from diagnosis to suppression and from infection to suppression (Cox regression) using demographic, clinical, and behavioral covariates. Between 2007 and 2016, the median time from HIV infection to diagnosis fell from 6.8 to 4.3 months (P = 0.001), from diagnosis to suppression fell from 22.7 to 3.2 months (P < 0.0001), and from infection to suppression fell from 49.0 to 9.6 months (P < 0.0001). Serological evidence of recent infection increased from 15.6 to 34.3% (P < 0.0001) of diagnoses. In the multivariate analyses, age, being recently arrived from a non-English speaking country, history of IDU, other sexually transmitted infections, and sexual risk were not associated with any of these measures. The duration of infectiousness in MSM diagnosed with HIV infection at Melbourne Sexual Health Centre in Victoria has fallen dramatically between 2007 and 2016 and the proportion diagnosed with serological evidence of recent infection has increased. This effect is observed across all population subgroups and marks a positive milestone for the treatment as prevention paradigm.

  2. Molecular evidence and functional expression of multidrug resistance associated protein (MRP) in rabbit corneal epithelial cells.

    Science.gov (United States)

    Karla, Pradeep K; Pal, Dananjay; Mitra, Ashim K

    2007-01-01

    Multidrug resistance associated protein (MRP) is a major family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify if the role of efflux transporters. MRP-2 is a major homologue of MRP family and found to express on the apical side of cell membrane. Cultured Rabbit Corneal Epithelial Cells (rCEC) were selected as an in vitro model for corneal epithelium. [14C]-erythromycin which is a proven substrate for MRP-2 was selected as a model drug for functional expression studies. MK-571, a known specific and potent inhibitor for MRP-2 was added to inhibit MRP mediated efflux. Membrane fraction of rCEC was used for western blot analysis. Polarized transport of [14C]-erythromycin was observed in rCEC and transport from B-->A was significantly high than from A-->B. Permeability's increased significantly from A-->B in the presence of MK-571 and ketoconozole. Uptake of [14C]-erythromycin in the presence of MK-571 was significantly higher than control in rCEC. RT-PCR analysis indicated a unique and distinct band at approximately 498 bp corresponding to MRP-2 in rCEC and MDCK11-MRP-2 cells. Immunoprecipitation followed by Western Blot analysis indicated a specific band at approximately 190 kDa in membrane fraction of rCEC and MDCK11-MRP-2 cells. For the first time we have demonstrated high expression of MRP-2 in rabbit corneal epithelium and its functional activity causing drug efflux. RT-PCR, immunoprecipitation followed by Western blot analysis further confirms the result.

  3. JS-K, a nitric oxide pro-drug, regulates growth and apoptosis through the ubiquitin-proteasome pathway in prostate cancer cells.

    Science.gov (United States)

    Tan, Guobin; Qiu, Mingning; Chen, Lieqian; Zhang, Sai; Ke, Longzhi; Liu, Jianjun

    2017-05-26

    In view of the fact that JS-K might regulate ubiquitin E3 ligase and that ubiquitin E3 ligase plays an important role in the mechanism of CRPC formation, the goal was to investigate the probable mechanism by which JS-K regulates prostate cancer cells. Proliferation inhibition by JS-K on prostate cancer cells was examined usingCCK-8 assays. Caspase 3/7 activity assays and flow cytometry were performed to examine whether JS-K induced apoptosis in prostate cancer cells. Western blotting and co-immunoprecipitation analyses investigated JS-K's effects on the associated apoptosis mechanism. Real time-PCR and Western blotting were performed to assess JS-K's effect on transcription of specific AR target genes. Western blotting was also performed to detect Siah2 and AR protein concentrations and co-immunoprecipitation to detect interactions of Siah2 and AR, NCoR1 and AR, and p300 and AR. JS-K inhibited proliferation and induced apoptosis in prostate cancer cells. JS-K increased p53 and Mdm2 concentrations and regulated the caspase cascade reaction-associated protein concentrations. JS-K inhibited transcription of AR target genes and down-regulated PSA protein concentrations. JS-K inhibited Siah2 interactions and also inhibited the ubiquitination of AR. With further investigation, JS-K was found to stabilize AR and NCoR1 interactions and diminish AR and p300 interactions. The present results suggested that JS-K might have been able to inhibit proliferation and induce apoptosis via regulation of the ubiquitin-proteasome degradation pathway, which represented a promising platform for the development of new compounds for PCa treatments.

  4. Synergistic suppression of the PI3K inhibitor CAL-101 with bortezomib on mantle cell lymphoma growth

    International Nuclear Information System (INIS)

    Qu, Fu-Lian; Xia, Bing; Li, Su-Xia; Tian, Chen; Yang, Hong-Liang; Li, Qian; Wang, Ya-Fei; Yu, Yong; Zhang, Yi-Zhuo

    2015-01-01

    To investigate the effects of CAL-101, particularly when combined with bortezomib (BTZ) on mantle cell lymphoma (MCL) cells, and to explore its relative mechanisms. MTT assay was applied to detect the inhibitory effects of different concentrations of CAL-101. MCL cells were divided into four groups: control group, CAL-101 group, BTZ group, and CAL-101/BTZ group. The expression of PI3K-p110σ, AKT, ERK, p-AKT and p-ERK were detected by Western blot. The apoptosis rates of CAL-101 group, BTZ group, and combination group were detected by flow cytometry. The location changes of nuclear factor kappa-B (NF-κB) of 4 groups was investigated by NF-κB Kit exploring. Western blot was applied to detect the levels of caspase-3 and the phosphorylation of AKT in different groups. CAL-101 dose- and time-dependently induced reduction in MCL cell viability. CAL-101 combined with BTZ enhanced the reduction in cell viability and apoptosis. Western blot analysis showed that CAL-101 significantly blocked the PI3K/AKT and ERK signaling pathway in MCL cells. The combination therapy contributed to the inactivation of NF-κB and AKT in MCL cell lines. However, cleaved caspase-3 was up-regulated after combined treatment. Our study showed that PI3K/p110σ is a novel therapeutic target in MCL, and the underlying mechanism could be the blocking of the PI3K/AKT and ERK signaling pathways. These findings provided a basis for clinical evaluation of CAL-101 and a rationale for its application in combination therapy, particularly with BTZ

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

    International Nuclear Information System (INIS)

    Dorner, G.

    1997-01-01

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

  6. Perceptions of the ankle brachial index amongst podiatrists registered in Western Australia

    Directory of Open Access Journals (Sweden)

    Chen Pamela Y

    2012-07-01

    Full Text Available Abstract Background The ankle brachial index (ABI is an objective diagnostic tool that is widely used for the diagnosis of peripheral arterial disease. Despite its usefulness, it is evident within the literature that many practitioners forgo using this screening tool due to limiting factors such as time. There is also no recommended technique for ABI measurement. The purpose of this study is to investigate the perceptions of the use of ABI clinically among Western Australian podiatrists. Methods This study was a cross sectional survey which evaluated the perceptions of the ABI amongst registered podiatrists in Western Australia. The study sample was obtained from the register of podiatrists listed with the Podiatrists Registration Board of Western Australia. Podiatrists were contacted by telephone and invited to participate in a telephone questionnaire. Chi-square tests were performed to determine if there was a statistically significant relationship between use of the ABI and podiatrists’ profile which included: sector of employment; geographical location; and length of time in practice. Results There is a statistically significant relationship (p=0.004 between podiatrists’ profile and the use of ABI, with higher usage in the tertiary hospital setting than in private practice. Length of time spent in practice had no significant impact on ABI usage (p=0.098. Time constraints and lack of equipment were key limiting factors to performing the ABI, and no preferred technique was indicated. Conclusion Western Australian podiatrists agree that the ABI is a useful tool for lower limb vascular assessment, however, various factors influence uptake in the clinical setting. This study suggests that a podiatrists’ profile has a significant influence on the use of the ABI, which may be attributed to different patient types across the various settings. The influence of time spent in practice on ABI usage may be attributed to differences in clinical

  7. Competitive edge of western coal

    International Nuclear Information System (INIS)

    Keith, R.D.

    1990-01-01

    This paper expresses views on the competitive advantages of one of the nation's most remarkable energy resources--Western coal. It covers utilization of Western coal, and its advantages. The Arkansas Power and Light Company and its demand for coal are also covered

  8. A Novel Mechanism of Estrogen Action in Breast Cancer Cells Mediated Through ER-FE65 Complex Formation

    Science.gov (United States)

    2013-03-01

    Western blot with anti-HA mAb, MMS-101P, Covance , 1:2000) or Anti-SIRT1(rabbit, D739, CST, 1:1000). β-actin blot was included to show even loading. 4...lipofectamine 2000 were bought from Invitrogen (Grand Island, NY). Anti- hemagglutinin (anti-HA.11; PRB-101P) antibody was obtained from Covance

  9. 78 FR 62350 - Lock+ Hydro Friends Fund III, LLC; Western Minnesota Municipal Power Agency; Notice Announcing...

    Science.gov (United States)

    2013-10-18

    .... 14540-000] Lock+ Hydro Friends Fund III, LLC; Western Minnesota Municipal Power Agency; Notice...+ Hydro Friends Fund III, LLC for Project No. 14539-000 and Western Minnesota Municipal Power Agency for... CFR 385.2001(a)(2) (2013). On October 21, 2013, at 10:00 a.m. (Eastern Time), the Secretary of the...

  10. 78 FR 70549 - Lock+ Hydro Friends Fund III, LLC, Western Minnesota Municipal Power Agency; Notice Announcing...

    Science.gov (United States)

    2013-11-26

    .... 14540-000] Lock+ Hydro Friends Fund III, LLC, Western Minnesota Municipal Power Agency; Notice...+ Hydro Friends Fund III, LLC for Project No. 14539-000 and Western Minnesota Municipal Power Agency for... CFR.385.2001(a)(2) (2013). On November 25, 2013, at 10:00 a.m. (Eastern Time), the Secretary of the...

  11. [Relationship between FoxO1 Expression and Wound Age during Skin Incised Wound Healing].

    Science.gov (United States)

    Chen, Y; Ji, X Y; Fan, Y Y; Yu, L S

    2018-02-01

    To investigate FoxO1 expression and its time-dependent changes during the skin incised wound healing. After the establishment of the skin incised wound model in mice, the FoxO1 expression of skin in different time periods was detected by immunohistochemistry and Western blotting. Immunohistochemistry staining showed that FoxO1 was weakly expressed in a few fibroblasts of epidermis, hair follicles, sebaceous glands, vessel endothelium and dermis in the control group. The FoxO1 expression was enhanced in the epidermis and skin appendages around the wound during 6-12 h after injury, which could be detected in the infiltrating neutrophils and a small number of monocytes. FoxO1 was mainly expressed in monocytes during 1-3 d after injury, and in neovascular endothelial cells and fibroblasts during 5-10 d. On the 14th day after injury, the FoxO1 expression still could be detected in a few fibroblasts. The Western blotting results showed that the FoxO1 expression quantity of the tissue samples in injury group was higher than in control group. The FoxO1 expression peaked at 12 h and 7 d after injury. FoxO1 is time-dependently expressed in skin wound healing, which can be a useful marker for wound age determination. Copyright© by the Editorial Department of Journal of Forensic Medicine.

  12. PANC-1 pancreatic cancer cell growth inhibited by cucurmosin alone and in combination with an epidermal growth factor receptor-targeted drug.

    Science.gov (United States)

    Wang, Congfei; Yang, Aiqin; Zhang, Baoming; Yin, Qiang; Huang, Heguang; Chen, Minghuang; Xie, Jieming

    2014-03-01

    To investigate the inhibition of PANC-1 pancreatic cancer cell growth by cucurmosin (CUS) and its possible mechanism. We observed the inhibition of PANC-1 cell growth by sulforhodamine B and colony-forming experiments in vitro and established nonobese diabetic/severe combined immunodeficiency mouse subcutaneous tumor models in vivo. We used Western blot to analyze protein levels related to apoptosis and epidermal growth factor receptor (EGFR) signaling pathways after drug intervention, whereas the messenger RNA expression of EGFR was analyzed by quantitative real-time polymerase chain reaction. Sulforhodamine B and colony-forming experiments indicated that CUS inhibited PANC-1 cell proliferation in a dose- and time-dependent manner. A stronger inhibitory effect was observed when CUS was combined with gefitinib. The subcutaneous tumor growth was also inhibited. Western blot showed that all the examined proteins decreased, except for 4E-BP1 and the active fragments of caspase 3 and caspase 9 increased. Epidermal growth factor receptor expression did not change significantly in quantitative real-time polymerase chain reaction. Cucurmosin can strongly inhibit the growth of PANC-1 cells in vitro and in vivo. Cucurmosin can down-regulate EGFR protein expression, but not at the messenger RNA level. Cucurmosin can also inhibit the ras/raf and phosphatidylinositol 3-kinase/Akt downstream signaling pathways and enhance the sensitivity of the EGFR-targeted drug gefitinib.

  13. Tumor necrosis factor alpha increases epithelial barrier permeability by disrupting tight junctions in Caco-2 cells

    Directory of Open Access Journals (Sweden)

    W. Cui

    2010-04-01

    Full Text Available The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-α on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell® microporous filters and treated with TNF-α (10 or 100 ng/mL for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-α treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-α decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-α did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-α increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.

  14. Maternal serum protein profile and immune response protein subunits as markers for non-invasive prenatal diagnosis of trisomy 21, 18, and 13

    KAUST Repository

    Narasimhan, Kothandaraman

    2013-02-01

    Objectives: To use proteomics to identify and characterize proteins in maternal serum from patients at high-risk for fetal trisomy 21, trisomy 18, and trisomy 13 on the basis of ultrasound and maternal serum triple tests. Methods: We performed a comprehensive proteomic analysis on 23 trisomy cases and 85 normal cases during the early second trimester of pregnancy. Protein profiling along with conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis/Tandem mass spectrometry analysis was carried out to characterize proteins associated with each trisomy condition and later validated using Western blot. Results: Protein profiling approach using surface enhanced laser desorption/ionization time-of-flight mass (SELDI-TOF/MS) spectrometry resulted in the identification of 37 unique hydrophobic proteomic features for three trisomy conditions. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Matrix Assisted Laser Desorption Ionization - Time of Flight/Time of Flight (MALDI-TOF/TOF) and western blot, glyco proteins such as alpha-1-antitrypsin, apolipoprotein E, apolipoprotein H, and serum carrier protein transthyretin were identified as potential maternal serum markers for fetal trisomy condition. The identified proteins showed differential expression at the subunit level. Conclusions: Maternal serum protein profiling using proteomics may allow non-invasive diagnostic testing for the most common trisomies and may complement ultrasound-based methods to more accurately determine pregnancies with fetal aneuploidies. © 2013 John Wiley & Sons, Ltd.

  15. Extracellular matrix metalloproteinase inducer (EMMPRIN) remodels the extracellular matrix through enhancing matrix metalloproteinases (MMPs) and inhibiting tissue inhibitors of MMPs expression in HPV-positive cervical cancer cells.

    Science.gov (United States)

    Xu, Q; Cao, X; Pan, J; Ye, Y; Xie, Y; Ohara, N; Ji, H

    2015-01-01

    PUPOSE OF INVESTIGATION: To study the expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), and tissue inhibitors of MMP (TIMPs) in uterine cervical cancer cell lines in vitro. EMMPRIN, MMPs, and TIMPs expression were assessed by Western blot and real-time RT-PCR from cervical carcinoma SiHa, HeLa, and C33-A cells. EMMPRIN recombinant significantly increased MMP-2, MMP-9 protein and mRNA expression in SiHa and Hela cells, but not in C33-A cells by Western blot analysis and real-time RT-PCR. EMMPRIN recombinant significantly inhibited TIMP-1 protein and mRNA levels in SiHa and Hela cells, but not in C33-A cells. There was no difference on the TIMP-2 expression in those cells with the treatment of EMMPRIN recombinant. EMMPRIN RNAi decreased MMP-2 and MMP-9 and increased TIMP-1 expression in SiHa and HeLa cells, but not in C33-A cells. There was no change on the expression of TIMP-2 mRNA levels in SiHa, HeLa and C33-A cells transfected with siEMMPRIN. EMMPRIN may induce MMP-2 and MMP-9, and downregulate TIMP-1 in HPV-positive cervical cancer cells in vitro.

  16. [Construction and selection of effective mouse Smad6 recombinant lenti-virus interference vectors].

    Science.gov (United States)

    Yu, Jing; Qi, Mengchun; Deng, Jiupeng; Liu, Gang; Chen, Huaiqing

    2010-10-01

    This experiment was designed to construct mouse Smad6 recombinant RNA interference vectors and determine their interference effects on bone marrow mesenchymal stem cells (BMSCs). Three recombinant Smad6 RNA interference vectors were constructed by molecular clone techniques with a lenti-virus vector expressing green fluorescent protein (GFP), and the correctness of recombinant vectors was verified by DNA sequencing. Mouse BMSCs were used for transfection experiments and BMP-2 was in use for osteogenic induction of MSCs. The transfection efficiency of recombinant vectors was examined by Laser confocal scanning microscope and the interference effect of recombinant vectors on Smad6 gene expression was determined by real-time RT-PCR and Western blot, respectively. Three Smad6 recombinant RNA interference vectors were successfully constructed and their correctness was proved by DNA sequencing. After transfection, GFPs were effectively expressed in MSCs and all of three recombinant vectors gained high transfection efficiency (> 95%). Both real-time PCR and Western blot examination indicated that among three recombinant vectors, No. 2 Svector had the best interference effect and the interference effect was nearly 91% at protein level. In conclusion, Mouse recombinant Smad6 RNA interference (RNAi) vector was successfully constructed and it provided an effective tool for further studies on BMP signal pathways.

  17. Advanced glycation end products promote ChREBP expression and cell proliferation in liver cancer cells by increasing reactive oxygen species.

    Science.gov (United States)

    Chen, Hanbei; Li, Yakui; Zhu, Yemin; Wu, Lifang; Meng, Jian; Lin, Ning; Yang, Dianqiang; Li, Minle; Ding, WenJin; Tong, Xuemei; Su, Qing

    2017-08-01

    The aim of the study was to elucidate the mechanism by which advanced glycation end products (AGEs) promote cell proliferation in liver cancer cells.We treated liver cancer HepG2 cells with 200 mg/L AGEs or bovine serum albumin (BSA) and assayed for cell viability, cell cycle, and apoptosis. We performed real-time PCR and Western blot analysis for RNA and protein levels of carbohydrate responsive element-binding protein (ChREBP) in AGEs- or BSA-treated HepG2 cells. We analyzed the level of reactive oxygen species (ROS) in HepG2 cells treated with AGEs or BSA.We found that increased S-phase cell percentage and decreased apoptosis contributed to AGEs-induced liver cancer cell proliferation. Real-time PCR and Western blot analysis showed that AGEs stimulated RNA and protein levels of ChREBP, a transcription factor promoting glycolysis and maintaining cell proliferation in liver cancer cells. Intriguingly, the level of ROS was higher in AGEs-treated liver cancer cells. Treating liver cancer cells with antioxidant N-acetyl cystein (NAC) partly blocked AGEs-induced ChREBP expression and cell proliferation.Our results suggest that the AGEs-ROS-ChREBP pathway plays a critical role in promoting ChREBP expression and liver cancer cell proliferation.

  18. BAG3 promoted starvation-induced apoptosis of thyroid cancer cells via attenuation of autophagy.

    Science.gov (United States)

    Li, Si; Zhang, Hai-Yan; Wang, Tian; Meng, Xin; Zong, Zhi-Hong; Kong, De-Hui; Wang, Hua-Qin; Du, Zhen-Xian

    2014-11-01

    BAG3 plays a regulatory role in a number of cellular processes. Recent studies have attracted much attention on its role in activation of selective autophagy. In addition, we have very recently reported that BAG3 is implicated in a BECN1-independent autophagy, namely noncanonical autophagy. The current study aimed to investigate the potential involvement of BAG3 in canonical autophagy triggered by Earle's Balanced Salt Solution (EBSS) starvation. Replacement of complete medium with EBSS was used to trigger canonical autophagy. BAG3 expression was measured using real-time RT-PCR and Western blot. Autophagy was monitored using LC3-II transition and p62/SQSTM1 accumulation by Western blot, as well as punctate distribution of LC3 by immunofluorescence staining. Cell growth and apoptotic cell death was investigated using real-time cell analyzer and flowcytometry, respectively. BAG3 expression was potently reduced by EBSS starvation. Forced expression of BAG3 suppressed autophagy and promoted apoptotic cell death of thyroid cancer cells elicited by starvation. In addition, in the presence of autophagy inhibitor, the enhancing effect of BAG3 on apoptotic cell death was attenuated. These results suggest that BAG3 promotes apoptotic cell death in starved thyroid cancer cells, at least in part by autophagy attenuation.

  19. How Western Does Business: An Explanation of Western's Products and Services

    Energy Technology Data Exchange (ETDEWEB)

    None

    2012-10-01

    The mission of the Western Area Power Administration is to market and deliver reliable, renewable, cost-based hydroelectric power and related services. This guide provides an overview of Western’s history and how Western carries out that mission and provides electrical, transmission and ancillary services. It also discusses how we develop plans for marketing our most valuable resources—long-term firm capacity and energy.

  20. Brains, language and the argumentative mind in Western and Eastern societies. The fertile differences between Western-Eastern argumentative traditions.

    Science.gov (United States)

    Vallverdú, Jordi

    2017-12-01

    The philosophical differences between Western and Eastern philosophy not only derive from general cultural ideas about reality, but as Nisbet writes (2003), are also methodological, ontological, and cognitively driven. Thus, we can see that strategies of thought and theory-generation are constrained and enabled by conceptual levels, and that the existence of differences and within these levels may be pragmatically combined in fruitful ways. At this point, I remark that there is not a single way to connect biology and culture, but at least we need to admit that brains allow the existence of minds and that these create languages, which also organize the world symbolically following a long set of (sometimes interconnected) heuristics. Throughout the paper we will see how fundamental, geographically located cultural perspectives have affected reasoning strategies and discourses, determining the main Western and Eastern Traditions. At the same time, we can conclude that different traditional perspectives allow more diversity for knowledge acquisition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Evidence of late Palaeocene-early Eocene equatorial rain forest refugia in southern Western Ghats, India.

    Science.gov (United States)

    Prasad, V; Farooqui, A; Tripathi, S K M; Garg, R; Thakur, B

    2009-11-01

    Equatorial rain forests that maintain a balance between speciation and extinction are hot-spots for studies of biodiversity. Western Ghats in southern India have gained attention due to high tropical biodiversity and endemism in their southern most area. We attempted to track the affinities of the pollen fl ora of the endemic plants of Western Ghat area within the fossil palynoflora of late Palaeocene-early Eocene (approximately 55-50 Ma) sedimentary deposits of western and northeastern Indian region. The study shows striking similarity of extant pollen with twenty eight most common fossil pollen taxa of the early Palaeogene. Widespread occurrences of coal and lignite deposits during early Palaeogene provide evidence of existence of well diversified rain forest community and swampy vegetation in the coastal low lying areas all along the western and northeastern margins of the Indian subcontinent. Prevalence of excessive humid climate during this period has been seen as a result of equatorial positioning of Indian subcontinent, superimposed by a long term global warming phase (PETM and EECO) during the early Palaeogene. The study presents clear evidence that highly diversifi ed equatorial rain forest vegetation once widespread in the Indian subcontinent during early Palaeogene times, are now restricted in a small area as a refugia in the southernmost part of the Western Ghat area. High precipitation and shorter periods of dry months seem to have provided suitable environment to sustain lineages of ancient tropical vegetation in this area of Western Ghats in spite of dramatic climatic changes subsequent to the post India-Asia collision and during the Quaternary and Recent times.

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

    Science.gov (United States)

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

    2015-10-01

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

  3. Implementation and Operational Research: A Time-Motion Analysis of HIV Transmission Prevention Counseling and Antiretroviral Adherence Messages in Western Kenya.

    Science.gov (United States)

    Were, Martin C; Kessler, Jason; Shen, Changyu; Sidle, John; Macharia, Stephen; Lizcano, John; Siika, Abraham; Wools-Kaloustian, Kara; Kurth, Ann

    2015-08-01

    Shortages of health workers and large number of HIV-infected persons in Africa mean that time to provide antiretroviral therapy (ART) adherence and other messages to patients is limited. Using time-motion methodology, we documented the intensity and nature of counseling delivered to patients. The study was conducted at a rural and an urban HIV clinic in western Kenya. We recorded all activities of 190 adult patients on ART during their return clinic visits to assess type, frequency, and duration of counseling messages. Mean visit length for patients at the rural clinic was 44.5 (SD = 27.9) minutes and at urban clinic was 78.2 (SD = 42.1) minutes. Median time spent receiving any counseling during a visit was 4.07 minutes [interquartile range (IQR), 1.57-7.33] at rural and 3.99 (IQR, 2.87-6.25) minutes at urban, representing 11% and 8% of total mean visit time, respectively. Median time patients received ART adherence counseling was 1.29 (IQR, 0.77-2.83) minutes at rural and 1.76 (IQR, 1.23-2.83) minutes at urban (P = 0.001 for difference). Patients received a median time of 0.18 (0-0.72) minutes at rural and 0.28 (IQR, 0-0.67) minutes at urban clinic of counseling regarding contraception and pregnancy. Most patients in the study did not receive any counseling regarding alcohol/substance use, emerging risks for ongoing HIV transmission. Although ART adherence was discussed with most patients, time was limited. Reproductive counseling was provided to only half of the patients, and "positive prevention" messaging was minimal. There are strategic opportunities to enhance counseling and information received by clients within HIV programs in resource-limited settings.

  4. Airborne measurements of western U.S. wildfire emissions: Comparison with prescribed burning and air quality implications: Western U.S. Wildfire Emissions

    International Nuclear Information System (INIS)

    Liu, Xiaoxi; University of Colorado, Boulder, CO; Huey, L. Gregory; Yokelson, Robert J.; Selimovic, Vanessa

    2017-01-01

    Wildfires emit significant amounts of pollutants that degrade air quality. Plumes from three wildfires in the western U.S. were measured from aircraft during the Studies of Emissions and Atmospheric Composition, Clouds and Climate Coupling by Regional Surveys (SEAC4RS) and the Biomass Burning Observation Project (BBOP), both in summer 2013. This study reports an extensive set of emission factors (EFs) for over 80 gases and 5 components of submicron particulate matter (PM 1 ) from these temperate wildfires. These include rarely, or never before, measured oxygenated volatile organic compounds and multifunctional organic nitrates. The observed EFs are compared with previous measurements of temperate wildfires, boreal forest fires, and temperate prescribed fires. Furthermore, the wildfires emitted high amounts of PM 1 (with organic aerosol (OA) dominating the mass) with an average EF that is more than 2 times the EFs for prescribed fires. The measured EFs were used to estimate the annual wildfire emissions of carbon monoxide, nitrogen oxides, total nonmethane organic compounds, and PM 1 from 11 western U.S. states. The estimated gas emissions are generally comparable with the 2011 National Emissions Inventory (NEI). However, our PM 1 emission estimate (1530 ± 570 Gg yr -1 ) is over 3 times that of the NEI PM2.5 estimate and is also higher than the PM2.5 emitted from all other sources in these states in the NEI. This study indicates that the source of OA from biomass burning in the western states is significantly underestimated. Additionally, our results indicate that prescribed burning may be an effective method to reduce fine particle emissions.

  5. [Environmental efficiency evaluation under carbon emission constraint in Western China].

    Science.gov (United States)

    Rong, Jian-bo; Yan, Li-jiao; Huang, Shao-rong; Zhang, Ge

    2015-06-01

    This research used the SBM model based on undesirable outputs to measure the static environmental efficiency of Western China under carbon emission constraint from 2000 to 2012. The researchers also utilized the Malmquist index to further analyze the change tendency of environmental efficiency. Additionally, Tobit regression analysis was used to study the factors relevant to environmental efficiency. Practical solutions to improve environmental quality in Western China were put forward. The study showed that in Western China, environmental efficiency with carbon emission constraint was significantly lower than that without carbon emission constraint, and the difference could be described as an inverse U-shaped curve which increased at first and then decreased. Guang-xi and Inner Mongolia, the two provinces met the effective environmental efficiency levels all the time under carbon emission constraint. However, the five provinces of Guizhou, Gansu, Qinghai, Ningxia and Xinjiang did not. Furthermore, Ningxia had the lowest level of environmental efficiency, with a score between 0.281-0.386. Although the environmental efficiency of most provinces was currently at an ineffective level, the environmental efficiency quality was gradually improving at an average speed of 6.6%. Excessive CO2 emission and a large amount of energy consumption were the primary factors causing environmental inefficiency in Western China, and energy intensity had the most negative impact on the environmental efficiency. The increase of import and export trade reduced the environmental efficiency significantly in Western China, while the increase of foreign direct investment had a positive effect on its environmental efficiency.

  6. Exhumation history of the western Kyrgyz Tien Shan: Implications for intramontane basin formation

    Science.gov (United States)

    Bande, Alejandro; Sobel, Edward R.; Mikolaichuk, Alexander; Schmidt, Alexander; Stockli, Daniel F.

    2017-01-01

    The dextral Talas-Fergana Fault separates the western from the central Tien Shan. Recent work has shed light on the Cenozoic evolution of the eastern and central Tien Shan; much less attention has been paid to the western Tien Shan. In this contribution we present new thermochronological ages for the Fergana and Alai ranges that, combined with the available data set, constrain the Cenozoic exhumation history of the western Tien Shan. Following a tectonically quiet early Cenozoic period, we suggest an onset of exhumation at 25 Ma. This early onset was followed by a period of slower exhumation and in some areas minor reheating. A final, strong late Miocene rapid cooling event is well represented in the western Tien Shan as in other sectors of the range. The early onset of uplift of the western Tien Shan dissected the previously continuous westernmost Parathethyan Sea, progressively isolating basins (e.g., Fergana, Tarim, and Alai basins) in the central Asian hinterland. Moreover, the coeval timing of late Miocene uplift along the length of entire Tien Shan implies that neither the Pamir nor Tarim can be the sole driver for exhumation of the entire range.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2014-01-01

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

  9. Myoblots: dystrophin quantification by in-cell western assay for a streamlined development of Duchenne muscular dystrophy (DMD) treatments.

    Science.gov (United States)

    Ruiz-Del-Yerro, E; Garcia-Jimenez, I; Mamchaoui, K; Arechavala-Gomeza, V

    2017-10-31

    New therapies for neuromuscular disorders are often mutation specific and require to be studied in patient's cell cultures. In Duchenne muscular dystrophy (DMD) dystrophin restoration drugs are being developed but as muscle cell cultures from DMD patients are scarce and do not grow or differentiate well, only a limited number of candidate drugs are tested. Moreover, dystrophin quantification by western blotting requires a large number of cultured cells; so fewer compounds are as thoroughly screened as is desirable. We aimed to develop a quantitative assessment tool using fewer cells to contribute in the study of dystrophin and to identify better drug candidates. An 'in-cell western' assay is a quantitative immunofluorescence assay performed in cell culture microplates that allows protein quantification directly in culture, allowing a higher number of experimental repeats and throughput. We have optimized the assay ('myoblot') to be applied to the study of differentiated myoblast cultures. After an exhaustive optimization of the technique to adapt it to the growth and differentiation rates of our cultures and the low intrinsic expression of our proteins of interests, our myoblot protocol allows the quantification of dystrophin and other muscle-associated proteins in muscle cell cultures. We are able to distinguish accurately between the different sets of patients based on their dystrophin expression and detect dystrophin restoration after treatment. We expect that this new tool to quantify muscle proteins in DMD and other muscle disorders will aid in their diagnosis and in the development of new therapies. © 2017 British Neuropathological Society.

  10. Geothermal resource assessment of western San Luis Valley, Colorado

    Energy Technology Data Exchange (ETDEWEB)

    Zacharakis, Ted G.; Pearl, Richard Howard; Ringrose, Charles D.

    1983-01-01

    The Colorado Geological Survey initiated and carried out a fully integrated assessment program of the geothermal resource potential of the western San Luis Valley during 1979 and 1980. The San Luis Valley is a large intermontane basin located in southcentral Colorado. While thermal springs and wells are found throughout the Valley, the only thermal waters found along the western part of the Valley are found at Shaw Warm Springs which is a relatively unused spring located approximately 6 miles (9.66 km) north of Del Norte, Colorado. The waters at Shaws Warm Spring have a temperature of 86 F (30 C), a discharge of 40 gallons per minute and contain approximately 408 mg/l of total dissolved solids. The assessment program carried out din the western San Luis Valley consisted of: soil mercury geochemical surveys; geothermal gradient drilling; and dipole-dipole electrical resistivity traverses, Schlumberger soundings, Audio-magnetotelluric surveys, telluric surveys, and time-domain electro-magnetic soundings and seismic surveys. Shaw Warm Springs appears to be the only source of thermal waters along the western side of the Valley. From the various investigations conducted the springs appear to be fault controlled and is very limited in extent. Based on best evidence presently available estimates are presented on the size and extent of Shaw Warm Springs thermal system. It is estimated that this could have an areal extent of 0.63 sq. miles (1.62 sq. km) and contain 0.0148 Q's of heat energy.

  11. How Ebola impacts genetics of Western lowland gorilla populations.

    Science.gov (United States)

    Le Gouar, Pascaline J; Vallet, Dominique; David, Laetitia; Bermejo, Magdalena; Gatti, Sylvain; Levréro, Florence; Petit, Eric J; Ménard, Nelly

    2009-12-18

    Emerging infectious diseases in wildlife are major threats for both human health and biodiversity conservation. Infectious diseases can have serious consequences for the genetic diversity of populations, which could enhance the species' extinction probability. The Ebola epizootic in western and central Africa induced more than 90% mortality in Western lowland gorilla population. Although mortality rates are very high, the impacts of Ebola on genetic diversity of Western lowland gorilla have never been assessed. We carried out long term studies of three populations of Western lowland gorilla in the Republic of the Congo (Odzala-Kokoua National Park, Lossi gorilla sanctuary both affected by Ebola and Lossi's periphery not affected). Using 17 microsatellite loci, we compared genetic diversity and structure of the populations and estimate their effective size before and after Ebola outbreaks. Despite the effective size decline in both populations, we did not detect loss in genetic diversity after the epizootic. We revealed temporal changes in allele frequencies in the smallest population. Immigration and short time elapsed since outbreaks could explain the conservation of genetic diversity after the demographic crash. Temporal changes in allele frequencies could not be explained by genetic drift or random sampling. Immigration from genetically differentiated populations and a non random mortality induced by Ebola, i.e., selective pressure and cost of sociality, are alternative hypotheses. Understanding the influence of Ebola on gorilla genetic dynamics is of paramount importance for human health, primate evolution and conservation biology.

  12. How Ebola impacts genetics of Western lowland gorilla populations.

    Directory of Open Access Journals (Sweden)

    Pascaline J Le Gouar

    2009-12-01

    Full Text Available Emerging infectious diseases in wildlife are major threats for both human health and biodiversity conservation. Infectious diseases can have serious consequences for the genetic diversity of populations, which could enhance the species' extinction probability. The Ebola epizootic in western and central Africa induced more than 90% mortality in Western lowland gorilla population. Although mortality rates are very high, the impacts of Ebola on genetic diversity of Western lowland gorilla have never been assessed.We carried out long term studies of three populations of Western lowland gorilla in the Republic of the Congo (Odzala-Kokoua National Park, Lossi gorilla sanctuary both affected by Ebola and Lossi's periphery not affected. Using 17 microsatellite loci, we compared genetic diversity and structure of the populations and estimate their effective size before and after Ebola outbreaks. Despite the effective size decline in both populations, we did not detect loss in genetic diversity after the epizootic. We revealed temporal changes in allele frequencies in the smallest population.Immigration and short time elapsed since outbreaks could explain the conservation of genetic diversity after the demographic crash. Temporal changes in allele frequencies could not be explained by genetic drift or random sampling. Immigration from genetically differentiated populations and a non random mortality induced by Ebola, i.e., selective pressure and cost of sociality, are alternative hypotheses. Understanding the influence of Ebola on gorilla genetic dynamics is of paramount importance for human health, primate evolution and conservation biology.

  13. HPLC and ELISA analyses of larval bile acids from Pacific and western brook lampreys

    Science.gov (United States)

    Yun, S.-S.; Scott, A.P.; Bayer, J.M.; Seelye, J.G.; Close, D.A.; Li, W.

    2003-01-01

    Comparative studies were performed on two native lamprey species, Pacific lamprey (Lampetra tridentata) and western brook lamprey (Lampetra richardsoni) from the Pacific coast along with sea lamprey (Petromyzon marinus) from the Great Lakes, to investigate their bile acid production and release. HPLC and ELISA analyses of the gall bladders and liver extract revealed that the major bile acid compound from Pacific and western brook larval lampreys was petromyzonol sulfate (PZS), previously identified as a migratory pheromone in larval sea lamprey. An ELISA for PZS has been developed in a working range of 20pg-10ng per well. The tissue concentrations of PZS in gall bladder were 127.40, 145.86, and 276.96??g/g body mass in sea lamprey, Pacific lamprey, and western brook lamprey, respectively. Releasing rates for PZS in the three species were measured using ELISA to find that western brook and sea lamprey released PZS 20 times higher than Pacific lamprey did. Further studies are required to determine whether PZS is a chemical cue in Pacific and western brook lampreys. ?? 2003 Elsevier Inc. All rights reserved.

  14. “The History and Culture of the Western Siberian Tatars”: Some Notes about the Book

    Directory of Open Access Journals (Sweden)

    Z.A. Tychinskikh

    2017-03-01

    Full Text Available The article presents a collective monograph “The History and Culture of the Western Siberian Tatars” published in 2015 by Sh. Marjani Institute of History of Tatarstan Academy of Sciences. This book is the first academic work, which integrated and multidimensionally accumulated almost all materials (of demography, genetics, linguistics, history, ethnography and ethnosociology available in modern science related to the formation and development of the Turkic-Tatar population of Western Siberia from ancient times to the early 21st century. The monograph is distinguished by the ethnoterritorial approach: the authors focus on the entire Turkic-Tatar population of Western Siberia, which was formed in different historical periods and includes two main components – population called in ethnographic science the “Siberian Tatars” and the group of Tatars who migrated (mainly from the Volga region to the Siberian region during the 16th–20th centuries. The book contains a large number of illustrations, which were collected during the expeditions of the authors in the rural settlements of the Tatars of Western Siberia, during archaeological excavations, in numerous archives and museums in Russia. Many photos are presented for the first time.

  15. Snag Dynamics in Western Oregon and Washington

    OpenAIRE

    Ohmann, Janet L

    2002-01-01

    To achieve desired amounts and characteristics of snags and down wood, managers require analytical tools for projecting changes in dead wood over time, and for comparing those changes to management objectives such as providing dead wood for wildlife and ecosystem processes. The following information on rates of snag recruitment, decay, and fall across forests of western Oregon and Washington may be useful in planning for future levels of dead wood. Eventually the information will be incorpora...

  16. Bootstrap inversion for Pn wave velocity in North-Western Italy

    Directory of Open Access Journals (Sweden)

    C. Eva

    1997-06-01

    Full Text Available An inversion of Pn arrival times from regional distance earthquakes (180-800 km, recorded by 94 seismic stations operating in North-Western Italy and surrounding areas, was carried out to image lateral variations of P-wave velocity at the crust-mantle boundary, and to estimate the static delay time at each station. The reliability of the obtained results was assessed using both synthetic tests and the bootstrap Monte Carlo resampling technique. Numerical simulations demonstrated the existence of a trade-off between cell velocities and estimated station delay times along the edge of the model. Bootstrap inversions were carried out to determine the standard deviation of velocities and time terms. Low Pn velocity anomalies are detected beneath the outer side of the Alps (-6% and the Western Po plain (-4% in correspondence with two regions of strong crustal thickening and negative Bouguer anomaly. In contrast, high Pn velocities are imaged beneath the inner side of the Alps (+4% indicating the presence of high velocity and density lower crust-upper mantle. The Ligurian sea shows high Pn velocities close to the Ligurian coastlines (+3% and low Pn velocities (-1.5% in the middle of the basin in agreement with the upper mantle velocity structure revealed by seismic refraction profiles.

  17. Centrosomal protein 55 activates NF-?B signalling and promotes pancreatic cancer cells aggressiveness

    OpenAIRE

    Peng, Tao; Zhou, Wei; Guo, Feng; Wu, He-shui; Wang, Chun-you; Wang, Li; Yang, Zhi-yong

    2017-01-01

    Centrosomal protein 55 (CEP55) is a microtubule-bundling protein that participants in cell mitosis. It is overexpressed in several solid tumours and promotes the growth and invasion of cancer cells. However, the role of CEP55 in pancreatic cancer (PANC) remains unclear. Herein, upregulated expression of CEP55 (associated with poor prognosis) was detected in PANC using quantitative real-time reverse transcription PCR, western blotting, and immunohistochemistry. Cell migration, colony formation...

  18. Espressione genica e proteica dell’albumina negli adipociti umani maturi. Relazione tra albuminemia e tessuto adiposo nei pazienti in trattamento dialitico.

    OpenAIRE

    Sirico, Maria Luisa

    2013-01-01

    Scopo: Lo scopo di questo lavoro era quello di indagare l’espressione genica dell’albumina negli adipociti umani maturi, la regolazione di tale espressione da parte dell’infiammazione e il contributo del tessuto adiposo ai livelli circolanti di albumina. Metodi: Campioni di tessuto adiposo sono stati prelevati da soggetti sani e infiammati. Sono state effettuate analisi di espressione genica (RT-PCR e Real-Time PCR) e proteica (Western Blot ) sugli adipociti e preadipociti estratti. Inoltr...

  19. Using Conventional Hydropower to Help Alleviate Variable Resource Grid Integration Challenges in the Western U.S

    Science.gov (United States)

    Veselka, T. D.; Poch, L.

    2011-12-01

    Integrating high penetration levels of wind and solar energy resources into the power grid is a formidable challenge in virtually all interconnected systems due to the fact that supply and demand must remain in balance at all times. Since large scale electricity storage is currently not economically viable, generation must exactly match electricity demand plus energy losses in the system as time unfolds. Therefore, as generation from variable resources such as wind and solar fluctuate, production from generating resources that are easier to control and dispatch need to compensate for these fluctuations while at the same time respond to both instantaneous change in load and follow daily load profiles. The grid in the Western U.S. is not exempt to grid integration challenges associated with variable resources. However, one advantage that the power system in the Western U.S. has over many other regional power systems is that its footprint contains an abundance of hydropower resources. Hydropower plants, especially those that have reservoir water storage, can physically change electricity production levels very quickly both via a dispatcher and through automatic generation control. Since hydropower response time is typically much faster than other dispatchable resources such as steam or gas turbines, it is well suited to alleviate variable resource grid integration issues. However, despite an abundance of hydropower resources and the current low penetration of variable resources in the Western U.S., problems have already surfaced. This spring in the Pacific Northwest, wetter than normal hydropower conditions in combination with transmission constraints resulted in controversial wind resource shedding. This action was taken since water spilling would have increased dissolved oxygen levels downstream of dams thereby significantly degrading fish habitats. The extent to which hydropower resources will be able to contribute toward a stable and reliable Western grid is

  20. Women and Islam in the Western Media

    NARCIS (Netherlands)

    Vintges, K.; Ennaji, M.

    2016-01-01

    This chapter focuses on recent debates on women and Islam as framed in the Western media. The first section discusses the way these debates are organized through the presentations and self-presentations of Muslim women in the Western media. The second section (titled “The Restyling of Western

  1. Atmospheric Nitrogen Deposition in the Western United States: Sources, Sinks and Changes over Time

    Science.gov (United States)

    Anderson, Sarah Marie

    Anthropogenic activities have greatly modified the way nitrogen moves through the atmosphere and terrestrial and aquatic environments. Excess reactive nitrogen generated through fossil fuel combustion, industrial fixation, and intensification of agriculture is not confined to anthropogenic systems but leaks into natural ecosystems with consequences including acidification, eutrophication, and biodiversity loss. A better understanding of where excess nitrogen originates and how that changes over time is crucial to identifying when, where, and to what degree environmental impacts occur. A major route into ecosystems for excess nitrogen is through atmospheric deposition. Excess nitrogen is emitted to the atmosphere where it can be transported great distances before being deposited back to the Earth's surface. Analyzing the composition of atmospheric nitrogen deposition and biological indicators that reflect deposition can provide insight into the emission sources as well as processes and atmospheric chemistry that occur during transport and what drives variation in these sources and processes. Chapter 1 provides a review and proof of concept of lichens to act as biological indicators and how their elemental and stable isotope composition can elucidate variation in amounts and emission sources of nitrogen over space and time. Information on amounts and emission sources of nitrogen deposition helps inform natural resources and land management decisions by helping to identify potentially impacted areas and causes of those impacts. Chapter 2 demonstrates that herbaria lichen specimens and field lichen samples reflect historical changes in atmospheric nitrogen deposition from urban and agricultural sources across the western United States. Nitrogen deposition increases throughout most of the 20 th century because of multiple types of emission sources until the implementation of the Clean Air Act Amendments of 1990 eventually decrease nitrogen deposition around the turn of

  2. Expression of JMJD2A in infiltrating duct carcinoma was markedly higher than fibroadenoma, and associated with expression of ARHI, p53 and ER in infiltrating duct carcinoma.

    Science.gov (United States)

    Li, Bei-Xu; Li, Jia; Luo, Cheng-Liang; Zhang, Ming-Chang; Li, Hui; Li, Li-Liang; Xu, Hong-Fei; Shen, Yi-Wen; Xue, Ai-Min; Zhao, Zi-Qin

    2013-03-01

    Jumonji Domain Containing 2A (JMJD2A) may be a cancer-associated gene involved in human breast cancer. With a view to investigating expression of JMJD2A in human breast cancer and benign lesion tissues as well as relationship between JMJD2A and tumor related proteins, histological and immunohistochemical analysis, Western blot and quantitative real-time PCR in infiltrating duct carcinoma and fibroadenoma for JMJD2A and immunohistochemical analysis and quantitative real-time PCR in infiltrating duct carcinoma for tumor related proteins (ARHI, p53, ER, PR and CerbB-2) were performed. Histological examination validated the clinical diagnosis. The JMJD2A positive rate of infiltrating duct carcinoma was significantly higher than fibroadenoma by immunohistochemical analysis. The mean optical density of JMJD2A in infiltrating duct carcinoma was higher than fibroadenoma by western blot. JMJD2A mRNA level in infiltrating duct carcinoma was higher than fibroadenoma by quantitative real-time PCR. Spearman correlation analysis revealed that the expression of JMJD2A was associated with ARHI, p53 and ER from immunohistochemical results respectively. Pearson correlation analysis revealed that the expression of JMJD2A was associated with ARHI, p53 and ER from quantitative real-time PCR results respectively. Expression of JMJD2A in infiltrating duct carcinoma was higher, and associated with ARHI, p53 and ER. The results may take JMJD2A as a potential diagnostic and therapeutic target in human breast cancer.

  3. The Western Balkans Geopolitics and Russian Energy Politics

    Directory of Open Access Journals (Sweden)

    Muhidin Mulalic

    2014-12-01

    Full Text Available The Energy politics is today at the center of the Russian and the Western Balkans relations. It is widely known that Russia has been playing significant role in world energy supply. Therefore, Russian energy companies have taken a leading role in the promotion of their businesses in the Western Balkans. The Western Balkans region has become geostrategic partner as a transit route for the integration of Russia into the global energy world market. With regards to the transportation of gas Western Balkans as a transit route is determined to fully realize South Stream project. In contrast to “a buffer zone” role, in the past few decades the Western Balkans gained an attention from Russia and became an important geopolitical bridge towards Western Europe. Such geopolitical and geostrategic planning of Russia is apparent with regards to gas and oil. Due to Russian South Stream gas pipeline project the Western Balkans became an important European energy transforming center. These new geostrategic games over the pipeline have also revived the Russian historical interests in the Balkans. This paper aims to analyze Russian-Western Balkans relations with special emphasis on energy politics and geopolitical and geostrategic interdependence of Russia and the Western Balkans.

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

    International Nuclear Information System (INIS)

    Lucher, L.A.; Lego, T.

    1989-01-01

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

  5. Role of Piezo Channels in Ultrasound-stimulated Dental Stem Cells.

    Science.gov (United States)

    Gao, Qianhua; Cooper, Paul R; Walmsley, A Damien; Scheven, Ben A

    2017-07-01

    Piezo1 and Piezo2 are mechanosensitive membrane ion channels. We hypothesized that Piezo proteins may play a role in transducing ultrasound-associated mechanical signals and activate downstream mitogen-activated protein kinase (MAPK) signaling processes in dental cells. In this study, the expression and role of Piezo channels were investigated in dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) after treatment with low-intensity pulsed ultrasound (LIPUS). Cell proliferation was evaluated by bromodeoxyuridine incorporation. Western blots were used to analyze the proliferating cell nuclear antigen as well as the transcription factors c-fos and c-jun. Enzyme-linked immunosorbent assay and Western blotting were used to determine the activation of MAPK after LIPUS treatment. Ruthenium red (RR), a Piezo ion channel blocker, was applied to determine the functional role of Piezo proteins in LIPUS-stimulated cell proliferation and MAPK signaling. Western blotting showed the presence of Piezo1 and Piezo2 in both dental cell types. LIPUS treatment significantly increased the level of the Piezo proteins in DPSCs after 24 hours; however, no significant effects were observed in PDLSCs. Treatment with RR significantly inhibited LIPUS-stimulated DPSC proliferation but not PDLSC proliferation. Extracellular signal-related kinase (ERK) 1/2 MAPK was consistently activated in DPSCs over a 24-hour time period after LIPUS exposure, whereas phosphorylated c-Jun N-terminal kinase and p38 mitogen-activated protein kinase MAPK were mainly increased in PDLSCs. RR affected MAPK signaling in both dental cell types with its most prominent effects on ERK1/2/MAPK phosphorylation levels; the significant inhibition of LIPUS-induced stimulation of ERK1/2 activation in DPSCs by RR suggests that stimulation of DPSC proliferation by LIPUS involves Piezo-mediated regulation of ERK1/2 MAPK signaling. This study for the first time supports the role of Piezo ion channels in

  6. Human T-cell lymphotropic virus type 1 provirus and phylogenetic analysis in patients with mycosis fungoides and their family relatives.

    Science.gov (United States)

    Shohat, M; Shohat, B; Mimouni, D; Pauli, G; Ellerbrok, H; David, M; Hodak, E

    2006-08-01

    Mycosis fungoides (MF) is a cutaneous T-cell lymphoma of unknown aetiology. A pathogenic role of human T-cell lymphotropic virus type 1 (HTLV-1) has been suggested but remains controversial. To determine whether MF is linked to HTLV-1. Blood samples were collected from 60 patients, 15 family relatives of patients with MF (MFRs), 20 healthy controls and 10 patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The presence of HTLV-1 antibodies in serum was tested by the Western blot rp21e-enhanced test. DNA was extracted from the blood with the Qiagen blood kit. We used 500 ng of DNA either in conventional HTLV-1-specific polymerase chain reaction (PCR) or in real-time PCR using primers sk43 and sk44 together with a tax-specific fluorescent probe. In Western blot, antibodies against three to four HTLV-1 antigens were detected in 52% of patients with MF. All of the patients with HAM/TSP were positive, while only 7% of the MFRs and none of the 20 healthy controls reacted with HTLV-1 antigens in Western blot. One of 60 patients with MF and one of 15 MFRs were positive in HTLV-1 PCR. These two PCR-positive samples which were quantified in real-time PCR showed that fewer than five in 10(6) cells were HTLV-1 infected. We succeeded in amplifying and sequencing the 5' end of the provirus from the blood of the PCR-positive MFR by seminested PCR. A positive result was also obtained in this test. Phylogenetic tree analyses revealed a high homology of this sequence with other HTLV-1 sequences from the Middle East. The above PCR-positive MFR was the brother of a PCR-negative patient with MF. These findings demonstrate that HTLV-1 is probably not the aetiological agent of MF. However, it may play a role in immunosuppression and in the spreading of the disease.

  7. Age and kinematics of ductile deformation in the Cerro Durazno area, NW Argentina: Significance for orogenic processes operating at the western margin of Gondwana during Ordovician-Silurian times

    OpenAIRE

    M. I. Wegmann; U. Riller; F. D. Hongn; Johannes Glodny; Onno Oncken

    2008-01-01

    The Cerro Durazno Pluton belongs to a suite of Paleozoic granitoid intrusions in NWArgentina, that are central for understanding the tectonic setting of the western margin of Gondwana in Ordovician and Silurian times. The pluton and its host rocks were tectonically overprinted by metamorphic mineral shape fabrics formed under middle greenschist-facies metamorphic conditions and associated with the nearby Agua Rosada Shear Zone. Kinematic analysis of the shear zone based on the geometric relat...

  8. Diagnostic-test evaluation of immunoassays for anti-Toxoplasma gondii IgG antibodies in a random sample of Mexican population.

    Science.gov (United States)

    Caballero-Ortega, Heriberto; Castillo-Cruz, Rocío; Murieta, Sandra; Ortíz-Alegría, Luz Belinda; Calderón-Segura, Esther; Conde-Glez, Carlos J; Cañedo-Solares, Irma; Correa, Dolores

    2014-05-14

    There are few articles on evaluation of Toxoplasma gondii serological tests. Besides, commercially available tests are not always useful and are expensive for studies in open population. The aim of this study was to evaluate in-house ELISA and western blot for IgG antibodies in a representative sample of people living in Mexico. Three hundred and five serum samples were randomly selected from two national seroepidemiological survey banks; they were taken from men and women of all ages and from all areas of the country. ELISA cut-off was established using the mean plus three standard deviations of negative samples. Western blots were analysed by two experienced technicians and positivity was established according to the presence of at least three diagnostic bands. A commercial ELISA kit was used as a third test. Two reference standards were built up: one using concordant results of two assays leaving the evaluated test out and the other in which the evaluated test was included (IN) with at least two concordant results to define diagnosis. the lowest values of diagnostic parameters were obtained with the OUT reference standards: in-house ELISA had 96.9% sensitivity, 62.1% specificity, 49.6% PPV, 98.1% NPV and 71.8% accuracy, while western blot presented 81.8%, 89.7%, 84.0%, 88.2% and 86.6% values and the best kappa coefficient (0.72-0.82). The in-house ELISA is useful for screening people of Mexico, due to its high sensitivity, while western blot may be used to confirm diagnosis. These techniques might prove useful in other Latin American countries.

  9. Protein and antigen profiles of Leptospira interrogans serovar Hardjo Perfil proteico e antigênico da Leptospira interrogans sorovariedade Hardjo

    Directory of Open Access Journals (Sweden)

    Bárbara Nobre Lafetá

    2009-12-01

    Full Text Available The protein profile of the outer membrane of Leptospira interrogans serovar Hardjo subtype hardjoprajitno associated with the bovine natural immune response was investigated. The outer membrane proteins were extracted utilizing Triton X114 and precipitated with acetone. The protein sample was then resolved by SDS-PAGE and reacted in western blot against sera from a hyperimmune rabbit and from naturally infected bovines. In silver stained gels, 14 protein bands were observed, among which four proteins, with 22, 29, 47 and 63kDa, appeared as major constituents. Western blot tests with hyperimmune rabbit antiserum detected bands corresponding to proteins with 35; 27; 24; 21; 17 and 14kDa, while 32kDa and 45kDa proteins were the most immunoreactive with sera from naturally infected bovines.Estudou-se o perfil proteico da membrana externa da Leptospira interrogans sorovariedade Hardjo, amostra hardjoprajitno, associado à resposta imune natural de bovinos infectados. Foram utilizados Triton X114 para a extração das proteínas de membrana externa e acetona para precipitá-las. As proteínas extraídas foram analisadas por SDS-PAGE e western blot contra soro de coelhos hiperimunes e de bovinos naturalmente infectados. Em géis corados com nitrato de prata, 14 bandas proteicas foram identificadas, e quatro dessas bandas, com 22, 29, 47 e 63kDa, foram as mais proeminentes. Os western blots com soro hiperimune de coelho detectaram bandas correspondentes a proteínas com pesos moleculares de 35, 27, 24, 21, 17 e 14kDa, e bandas de 32 e 45kDa destacaram-se nos testes com soros de bovinos naturalmente infectados.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1981-08-28

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

  12. Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Davoud Koolivand

    2016-10-01

    Full Text Available The genomic region of Grapevine fanleaf virus (GFLV encoding the movement protein (MP was cloned into pET21a and transformed into Escherichia coli strain BL21 (DE3 to express the protein. Induction was made with a wide range of isopropyl-β-D-thiogalactopyranoside (IPTG concentrations (1, 1.5, and 2 mM each for duration of 4, 6, or 16 h. However, the highest expression level was achieved with 1 mM IPTG for 4 h. Identity of the expressed protein was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE followed by Western blotting. The expressed 41 kDa protein was purified under denaturing condition by affinity chromatography, reconfirmed by Western blotting and plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA before being used as a recombinant antigen to raise polyclonal antibodies in rabbits. Purified anti-GFLV MP immunoglobulines (IgGs and conjugated IgGs detected the expressed MP and GFLV virions in infected grapevines when used in PTA-ELISA, double antibody sandwich-ELISA, and Western blotting. This is the first report on the production of anti-GFLV MP polyclonal antibodies and application for the virus detection.

  13. A Proteomic Approach for the Identification of Up-Regulated Proteins Involved in the Metabolic Process of the Leiomyoma.

    Science.gov (United States)

    Ura, Blendi; Scrimin, Federica; Arrigoni, Giorgio; Franchin, Cinzia; Monasta, Lorenzo; Ricci, Giuseppe

    2016-04-09

    Uterine leiomyoma is the most common benign smooth muscle cell tumor of the uterus. Proteomics is a powerful tool for the analysis of complex mixtures of proteins. In our study, we focused on proteins that were upregulated in the leiomyoma compared to the myometrium. Paired samples of eight leiomyomas and adjacent myometrium were obtained and submitted to two-dimensional gel electrophoresis (2-DE) and mass spectrometry for protein identification and to Western blotting for 2-DE data validation. The comparison between the patterns revealed 24 significantly upregulated (p leiomyoma and not with the normal myometrium. The overexpression of seven proteins involved in the metabolic processes of the leiomyoma was further validated by Western blotting and 2D Western blotting. Four of these proteins have never been associated with the leiomyoma before. The 2-DE approach coupled with mass spectrometry, which is among the methods of choice for comparative proteomic studies, identified a number of proteins overexpressed in the leiomyoma and involved in several biological processes, including metabolic processes. A better understanding of the mechanism underlying the overexpression of these proteins may be important for therapeutic purposes.

  14. Time and dose effects of cigarette smoke and acrolein on protein carbonyl formation in HaCaT keratinocytes.

    Science.gov (United States)

    Avezov, K; Reznick, A Z; Aizenbud, D

    2015-01-01

    Cigarette smoke (CS) is an important environmental source of human exposure to a highly toxic and chemically active α,β-unsaturated aldehyde: acrolein. It is capable of causing protein carbonylation and dysfunction, especially in oral tissues of smokers, constantly exposed to CS toxic constituents. The foremost damage is considered to be cumulative, but even a short exposure can be potentially harmful. The objectives of the current study were to examine the short time and dose effects of direct CS and acrolein exposure on intracellular protein carbonylation in epithelial cells. HaCaT-keratinocytes were exposed to different doses of acrolein and whole phase CS using a unique smoking simulator apparatus that mimics the exposure in smokers. The rate of intracellular protein carbonyl modification was examined 10-60 min after the exposure by Western blot. In addition, the effect of pre-incubation with a thiol scavenger N-acetylcysteine (NAC) was also assessed. We found that intracellular protein carbonyls increased as fast as 10 min after CS exposure and their concentration doubled after 20 min, with a slight elevation afterwards. Also, carbonyl levels increased gradually as CS and acrolein doses were elevated. Addition of 1 mM NAC neutralized part of the damage. We conclude that CS and acrolein intracellular protein carbonylation is dose- and time- dependent. Even a short time exposure to CS and its aldehydic constituents can be potentially harmful.

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  16. Integrated ecosystem assessment for western development of China

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The objectives of Integrated Ecosystem Assessment for Western Development of China includes: (1) providing scientific basis for ecosystem protection, ecosystem management and ecological construction in the western development; (2) developing complete database and analytical tools and strengthening decision-making support capacity; and (3) improving ecosystem management in China, spreading ecological knowledge to the public, serving decision-making of local and central governments, and promoting socio-economic sustainable development. The design and implementation of the project are of significance under the macro background of western development of China. By the integrated assessment of western China, we can get the first-hand data covering all the environmental factors as well as disclose the situations and their changing trends of ecosystem in the western part of China, which will benefit the decision-making for the central and local governments in the implementation of the western development strategy. In other words, the implementation of the project, to a certain extent, can guarantee the regional sustainable development of western China.

  17. Disentangling the control of tectonics, eustasy, trophic conditions and climate on shallow-marine carbonate production during the Aalenian-Oxfordian interval: From the western France platform to the western Tethyan domain

    Science.gov (United States)

    Andrieu, Simon; Brigaud, Benjamin; Barbarand, Jocelyn; Lasseur, Eric; Saucède, Thomas

    2016-11-01

    The objective of this work is to improve our understanding of the processes controlling changes in the architecture and facies of intracontinental carbonate platforms. We examined the facies and sequence stratigraphy of Aalenian to Oxfordian limestones of western France. Seventy-seven outcrop sections were studied and thirty-one sedimentary facies identified in five depositional environments ranging from lower offshore to backshore. Platform evolution was reconstructed along a 500 km cross-section. Twenty-two depositional sequences were identified on the entire western France platform and correlated with European third-order sequences at the biozone level, demonstrating that eustasy was the major factor controlling the cyclic trend of accommodation. The tectonic subsidence rate was computed from accommodation measurements from the Aalenian to the Oxfordian in key localities. Tectonism controlled the sedimentation rate and platform architecture at a longer time scale. Tectonic subsidence triggered the demise of carbonate production at the Bathonian/Callovian boundary while the uplift made possible the recovery of carbonate platform from Caen to Le Mans during the mid Oxfordian. Topography of the Paleozoic basement mainly controlled lateral variations of paleodepth within the western France platform until the mid Bathonian. A synthesis of carbonate production in the western Tethyan domain at that time was conducted. Stages of high carbonate production during the Bajocian/Bathonian and the middle to late Oxfordian are synchronous with low δ13C, high eccentricity intervals, and rather dry climate promoting (1) evaporation and carbonate supersaturation, and (2) oligotrophic conditions. Periods of low carbonate production during the Aalenian and from the middle Callovian to early Oxfordian correlate with high δ13C and low eccentricity intervals, characterized by wet climate and less oligotrophic conditions. Such conditions tend to diminish growth potential of carbonate

  18. Fed-batch production of hydrophobin RodB from Aspergillus fumigatus in host Pichia pastoris

    DEFF Research Database (Denmark)

    Pedersen, Mona Højgaard; Borodina, Irina; Frisvad, Jens Christian

    was dependent on the methanol-induced AOX1 promoter. Later production was scaled up to a 2 L fed-batch fermentor. Protein production was analyzed by SDS-PAGE, coomassie and silver-stained, as well as western blotting using an anti-his detection antibody. RodB was purified using His-select Nickel Affinity gel....... The emulsifying property of rRodB was investigated using olive oil stained with Sudan black suspended in tris-buffer. The stability of oil micelles were studied by light microscopy. Results: Protein bands of expected size were detected by SDS-PAGE and western blotting in both the fermentation broth and excess...

  19. Ideologies in conflict in XXI century: Islamophobia vs Western phobia

    Directory of Open Access Journals (Sweden)

    José Manuel Bermejo Laguna

    2016-06-01

    Full Text Available Recently started the new millennium, the West is witness a conflict between two ideologies that converge to use Islam as a weapon of choice with which declared opponents. To Islamophobia, that joins large sectors of the Western population and whose strategy involves systematically revile the Islamic civilization, with the purpose of ratting as Muslim enemies to men and women who reside in Western societies, opposes the Western phobia which is monopolized by native radical ideologies of the Islamic world. Identified these two philosophies, the aim of this research pursues the separately analysis from both to come to conclude as necessary the alteration of the foundations that are supporting, to avoid being affected as sensitive areas as the personal and social safety. The methodology, consisting in the exposition of causes that may be indicative of the mutual resentment that his followers profess themselves, comes to demostrate the impact that those speeches have the time to build an enemy; fed, on the one hand, of imaginary fears, the rejection of all meaning of multicultural theory of modern societies, and therefore, negative cultural concessions to people who do not promote them in their societies of origin. The Western phobia, sponsored by the Islamist ideology, comes to introduce Islam in the early stages as the resource with which to face the Western supremacy, and from there to promote violence against those who have usurped their land, and are reproaching their lano interest in addressing proposals for modern political, social or family. Only proposals of intercultural harmony and rejection of the discourses that make Islam a political and military ideology may prevent that these ideologies continue gaining accessions.

  20. Annosus Root disease of Western Conifers (FIDL)

    Science.gov (United States)

    Craig L. Schmitt; John R. Parmeter; John T. Kliejunas

    2000-01-01

    Annosus root disease is found on all western conifer species but is of most concern on true firs, hemlocks, and pines. Incense cedar, coast redwood and sequoia are sometimes infected in California. Western juniper is infected throughout its range. Annosus is common and causes extensive decay in old-growth western and mountain hemlock stands. Many mixed conifer stands...

  1. Homicides in Western Norway, 1985-2009, time trends, age and gender differences.

    Science.gov (United States)

    Kristoffersen, S; Lilleng, P K; Mæhle, B O; Morild, I

    2014-05-01

    This retrospective study from Western Norway is based on the cases of 196 homicide victims from 1985 to 2009. The median age of the victims was 35 years, in both genders. Within the cases, 113 of the victims were male and 83 female, 28 victims were under the age of 18, and 19 victims were not native Norwegians. Ethanol was detected in the blood of a higher proportion of male compared to female victims, whereas a higher proportion of female compared to male victims had both illegal/legal drugs detected in their blood. Most perpetrators were male. Men were most often killed by an acquaintance, women by their present or former intimate partner. In 14 cases of intimate partner homicide the perpetrator committed suicide after killing their female partner. The dominant scene of crime was private homes. Most victims were killed by blunt force, sharp force or gunshot. The head was the body region most often injured in the homicide victims. Female victims were more often killed by manual strangulation than male victims. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  2. Factors affecting the use of prenatal and postnatal care by women of non-western immigrant origin in industrialized western countries.

    NARCIS (Netherlands)

    Boerleider, A.W.; Devillé, W.L.J.M.; Francke, A.L.; Wiegers, T.A.

    2011-01-01

    Background: In many industrialized western countries immigrants constitute a substantial part of the population, which is also seen in the prenatal and postnatal care client population. Research in several industrialized western countries has shown that women of non-western immigrant origin make

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

    Science.gov (United States)

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

    2010-06-01

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

  4. Caracterização de anticorpos monoclonais contra rotavírus bovino e suas aplicações como ferramenta de diagnóstico Characterization of monoclonal antibodies against bovine rotavirus and their diagnostic applications

    Directory of Open Access Journals (Sweden)

    P.A. Beck

    2007-06-01

    Full Text Available Anticorpos monoclonais (AcM para rotavírus bovino foram caracterizados para sua aplicação como ferramenta de diagnóstico, utilizando-se as técnicas de isotipificação, dot-blot, western-blot, imunofluorescência indireta (IFI e ELISA de captura. A caracterização imunoquímica demonstrou que os cinco AcM 1G5, 4F7, 1E12, 4F3 e 3C12 foram do isótipo IgG2a. Pela técnica de dot-blot, os AcM 1G5, 4F7, 1E12, 4F3 detectaram antígenos do rotavírus, em diferentes concentrações, e dois AcM (1E12 e 4F3 reconheceram proteínas virais pela técnica de western-blot. Todos os AcM reagiram positivamente na técnica de IFI em cultivo celular e foram capazes de detectar antígeno viral em amostras fecais bovinas e humanas, pela técnica de ELISA de captura. Identificaram-se dois grupos de AcM, um deles formado pelos AcM 4F7, 1E12 e 1G5, para seu possível uso na detecção de antígeno viral em fezes por meio do ELISA de captura ou dot-blot e outro pelos 4F3 e 3C12, que podem ser usados para detectar antígeno viral em culturas de células por meio de IFI.This work was carried out to characterize and evaluate five bovine rotavirus, monoclonal antibodies (MAbs, as a diagnosis tool, by isotyping, dot-blot, western-blot, indirect immunofluorescence (IFI and ELISA techniques. The immunochemistry characterization showed that all five MAbs (4F7, 4F3, 1G5, 1E12 and 3C12 were IgG2a isotype. The dot-blot immunoassay showed that 1G5, 4F7, 1E12 and 4F3 detected viral antigen in different concentrations and two MAbs (1E12 and 4F3 recognized viral proteins by western-blot. All MAbs detected viral antigen in bovine and human fecal samples by capture ELISA technique and viral antigen in infected MA-104 cell culture by IFI. In conclusion, two groups of Mabs were indetified: one with Mabs 4F7, 1E12 and 1G5 showed the best results to detect rotavirus antigen in fecal samples by capture ELISA or dot-blot techniques assay and other with 4F3 and 3C12 which may be used to

  5. Comparative Analysis of Music Recordings from Western and Non-Western traditions by Automatic Tonal Feature Extraction

    Directory of Open Access Journals (Sweden)

    Emilia Gómez

    2008-09-01

    Full Text Available The automatic analysis of large musical corpora by means of computational models overcomes some limitations of manual analysis, and the unavailability of scores for most existing music makes necessary to work with audio recordings. Until now, research on this area has focused on music from the Western tradition. Nevertheless, we might ask if the available methods are suitable when analyzing music from other cultures. We present an empirical approach to the comparative analysis of audio recordings, focusing on tonal features and data mining techniques. Tonal features are related to the pitch class distribution, pitch range and employed scale, gamut and tuning system. We provide our initial but promising results obtained when trying to automatically distinguish music from Western and non- Western traditions; we analyze which descriptors are most relevant and study their distribution over 1500 pieces from different traditions and styles. As a result, some feature distributions differ for Western and non-Western music, and the obtained classification accuracy is higher than 80% for different classification algorithms and an independent test set. These results show that automatic description of audio signals together with data mining techniques provide means to characterize huge music collections from different traditions and complement musicological manual analyses.

  6. Northern blot analysis to investigate the abundance of microorganisms

    International Nuclear Information System (INIS)

    Krause, D.O.

    2005-01-01

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

  7. Conservation Motivation, Social Equality and Left-Right Ideological Preferences in Western and Eastern Europe.

    Science.gov (United States)

    Hadarics, Márton

    2017-05-01

    We investigated how attitudes towards social equality can influence the relationship between conservation motivation (or openness) and personal ideological preferences on the left-right dimension, and how this relationship pattern differs between Western and Central & Eastern European (CEE) respondents. Using data from the European Social Survey (2012) we found that individual-level of conservation motivation reduces cultural egalitarianism in both the Western European and the CEE regions, but its connection with economic egalitarianism is only relevant in the CEE region where it fosters economic egalitarianism. Since both forms of egalitarianism were related to leftist ideological preferences in Western Europe, but in the CEE region only economic egalitarianism was ideologically relevant, we concluded that the classic "rigidity of the right" phenomenon is strongly related to cultural (anti)egalitarianism in Western Europe. At the same time, conservation motivation serves as a basis for the "rigidity of the left" in the post-socialist CEE region, in a great part due to the conventional egalitarian economic views.

  8. Cooperation and Conflict: Faction Problem of Western Medicine Group in Modern China

    Directory of Open Access Journals (Sweden)

    Jeongeun JO

    2016-08-01

    Full Text Available After the defeat of the Opium War and the Sino-Japanese War, China’s intellectuals realized necessity of modernization (Westernization to survive in the imperial order of the survival of the fittest. In particular, it was urgent to accept Western medicine and train the doctors who learned Western medicine to change the sick and weary Chinese to be robust. Thus, new occupations of the Western Medicine Group (xiyi, doctors who learned Western medicine emerged in China. As with the first profession, the new Western Medicine Group tried to define standards of Western medicine and medical profession; however, it was difficult in the absence of the strong central government. In addition, they formed a faction by the country where they studied or the language they learned. The factions included the Britain - America faction(yingmeipai consisting of the Britain - America studied doctors or graduates from Protestant missions based medical schools, and the Germany - Japan faction(deripai, graduates from medical schools by Japanese or German government and the Chinese government. In 1915, they founded the National Medical Association of China mainly consisting of the Britain - America faction and the National Medical and Pharmaceutical Association of China led by the Germany – Japan faction. Initially, exchanges were active so most of eminent doctors belonged the two associations at the same time. They had a consciousness of a common occupation group as a doctor who had learned Western medicine. Thus, they actively cooperated to keep their profits against Chinese medicine and enjoy their reputation. Their cooperation emitted light particularly in translation of medical terms and unified works. Thanks to cooperation, the two associations selected medical terminologies by properly using the cases of the West and Japan. Additionally, medical schools of the Britain - America faction and the Germany – Japan faction produced various levels of the Western

  9. Involvement of Resveratrol and ω-3 Polyunsaturated Fatty Acids on Sirtuin 1 Gene Expression in THP1 Cells.

    Science.gov (United States)

    Tsuchiya, Takafumi; Endo, Ayano; Tsujikado, Kyoko; Inukai, Toshihiko

    2017-10-01

    Resveratrol, a kind of polyphenol, has the potential to activate the longevity gene in several cells, in the same manner as calorie restriction. We investigated the effect of resveratrol and ω-3-line polyunsaturated fatty acid on surtuin 1 (SIRT1) gene expression in human monocytes (THP1) cells. We examined the gene expression of THP1 cells using real-time polymerase chain reaction and Western blotting analysis. Resveratol, eicosapentaenoic acid (EPA) and docosahexaeanoic acid (DHA) as n-3 polyunsaturated fatty acid were added on THP1 cells. We observed the changes in the SIRT1 gene expression in those cells, under various doses of agents and in time courses. Then, we examined the interaction of glucose and mannitol on those agents׳ effect of the gene expression. The concentration range of glucose and mannitol was from 5-20mM, respectively. The SIRT1 gene expression could be defined in 24 and 48 hours both in real-time polymerase chain reaction analysis and in Western blotting. Resveratrol showed SIRT1 gene expression in a dose-dependent manner in the range of 0-20μM in both analyses. Although EPA at 10μM showed marked increase in SIRT1 gene expression compared to control condition in Western blotting, this phenomenon was not in dose-dependent manner. DHA did not exhibit any augmentation of SIRT1 gene expression in a dose-dependent manner in the range of 0-20μM in both analyses. We refined the dose-dependent inhibition of the SIRT1 gene expression within 20mM glucose medium. Although 20mM did not exhibit any inhibition, 10μM resveratrol induced the gene expression compared to control medium. Both 5 and 15mM mannitol medium did not significantly alter basic gene expression and 10μM resveratrol-induced gene expression. The present results suggest that resveratrol and EPA, but not DHA, markedly activated the SIRT1 gene expression in THP1 cells, and that high glucose medium could inhibit the basic gene expression, but not powerful resveratrol-induced gene

  10. Presence and seeding activity of pathological prion protein (PrP(TSE in skeletal muscles of white-tailed deer infected with chronic wasting disease.

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    Martin L Daus

    Full Text Available Chronic wasting disease (CWD is a contagious, rapidly spreading transmissible spongiform encephalopathy (TSE, or prion disease, occurring in cervids such as white tailed-deer (WTD, mule deer or elk in North America. Despite efficient horizontal transmission of CWD among cervids natural transmission of the disease to other species has not yet been observed. Here, we report for the first time a direct biochemical demonstration of pathological prion protein PrP(TSE and of PrP(TSE-associated seeding activity, the static and dynamic biochemical markers for biological prion infectivity, respectively, in skeletal muscles of CWD-infected cervids, i. e. WTD for which no clinical signs of CWD had been recognized. The presence of PrP(TSE was detected by Western- and postfixed frozen tissue blotting, while the seeding activity of PrP(TSE was revealed by protein misfolding cyclic amplification (PMCA. Semi-quantitative Western blotting indicated that the concentration of PrP(TSE in skeletal muscles of CWD-infected WTD was approximately 2000-10,000-fold lower than in brain tissue. Tissue-blot-analyses revealed that PrP(TSE was located in muscle-associated nerve fascicles but not, in detectable amounts, in myocytes. The presence and seeding activity of PrP(TSE in skeletal muscle from CWD-infected cervids suggests prevention of such tissue in the human diet as a precautionary measure for food safety, pending on further clarification of whether CWD may be transmissible to humans.

  11. Anti-inflammatory effect of garlic 14-kDa protein on LPS-stimulated-J774A.1 macrophages.

    Science.gov (United States)

    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.

  12. The major antigenic membrane protein of "Candidatus Phytoplasma asteris" selectively interacts with ATP synthase and actin of leafhopper vectors.

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    Luciana Galetto

    Full Text Available Phytoplasmas, uncultivable phloem-limited phytopathogenic wall-less bacteria, represent a major threat to agriculture worldwide. They are transmitted in a persistent, propagative manner by phloem-sucking Hemipteran insects. Phytoplasma membrane proteins are in direct contact with hosts and are presumably involved in determining vector specificity. Such a role has been proposed for phytoplasma transmembrane proteins encoded by circular extrachromosomal elements, at least one of which is a plasmid. Little is known about the interactions between major phytoplasma antigenic membrane protein (Amp and insect vector proteins. The aims of our work were to identify vector proteins interacting with Amp and to investigate their role in transmission specificity. In controlled transmission experiments, four Hemipteran species were identified as vectors of "Candidatus Phytoplasma asteris", the chrysanthemum yellows phytoplasmas (CYP strain, and three others as non-vectors. Interactions between a labelled (recombinant CYP Amp and insect proteins were analysed by far Western blots and affinity chromatography. Amp interacted specifically with a few proteins from vector species only. Among Amp-binding vector proteins, actin and both the α and β subunits of ATP synthase were identified by mass spectrometry and Western blots. Immunofluorescence confocal microscopy and Western blots of plasma membrane and mitochondrial fractions confirmed the localisation of ATP synthase, generally known as a mitochondrial protein, in plasma membranes of midgut and salivary gland cells in the vector Euscelidius variegatus. The vector-specific interaction between phytoplasma Amp and insect ATP synthase is demonstrated for the first time, and this work also supports the hypothesis that host actin is involved in the internalization and intracellular motility of phytoplasmas within their vectors. Phytoplasma Amp is hypothesized to play a crucial role in insect transmission specificity.

  13. Ghrelin Attenuated Lipotoxicity via Autophagy Induction and Nuclear Factor-κB Inhibition

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    Yuqing Mao

    2015-09-01

    Full Text Available Background/Aims: Nonalcoholic fatty liver disease (NAFLD is the most common chronic liver disease worldwide. Autophagy is associated with NAFLD. Ghrelin is a gut hormone with various functions including energy metabolism and inflammation inhibition. We investigated the therapeutic effect of ghrelin on NAFLD and its association with autophagy. Methods: C57bl/6 mice were fed a high-fat diet for 8 weeks to induce a model of chronic NAFLD, with ghrelin (10 µg/kg administrated subcutaneously twice weekly from weeks 6 to 8. LO2 cells were pretreated with ghrelin (10-8 M before stimulation with free fatty acid (palmitic and oleic acids; 1 mM. Lipid droplets were identified by hematoxylin and eosin and Red O staining and quantified by triglyceride test kits. LC3I/II, an important biomarker protein of autophagy was detected by western blotting, real-time polymerase chain reaction, immunohistochemistry and immunofluorescence. Tumor necrosis factor (TNF-a and interleukin (IL-6 were detected by ELISA and immunohistochemistry. Nuclear factor (NF-κB p65 was detected by western blotting and immunofluorescence. AMP-activated protein kinase (AMPK and mammalian target of rapamycin (mTOR were detected by western blotting. Results: Ghrelin reduced the triglyceride content in high fat diet (HFD group in vivo and free fatty acid (FFA group in vitro. TNF-a and IL-6 were significantly reduced in the ghrelin-treated mice compared with the control group. Autophagy induction was accompanied with intracellular lipid reduction in ghrelin-treated mice. Ghrelin upregulated autophagy via AMPK/mTOR restoration and inhibited translocation of NF-κB into the nucleus. Conclusions: The results indicate that ghrelin attenuates lipotoxicity by autophagy stimulation and NF-κB inhibition.

  14. TGF-β1 Induces EMT in Bovine Mammary Epithelial Cells Through the TGFβ1/Smad Signaling Pathway

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    Qing Chen

    2017-08-01

    Full Text Available Background/Aims: Transforming growth factor-β1 (TGF-β1 plays a crucial role in chronic inflammation in various tissues, and is related to inflammation-caused organ fibrogenesis associated with the epithelial-mesenchymal transition (EMT and the deposition of the extracellular matrix (ECM. However, the effect of TGF-β1 on bovine mammary epithelial cells (BMECs with mastitis, and its mechanism, remain unknown. Methods: We analyzed the level of TGF-β1 in inflamed mammary tissues and cells using western blotting. BMECs were treated with TGF-β1, and EMT-related gene and protein expression changes were evaluated using quantitative real-time polymerase chain reaction (qPCR, western blotting, and immunofluorescence. We also inhibited the TGF/Smad signaling pathway using a receptor inhibitor, and analyzed EMT-related protein expression by western blotting. In addition, we injected TGF-β1 into mice mammary glands to investigate whether it can cause mammary fibrosis in vivo. Results: The TGF-β1 level was up-regulated in mammary tissues with mastitis and in inducible inflammatory BMECs. TGF-β1 treatment activated the TGF/ Smad signaling pathway in BMECs during their transition to the EMT phenotype, as indicated by morphological changes from a cobblestone-like shape to a spindle-like one. TGF-β1 treatment also up-regulated the expression of α-smooth muscle actin, vimentin, and collagen I, albumin, and down-regulated the expression of E-cadherin both in mRNA level and protein level. Furthermore, TGF-β1 enhanced the gene expressions of MMP2, MMP7, and fibronectin in BMECs. TGF-β1 injection induced mice mammary infection and fibrosis. Conclusion: These findings suggested that aberrant up-regulation of TGF-β1 in bovine mastitic mammary glands might play an important role in bovine mammary fibrosis caused by unresolved inflammation.

  15. Curcumin increased the differentiation rate of neurons in neural stem cells via wnt signaling in vitro study.

    Science.gov (United States)

    Chen, Fei; Wang, Haoxiang; Xiang, Xin; Yuan, Jichao; Chu, Weihua; Xue, Xingsen; Zhu, Haitao; Ge, Hongfei; Zou, Mingming; Feng, Hua; Lin, Jiangkai

    2014-12-01

    The objective of the present study was to clarify the relationship between the neuroprotective effects of curcumin and the classical wnt signaling pathway. Using Sprague-Dawley rats at a gestational age of 14.5 d, we isolated neural stem cells from the anterior two-thirds of the fetal rat brain. The neural stem cells were passaged three times using the half media replacement method and identified using cellular immunofluorescence. After passaging for three generations, we cultured cells in media without basic fibroblast growth factor and epidermal growth factor. Then we treated cells in five different ways, including a blank control group, a group treated with IWR1 (10 μmol/L), a group treated with curcumin (500 nmol/L), a group treated with IWR1 + curcumin, and a group treated with dimethyl sulfoxide (10 μmol/L). We then measured the protein and RNA expression levels for wnt3a and β-catenin using Western blotting and Reverse transcription-polymerase chain reaction (RT-PCR). Western-blotting: after the third generation of cells had been treated for 72 h, we observed that wnt3a and β-catenin expression was significantly increased in the group receiving 500 nmol/L curcumin but not in the other groups. Furthermore, cells in the IWR1-treated group showed decreased wnt3a and β-catenin expression, and wnt3a and β-catenin was also decreased in the IWR1 + 500 nmol/L curcumin group. No obvious change was observed in the dimethyl sulfoxide group. RT-PCR showed similar changes to those observed with the Western blotting experiments. Our study suggests that curcumin can activate the wnt signaling pathway, which provides evidence that curcumin exhibits a neuroprotective effect through the classical wnt signaling pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Anti-oxidative and anti-inflammatory effects of cinnamaldehyde on protecting high glucose-induced damage in cultured dorsal root ganglion neurons of rats.

    Science.gov (United States)

    Yang, Dan; Liang, Xiao-Chun; Shi, Yue; Sun, Qing; Liu, Di; Liu, Wei; Zhang, Hong

    2016-01-01

    To examine the mechanism underlying the beneficial role of cinnamaldehyde on oxidative damage and apoptosis in high glucose (HG)-induced dorsal root ganglion (DRG) neurons in vitro. HG-treated DRG neurons were developed as an in vitro model of diabetic neuropathy. The neurons were randomly divided into five groups: the control group, the HG group and the HG groups treated with 25, 50 and 100 nmol/L cinnamaldehyde, respectively. Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and apoptosis rate was evaluated by the in situ TdT-mediated dUTP nick end labeling (TUNEL) assay. The intracellular level of reactive oxygen species (ROS) was measured with flow cytometry. Expression of nuclear factor-kappa B (NF-κB), inhibitor of κB (IκB), phosphorylated IκB (p-IκB), tumor necrosis factor (TNF)-α, interleukin-6 (IL-6) and caspase-3 were determined by western blotting and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1) were also measured by western blotting. Cinnamaldehyde reduced HG-induced loss of viability, apoptosis and intracellular generation of ROS in the DRG neurons via inhibiting NF-κB activity. The western blot assay results showed that the HG-induced elevated expressions of NF-κB, IκB and p-IκB were remarkably reduced by cinnamaldehyde treatment in a dose-dependent manner (P neurons, but also lowered the elevated IL-6, TNF-α, cyclo-oxygenase and inducible nitric oxide synthase levels, indicating a reduction in inflammatory damage. Cinnamaldehyde protected DRG neurons from the deleterious effects of HG through inactivation of NF-κB pathway but not through activation of Nrf2/HO-1. And thus cinnamaldehyde may have potential application as a treatment for DPN.

  17. Exosomal MicroRNA MiR-1246 Promotes Cell Proliferation, Invasion and Drug Resistance by Targeting CCNG2 in Breast Cancer

    OpenAIRE

    Xiu Juan Li; Zhao Jun Ren; Jin Hai Tang; Qiao Yu

    2017-01-01

    Background/Aims: Treatment of breast cancer remains a clinical challenge. This study aims to validate exosomal microRNA-1246 (miR-1246) as a serum biomarker for breast cancer and understand the underlying mechanism in breast cancer progression. Methods: The expression levels of endogenous and exosomal miRNAs were examined by real time PCR, and the expression level of the target protein was detected by western blot. Scanning electron and confocal microscopy were used to characterize exosomes a...

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

    International Nuclear Information System (INIS)

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

    1996-01-01

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

  19. Development of a dot blot assay with antibodies to recombinant “core” 14-3-3 protein: Evaluation of its usefulness in diagnosis of Creutzfeldt–Jakob disease

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    Sarada Subramanian

    2016-01-01

    Full Text Available Background and Purpose: Definitive diagnosis of Creutzfeldt–Jakob disease (CJD requires demonstration of infective prion protein (PrPSc in brain tissues by immunohistochemistry or immunoblot, making antemortem diagnosis of CJD difficult. The World Health Organization (WHO recommends detection of 14-3-3 protein in cerebrospinal fluid (CSF in cases of dementia, with clinical correlation, as a useful diagnostic marker for CJD, obviating the need for brain biopsy.This facility is currently available in only a few specialized centers in the West and no commercial kit is available for clinical diagnostic use in India. Hence the objective of this study was to develop an in-house sensitive assay for quantitation of 14-3-3 protein and to evaluate its diagnostic potential to detect 14-3-3 proteins in CSF as a biomarker in suspected cases of CJD. Materials and Methods: A minigene expressing the “core” 14-3-3 protein was synthesized by overlapping polymerase chain reaction (PCR and the recombinant protein was produced by employing a bacterial expression system. Polyclonal antibodies raised in rabbit against the purified recombinant protein were used for developing a dot blot assay with avidin-biotin technology for signal amplification and quantitation of 14-3-3 protein in CSF. Results: The results in the present study suggest the diagnostic potential of the dot blot method with about 10-fold difference (P< 0.001 in the CSF levels of 14-3-3 protein between the CJD cases (N= 50 and disease controls (N= 70. The receiver operating characteristic (ROC analysis of the results suggested an optimal cutoff value of 2 ng/mL. Conclusions: We have developed an indigenous, economical, and sensitive dot blot method for the quantitation of 14-3-3 protein in CSF.

  20. The western diet and lifestyle and diseases of civilization

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    Pedro Carrera-Bastos

    2011-03-01

    Full Text Available Pedro Carrera-Bastos1, Maelan Fontes-Villalba1, James H O’Keefe2, Staffan Lindeberg1, Loren Cordain31Center for Primary Health Care Research, Faculty of Medicine at Lund University, Malmö, Sweden; 2Mid America Heart and Vascular Institute/University of Missouri-Kansas City, Kansas City, Missouri, USA; 3Department of Health and Exercise Science, Colorado State University, Fort Collins, Colorado, USAAbstract: It is increasingly recognized that certain fundamental changes in diet and lifestyle that occurred after the Neolithic Revolution, and especially after the Industrial Revolution and the Modern Age, are too recent, on an evolutionary time scale, for the human genome to have completely adapted. This mismatch between our ancient physiology and the western diet and lifestyle underlies many so-called diseases of civilization, including coronary heart disease, obesity, hypertension, type 2 diabetes, epithelial cell cancers, autoimmune disease, and osteoporosis, which are rare or virtually absent in hunter–gatherers and other non-westernized populations. It is therefore proposed that the adoption of diet and lifestyle that mimic the beneficial characteristics of the preagricultural environment is an effective strategy to reduce the risk of chronic degenerative diseases.Keywords: Paleolithic, hunter–gatherers, Agricultural Revolution, modern diet, western lifestyle and diseases

  1. Characterization of immunogenic proteins of Cysticercus tenuicollis of goats

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    A. Goswami

    2013-10-01

    Full Text Available Aim: To identify immunodominant proteins of cystic fluid antigens and whole cyst lysate antigens of Cysticercus tenuicollis, the larval stage of the canine tapeworm Taenia hydatigena. Materials and Methods: Three numbers of cysts of C. tenuicollis were collected from the mesentry of small intestine of goats after slaughter. C. tenuicollis cysts of each sample were washed thoroughly with PBS (pH 7.4. Two types of antigens i.e. cystic fluid antigens and whole cyst lysate antigens were prepared from each sample. Polypeptide profiles of cystic fluid antigens and whole cyst lysate antigens preparations of C. tunicollis were analysed by Sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE containing 10% gel. C. tenuicollis proteins resolved by SDS-PAGE were electrophoretically transferred from the gel to a nitrocellulose membrane and probed by western blotting using natural immune sera obtained from infected goats. C. tenuicollis proteins that were resolved by SDS-PAGE and reactive by immune sera of goat was estimated by a Soft ware programme named KODAK 1D Image Analysis Software. Results: A total of eight major polypeptides of molecular weight (Mr 149.4 kDa, 92.9 kDa, 74.2 kDa, 63.5 kDa, 36.2 kDa, 23.9 kDa, 15.7 kDa and 9.6 kDa were resolved by SDS-PAGE with minor variations. Out of these, three polypeptides of Mr 36.2 kDa, 23.9 kDa and 9.6 kDa were recognized as immunodominant polypeptides after western blotting. Both cystic fluid antigens and whole cyst lysate antigens resolved same Mr polypeptides by SDS-PAGE and identified same immunodominant polypeptides after western blotting. The immunodominant polipeptides of Mr 23.9 kDa and 9.6 kDa identified for the first time from both cystic fluid antigens and whole cyst lysate antigens prepared from Cysticercus tenuicollis. Conclusion: Eight polypeptides were resolved from cystic fluid antigens and whole cyst lysate antigens of Cysticercus tenuicollis by SDS-PAGe of which polypeptides of

  2. Overexpression of B7-H3 augments anti-apoptosis of colorectal cancer cells by Jak2-STAT3.

    Science.gov (United States)

    Zhang, Ting; Jiang, Bo; Zou, Shi-Tao; Liu, Fen; Hua, Dong

    2015-02-14

    To investigate the role of the overexpression of B7-H3 in apoptosis in colorectal cancer cell lines and the underlying molecular mechanisms. SW620 cells that highly overexpressed B7-H3 (SW620-B7-H3-EGFP) and HCT8 cells stably transfected with B7-H3 shRNA (HCT8-shB7-H3) were previously constructed in our laboratory. Cells transfected with pIRES2-EGFP were used as negative controls (SW620-NC and HCT8-NC). Real-time PCR and western blotting analysis were used to detect the mRNA and protein expressions of the apoptosis regulator proteins Bcl-2, Bcl-xl and Bax. A cell proliferation assay was used to evaluate the survival rate and drug sensitivity of the cells. The effect of drug resistance was detected by a cell cycle assay. Active caspase-3 western blotting was used to reflect the anti-apoptotic ability of cells. Western blotting was also performed to determine the expression of proteins associated with the Jak2-STAT3 signaling pathway and the apoptosis regulator proteins after the treatment with AG490, a Jak2 specific inhibitor, in B7-H3 overexpressing cells. The data were analyzed by GraphPad Prism 6 using a non-paired t-test. Whether by overexpression in SW620 cells or downregulation in HCT8, B7-H3 significantly affected the expression of anti- and pro-apoptotic proteins, at both the transcriptional and translational levels, compared with the negative control (P overexpression increased the drug resistance of cells and resulted in a higher survival rate (P overexpression inhibited apoptosis in colorectal cancer cell lines (P overexpression improved Jak2 and STAT3 phosphorylation and, in turn, increased the expression of the downstream anti-apoptotic proteins B-cell CLL/lymphoma 2 (Bcl-2) and Bcl-xl, based on western blotting (P overexpressing cells with the Jak2-specific inhibitor AG490, the phosphorylation of Jak2 and STAT3, and the expression of Bcl-2 and Bcl-xl, decreased accordingly (P overexpression of B7-H3 induces resistance to apoptosis in colorectal cancer

  3. IGNORING CHILDREN'S BEDTIME CRYING: THE POWER OF WESTERN-ORIENTED BELIEFS.

    Science.gov (United States)

    Maute, Monique; Perren, Sonja

    2018-03-01

    Ignoring children's bedtime crying (ICBC) is an issue that polarizes parents as well as pediatricians. While most studies have focused on the effectiveness of sleep interventions, no study has yet questioned which parents use ICBC. Parents often find children's sleep difficulties to be very challenging, but factors such as the influence of Western approaches to infant care, stress, and sensitivity have not been analyzed in terms of ICBC. A sample of 586 parents completed a questionnaire to investigate the relationships between parental factors and the method of ICBC. Data were analyzed using structural equation modeling. Latent variables were used to measure parental stress (Parental Stress Scale; J.O. Berry & W.H. Jones, 1995), sensitivity (Situation-Reaction-Questionnaire; Y. Hänggi, K. Schweinberger, N. Gugger, & M. Perrez, 2010), Western-oriented parental beliefs (Rigidity), and children's temperament (Parenting Stress Index; H. Tröster & R.R. Abidin). ICBC was used by 32.6% (n = 191) of parents in this study. Parents' Western-oriented beliefs predicted ICBC. Attitudes such as feeding a child on a time schedule and not carrying it out to prevent dependence were associated with letting the child cry to fall asleep. Low-sensitivity parents as well as parents of children with a difficult temperament used ICBC more frequently. Path analysis shows that parental stress did not predict ICBC. The results suggest that ICBC has become part of Western childrearing tradition. © 2018 Michigan Association for Infant Mental Health.

  4. Retinoid X receptor gene expression and protein content in tissues of the rock shell Thais clavigera

    Energy Technology Data Exchange (ETDEWEB)

    Horiguchi, Toshihiro [Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 (Japan)], E-mail: thorigu@nies.go.jp; Nishikawa, Tomohiro [Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 (Japan); Ohta, Yasuhiko [Department of Veterinary Science, Faculty of Agriculture, Tottori University, 4-101 Koyamacho-Minami, Tottori 680-8553 (Japan); Shiraishi, Hiroaki; Morita, Masatoshi [Research Center for Environmental Risk, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506 (Japan)

    2007-10-15

    To elucidate the role of retinoid X receptor (RXR) in the development of imposex caused by organotin compounds in gastropod molluscs, we investigated RXR gene expression and RXR protein content in various tissues of male and female wild rock shells (Thais clavigera). Quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry with a commercial antibody against human RXR {alpha} revealed that RXR gene expression was significantly higher in the penises of males and imposex-exhibiting females than in the penis-forming areas of normal females (P < 0.01 and P < 0.05, respectively). Western blotting demonstrated that the antibody could detect rock shell RXR and showed that the male penis had the highest content of RXR protein among the analyzed tissues of males and normal females. Immunohistochemical staining revealed nuclear localization of RXR protein in the epithelial and smooth muscle cells of the vas deferens and in the interstitial or connective tissues and epidermis of the penis in males and imposex-exhibiting females. RXR could be involved in the mechanism of induction of male-type genitalia (penis and vas deferens) by organotin compounds in female rock shells.

  5. Antimetastatic effects of Rheum palmatum L. extract on oral cancer cells.

    Science.gov (United States)

    Chen, Yang-Yu; Hsieh, Ming-Ju; Hsieh, Yih-Shou; Chang, Yu-Chao; Chen, Pei-Ni; Yang, Shun-Fa; Ho, Hsin-Yu; Chou, Ying-Erh; Lin, Chiao-Wen

    2017-10-01

    Rheum palmatum L., a traditional Chinese medication, has been used for the treatment of various disorders. However, the detailed impacts and underlying mechanisms of R. palmatum L. extracts (RLEs) on human oral cancer cell metastasis are still unclear. Here, we tested the hypothesis that an RLE has antimetastatic effects on SCC-9 and SAS human oral cancer cells. Gelatin zymography, Western blot, real-time polymerase chain reaction, and luciferase assay were used to explore the underlying mechanisms involved in the antimetastatic effects on oral cancer cells. Our results revealed that the RLE (up to 20 μg/mL, without cytotoxicity) attenuated SCC-9 and SAS cell motility, invasiveness, and migration by reducing matrix metalloproteinase (MMP)-2 enzyme activities. Western blot analysis of the MAPK signaling pathway indicated that the RLE significantly decreased phosphorylated ERK1/2 levels but not p38 and JNK levels. In conclusion, RLEs exhibit antimetastatic activity against oral cancer cells through the transcriptional repression of MMP-2 via the Erk1/2 signaling pathways. Thus, RLEs may be potentially useful as antimetastatic agents for oral cancer chemotherapy. © 2017 Wiley Periodicals, Inc.

  6. The role of Rad 51 protein in radioresistance of spheroid model of Du 145 prostate carcinoma cell line

    International Nuclear Information System (INIS)

    Taghizadeh, M.; Khoei, S.; Nikoofar, A. R.; Ghamsari, L.; Goliaei, B.

    2009-01-01

    Rad 51 is a protein with critical role in double strand break repair. Down-regulation of this protein has a significant effect in radiosensitivity of some cell lines like prostate carcinoma. Compared to monolayer cell culture model, the spheroids are more resistant to radiation. The aim of the current study was to determine the Rad 51 protein level in Du 145 spheroids, and monolayer cells before and after exposure to gamma irradiation. Materials and Methods: In the present study, western blot was used to determine the level of Rad 51 protein in Du 145 cell line grown as monolayer and spheroid. Results: Western blot analysis showed that in the spheroid cells, Rad 51 had an elevated level before and after radiation in comparison with monolayer cells. Higher doses of radiation induced elevated expression of Rad 51 protein in both culture models.The level of at protein after exposure to gamma rays had been time-dependent. Conclusion: Rad 51 might act as a mediator of radiation resistance in tumor cells. Repression of Rad 51 activity could be a prominent strategy to overcome radiation resistance of tumors.

  7. Generation of a monoclonal antibody against the glycosylphosphatidylinositol-linked protein Rae-1 using genetically engineered tumor cells.

    Science.gov (United States)

    Hu, Jiemiao; Vien, Long T; Xia, Xueqing; Bover, Laura; Li, Shulin

    2014-02-04

    Although genetically engineered cells have been used to generate monoclonal antibodies (mAbs) against numerous proteins, no study has used them to generate mAbs against glycosylphosphatidylinositol (GPI)-anchored proteins. The GPI-linked protein Rae-1, an NKG2D ligand member, is responsible for interacting with immune surveillance cells. However, very few high-quality mAbs against Rae-1 are available for use in multiple analyses, including Western blotting, immunohistochemistry, and flow cytometry. The lack of high-quality mAbs limits the in-depth analysis of Rae-1 fate, such as shedding and internalization, in murine models. Moreover, currently available screening approaches for identifying high-quality mAbs are excessively time-consuming and costly. We used Rae-1-overexpressing CT26 tumor cells to generate 60 hybridomas that secreted mAbs against Rae-1. We also developed a streamlined screening strategy for selecting the best anti-Rae-1 mAb for use in flow cytometry assay, enzyme-linked immunosorbent assay, Western blotting, and immunostaining. Our cell line-based immunization approach can yield mAbs against GPI-anchored proteins, and our streamlined screening strategy can be used to select the ideal hybridoma for producing such mAbs.

  8. Expression of the Pokemon proto-oncogene in nasopharyngeal carcinoma cell lines and tissues.

    Science.gov (United States)

    Jiao, Wei; Liu, Fei; Tang, Feng-Zhu; Lan, Jiao; Xiao, Rui-Ping; Chen, Xing-Zhou; Ye, Hui-Lan; Cai, Yong-Lin

    2013-01-01

    To study the differentiated expression of the proto-oncogene Pokemon in nasopharyngeal carcinoma (NPC) cell lines and tissues, mRNA and protein expression levels of CNE1, CNE2, CNE3 and C666-1 were detected separately by reverse transcription polymerase chain reaction (RT-PCR), real-time PCR and Western-blotting. The immortalized nasopharyngeal epithelial cell line NP69 was used as a control. The Pokemon protein expression level in biopsy specimens from chronic rhinitis patients and undifferentiated non keratinizing NPC patients was determined by Western-blotting and arranged from high to low: C666-1>CNE1>CNE2> CNE3>NP69. The Pokemon mRNA expression level was also arranged from high to low: CNE1>CNE2>NP69>C666-1>CNE3. Pokemon expression of NP69 and C666-1 obviously varied from mRNA to protein. The Pokemon protein level of NPC biopsy specimens was obviously higher than in chronic rhinitis. The data suggest that high Pokemon protein expression is closely associated with undifferentiated non-keratinizing NPC and may provide useful information for NPC molecular target therapy.

  9. The impairment of learning and memory and synaptic loss in mouse after chronic nitrite exposure.

    Science.gov (United States)

    Chen, Yongfang; Cui, Zhanjun; Wang, Lai; Liu, Hongliang; Fan, Wenjuan; Deng, Jinbo; Deng, Jiexin

    2016-12-01

    The objective of this study is to understand the impairment of learning and memory in mouse after chronic nitrite exposure. The animal model of nitrite exposure in mouse was created with the daily intubation of nitrite in adult healthy male mice for 3 months. Furthermore, the mouse's learning and memory abilities were tested with Morris water maze, and the expression of Synaptophysin and γ-Synuclein was visualized with immunocytochemistry and Western blot. Our results showed that nitrite exposure significantly prolonged the escape latency period (ELP) and decreased the values of the frequency across platform (FAP) as well as the accumulative time in target quadrant (ATITQ) compared to control, in dose-dependent manner. In addition, after nitrite exposure, synaptophysin (SYN) positive buttons in the visual cortex was reduced, in contrast the increase of γ-synuclein positive cells. The results above were supported by Western blot as well. We conclude that nitrite exposure could lead to a decline in mice's learning and memory. The overexpression of γ-synuclein contributed to the synaptic loss, which is most likely the cause of learning and memory impairment. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1720-1730, 2016. © 2015 Wiley Periodicals, Inc.

  10. Novel flow cytometric analysis of the progress and route of internalization of a monoclonal anti-carcinoembryonic antigen (CEA) antibody.

    Science.gov (United States)

    Ford, C H; Tsaltas, G C; Osborne, P A; Addetia, K

    1996-03-01

    A flow cytometric method of studying the internalization of a monoclonal antibody (Mab) directed against carcinoembryonic antigen (CEA) has been compared with Western blotting, using three human colonic cancer cell lines which express varying amounts of the target antigen. Cell samples incubated for increasing time intervals with fluoresceinated or unlabelled Mab were analyzed using flow cytometry or polyacrylamide gel electrophoresis and Western blotting. SDS/PAGE analysis of cytosolic and membrane components of solubilized cells from the cell lines provided evidence of non-degraded internalized anti-CEA Mab throughout seven half hour intervals, starting at 5 min. Internalized anti-CEA was detected in the case of high CEA expressing cell lines (LS174T, SKCO1). Very similar results were obtained with an anti-fluorescein flow cytometric assay. Given that these two methods consistently provided comparable results, use of flow cytometry for the detection of internalized antibody is suggested as a rapid alternative to most currently used methods for assessing antibody internalization. The question of the endocytic route followed by CEA-anti-CEA complexes was addressed by using hypertonic medium to block clathrin mediated endocytosis.

  11. Analysis of vegetation and land cover dynamics in north-western Morocco during the last decade using MODIS NDVI time series data

    Directory of Open Access Journals (Sweden)

    C. Höpfner

    2011-11-01

    Full Text Available Vegetation phenology as well as the current variability and dynamics of vegetation and land cover, including its climatic and human drivers, are examined in a region in north-western Morocco that is nearly 22 700 km2 big. A gapless time series of Normalized Differenced Vegetation Index (NDVI composite raster data from 29 September 2000 to 29 September 2009 is utilised. The data have a spatial resolution of 250 m and were acquired by the Moderate Resolution Imaging Spectroradiometer (MODIS sensor.

    The presented approach allows to compose and to analyse yearly land cover maps in a widely unknown region with scarce validated ground truth data by deriving phenological parameters. Results show that the high temporal resolution of 16 d is sufficient for (a determining local land cover better than global land cover classifications of Plant Functional Types (PFT and Global Land Cover 2000 (GLC2000 and (b for drawing conclusions on vegetation dynamics and its drivers. Areas of stably classified land cover types (i.e. areas that did not change their land cover type show climatically driven inter- and intra-annual variability with indicated influence of droughts. The presented approach to determine human-driven influence on vegetation dynamics caused by agriculture results in a more than ten times larger area compared with stably classified areas. Change detection based on yearly land cover maps shows a gain of high-productive vegetation (cropland of about 259.3 km2. Statistically significant inter-annual trends in vegetation dynamics during the last decade could however not be discovered. A sequence of correlations was respectively carried out to extract the most important periods of rainfall responsible for the production of green biomass and for the extent of land cover types. Results show that mean daily precipitation from 1 October to 15 December has high correlation results (max. r2=0.85 on an intra

  12. Factors affecting the use of prenatal care by non-western women in industrialized western countries: a systematic review.

    Science.gov (United States)

    Boerleider, Agatha W; Wiegers, Therese A; Manniën, Judith; Francke, Anneke L; Devillé, Walter L J M

    2013-03-27

    Despite the potential of prenatal care for addressing many pregnancy complications and concurrent health problems, non-western women in industrialized western countries more often make inadequate use of prenatal care than women from the majority population do. This study aimed to give a systematic review of factors affecting non-western women's use of prenatal care (both medical care and prenatal classes) in industrialized western countries. Eleven databases (PubMed, Embase, PsycINFO, Cochrane, Sociological Abstracts, Web of Science, Women's Studies International, MIDIRS, CINAHL, Scopus and the NIVEL catalogue) were searched for relevant peer-reviewed articles from between 1995 and July 2012. Qualitative as well as quantitative studies were included. Quality was assessed using the Mixed Methods Appraisal Tool. Factors identified were classified as impeding or facilitating, and categorized according to a conceptual framework, an elaborated version of Andersen's healthcare utilization model. Sixteen articles provided relevant factors that were all categorized. A number of factors (migration, culture, position in host country, social network, expertise of the care provider and personal treatment and communication) were found to include both facilitating and impeding factors for non-western women's utilization of prenatal care. The category demographic, genetic and pregnancy characteristics and the category accessibility of care only included impeding factors.Lack of knowledge of the western healthcare system and poor language proficiency were the most frequently reported impeding factors. Provision of information and care in women's native languages was the most frequently reported facilitating factor. The factors found in this review provide specific indications for identifying non-western women who are at risk of not using prenatal care adequately and for developing interventions and appropriate policy aimed at improving their prenatal care utilization.

  13. Herpes simplex virus type 1 strain KOS carries a defective US9 and a mutated US8A gene.

    Science.gov (United States)

    Negatsch, Alexandra; Mettenleiter, Thomas C; Fuchs, Walter

    2011-01-01

    The membrane protein encoded by the US9 gene of alphaherpesviruses plays an important role during virion assembly and transport in neurons. Here, we demonstrate that in herpes simplex virus type 1 (HSV-1) strain KOS, due to base substitutions, the predicted TATA-box of US9 is mutated, and a premature stop is present at codon 58 of US9, which contains 91 codons in other HSV-1 strains. The TATA-box mutation also removes the native stop codon of the adjacent US8A gene, leading to extension of the coding region from 160 to 191 codons. Northern blot analyses revealed reduced transcription of US9 in cells infected with HSV-1 KOS. Moreover, a US9-specific antiserum did not detect any gene products in Western blot and immunofluorescence analyses of KOS-infected cells, indicating that the truncated protein is not stable. In contrast, Western blot reactions of a pUS8A-specific antiserum confirmed enlargement of this protein in HSV-1 KOS.

  14. Targeting and Therapy of Glioblastoma in a Mouse Model Using Exosomes Derived From Natural Killer Cells

    Directory of Open Access Journals (Sweden)

    Liya Zhu

    2018-04-01

    Full Text Available ObjectiveGlioblastoma is a highly aggressive primary brain tumor that is resistant to radiotherapy and chemotherapy. Natural killer (NK cells have been used to treat incurable cancers. Recent studies have investigated the effectiveness of NK-cell-derived exosomes (NK-Exo for treating incurable cancers such as melanoma, leukemia, and neuroblastoma; however, NK-Exo have not been used to treat glioblastoma. In the present study, we investigated the antitumor effects of NK-Exo against aggressive glioblastoma both in vitro and in vivo and determined the tumor-targeting ability of NK-Exo by performing fluorescence imaging.MethodsU87/MG cells were transfected with the enhanced firefly luciferase (effluc and thy1.1 genes; thy1.1-positive cells were selected using microbeads. U87/MG/F cells were assessed by reverse transcription polymerase chain reaction (RT-PCR, western blotting, and luciferase-activity assays. NK-Exo were isolated by ultracentrifugation, purified by density gradient centrifugation, and characterized by transmission electron microscopy, dynamic light scattering (DLS, nanoparticle-tracking analysis (NTA, and western blotting. Cytokine levels in NK-Exo were compared to those in NK cells and NK-cell medium by performing an enzyme-linked immunosorbent assay (ELISA. NK-Exo-induced apoptosis of cancer cells was confirmed by flow cytometry and western blotting. In vivo therapeutic effects and specificity of NK-Exo against glioblastoma were assessed in a xenograft mouse model by fluorescence imaging. Xenograft mice were treated with NK-Exo, which was administered seven times through the tail vein. Tumor growth was monitored by bioluminescence imaging (BLI, and tumor volume was measured by ultrasound imaging. The mice were intraperitoneally injected with dextran sulfate 2 h before NK-Exo injection to decrease the liver uptake and increase the tumor specificity of NK-Exo.ResultsRT-PCR and western blotting confirmed the gene and protein

  15. Global Communication and Cultural Desensitisation: Repackaging Western Values for Non-Western Markets

    Directory of Open Access Journals (Sweden)

    Mahmoud M. Galander

    2008-06-01

    Full Text Available Abstract: Global communication is widely perceived as an instrument to disseminate Western values in the developing world. The “Wheel of Fortune” and “Who Wants to be a Millionnaire” licensed to Malaysian Television stations, though the language and the word puzzles were localised, carried the same format of the original (American show. They promote consumerism, gambling and the images of usury, the style of wealth accumulation forbidden in Islam. For the Malaysian audience whose priorities are those of contentment, modesty and humility, such emphasis on material desires breeds internal contradictions that may lead the audience to succumb to the new Western values.

  16. Expert Western Classical Music Improvisers' Strategies

    Science.gov (United States)

    Després, Jean-Philippe; Burnard, Pamela; Dubé, Francis; Stévance, Sophie

    2017-01-01

    The growing interest in musical improvisation is exemplified by the body of literatures evidencing the positive impacts of improvisation learning on the musical apprentice's aptitudes and the increasing presence of improvisation in Western classical concert halls and competitions. However, high-level Western classical music improvisers' thinking…

  17. Body image in non-western societies

    NARCIS (Netherlands)

    Edmonds, A.; Cash, T.

    2012-01-01

    This article discusses a range of body modification and conceptions of the body in non-Western societies. It also analyzes difficulties in applying the primarily Western psychological notion of body image to different societies. Body modification is a near human universal, but has many meanings and

  18. Effect of propofol in the immature rat brain on short- and long-term neurodevelopmental outcome.

    Directory of Open Access Journals (Sweden)

    Tanja Karen

    Full Text Available BACKGROUND: Propofol is commonly used as sedative in newborns and children. Recent experimental studies led to contradictory results, revealing neurodegenerative or neuroprotective properties of propofol on the developing brain. We investigated neurodevelopmental short- and long-term effects of neonatal propofol treatment. METHODS: 6-day-old Wistar rats (P6, randomised in two groups, received repeated intraperitoneal injections (0, 90, 180 min of 30 mg/kg propofol or normal saline and sacrificed 6, 12 and 24 hrs following the first injection. Cortical and thalamic areas were analysed by Western blot and quantitative real-time PCR (qRT-PCR for expression of apoptotic and neurotrophin-dependent signalling pathways. Long-term effects were assessed by Open-field and Novel-Object-Recognition at P30 and P120. RESULTS: Western blot analyses revealed a transient increase of activated caspase-3 in cortical, and a reduction of active mitogen-activated protein kinases (ERK1/2, AKT in cortical and thalamic areas. qRT-PCR analyses showed a down-regulation of neurotrophic factors (BDNF, NGF, NT-3 in cortical and thalamic regions. Minor impairment in locomotive activity was observed in propofol treated adolescent animals at P30. Memory or anxiety were not impaired at any time point. CONCLUSION: Exposing the neonatal rat brain to propofol induces acute neurotrophic imbalance and neuroapoptosis in a region- and time-specific manner and minor behavioural changes in adolescent animals.

  19. Neurotrophins and Neurotrophin Receptors in Proliferative Diabetic Retinopathy

    Science.gov (United States)

    Abu El-Asrar, Ahmed M.; Mohammad, Ghulam; De Hertogh, Gert; Nawaz, Mohd Imtiaz; Van Den Eynde, Kathleen; Siddiquei, Mohammad Mairaj; Struyf, Sofie; Opdenakker, Ghislain; Geboes, Karel

    2013-01-01

    Neurotrophins (NTs) are emerging as important mediators of angiogenesis and fibrosis. We investigated the expression of the NTs nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) and their receptors TrkA, TrkB, and TrkC in proliferative diabetic retinopathy (PDR). As a comparison, we examined the expression of NTs and their receptors in the retinas of diabetic rats. Vitreous samples from 16 PDR and 15 nondiabetic patients were studied by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Epiretinal membranes from 17 patients with PDR were studied by immunohistochemistry. Rats were made diabetic with a single high dose of streptozotocin and retinas of rats were examined by Western blot analysis. Western blot analysis revealed a significant increase in the expression of NT-3 and NT-4 and the shedding of receptors TrkA and TrkB in vitreous samples from PDR patients compared to nondiabetic controls, whereas NGF and BDNF and the receptor TrkC were not detected with the use of Western blot analysis and ELISA. In epiretinal membranes, vascular endothelial cells and myofibroblasts expressed NT-3 and the receptors TrkA, TrkB and TrkC in situ, whereas NT-4 was not detected. The expression levels of NT-3 and NT-4 and the receptors TrkA and TrkB, both in intact and solubilized forms, were upregulated in the retinas of diabetic rats, whereas the receptor TrkC was not detected. Co-immunoprecipitation studies revealed binding between NT-3 and the receptors TrkA and TrkB in the retinas of diabetic rats. Our findings in diabetic eyes from humans and rats suggest that the increased expression levels within the NT-3 and NT-4/Trk axis are associated with the progression of PDR. PMID:23762379

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

    Science.gov (United States)

    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

  1. [Health care in today's Western Belarus in the inter-war period (1921-1939)].

    Science.gov (United States)

    Tiszczenko, E M

    2001-01-01

    During the inter-war period, today's Western Belarus, which under the Treaty of Riga constituted part of the Second Polish Republic, stood out for its higher morbidity in various disease categories than other regions of the country. Heightened threats of epidemics prevailed in that area, and typhoid fever, trachoma, tuberculois and venereal disease were serious health problems. Specific features of the health care of today's Western Belarus could be observed that set it apart from both that of Eastern Belarus forming a part of the Soviet Union as well as from the central and western provinces of Poland. Compared with Eastern Belarus, there was a lack of health services, including anti-tuberculosis, anti-venereal and paediatric dispensaries, or they were in the process of being created. But physicians' self-government bodies functioned, as did out-patient care with dispensary-like elements and health care provided by local health centres. There also existed the institution of full-time family doctors within the insured health-care system - something lacking in Eastern Belarus. As in other voivodships (provinces) of the Second Republic, there existed multi-sector health care: state, local, insurance-based and private. The Western Belarus had Poland's smallest number of physicians, new hospital beds and pharmacies. On the other hand, today's Western Belarus stands out by virtue of maintaining field-medicine traditions. To this day, assistant medical officers provide health care to the rural population - an arrangement not typical of the central and western provinces fo pre-war Poland.

  2. Land-surface evolution at the continental time-scale: An example from interior Western Australia

    Science.gov (United States)

    Mabbutt, J. A.

    1988-12-01

    The interior plateau of Western Australia in the Wiluna—Meekatharra area is in part an exhumed pre-Permian surface of low relief and to that extent of Gondwana age. A lateritic duricrust on interfluvial remnants of an Old Plateau surface is probably the outcome of several cycles of weathering and stripping, rather than of a single geomorphic episode. Landforms above the Old Plateau have maintained their relief during this circumdenudation and there is no regional evidence of their isolation by major escarpment retreat. A New Plateau surface has extended by stripping of saprolite and is an etchplain, as also is the Old Plateau under the genesis postulated. The New Plateau cycle was initiated by general drainage rejuvenation whilst lateritic weathering still continued, but its extension was halted through increasing climatic aridity, probably during the Miocene.

  3. Biochemical Neuroscience Laboratory

    Data.gov (United States)

    Federal Laboratory Consortium — This biochemistry lab is set up for protein analysis using Western blot, enzyme linked immunosorbent assays, immunohistochemistry, and bead-based immunoassays. The...

  4. Western Australian natural gas

    International Nuclear Information System (INIS)

    Harman, Frank

    1994-01-01

    Western Australia has 80% of Australia's natural gas resources. These are currently exploited to supply the Western Australian market and LNG to Japan. Growth in the market is dependent on limited prospects for power generation and mineral resource processing. Future exploitation of gas resources will require new export LNG markets and/or the installations of a transcontinental pipeline to eastern Australia. The transcontinental option should only be considered after other options for energy supply in eastern Australia are eliminated. Competition to meet market growth in North-east Asia will be considerable and Australia lacks the policies to underpin future LNG capacity. (author)

  5. The role of EMMPRIN expression in ovarian epithelial carcinomas.

    Science.gov (United States)

    Zhao, Yang; Chen, Shuo; Gou, Wen-feng; Niu, Zhe-feng; Zhao, Shuang; Xiao, Li-jun; Takano, Yasuo; Zheng, Hua-chuan

    2013-09-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN) was reported to involve in the invasion and metastasis of malignancies by regulating the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) in stromal and cancer cells. The study aimed to clarify the role of EMMPRIN expression in tumorigenesis and progression of ovarian epithelial carcinomas. EMMPRIN siRNA were transfected into ovarian carcinoma cells with the phenotypes and their related molecules examined. EMMPRIN expression was determined in ovarian normal tissue, benign and borderline tumors, and epithelial carcinomas by real-time PCR, western blot, and immunohistochemistry. EMMPRIN siRNA treatment resulted in a lower growth, G 1 arrest, apoptotic induction, decreased migration, and invasion. The transfectants showed reduced expression of Wnt5a, Akt, p70s6k, Bcl-xL, survivin, VEGF, and MMP-9 than mock and control cells at both mRNA and protein levels. According to real-time PCR and western blot, EMMPRIN mRNA or protein level was higher in ovarian borderline tumor and carcinoma than normal ovary and benign tumors (PEMMPRIN expression was positively correlated with FIGO staging, dedifferentiation, Ki-67 expression, the lower cumulative and relapse-free survival rate (PEMMPRIN protein and mRNA might be involved in the pathogenesis, differentiation, and progression of ovarian carcinomas, possibly by modulating cellular events, such as proliferation, cell cycle, apoptosis, migration, and invasion.

  6. Analysis of the physical activity effects and measurement of pro-inflammatory cytokines in irradiated lungs in rats

    Energy Technology Data Exchange (ETDEWEB)

    Bianchi, Renata Cristiane Gennari; Katashima, Carlos Kiyoshi [Faculty of Medical Sciences, UNICAMP, Campinas, SP (Brazil); Ropelle, Eduardo Rochete [School of Applied Sciences, University of Campinas, UNICAMP, Limeira, SP (Brazil); Carvalheira, Jose Barreto Campello [Department of Internal Medicine, UNICAMP, Campinas, SP (Brazil); Lopes, Luiz Roberto; Andreollo, Nelson Adami [Department of Surgery, UNICAMP, Campinas, SP (Brazil)

    2012-03-15

    Purpose: To study if the pre-radiotherapy physical activity has radio-protective elements, by measuring the radio-induced activation of pro-inflammatory cytokines as interleukin-6 (il-6), transforming growth factor -{beta} (tgf -{beta}), tumor necrosis factor -a (tnf-a) and protein beta kinase {beta} (ikk{beta}), through western blotting analysis. Methods: A randomized study with 28 Wistar Hannover rats, males, with a mean age of 90 days and weighing about 200 grams. The animals were divided into three groups: (GI, GII and GIII). GIII group were submitted to swimming for eight weeks (zero load, three times a week, about 30 minutes). Then, the groups (except the control group) were submitted to irradiation by cobalt therapy, single dose of 3.5 gray in the whole body. All animals were sacrificed by overdose of pentobarbital, according to the time for analysis of cytokines, and then a fragment of the lower lobe of the right lung went to western blotting analysis. Results: The cytokines IKK{beta}, TNF-{alpha} and IL-6 induced by radiation in the lung were lower in the exercised animals. However, exercise did not alter the radiation-induced increase in tgf-{beta}. Conclusion: The results show a lower response in relation to inflammatory cytokines in the group that practiced the exercise preradiotherapy, showing that exercise can protect tissues from tissue damage due to irradiation. (author)

  7. Effect of Negative Pressure Wound Therapy on Cellular Fibronectin and Transforming Growth Factor-β1 Expression in Diabetic Foot Wounds.

    Science.gov (United States)

    Yang, Shao Ling; Zhu, Lv Yun; Han, Rui; Sun, Lei Lei; Dou, Jing Tao

    2017-08-01

    Chronic diabetic foot wounds are a leading cause of amputation, morbidity, and hospitalization for patients with diabetes. Negative-pressure wound therapy (NPWT) can putatively facilitate wound healing, but the underlying mechanisms remain unclear. Cellular fibronectin (cFN) and transforming growth factor-β1 (TGF-β1) play an important role in wound healing. This prospective randomized controlled trial evaluated the effects of NPWT on the production of cFN and the expression of TGF-β1 in diabetic foot wounds of patients. From January 2012 to January 2015, 40 patients with diabetic foot wounds were randomly and equally apportioned to receive either NPWT or advanced moist wound therapy (control) for 7 days. Granulation tissue was harvested before and after treatment. Immunohistochemistry and Western blot were performed to evaluate protein levels of cFN and TGF-β1, and real-time polymerase chain reaction (PCR) to measure corresponding mRNA expressions. NPWT facilitated the expression of cFN and TGF-β1 in diabetic foot wounds. Immunohistochemical analysis revealed higher levels of cFN and TGF-β1 in the NPWT group than in the control group. Western blot and real-time PCR analysis further showed that protein and mRNA levels of cFN or TGF-β1 were higher in the NPWT group than that in the control group ( P diabetic foot ulcers. Level I, randomized controlled study.

  8. Attenuation of TRPV1 and TRPV4 Expression and Function in Mouse Inflammatory Pain Models Using Electroacupuncture

    Directory of Open Access Journals (Sweden)

    Wei-Hsin Chen

    2012-01-01

    Full Text Available Although pain is a major human affliction, our understanding of pain mechanisms is limited. TRPV1 (transient receptor potential vanilloid subtype 1 and TRPV4 are two crucial receptors involved in inflammatory pain, but their roles in EA- (electroacupuncture- mediated analgesia are unknown. We injected mice with carrageenan (carra or a complete Freund’s adjuvant (CFA to model inflammatory pain and investigated the analgesic effect of EA using animal behavior tests, immunostaining, Western blotting, and a whole-cell recording technique. The inflammatory pain model mice developed both mechanical and thermal hyperalgesia. Notably, EA at the ST36 acupoint reversed these phenomena, indicating its curative effect in inflammatory pain. The protein levels of TRPV1 and TRPV4 in DRG (dorsal root ganglion neurons were both increased at day 4 after the initiation of inflammatory pain and were attenuated by EA, as demonstrated by immunostaining and Western blot analysis. We verified DRG electrophysiological properties to confirm that EA ameliorated peripheral nerve hyperexcitation. Our results indicated that the AP (action potential threshold, rise time, and fall time, and the percentage and amplitude of TRPV1 and TRPV4 were altered by EA, indicating that EA has an antinociceptive role in inflammatory pain. Our results demonstrate a novel role for EA in regulating TRPV1 and TRPV4 protein expression and nerve excitation in mouse inflammatory pain models.

  9. Downregulation of Clusterin Expression in Human Testicular Seminoma

    Directory of Open Access Journals (Sweden)

    Bianjiang Liu

    2013-11-01

    Full Text Available Background: Clusterin, a heterodimeric glycoprotein of approximately 80 kDa, exists extensively in human body fluids. The abnormal expression of clusterin is closely related to the occurrence, progression, and prognosis of tumors. Up to now, few studies have focused on clusterin in human testicular cancer. This study describes an extensive exploration of the presence and expression of clusterin in testicular seminoma. Methods: Tumor tissues and normal testis tissues were collected from 13 patients with testicular seminoma and 16 patients undergoing surgical castration for prostate cancer. Real-time polymerase chain reaction (PCR was performed to detect the expression difference of clusterin mRNA between testicular seminoma and normal testis. Western blot and immunohistochemical analysis were performed to detect the presence and expression difference of clusterin protein between two groups. Results: Real-time PCR showed the expression of clusterin mRNA in testicular seminoma to be significantly lower than in normal testis (only 13% relative quantification. Western blot analysis indicated marked reductions in the expression of clusterin protein in testicular seminoma. Similar results were observed upon immunohistochemical analysis. Conclusion: In testicular seminoma and normal testis, clusterin exists in its heterodimeric secretory isoform. Clusterin expression is significantly lower in testicular seminoma than in normal testis. This is the first comprehensive study of the presence and expression of clusterin in human testicular cancer.

  10. Persistent Dystrophin Protein Restoration 90 Days after a Course of Intraperitoneally Administered Naked 2′OMePS AON and ZM2 NP-AON Complexes in mdx Mice

    Directory of Open Access Journals (Sweden)

    Elena Bassi

    2012-01-01

    Full Text Available In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week of 2′-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage. In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdx mice. Our data therefore demonstrate the long-term residual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules.

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

    DEFF Research Database (Denmark)

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

    2001-01-01

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

  12. Evaluation of the seasonal and annual abortifacient risk of western juniper trees on Oregon rangelands: Abortion risk of western juniper trees

    Science.gov (United States)

    Western juniper trees can cause late term abortions in cattle, similar to ponderosa pine trees. Analyses of western juniper trees from 35 locations across the state of Oregon suggest that western juniper trees in all areas present an abortion risk in pregnant cattle. Results from this study demonstr...

  13. Western teachers of science or teachers of Western science: On the influence of Western modern science in a post-colonial context

    Science.gov (United States)

    Burke, Lydia E. Carol-Ann

    An expanding body of research explores the social, political, cultural and personal challenges presented by the Western emphasis of curricula around the world. The aim of my study is to advance this field of inquiry by gaining insight into perceptions of Western modern science presented by students, teachers and administrators in a given Caribbean setting. Through this study I asked how my research participants described the nature of scientific knowledge, how they related scientific knowledge to other culturally-valued knowledges and the meanings they attached to the geographic origins of science teachers. Situating this work firmly within the practice of Foucauldian critical discourse analysis, I have utilised a conceptual framework defined by the power/knowledge and complicity/resistance themes of post-colonial theory to support my interpretation of participant commentary in an overall quest that is concerned about the ways in which Western modern science might be exerting a colonising influence. Fourteen students, nine teachers (both expatriate and local) and three administrators participated in the study. I combined a semi-structured question and answer interview format with a card sort activity. I used a procedure based on my own adaptation of Stephenson's Q methodology, where the respondents placed 24 statements hierarchically along a continuum of increasing strength of agreement, presenting their rationalisations, personal stories and illustrations as they sorted. I used an inverse factor analysis, in combination with the interview transcripts, to assist me in the identification of three discourse positions described by my research participants: The truth value of scientific knowledge, The pragmatic use of science to promote progress, and The priority of cultural preservation. The interview transcripts were also analysed for emergent themes, providing an additional layer of data interpretation. The research findings raise concerns regarding the hegemonic

  14. Clovis age Western stemmed projectile points and human coprolites at the Paisley Caves

    DEFF Research Database (Denmark)

    Jenkins, Dennis L.; Davis, Loren G.; Stafford jr., Thomas

    2012-01-01

    The Paisley Caves in Oregon record the oldest directly dated human remains (DNA) in the Western Hemisphere. More than 100 high-precision radiocarbon dates show that deposits containing artifacts and coprolites ranging in age from 12,450 to 2295 C years ago are well stratified. Western Stemmed...... projectile points were recovered in deposits dated to 11,070 to 11,340 C years ago, a time contemporaneous with or preceding the Clovis technology. There is no evidence of diagnostic Clovis technology at the site. These two distinct technologies were parallel developments, not the product of a unilinear...

  15. Direct analysis of the secretions of the potato cyst nematode Globodera rostochiensis.

    Science.gov (United States)

    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.

  16. Transfer in SDS of biotinylated proteins from acrylamide gels to an avidin-coated membrane filter.

    Science.gov (United States)

    Karlin, Arthur; Wang, Chaojian; Li, Jing; Xu, Qiang

    2004-06-01

    Avidin was covalently linked to aldehyde-derivatized polyethersulfone membrane filters. These filters were used in Western blot analysis of proteins reacted with biotinylation reagents and electrophoresed in sodium dodecyl sulfate (SDS) on polyacrylamide gels. Electrophoretic transfer from the gels to these filters was in 0.1% SDS, in which the covalently bound avidin retained its biotin-binding capacity. We compared Western blots on avidin-coated membrane filters of biotinylated and nonbiotinylated forms of mouse immunoglobulin G (IgG), mouse IgG heavy chain, muscle-type acetylcholine receptor alpha subunit, and fused alpha and beta subunits of receptor. Biotinylated proteins were captured with high specificity compared to their nonbiotinylated counterparts and sensitively detected on the avidin-coated membranes.

  17. Secretagogin is a novel marker for neuroendocrine differentiation

    DEFF Research Database (Denmark)

    Birkenkamp-Demtröder, Karin; Wagner, Ludwig; Brandt Sørensen, Flemming

    2005-01-01

    Our previous microarray-based studies identified secretagogin to be highly expressed in normal colon mucosa compared to basal expression in colon adenocarcinomas. The aim of this study was to analyze the differential expression of secretagogin in normal mucosa, adenocarcinomas, and neuroendocrine...... tumors. Western blotting, immunohistochemistry, immunofluorescence microscopy and ELISA were applied. Western blot analysis detected a 32-kDa secretagogin band in samples from normal mucosa. Immunohistochemical analyses on tissue specimens showed that secretagogin is exclusively expressed...... and adrenal gland. Secretagogin was detected in plasma from carcinoid patients with distant metastasis. Combined immunohistochemical analysis of secretagogin and FK506-binding protein 65, a protein de novo synthesized in adenocarcinomas, distinguished well-differentiated carcinoids, adenocarcinoids...

  18. Anticuerpos policlonales contra la proteína recombinante NS3 del virus del dengue

    Directory of Open Access Journals (Sweden)

    Liliana Morales

    2017-01-01

    Resultados. Los anticuerpos producidos fueron útiles en ensayos de Western blot e inmunofluorescencia y se reportó por primera vez un anticuerpo policlonal anti-NS3 que permitió la inmunoprecipitación de la proteína viral y la detecta con Western blot sin necesidad de inducir sobreexpresión de NS3 o de usar extractos de células marcados metabólicamente con radioisótopos. Conclusión. Las proteínas recombinantes expresadas y los anticuerpos producidos constituyen herramientas valiosas para estudiar procesos infecciosos del DENV que involucren a la proteína NS3 y evaluar pruebas dirigidas a interferir las funciones de esta proteína.

  19. Etoposide induces apoptosis via the mitochondrial- and caspase-dependent pathways and in non-cancer stem cells in Panc-1 pancreatic cancer cells.

    Science.gov (United States)

    Zhang, She-Hong; Huang, Qian

    2013-12-01

    Pancreatic cancer is a highly aggressive malignant tumor. In the present study, we performed several methods, including CCK-8 assay, immunofluorescence technique, western blotting and flow cytometry, to determine the effects of VP16 (etoposide) on Panc-1 pancreatic cancer cells. The results demonstrated that VP16 inhibited the growth of and induced apoptosis in Panc-1 cells. Western blot analysis showed that VP16 inhibited the expression of Bcl-2 and enhanced the expression of Bax, caspases-3 and -9, cytochrome c and PARP. Notably, a strong inhibitory effect of VP16 on Panc-1 cells mainly occurred in non-CSCs. These data provide a new strategy for the therapy of pancreatic cancer.

  20. Homogentisate 1,2 dioxygenase is expressed in human osteoarticular cells: implications in alkaptonuria.

    Science.gov (United States)

    Laschi, Marcella; Tinti, Laura; Braconi, Daniela; Millucci, Lia; Ghezzi, Lorenzo; Amato, Loredana; Selvi, Enrico; Spreafico, Adriano; Bernardini, Giulia; Santucci, Annalisa

    2012-09-01

    Alkaptonuria (AKU) results from defective homogentisate1,2-dioxygenase (HGD), causing degenerative arthropathy. The deposition of ochronotic pigment in joints is so far attributed to homogentisic acid produced by the liver, circulating in the blood and accumulating locally. Human normal and AKU osteoarticular cells were tested for HGD gene expression by RT-PCR, mono- and 2D-Western blotting. HGD gene expression was revealed in chondrocytes, synoviocytes, osteoblasts. Furthermore, HGD expression was confirmed by Western blotting, that also revealed the presence of five enzymatic molecular species. Our findings indicate that AKU osteoarticular cells produce the ochronotic pigment in loco and this may strongly contribute to induction of ochronotic arthropathy. Copyright © 2011 Wiley Periodicals, Inc.

  1. Factors affecting the use of prenatal care by non-western women in industrialized western countries: a systematic review

    Science.gov (United States)

    2013-01-01

    Background Despite the potential of prenatal care for addressing many pregnancy complications and concurrent health problems, non-western women in industrialized western countries more often make inadequate use of prenatal care than women from the majority population do. This study aimed to give a systematic review of factors affecting non-western women’s use of prenatal care (both medical care and prenatal classes) in industrialized western countries. Methods Eleven databases (PubMed, Embase, PsycINFO, Cochrane, Sociological Abstracts, Web of Science, Women’s Studies International, MIDIRS, CINAHL, Scopus and the NIVEL catalogue) were searched for relevant peer-reviewed articles from between 1995 and July 2012. Qualitative as well as quantitative studies were included. Quality was assessed using the Mixed Methods Appraisal Tool. Factors identified were classified as impeding or facilitating, and categorized according to a conceptual framework, an elaborated version of Andersen’s healthcare utilization model. Results Sixteen articles provided relevant factors that were all categorized. A number of factors (migration, culture, position in host country, social network, expertise of the care provider and personal treatment and communication) were found to include both facilitating and impeding factors for non-western women’s utilization of prenatal care. The category demographic, genetic and pregnancy characteristics and the category accessibility of care only included impeding factors. Lack of knowledge of the western healthcare system and poor language proficiency were the most frequently reported impeding factors. Provision of information and care in women’s native languages was the most frequently reported facilitating factor. Conclusion The factors found in this review provide specific indications for identifying non-western women who are at risk of not using prenatal care adequately and for developing interventions and appropriate policy aimed at

  2. Risk assessment for yellow fever in western and North-Western provinces of Zambia

    Directory of Open Access Journals (Sweden)

    Olusegun A Babaniyi

    2015-01-01

    Full Text Available Background: North-Western and Western provinces of Zambia were reclassified as low-risk areas for yellow fever (YF. However, the current potential for YF transmission in these areas is unclear. Aims: To determine the current potential risk of YF infection. Setting and Design: A cross sectional study was conducted in North-Western and Western provinces of Zambia. Materials and Methods: Samples were tested for both YF virus-specific IgG and IgM antibodies by the ELISA and YF virus confirmation was done using Plaque Reduction Neutralization Test. The samples were also tested for IgG and IgM antibodies against other flaviviruses. Results: Out of the 3625 respondents who participated in the survey, 46.7% were males and 9.4% were aged less than 5 years. Overall, 58.1% of the participants slept under an impregnated insecticide-treated net and 20.6% reported indoor residual spraying of insecticides. A total of 616 (17.0% samples were presumptive YF positive. The prevalence for YF was 0.3% for long-term infection and 0.2% for recent YF infection. None of the YF confirmed cases had received YF vaccine. Prevalence rates for other flaviviruses were 149 (4.1% for Dengue, 370 (10.2% for West Nile and 217 (6.0% for Zika. Conclusion: There is evidence of past and recent infection of YF in both provinces. Hence, they are at a low risk for YF infection. Yellow fever vaccination should be included in the EPI program in the two provinces and strengthen surveillance with laboratory confirmation.

  3. Western forests and air pollution

    International Nuclear Information System (INIS)

    Olson, R.K.; Binkley, D.; Boehm, M.

    1992-01-01

    The book addresses the relationships between air pollution in the western United States and trends in the growth and condition of Western coniferous forests. The major atmospheric pollutants to which forest in the region are exposed are sulfur and nitrogen compounds and ozone. The potential effects of atmospheric pollution on these forests include foliar injury, alteration of growth rates and patterns, soil acidification, shifts in species composition, and modification of the effects of natural stresses

  4. Malaria in Sokoto, North Western Nigeria

    African Journals Online (AJOL)

    STORAGESEVER

    2009-12-15

    Dec 15, 2009 ... Malaria prevalence studies had been undertaken in many parts of Nigeria but there is probably no data available from the far North Western region. This research study was undertaken to determine the prevalence, monthly distribution of malaria in Sokoto, North Western Nigeria in order to generate base-.

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

    Science.gov (United States)

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

    2014-06-01

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

  6. Preliminary Strategic Environmental Assessment of the Great Western Development Strategy: Safeguarding Ecological Security for a New Western China

    Science.gov (United States)

    Li, Wei; Liu, Yan-Ju; Yang, Zhifeng

    2012-02-01

    The Great Western Development Strategy (GWDS) is a long term national campaign aimed at boosting development of the western area of China and narrowing the economic gap between the western and the eastern parts of China. The Strategic Environmental Assessment (SEA) procedure was employed to assess the environmental challenges brought about by the western development plans. These plans include five key developmental domains (KDDs): water resource exploitation and use, land utilization, energy generation, tourism development, and ecological restoration and conservation. A combination of methods involving matrix assessment, incorporation of expert judgment and trend analysis was employed to analyze and predict the environmental impacts upon eight selected environmental indicators: water resource availability, soil erosion, soil salinization, forest destruction, land desertification, biological diversity, water quality and air quality. Based on the overall results of the assessment, countermeasures for environmental challenges that emerged were raised as key recommendations to ensure ecological security during the implementation of the GWDS. This paper is intended to introduce a consensus-based process for evaluating the complex, long term pressures on the ecological security of large areas, such as western China, that focuses on the use of combined methods applied at the strategic level.

  7. A mini-review on econophysics: Comparative study of Chinese and western financial markets

    International Nuclear Information System (INIS)

    Zheng Bo; Jiang Xiong-Fei; Ni Peng-Yun

    2014-01-01

    We present a review of our recent research in econophysics, and focus on the comparative study of Chinese and western financial markets. By virtue of concepts and methods in statistical physics, we investigate the time correlations and spatial structure of financial markets based on empirical high-frequency data. We discover that the Chinese stock market shares common basic properties with the western stock markets, such as the fat-tail probability distribution of price returns, the long-range auto-correlation of volatilities, and the persistence probability of volatilities, while it exhibits very different higher-order time correlations of price returns and volatilities, spatial correlations of individual stock prices, and large-fluctuation dynamic behaviors. Furthermore, multi-agent-based models are developed to simulate the microscopic interaction and dynamic evolution of the stock markets. (topical review - statistical physics and complex systems)

  8. Immunohistochemical localization of CYP1A, vitellogenin and Zona radiata proteins in the liver of swordfish (Xiphias gladius L.) taken from the Mediterranean Sea, South Atlantic, South Western Indian and Central North Pacific Oceans

    International Nuclear Information System (INIS)

    Desantis, S.; Corriero, A.; Cirillo, F.; Deflorio, M.; Brill, R.; Griffiths, M.; Lopata, A.L.; Serna, J.M. de la; Bridges, C.R.; Kime, D.E.; De Metrio, G.

    2005-01-01

    Cytochrome P4501A (CYP1A) monoxygenase, vitellogenin (Vtg) and Zona radiata proteins (Zrp) are frequently used as biomarkers of fish exposure to organic contaminants. In this work, swordfish liver sections obtained from the Mediterranean Sea, the South African coasts (South Atlantic and South Western Indian Oceans) and the Central North Pacific Ocean were immunostained with antisera against CYP1A, Zrp, and Vtg. CYP1A induction was found in hepatocytes, epithelium of the biliary ductus and the endothelium of large blood vessels of fish from the Mediterranean Sea and South African waters, but not from the Pacific Ocean. Zrp and Vtg were immunolocalized in hepatocytes of male swordfish from the Mediterranean Sea and from South African waters. Plasma Dot-Blot analysis, performed in Mediterranean and Pacific specimens, revealed the presence of Zrp and Vtg in males from Mediterranean but not from Pacific. These results confirm previous findings about the potential exposure of Mediterranean swordfish to endocrine, disrupting chemicals and raise questions concerning the possible presence of xenobiotic contaminants off the Southern coasts of South Africa in both the South Atlantic and South Western Indian Oceans

  9. Air quality analysis for the Western Area Power Administration's 2004 Power Marketing Plan Environmental Impact Statement

    International Nuclear Information System (INIS)

    Glantz, C.S.; Dagle, J.E.; Bilyard, G.R.

    1997-01-01

    The Western Area Power Administration (Western) markets and transmits electric power throughout 15 western states. Western's Sierra Nevada Customer Service Region (Sierra Nevada Region) markets approximately 1,480 megawatts (MW) of firm power (plus 100 MW of seasonal peaking capacity) from the Central Valley Project (CVP) and other resources. Western's mission is to sell and deliver electricity generated from these resources. Western's capacity and energy sales must be in conformance with the laws that govern its sale of electrical power. Further, Western's hydropower operations at each facility must comply with minimum and maximum flows and other constraints set by other regulatory agencies. The Sierra Nevada Region proposes to develop a marketing plan that defines the products and services it would offer beyond the year 2004 and the eligibility and allocation criteria for its electric power resources. Because determining levels of long-term firm power resources to be marketed and subsequently entering into contracts for the delivery of related products and services could be a major Federal action with potentially significant impacts to the human environment, the 2004 Power Marketing Plan Environmental Impact Statement (2004 EIS) is being prepared. Decisions made by the Sierra Nevada Region on how and when to supply power to its customers would influence the operation of power plants within the Western Systems Coordinating Council (WSCC). If the resources affected are thermal resources, this could in turn affect the amount, timing, and location of pollutant emissions to the air at locations throughout the western United States. This report has been produced in conjunction with the 2004 EIS to provide a more detailed discussion of the air quality implications of the 2004 power marketing plan

  10. Different responsiveness to a high-fat/cholesterol diet in two inbred mice and underlying genetic factors: a whole genome microarray analysis

    Directory of Open Access Journals (Sweden)

    Jin Gang

    2009-10-01

    Full Text Available Abstract Background To investigate different responses to a high-fat/cholesterol diet and uncover their underlying genetic factors between C57BL/6J (B6 and DBA/2J (D2 inbred mice. Methods B6 and D2 mice were fed a high-fat/cholesterol diet for a series of time-points. Serum and bile lipid profiles, bile acid yields, hepatic apoptosis, gallstones and atherosclerosis formation were measured. Furthermore, a whole genome microarray was performed to screen hepatic genes expression profile. Quantitative real-time PCR, western blot and TUNEL assay were conducted to validate microarray data. Results After fed the high-fat/cholesterol diet, serum and bile total cholesterol, serum cholesterol esters, HDL cholesterol and Non-HDL cholesterol levels were altered in B6 but not significantly changed in D2; meanwhile, biliary bile acid was decreased in B6 but increased in D2. At the same time, hepatic apoptosis, gallstones and atherosclerotic lesions occurred in B6 but not in D2. The hepatic microarray analysis revealed distinctly different genes expression patterns between B6 and D2 mice. Their functional pathway groups included lipid metabolism, oxidative stress, immune/inflammation response and apoptosis. Quantitative real time PCR, TUNEL assay and western-blot results were consistent with microarray analysis. Conclusion Different genes expression patterns between B6 and D2 mice might provide a genetic basis for their distinctive responses to a high-fat/cholesterol diet, and give us an opportunity to identify novel pharmaceutical targets in related diseases in the future.

  11. Seroprevalence of simian immunodeficiency virus in wild and captive born Sykes' monkeys (Cercopithecus mitis in Kenya

    Directory of Open Access Journals (Sweden)

    Otsyula Moses G

    2004-10-01

    Full Text Available Abstract Background The Sykes' monkey and related forms (Cercopithecus mitis make up an abundant, widespread and morphologically diverse species complex in eastern Africa that naturally harbors a distinct simian immunodeficiency virus (SIVsyk. We carried out a retrospective serological survey of SIV infection from both wild and captive Sykes' monkeys from Kenya. We compared two commercially available, cross-reactive ELISA tests using HIV antigens with a novel SIVsyk antigen-specific Western blot assay and analyzed the data by origin, subspecies, age and sex. Results The SIVsyk antigen-specific Western blot assay detected more serum samples as positive than either of the cross-reactive ELISA assays. Using this assay, we found that seroprevalence is higher than previously reported, but extremely variable in wild populations (from 0.0 to 90.9%. Females were infected more often than males in both wild and captive populations. Seropositive infants were common. However, no seropositive juveniles were identified. Conclusion We have developed a specific and sensitive Western blot assay for anti-SIVsyk antibody detection. Sykes' monkeys are commonly infected with SIVsyk, but with extremely variable prevalence in the wild. Higher infection prevalence in females suggests predominantly sexual transmission. High infection prevalence in infants, but none in juveniles, suggests maternal antibodies, but little or no vertical transmission.

  12. Generation and Characterization of Anti-CD34 Monoclonal Antibodies that React with Hematopoietic Stem Cells

    Science.gov (United States)

    Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Movassaghpour, Aliakbar; Abdolalizadeh, Jalal

    2014-01-01

    CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies (mAbs) directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin (KLH)-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by the limiting dilution (L.D) method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the mAbs (3D5) was strongly reactive against the CD34 peptide and with native CD34 from human umbilical cord blood cells (UCB) in ELISA and Western blotting analyses. The results have shown that this antibody is highly specific and functional in biomedical applications such as ELISA and Western blot assays. This monoclonal antibodies (mAb) can be a useful tool for isolation and purification of human hematopoietic stem cells (HSCs). PMID:24611141

  13. 7, 8, 3′-Trihydroxyflavone Promotes Neurite Outgrowth and Protects Against Bupivacaine-Induced Neurotoxicity in Mouse Dorsal Root Ganglion Neurons

    Science.gov (United States)

    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

  14. Antigenicity analysis of Vibrio harveyi TS-628 strain

    Institute of Scientific and Technical Information of China (English)

    QIN Yingxue; WANG Jun; WANG Shifeng; YAN Qingpi

    2007-01-01

    Vibrio harveyi,the major causative agent of vibriosis,affects a diverse range of marine cultured organisms over a wide geographical area.However,reports about screening the effective antigen and research on vaccines of V.harveyi are scarce.Flagellin,lipopolysaccharide (LPS) and outer membrane proteins (OMP) are major immunogenic antigens in many Gram-negative bacteria.In this study,the flagellin,OMP and LPS of the V.harveyi TS-628 strain isolated from infected groupers were extracted and Western blot analysis was used to detect the antigenicity of these extractions.Results of the Western blot assay reveal that there are four positive flagellin bands:35 kDa,38 kDa,43 kDa,and 52 kDa,of which the 43 kDa and 52 kDa bands displayed the strongest positive reaction.There are five positive OMP bands about 35 kDa,38 kDa,43 kDa,47 kDa,and 52 kDa,of which the 43 kDa appeared to have the strongest positive reaction although the other four proteins also displayed strong reactions.However,LPS is Western blot-negative.These results indicate that the 43 kDa and 52 kDa flagellin and OMP of size 43 kDa,52 kDa can be candidates for developing vaccines against V.harveyi.

  15. Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

    Science.gov (United States)

    Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Chang, Yao-Wen; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

    2017-12-01

    Human epidermal growth factor receptor 2 (HER2) is overexpressed in breast cancer and is associated with poor clinical outcomes. In addition, HER2 expression has been reported in other cancers, such as gastric, colorectal, lung, and pancreatic cancers. An anti-HER2 humanized antibody, trastuzumab, leads to significant survival benefits in patients with HER2-overexpressing breast cancers and gastric cancers. Herein, we established a novel anti-HER2 monoclonal antibody (mAb), H 2 Mab-119 (IgG 1 , kappa), and characterized its efficacy against pancreatic cancers using flow cytometry, Western blot, and immunohistochemical analyses. H 2 Mab-119 reacted with pancreatic cancer cell lines, such as KLM-1, Capan-2, and MIA PaCa-2, but did not react with PANC-1 in flow cytometry analysis. Western blot analysis also revealed a moderate signal for KLM-1 and a weak signal for MIA PaCa-2, although H 2 Mab-119 reacted strongly with LN229/HER2 cells. Finally, immunohistochemical analyses with H 2 Mab-119 revealed sensitive and specific reactions against breast and colon cancers but did not react with pancreatic cancers, indicating that H 2 Mab-119 is useful for detecting HER2 overexpression in pancreatic cancers using flow cytometry and Western blot analyses.

  16. H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer.

    Science.gov (United States)

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

    2017-08-01

    Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H 2 Mab-77 (IgG 1 , kappa) was selected. Finally, immunohistochemical analyses with H 2 Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H 2 Mab-77 to detect HER2 in pathological analyses of breast cancers.

  17. Development and Application of an Indirect Enzyme-Linked Immunosorbent Assay Using Recombinant Mag1 for Serodiagnosis of Toxoplasma gondii In Dogs.

    Science.gov (United States)

    Zhuo, Xunhui; Sun, Hongchao; Zhang, Zhi; Luo, Jiaqing; Shan, Ying; Du, Aifang

    2017-06-01

    Serologic tests are widely accepted and applied as means to detect anti- Toxoplasma gondii immunoglobulin G antibodies. In this study, recombinant matrix antigen (rMAG1) was induced by isopropyl-β-d-thiogalactoside and purified by nickel-nitrilotriacetic acid purification system. We then developed and optimized an indirect enzyme-linked immunosorbent assay (ELISA) through checkerboard assays using serial dilutions of antigens and sera to assess the potential use of rMAG1 in serologic detection of T. gondii infection in dogs. Serum samples from 93 domestic dogs were analyzed by western blot and rMAG1-ELISA. The results were compared with those obtained from an ELISA with the soluble Toxoplasma lysate antigens (TLA). We found that although yielding an excellent agreement (96.7%) with western blot data (κ = 0.9659), rMAG1-ELISA produced higher sensitivity (93.9% vs. 87.8%) and specificity (98.3% vs. 96.7%) than TLA-ELISA. In addition, receiver operating characteristic analysis also revealed that rMAG1-ELISA is in more agreement with western blot (area under the curve [AUC] = 0.985) relative to TLA-ELISA (AUC = 0.955). These results indicated that the rMAG1-ELISA established in this study provides a promising and reliable tool for serologic detection of T. gondii infection in dogs.

  18. A reconstructed database of historic bluefin tuna captures in the Gibraltar Strait and Western Mediterranean

    Directory of Open Access Journals (Sweden)

    Josué M. Polanco-Martínez

    2018-02-01

    Full Text Available This data paper presents a reconstruction of a compilation of a small but consistent database of historical capture records of bluefin tuna (Thunnus thynnus; BFT hereafter from the Gibraltar Strait and Western Mediterranean (Portugal, Spain and Italy. The compilation come from diverse historical and documentary sources and span the time interval from 1525 to 1936 covering a period of 412 years. There is a total of 3074 datum, which reach up to 67.83% of the total implying a 32.17% of missing data. However, we have only reconstructed the captures for the time interval 1700–1936 and we provide these reconstructions only for this time interval and for 9 out of 11 series due to the scarcity and inhomogeneity of the two oldest capture time series. This reconstructed database provides an invaluable opportunity for fisheries and marine research as well as for multidisciplinary research in climate change. Keywords: Data reconstructions, DINEOF, Missing data, Historic Bluefin tuna captures, Gibraltar Strait and Western Mediterranean

  19. Matrix-assisted laser desorption-ionization mass spectrometry peptide mass fingerprinting for proteome analysis: identification efficiency after on-blot or in-gel digestion with and without desalting procedures.

    Science.gov (United States)

    Lamer, S; Jungblut, P R

    2001-03-10

    In theory, peptide mass fingerprinting by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS) has the potential to identify all of the proteins detected by silver staining on gels. In practice, if the genome of the organism investigated is completely sequenced, using current techniques, all proteins stained by Coomassie Brilliant Blue can be identified. This loss of identification sensitivity of ten to hundred-fold is caused by loss of peptides by surface contacts. Therefore, we performed digestion and transfer of peptides in the lower microl range and reduced the number of steps. The peptide mix obtained from in-gel or on-blot digestion was analyzed directly after digestion or after concentration on POROS R2 beads. Eight protein spots of a 2-DE gel from Mycobacterium bovis BCG were identified using these four preparation procedures for MALDI-MS. Overall, on-blot digestion was as effective as in-gel digestion. Whereas higher signal intensities resulted after concentration, hydrophilic peptides are better detected by direct measurement of the peptide mix without POROS R2 concentration.

  20. Interactions between traditional Chinese medicine and western drugs in Taiwan: A population-based study.

    Science.gov (United States)

    Chen, Kuan Chen; Lu, Richard; Iqbal, Usman; Hsu, Ko-Ching; Chen, Bi-Li; Nguyen, Phung-Anh; Yang, Hsuan-Chia; Huang, Chih-Wei; Li, Yu-Chuan Jack; Jian, Wen-Shan; Tsai, Shin-Han

    2015-12-01

    MA SHING GAN SHYR TANG (23.1%), followed by SHEAU CHING LONG TANG (15.5%) and DINQ CHUAN TANG (13.2%). The prevalence of herb-drug interactions related to Angelicae Sinensis Radix, Angelicae Dahuricae Radix was 4.59%. The most common traditional Chinese compound formula were TSANG EEL SAAN (32%), followed by HUOH SHIANG JENQ CHIH SAAN (31.4%) and SHY WUH TANG (10.7%). Once the multi-herb drug interaction database was deployed in a hospital system, there were 480 prescriptions that indicated a TCM-western drug interaction. Physicians were alerted 24 times during two months. These alerts resulted in a prescription change four times (16.7%). Due to the unique cultural factors that have resulted in widespread acceptance of both western and traditional Chinese medicine, Taiwan stands well positioned to report on the prevalence of interactions between western drugs and traditional Chinese medicine and devise ways to reduce their incidence. This study built a multi-herb/western drug interactions database, embedded inside a hospital clinical information system, and then examined the effects that drug interaction alerts had on clinician prescribing behaviour. The results demonstrated that western drug/traditional Chinese medicine interactions are prevalent and that western-trained physicians tend to change their prescribing behaviour more than traditional Chinese medicine physicians in their response to medication interaction alerts. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. Seabird Colonies in Western Greenland

    DEFF Research Database (Denmark)

    Boertmann, D.; Mosbech, A.; Falk, K.

    About 1 million seabirds (indvs) breed in 1032 colonies distributed along the coasts of western Greenland (Fig. 1). However, this figure does not include the little auk colonies in Avanersuaq. These colonies are roughly estimated to hold about 20 mill. pairs. All the basic information on seabird...... colonies in Greenland is compiled in a database maintained by NERI-AE. This report presents data on distribution, population numbers and population trends of 19 species of breeding colonial seabirds in western Greenland. Distributions are depicted on maps in Fig. 18-39. It is apparent that the major...... colonies are found in the northern part of the region, viz. Upernavik and Avanersuaq. The numbers of birds recorded in the database for each species are presented in Tab. 4, and on the basis of these figures estimates of the populations in western Greenland are given (Tab. 5). The most numerous species...

  2. Rethinking Education Research methodologies-Dominance of Western perspectives?

    Directory of Open Access Journals (Sweden)

    Yu Sing Ong

    2016-11-01

    Full Text Available This paper examines the different approaches and methods in comparative education research. It begins with a broad overview of the influences of Western philosophies and how Western education researchers approach social situations. Its differences with Chinese philosophies were highlighted with the concept of dao and Confucianism. More than just an epistemological discussion, this paper argues that Western researchers’ biasness towards Asian researchers may be due to their failure or lack of experience in making the connections with Asian social systems. It attempts to provide some plausible explanations of the prevalence of biasness by peer reviewers of Western journals.

  3. Response of predators to Western Sandpiper nest exclosures

    Science.gov (United States)

    Niehaus, Amanda C.; Ruthrauff, Daniel R.; McCaffery, Brian J.

    2004-01-01

    In 2001, predator exclosures were used to protect nests of the Western Sandpiper (Calidris mauri) in western Alaska. During the exclosure experiment, nest contents in exclosures had significantly higher daily survival rates than control nests, however, late in the study predators began to cue in on exclosures and predate the nest contents. An Arctic Fox (Alopex lagopus) dug under one exclosure and took the newly hatched chicks, and Long-tailed Jaegers (Stercorarius longicaudus) learned to associate exclosures with active nests and repeatedly visited them. The jaegers attempted to gain access to exclosed nests and pursued adult sandpipers as they emerged from the exclosures. The exclosures were removed to reduce potential mortality to adult and young sandpipers, but subsequently, post-exclosure nests had lower daily survival rates than controls during the same time period. Predation of post-exclosure eggs and chicks highlighted the lasting influence of the exclosure treatment on offspring survival because predators probably remembered nest locations. Researchers are urged to use caution when considering use of predator exclosures in areas where jaegers occur.

  4. The Western Denmark Cardiac Computed Tomography Registry

    DEFF Research Database (Denmark)

    Nielsen, Lene Hüche; Nørgaard, Bjarne Linde; Tilsted, Hans-Henrik

    2014-01-01

    BACKGROUND: As a subregistry to the Western Denmark Heart Registry (WDHR), the Western Denmark Cardiac Computed Tomography Registry (WDHR-CCTR) is a clinical database established in 2008 to monitor and improve the quality of cardiac computed tomography (CT) in Western Denmark. OBJECTIVE: We...... examined the content, data quality, and research potential of the WDHR-CCTR. METHODS: We retrieved 2008-2012 data to examine the 1) content; 2) completeness of procedure registration using the Danish National Patient Registry as reference; 3) completeness of variable registration comparing observed vs...

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

    Directory of Open Access Journals (Sweden)

    Osmir Cabrera-Blanco

    2017-04-01

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

  6. Early false-belief understanding in traditional non-Western societies.

    Science.gov (United States)

    Barrett, H Clark; Broesch, Tanya; Scott, Rose M; He, Zijing; Baillargeon, Renée; Wu, Di; Bolz, Matthias; Henrich, Joseph; Setoh, Peipei; Wang, Jianxin; Laurence, Stephen

    2013-03-22

    The psychological capacity to recognize that others may hold and act on false beliefs has been proposed to reflect an evolved, species-typical adaptation for social reasoning in humans; however, controversy surrounds the developmental timing and universality of this trait. Cross-cultural studies using elicited-response tasks indicate that the age at which children begin to understand false beliefs ranges from 4 to 7 years across societies, whereas studies using spontaneous-response tasks with Western children indicate that false-belief understanding emerges much earlier, consistent with the hypothesis that false-belief understanding is a psychological adaptation that is universally present in early childhood. To evaluate this hypothesis, we used three spontaneous-response tasks that have revealed early false-belief understanding in the West to test young children in three traditional, non-Western societies: Salar (China), Shuar/Colono (Ecuador) and Yasawan (Fiji). Results were comparable with those from the West, supporting the hypothesis that false-belief understanding reflects an adaptation that is universally present early in development.

  7. Management strategies for sustainable western water

    Science.gov (United States)

    Scott Tyler; Sudeep Chandra; Gordon Grant

    2017-01-01

    With the effects of the dramatic western US drought still reverberating through the landscape, researchers gathered in advance of the 20th annual Lake Tahoe Summit to discuss western US water issues in the 21st century. This two-day workshop brought together ~40 researchers from universities and agencies (federal and state) to discuss the prospects that...

  8. Western Area Power Administration. Combined power system financial statements

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1998-02-26

    This report presents the results of the independent certified public accountants` audit of the Western Area Power Administration`s combined power system statements of assets, Federal investment and liabilities, and the related combined statements of revenues, expenses and accumulated net revenues, and cash flows. The auditors` report on Westerns internal control structure disclosed three new reportable conditions concerning the lack of: (1) a reconciliation of stores inventory from subsidiary ledgers to summary financial information, (2) communication of interest during construction and related adjustments to interest on Federal investment, and (3) a system to prevent and detect power billing errors. None of the conditions were considered to be material weaknesses. Western provided concurrence and corrective action plans. The auditors` report on Western`s compliance with laws and regulations also disclosed two new instances of noncompliance. Western failed to calculate nonreimbursable expenses in accordance with the Grand Canyon Protection Act and had an unexplained difference in gross Federal investment balances used to calculate interest on Federal investment. Western provided concurrence and corrective action plans for the instances.

  9. A comparison of multivariate and univariate time series approaches to modelling and forecasting emergency department demand in Western Australia.

    Science.gov (United States)

    Aboagye-Sarfo, Patrick; Mai, Qun; Sanfilippo, Frank M; Preen, David B; Stewart, Louise M; Fatovich, Daniel M

    2015-10-01

    To develop multivariate vector-ARMA (VARMA) forecast models for predicting emergency department (ED) demand in Western Australia (WA) and compare them to the benchmark univariate autoregressive moving average (ARMA) and Winters' models. Seven-year monthly WA state-wide public hospital ED presentation data from 2006/07 to 2012/13 were modelled. Graphical and VARMA modelling methods were used for descriptive analysis and model fitting. The VARMA models were compared to the benchmark univariate ARMA and Winters' models to determine their accuracy to predict ED demand. The best models were evaluated by using error correction methods for accuracy. Descriptive analysis of all the dependent variables showed an increasing pattern of ED use with seasonal trends over time. The VARMA models provided a more precise and accurate forecast with smaller confidence intervals and better measures of accuracy in predicting ED demand in WA than the ARMA and Winters' method. VARMA models are a reliable forecasting method to predict ED demand for strategic planning and resource allocation. While the ARMA models are a closely competing alternative, they under-estimated future ED demand. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Factors affecting the use of prenatal care by non-western women in industrialized western countries: a systematic review

    NARCIS (Netherlands)

    Boerleider, A.W.; Wiegers, T.A.; Manniën, J.; Francke, A.L.; Devillé, W.L.J.M.

    2013-01-01

    Background Despite the potential of prenatal care for addressing many pregnancy complications and concurrent health problems, non-western women in industrialized western countries more often make inadequate use of prenatal care than women from the majority population do. This study aimed to give a

  11. Factors affecting the use of prenatal care by non-western women in industrialized western countries: a systematic review.

    NARCIS (Netherlands)

    Boerleider, A.W.; Wiegers, T.A.; Manniën, J.; Francke, A.L.; Devillé, W.L.J.M.

    2013-01-01

    Background: Despite the potential of prenatal care for addressing many pregnancy complications and concurrent health problems, non-western women in industrialized western countries more often make inadequate use of prenatal care than women from the majority population do. This study aimed to give a

  12. Factors affecting the use of prenatal care by non-western women in industrialized western countries: a systematic review

    NARCIS (Netherlands)

    Boerleider, A.W.; Wiegers, T.A.; Manniën, J.; Francke, A.L.; Deville, W.L.J.M.

    2013-01-01

    Background: Despite the potential of prenatal care for addressing many pregnancy complications and concurrent health problems, non-western women in industrialized western countries more often make inadequate use of prenatal care than women from the majority population do. This study aimed to give a

  13. The Western Denmark Heart Registry

    DEFF Research Database (Denmark)

    Schmidt, Morten; Maeng, Michael; Madsen, Morten

    2018-01-01

    The WDHR (Western Denmark Heart Registry) is a seminational, multicenter-based registry with longitudinal registration of detailed patient and procedure data since 1999. The registry includes as of January 1, 2017 approximately 240,000 coronary angiographies, 90,000 percutaneous coronary interven......The WDHR (Western Denmark Heart Registry) is a seminational, multicenter-based registry with longitudinal registration of detailed patient and procedure data since 1999. The registry includes as of January 1, 2017 approximately 240,000 coronary angiographies, 90,000 percutaneous coronary...

  14. Webspinners in Early Eocene amber from western India (Insecta, Embiodea

    Directory of Open Access Journals (Sweden)

    Michael Engel

    2011-11-01

    Full Text Available The family Scelembiidae (Neoembiodea: Embiomorpha: Archembioidea is recorded from Asia for the first time, based on two individuals preserved in Early Eocene amber from the Cambay Basin, western India. Kumarembia hurleyi Engel & Grimaldi, gen. n. et sp. n., is described, figured, and distinguished from other archembioid genera. The genus shares male genitalic features with scelembiids, otherwise known from South America and Africa.

  15. Stochastic approaches for time series forecasting of boron: a case study of Western Turkey.

    Science.gov (United States)

    Durdu, Omer Faruk

    2010-10-01

    In the present study, a seasonal and non-seasonal prediction of boron concentrations time series data for the period of 1996-2004 from Büyük Menderes river in western Turkey are addressed by means of linear stochastic models. The methodology presented here is to develop adequate linear stochastic models known as autoregressive integrated moving average (ARIMA) and multiplicative seasonal autoregressive integrated moving average (SARIMA) to predict boron content in the Büyük Menderes catchment. Initially, the Box-Whisker plots and Kendall's tau test are used to identify the trends during the study period. The measurements locations do not show significant overall trend in boron concentrations, though marginal increasing and decreasing trends are observed for certain periods at some locations. ARIMA modeling approach involves the following three steps: model identification, parameter estimation, and diagnostic checking. In the model identification step, considering the autocorrelation function (ACF) and partial autocorrelation function (PACF) results of boron data series, different ARIMA models are identified. The model gives the minimum Akaike information criterion (AIC) is selected as the best-fit model. The parameter estimation step indicates that the estimated model parameters are significantly different from zero. The diagnostic check step is applied to the residuals of the selected ARIMA models and the results indicate that the residuals are independent, normally distributed, and homoscadastic. For the model validation purposes, the predicted results using the best ARIMA models are compared to the observed data. The predicted data show reasonably good agreement with the actual data. The comparison of the mean and variance of 3-year (2002-2004) observed data vs predicted data from the selected best models show that the boron model from ARIMA modeling approaches could be used in a safe manner since the predicted values from these models preserve the basic

  16. 37 CFR 1.84 - Standards for drawings.

    Science.gov (United States)

    2010-07-01

    ...: electrophoresis gels, blots (e.g., immunological, western, Southern, and northern), autoradiographs, cell cultures... loss in facility of understanding the view. Partial views drawn on separate sheets must always be...

  17. Reconciling the geological history of western Turkey with plate circuits and mantle tomography

    Science.gov (United States)

    Kaymakci, N.; van Hinsbergen, D. J.; Spakman, W.; Torsvik, T. H.

    2010-12-01

    We place the geological history since Cretaceous times in western Turkey in a context of convergence, subduction, collision and slab break-off. To this end, we compare the west Anatolian geological history with amounts of Africa-Europe convergence calculated from the Atlantic plate circuit, and the seismic tomography images of the west Anatolian mantle structure. Western Turkish geology reflects the convergence between the Sakarya continent (here treated as Eurasia) in the north and Africa in the south, with the Anatolide-Tauride Block (ATB) between two strands of the Neotethyan ocean. Convergence between the Sakarya and the ATB started at least ~95-90Myr ago, marked by ages of metamorphic soles of ophiolites that form the highest structural unit below Sakarya. These are underlain by high-pressure, low-temperature metamorphic rocks of the Tavsanli and Afyon zones, and the Ören Unit, which in turn are underlain by the Menderes Massif derived from the ATB. Underthrusting of the ATB below Sakarya was since ~50Ma, associated with high-temperature metamorphism and widespread granitic magmatism. Thrusting in the Menderes Massif continued until 35 Ma, after which there is no record of accretion in western Turkey. Plate circuits show that since 90 Ma, ~1400 km of Africa-Europe convergence occurred, of which ~700 km since 50 Ma and ~450 km since 35Ma. Seismic tomography shows that the African slab under western Turkey is decoupled from the African Plate. This detached slab is a single, coherent body, representing the lithosphere consumed since 90 Ma. There was no subduction re-initiation after slab break-off. ATB collision with Europe therefore did not immediately lead to slab break-off but instead to delamination of subducting lithospheric mantle from accreting ATB crust, while staying attached to the African Plate. This led to asthenospheric inflow below the ATB crust, high-temperature metamorphism and felsic magmatism. Slab break-off in western Turkey probably occurred

  18. Snow and Ice Climatology of the Western United States and Alaska from MODIS

    Science.gov (United States)

    Rittger, K. E.; Painter, T. H.; Mattmann, C. A.; Seidel, F. C.; Burgess, A.; Brodzik, M.

    2013-12-01

    The climate and hydroclimate of the Western US and Alaska are tightly coupled to their snow and ice cover. The Western US depends on mountain snowmelt for the majority of its water supply to agriculture, industrial and urban use, hydroelectric generation, and recreation, all driven by increasing population and demand. Alaskan snow and glacier cover modulate regional climate and, as with the Western US, dominate water supply and hydroelectric generation in much of the state. Projections of climate change in the Western US and Alaska suggest that the most pronounced impacts will include reductions of mountain snow and ice cover, earlier runoff, and a greater fraction of rain instead of snow. We establish a snow and ice climatology of the Western US and Alaska using physically based MODIS Snow Covered Area and Grain size model (MODSCAG) for fractional snow cover, the MODIS Dust Radiative Forcing in Snow model (MODDRFS) for radiative forcing by light absorbing impurities in snow, and the MODIS Permanent Ice model (MODICE) for annual minimum exposed snow. MODSCAG and MODDRFS use EOS MOD09GA historical reflectance data (2000-2012) to provide daily and 8-day composites and near real time products since the beginning of 2013, themselves ultimately composited to 8-day products. The compositing method considers sensor-viewing geometry, solar illumination, clouds, cloud shadows, aerosols and noisy detectors in order to select the best pixel for an 8-day period. The MODICE annual minimum exposed snow and ice product uses the daily time series of fractional snow and ice from MODSCAG to generate annual maps. With this project we have established an ongoing, national-scale, consistent and replicable approach to assessing current and projected climate impacts and climate-related risk in the context of other stressors. We analyze the products in the Northwest, Southwest, and Alaska/Arctic regions of the National Climate Assessment for the last decade, the nation's hottest on record

  19. Skin disease affecting the conservation of the western swamp tortoise (Pseudemydura umbrina)

    Science.gov (United States)

    Ladyman, J M; Kuchling, G; Burford, D; Boardman, W; Raidal, S R

    1998-11-01

    To review the present position of the western swamp tortoise (Pseudemydura umbrina) as an endangered species and significant health issues affecting efforts to save it from extinction. A retrospective analysis of the husbandry, hospital and pathology records of the western swamp tortoise captive breeding program at Perth Zoo. In 1987 a captive breeding project was developed to prevent the extinction of the western swamp tortoise but an outbreak of a necrotising dermatitis in 1989 threatened the survival of the captive bred hatchlings. Less severe outbreaks occurred in 1990 and 1993, with isolated cases in between. Of 283 tortoises that were born in captivity or came into captivity from the wild, 37 (13.1%) were affected, comprising 37% of all males, 26% of all females and 13% of animals of unknown gender. Of the affected animals, 70% were less than 2 years of age and 29% were older. Males were 1.6 times more likely to be infected than females but this difference was not statistically significant (P = 0.27). Culture of the lesions consistently yielded unidentified Pseudomonas sp. Improved husbandry, such as strict maintenance of water quality and temperature conditions similar to that of the animal's natural habitat, and monitoring the health of individual tortoises have successfully controlled skin disease in the captive breeding of the western swamp tortoise.

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

    Science.gov (United States)

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

    1999-01-01

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