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.

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

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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…

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

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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.

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

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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.

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.

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

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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.

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

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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.

Determination of Diagnostic Antigens in Cattle Amphistomiasis Using Western Blotting

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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.

Routine Western blot to check autophagic flux : Cautions and recommendations

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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

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

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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)

Multi-strip Western blotting to increase quantitative data output

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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...

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...

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

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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...

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...

Protein blotting protocol for beginners.

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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.

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

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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.

The early days of blotting.

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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.

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

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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.

Acid-Urea Gel Electrophoresis and Western Blotting of Histones.

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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.

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

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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.

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

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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.

Proteínas inmunodominantes de Brucella Melitensis evaluadas por Western Blot

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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.

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

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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.

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

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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.

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

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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

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

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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.

Cy5 total protein normalization in Western blot analysis.

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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.

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

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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

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

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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

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

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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.

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

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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

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

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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.

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...

Characterization of Nora Virus Structural Proteins via Western Blot Analysis.

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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.

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

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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.

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

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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.

Detection of proteins on blot transfer membranes.

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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.

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

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

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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

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.

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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.

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

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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.’

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

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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.

[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.

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

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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.

Other notable protein blotting methods: a brief review.

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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.

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.

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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.

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

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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.

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

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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.

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

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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.

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

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Hermes Escalante

2011-09-01

finding of parasite eggs in the stool microscopy. Antigens of 10, 12, 17, 23, 27, 30, 36, 43, 66 and 136 kDa were detected and used to develop the Western blot technique. The sensitivity was evaluated using sera from 67 fasciolosis patients, and the specificity using sera from 57 patients with other parasitic diseases, and 10 from healthy individuals. Results. Out of the 67 sera, 64 reacted with the 23 kDa band and 61 with the one of 17 kDa. These two bands were not detected in sera from patients with other parasitic diseases or in those from healthy volunteers and thus could be considered specific and diagnostic. Conclusions. The sensitivity of the test, using the bands of 17 and 23 kDa, was 95.5% for positive reactions to at least one of these two bands, being its specificity 100% with a positive predictive value of 100% and negative predictive value of 95.71%.

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

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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).

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

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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.

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

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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.

A brief review of other notable protein blotting methods.

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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.

Northern blotting analysis

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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...

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

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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.

Southern blotting.

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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

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

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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.

Far Western: probing membranes.

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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.

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

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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.

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

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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.

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

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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

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

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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.

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...

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

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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).

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

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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.

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.

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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.

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

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

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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.

Introduction to protein blotting.

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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.

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

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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.

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.

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

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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.

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”.

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

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

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.

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.

Automated design of genomic Southern blot probes

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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

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.

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.

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

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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.

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

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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.

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

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?

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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.

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

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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

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

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.

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

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.

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

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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.

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

Immunological diagnosis of human hydatid cyst using Western immunoblotting technique

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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.

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

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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.

Standardization of Licorice and TCM Formulations Using Eastern Blot Fingerprinting Analysis

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Yukihiro Shoyama

2013-01-01

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

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...

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

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...

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...

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

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

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.

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

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.

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.

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.

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).

P27Kip1, regulated by glycogen synthase kinase-3β, results in HMBA-induced differentiation of human gastric cancer cells

International Nuclear Information System (INIS)

Wei, Min; Gu, Qinlong; Wang, Zhiwei; Yao, Hongliang; Yang, Zhongyin; Zhang, Qing; Liu, Bingya; Yu, Yingyan; Su, Liping; Zhu, Zhenggang

2011-01-01

Gastric cancer is the second most common cause of global cancer-related mortality. Although dedifferentiation predicts poor prognosis in gastric cancer, the molecular mechanism underlying dedifferentiation, which could provide fundamental insights into tumor development and progression, has yet to be elucidated. Furthermore, the molecular mechanism underlying the effects of hexamethylene bisacetamide (HMBA), a recently discovered differentiation inducer, requires investigation and there are no reported studies concerning the effect of HMBA on gastric cancer. Based on the results of FACS analysis, the levels of proteins involved in the cell cycle or apoptosis were determined using western blotting after single treatments and sequential combinations of HMBA and LiCl. GSK-3β and proton pump were investigated by western blotting after up-regulating Akt expression by Ad-Akt infection. To investigate the effects of HMBA on protein localization and the activities of GSK-3β, CDK2 and CDK4, kinase assays, immunoprecipitation and western blotting were performed. In addition, northern blotting and RNase protection assays were carried out to determine the functional concentration of HMBA. HMBA increased p27Kip1 expression and induced cell cycle arrest associated with gastric epithelial cell differentiation. In addition, treating gastric-derived cells with HMBA induced G0/G1 arrest and up-regulation of the proton pump, a marker of gastric cancer differentiation. Moreover, treatment with HMBA increased the expression and activity of GSK-3β in the nucleus but not the cytosol. HMBA decreased CDK2 activity and induced p27Kip1 expression, which could be rescued by inhibition of GSK-3β. Furthermore, HMBA increased p27Kip1 binding to CDK2, and this was abolished by GSK-3β inhibition. The results presented herein suggest that GSK-3β functions by regulating p27Kip1 assembly with CDK2, thereby playing a critical role in G0/G1 arrest associated with HMBA-induced gastric epithelial

Western gulf culture-density study-early results

Science.gov (United States)

Mohd S. Rahman; Michael G. Messina; Richard F. Fisher; Alan B. Wilson; Nick Chappell; Conner Fristoe; Larry Anderson

2006-01-01

The Western Gulf Culture-Density Study is a collaborative research effort between Texas A&M University and five forest products companies to examine the effects of early silvicultural treatment intensity and a wide range of both densities and soil types on performance of loblolly pine. The study tests 2 silvicultural intensities, 5 planting densities (200 to 1,200...

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.

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

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

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

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.

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...

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…

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

Regional economic impacts of changes in electricity rates resulting from Western Area Power Administration`s power marketing alternatives

Energy Technology Data Exchange (ETDEWEB)

Allison, T.; Griffes, P.; Edwards, B.K.

1995-03-01

This technical memorandum describes an analysis of regional economic impacts resulting from changes in retail electricity rates due to six power marketing programs proposed by Western Area Power Administration (Western). Regional economic impacts of changes in rates are estimated in terms of five key regional economic variables: population, gross regional product, disposable income, employment, and household income. The REMI (Regional Impact Models, Inc.) and IMPLAN (Impact Analysis for Planning) models simulate economic impacts in nine subregions in the area in which Western power is sold for the years 1993, 2000, and 2008. Estimates show that impacts on aggregate economic activity in any of the subregions or years would be minimal for three reasons. First, the utilities that buy power from Western sell only a relatively small proportion of the total electricity sold in any of the subregions. Second, reliance of Western customers on Western power is fairly low in each subregion. Finally, electricity is not a significant input cost for any industry or for households in any subregion.

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

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.

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.

Utility of Western Blot Analysis for the Diagnosis of Cutaneous Leishmaniasis

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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.

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.

[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.

Production of hybrids between western gray wolves and western coyotes.

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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.

Pre-bomb marine reservoir ages in the western north Pacific: Preliminary result on Kyoto University collection

International Nuclear Information System (INIS)

Yoneda, Minoru; Kitagawa, Hiroyuki; Plicht, Johannes van der; Uchida, Masao; Tanaka, Atsushi; Uehiro, Takashi; Shibata, Yasuyuki; Morita, Masatoshi; Ohno, Terufumi

2000-01-01

The calibration of radiocarbon dates on marine materials involves a global marine calibration with regional corrections. The marine reservoir ages in the Western North Pacific have not been discussed, while it is quite important to determine the timing of palaeo-environmental changes as well as archaeological interpretation around this region. The lack of adequate collection of the pre-bomb shell from western north Pacific was the biggest problem. Recently we had a chance to examine specimens from an old shell collection stored in Kyoto University, including shell specimens from Japan, Korea, Taiwan and the Micronesia of 1920s and 1930s. We explored the possibility for usage of specimen without clear evidence of live collection by measuring 30 apparent radiocarbon ages of pre-bomb mollusk shells from 18 sites in Western North Pacific. The preliminary results showed several discrepancies with previously reported results and with each other. We have to carefully select the shell specimen that has biological signs such as articulating fulcrum. In order to exploit this big resource of pre-bomb shell collection, the new technique to distinguish fossils from live collected samples should be developed by using chemical and physical methods

Characterization of a polymorphism in the coding sequence of FCN3 resulting in a Ficolin-3 (Hakata antigen) deficiency state

DEFF Research Database (Denmark)

Munthe-Fog, Lea; Hummelshøj, Tina; Ma, Ying Jie

2008-01-01

Ficolin-3 (Hakata antigen or H-ficolin) is a soluble pattern recognition molecule in the lectin complement pathway. We speculated whether common genetic variations in the FCN3 gene contribute to deficiency of Ficolin-3. The FCN3 gene was sequenced in 237 healthy Danish Caucasians. The relevance...... of polymorphisms was assessed with antibodies against Ficolin-3 in a novel ELISA system and by production of recombinant Ficolin-3 variants. Ficolin-3 serum profiles were analyzed by SDS-PAGE and western blotting. Ficolin-3 serum concentration varied 10-fold (median, 24microg/ml; range, 3-54microg/ml). Out.......025). SDS-PAGE and western blotting of serum revealed a weak band corresponding to the truncated molecule in addition to the normal Ficolin-3 pattern. Characterization of recombinant Ficolin-3 derived from FCN3+1637delC showed that in the homozygous situation this allelic variant would lead to Ficolin-3...

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...

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

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

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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.

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.

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.

Women Entrepreneurship in the Western Romania. Research Results and Policy Recommendations

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Anca DODESCU

2011-01-01

Full Text Available Starting from the results of the empirical, quantitative, and qualitative research performed in the AntrES project, between 5.01.2009 - 4.01.2011, in the Western Romania, referring to the existence of obvious gender gaps regarding the start up of new businesses and the ownership of the businesses; the characteristics, the motivations, and the difficulties of the women entrepreneurs and potentially enterprising women; the successful women entrepreneurs' perceptions on the sex equality in the field of entrepreneurship - this paper presents arguments and recommendations of specific policies meant to support the potential women entrepreneurship in Romania, as an engine to overcome the economic crisis and to reconnect to the regional development mechanism facilitated by the European Union's regional policy.

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

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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.

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.

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 .

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

Impacts on irrigated agriculture of changes in electricity costs resulting from Western Area Power Administration's power marketing alternatives

International Nuclear Information System (INIS)

Edwards, B.K.; Flaim, S.J.; Howitt, R.E.; Palmer, S.C.

1995-03-01

Irrigation is a major factor in the growth of US agricultural productivity, especially in western states, which account for more than 85% of the nation's irrigated acreage. In some of these states, almost all cropland is irrigated, and nearly 50% of the irrigation is done with electrically powered pumps. Therefore, even small increases in the cost of electricity could have a disproportionate impact on irrigated agriculture. This technical memorandum examines the impacts that could result from proposed changes in the power marketing programs of the Western Area Power Administration's Salt Lake City Area Office. The changes could increase the cost of power to all Western customers, including rural municipalities and irrigation districts that rely on inexpensive federal power to pump water. The impacts are assessed by translating changes in Western's wholesale power rate into changes in the cost of pumping water as an input for agricultural production. Farmers can adapt to higher electricity prices in many ways, such as (1) using different pumping fuels, (2) adding workers and increasing management to irrigate more efficiently, and (3) growing more drought-tolerant crops. This study projects several responses, including using less groundwater and planting fewer waterintensive crops. The study finds that when dependence on Western's power is high, the cost of power can have a major effect on energy use, agricultural practices, and the distribution of planted acreage. The biggest percentage changes in farm income would occur (1) in Nevada and Utah (however, all projected changes are less than 2% of the baseline) and (2) under the marketing alternatives that represent the lowest capacity and energy offer considered in Western's Electric Power Marketing Environmental Impact Statement. The aggregate impact on farm incomes and the value of total farm production would be much smaller than that suggested by the changes in water use and planted acreage

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.

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

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

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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.

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

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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.

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...

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.

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.

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.

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.

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....

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.

Developing priority criteria for magnetic resonance imaging: results from the Western Canada Waiting List project

International Nuclear Information System (INIS)

Hadorn, D.C.

2002-01-01

The Western Canada Waiting List (WCWL) Project is a federally funded partnership of 19 organizations, including medical associations, health authorities, ministries of health and research organizations, that was created to develop tools to assist in assessing the relative urgency and priority of patients on waiting lists. The WCWL panel on magnetic resonance imaging (MRI) was 1 of 5 panels constituted under this project. The panel developed and tested a set of standardized clinical criteria for setting priorities among patients awaiting MRI. The criteria were applied to 407 patients in the 4 western provinces. Regression analysis was used to determine the set of criteria weights that collectively best predicted clinicians' overall ratings of patients' urgency for MRI. Reliability was assessed using clinicians' ratings of 6 hypothetical paper cases. The resulting weighted criteria accounted for about two-fifths of the observed variance in overall urgency ratings (R 2 = 39.9%). The panel then modified the criteria on the basis of regression results and clinical judgment. Most of the revised criteria items showed poor inter-rater reliability, but test-retest reliability (over a 2-month interval) was relatively good. Criteria items requiring probability judgments were a challenge for clinicians. Further development and testing of the tool appears warranted, although considerable question remains concerning the utility of priority criteria for MRI and other diagnostic services. (author)

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 ...

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.

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.

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

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

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....

Electron spin resonance dating of teeth from Western Brazilian megafauna - preliminary results

Energy Technology Data Exchange (ETDEWEB)

Kinoshita, Angela, E-mail: angela.kinoshita@usc.br [Departamento de Fisica, FFCLRP, Universidade de Sao Paulo, 14040-901 Ribeirao Preto-SP (Brazil); Universidade Sagrado Coracao, Rua Irma Arminda 10-50, 17011-160 Bauru - Sao Paulo (Brazil); Jose, Flavio A. [Departamento de Fisica, FFCLRP, Universidade de Sao Paulo, 14040-901 Ribeirao Preto-SP (Brazil); Sundaram, Dharani; Paixao, Jesus da S.; Soares, Isabella R.M. [Universidade Federal de Mato Grosso, Departamento de Geologia Geral, 78090-000 Cuiaba-MT (Brazil); Figueiredo, Ana Maria [Instituto de Pesquisas Energeticas e Nucleares (IPEN), 05422-970 Sao Paulo-SP (Brazil); Baffa, Oswaldo [Departamento de Fisica, FFCLRP, Universidade de Sao Paulo, 14040-901 Ribeirao Preto-SP (Brazil)

2011-09-15

Electron Spin Resonance (ESR) was applied to determine ages of Haplomastodon teeth from Western Brazilian Megafauna. The Equivalent Doses (D{sub e}) of (1.3 {+-} 0.2)kGy, (800 {+-} 100)Gy and (140 {+-} 20)Gy were found and the software ROSY ESR dating was employed to convert D{sub e} in age, using isotope concentrations determined by neutron activation analysis (NAA) and other information, resulting in (500 {+-} 100)ka, (320 {+-} 50) and (90 {+-} 10)ka considering the Combination Uptake (CU) model for Uranium uptake, set as an Early Uptake (EU) for dentine and Linear Uptake (LU) for enamel. There are scarce reports about Pleistocene Megafauna in this area. This paper presents the first dating of megafauna tooth and this study could contribute to improve the knowledge about the paleoclimate and paleoenvironment of this region and prompt more investigations in this area.

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.

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

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...

Estimating mercury emissions resulting from wildfire in forests of the Western United States

Science.gov (United States)

Webster, Jackson; Kane, Tyler J.; Obrist, Daniel; Ryan, Joseph N.; Aiken, George R.

2016-01-01

Understanding the emissions of mercury (Hg) from wildfires is important for quantifying the global atmospheric Hg sources. Emissions of Hg from soils resulting from wildfires in the Western United States was estimated for the 2000 to 2013 period, and the potential emission of Hg from forest soils was assessed as a function of forest type and soil-heating. Wildfire released an annual average of 3100 ± 1900 kg-Hg y− 1 for the years spanning 2000–2013 in the 11 states within the study area. This estimate is nearly 5-fold lower than previous estimates for the study region. Lower emission estimates are attributed to an inclusion of fire severity within burn perimeters. Within reported wildfire perimeters, the average distribution of low, moderate, and high severity burns was 52, 29, and 19% of the total area, respectively. Review of literature data suggests that that low severity burning does not result in soil heating, moderate severity fire results in shallow soil heating, and high severity fire results in relatively deep soil heating ( wood > foliage > litter > branches.

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.

Patterns of Enterprise Flexibility: IAB Establishment Panel Results, Western Germany, 1993-95.

Science.gov (United States)

Bellmann, Lutz; Dull, Herbert; Kuhl, Jurgen; Lahner, Manfred; Lehmann, Udo

1997-01-01

Data on flexibility in German companies were compiled through interviews and followed up annually. Employment trends in western Germany indicated a historic peak of 29.5 million employed in 1992 followed by a steady decline of about 1 million to 28.5 million in 1995. Employment losses continued in 1996; 1997 showed some signs of stability.…

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.

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...

Residual densities affect growth of overstory trees and planted Douglas-fir, western hemlock, and western redcedar: results from the first decade

Science.gov (United States)

Leslie Chandler Brodie; Dean S. DeBell

2013-01-01

In recent years, interest has increased in silvicultural systems and harvest cuts that retain partial overstories, but there are few data available on the growth of the understory trees in such stands. We studied the response of overstory trees and underplanted seedlings, Douglas-fir (Pseudotsuga menziesii), western hemlock (Tsuga...

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

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.

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...

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

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.

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.

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...

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)....

Two novel variants of human medium chain acyl-CoA dehydrogenase (MCAD). K364R, a folding mutation, and R256T, a catalytic-site mutation resulting in a well-folded but totally inactive protein

DEFF Research Database (Denmark)

O'Reilly, Linda P; Andresen, Brage S; Engel, Paul C

2005-01-01

was again totally inactive. Neither mutant showed marked depletion of FAD. The pure K364R protein was considerably less thermostable than wild-type MCAD. Western blots indicated that, although the R256T mutant protein is less thermostable than normal MCAD, it is much more stable than K364R. Though......Two novel rare mutations, MCAD approximately 842G-->C (R256T) and MCAD approximately 1166A-->G (K364R), have been investigated to assess how far the biochemical properties of the mutant proteins correlate with the clinical phenotype of medium chain acyl-CoA dehydrogenase (MCAD) deficiency. When...... the gene for K364R was overexpressed in Escherichia coli, the synthesized mutant protein only exhibited activity when the gene for chaperonin GroELS was co-overexpressed. Levels of activity correlated with the amounts of native MCAD protein visible in western blots. The R256T mutant, by contrast, displayed...

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...

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

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

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

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.

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.

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

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

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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.

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...

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.

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

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

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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.

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...

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

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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

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.

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...

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.

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.

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.

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

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

Directory of Open Access Journals (Sweden)

William H Roldán

2009-05-01

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

Suaeda japonica Makino Attenuates Lipopolysaccharide- Induced ...

African Journals Online (AJOL)

HP

Tropical Journal of Pharmaceutical Research June 2013; 12 (3): 351-356 ... expression and production of inflammatory mediators determined by Western blot analysis. Results: SJE significantly ..... This work was supported by Business for.

NEOLITHIC PLANT USE IN THE WESTERN MEDITERRANEAN REGION: PRELIMINARY RESULTS FROM THE AGRIWESTMED PROJECT

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L. Peña-Chocarro

2013-04-01

Full Text Available This contribution focuses on the preliminary results of the AGRIWESTMED project which focuses on the archaeobotanical analyses of early Neolithic sites in the western Mediterranean region (both in Iberia and in northern Morocco. A large number of sites has been studied producing an interesting dataset of plant remains which places the earliest examples of domesticated plants in the second half of the 6th millennium cal BC. Plant diversity is high as it is shown by the large number of species represented: hulled and naked wheats, barley, peas, fava beans, vetches, lentils and grass peas. To more crops, poppy and flax, are also part of the first agricultural crops of the area. Although agriculture seems to occupy a first place in the production of food, gathering is well represented in the Moroccan sites where a large number of species has been identified. 

Twenty-year-old results from a bottomland oak species comparison trial in western Kentucky

Science.gov (United States)

Randall J. Rousseau

2008-01-01

A 20-year-old trial of five bottomland oak species (cherrybark, Nuttall, pin, water, and willow oaks) located in western Kentucky showed little difference in survival and growth but considerable difference in form characteristics. Mortality was highest between ages 1 and 3 years during plantation establishment until tree-to-tree competition began increasing between the...

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 ...

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.

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).

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.

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

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.

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.

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

Cloning and Expression of Recombinant Nucleoprotein of Influenza H1N1

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Somaie Tavakoli

2015-04-01

Full Text Available Background: Influenza virus is the major cause of lower respiratory tract illnesses on the worldwide. Vaccination can be an effective tool to prevent its outbreak. Highly conserved viral nucleoprotein is an effective vaccine candidate to provide heterosubtypic immunity, offering resistance against various influenza virus strains.Materials and Methods: In present research NP gene was inserted in pET-22b expression vector. New construct (pET-22b/NP was transformed into E. coli BL21 (DE3 strain and the expression of nucleoprotein was induced by IPTG. It was analyzed by SDS-PAGE and confirmed by Western blotting.Results: Western blotting confirmed the expression and production of recombinant Influenza nucleoprotein.Conclusion: These results suggest that the codon-optimized influenza A virus NP gene can be efficiently expressed in E. coli.

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

Mathematics in middle schools in Western European countries

NARCIS (Netherlands)

Pelgrum, W.J.

1988-01-01

The International Association for the Evaluation of Educational Achievement has conducted a number of cross-national studies in which Western European countries participated. Results from the Second International Mathematics Study regarding the content and outcomes of this study in some Western

Expert Western Classical Music Improvisers' Strategies

OpenAIRE

Despres, JP; Burnard, Pamela Anne; Dube, F; Stevance, S

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 processes are not yet thoroughly documented. As a result of this gap, our research addresses the following question: What strategies are implement...

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 ...

Effects of tMa-Xin-Di-Tan decoction on ovalbumin-induced allergic ...

African Journals Online (AJOL)

box expressed in T cells (T-bet) levels were determined in lung tissues by western blot analysis. Results: MXDT ... airway inflammation, airway hyperreactivity, shortness of breath, and ..... polysaccharide on activation of mast cells. Evid Based.

Results of a pediatric vision screening program in western South Dakota.

Science.gov (United States)

Terveen, Daniel C; Moser, Jess M; Spencer, Terrence S

2015-03-01

South Dakota is one of eight states that do not require any vision screening for children. This study describes the results of the first children's vision screening program in the state. Children ages 6 months to 12 years were screened using the SPOT photoscreener by lay volunteers as part of the Northern Plains Eye Foundation's Western South Dakota Children's Vision Screening Initiative (CVSI). Referral criteria were based on the recommendations of the manufacturer. Data was stratified by age group, sex, and percentage of children referred for hyperopia, myopia, astigmatism, anisocoria, anisometropia, and ocular misalignment. The cost benefit of amblyopia treatment in South Dakota was also calculated. Screenings were completed on 4,784 children from August 2012 to May 2014 with 62 excluded due to age. Mean age of the 4,722 (2,373 females) subjects was 6 years 7 months. Overall, the SPOT device referred 563 (11.9 percent) children. There was no significant difference in referral rate based on sex (p = 0.598). Children aged 73-144 months had the highest referral rate (12.2 percent) and children aged 12-30 months had the lowest referral rate (7.9 percent). The suspected reasons for referral based upon the screenings were as follows: 371 (7.9 percent) astigmatism, 24 (0.5 percent) ocular misalignment, 101 (2.1 percent) anisometropia, 135 (2.9 percent) myopia, 36 (0.8 percent) hyperopia, and 16 (0.3 percent) anisocoria. The SPOT photoscreener yielded an acceptable referral rate of 11.9 percent. This study represents an effective model for pediatric vision screening in South Dakota.

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

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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

[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.

Elevated c-Src and c-Yes expression in malignant skin cancers

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Lee Jang

2010-08-01

Full Text Available Abstracts Background Src family kinases (SFKs play an important role in cancer proliferation, survival, motility, invasiveness, metastasis, and angiogenesis. Among the SFKs, c-Src and c-Yes are particularly over-expressed or hyper-activated in many human epithelial cancers. However, only a few studies have attempted to define the expression and role of c-Src and c-Yes in cutaneous carcinomas. Objectives To investigate the expression of c-Src and c-Yes in cutaneous carcinomas to include malignant melanoma (MM, squamous cell carcinoma (SCC and basal cell carcinoma (BCC. Methods We examined 6 normal skin tissues and 18 malignant skin tumor tissues using western blotting for the expression of c-Src and c-Yes. In another set, 16 specimens of MM, 16 SCCs and 16 BCCs were analyzed for the expression of c-Src and c-Yes using immunohistochemical staining. Results Western blotting showed that c-Src was expressed in all malignant skin tumors, but not in normal skin, while c-Yes was expressed in MM and SCC, but not in BCC and normal skin. Immunohistochemical staining results of c-Src and c-Yes in MM, SCC, and BCC mirrored those of the western blot analysis. Conclusions c-Src, rather than c-Yes, plays a key role in the proliferation and progression of malignant skin cancers.

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.

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.

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.

Estimating Western U.S. Reservoir Sedimentation

Science.gov (United States)

Bensching, L.; Livneh, B.; Greimann, B. P.

2017-12-01

Reservoir sedimentation is a long-term problem for water management across the Western U.S. Observations of sedimentation are limited to reservoir surveys that are costly and infrequent, with many reservoirs having only two or fewer surveys. This work aims to apply a recently developed ensemble of sediment algorithms to estimate reservoir sedimentation over several western U.S. reservoirs. The sediment algorithms include empirical, conceptual, stochastic, and processes based approaches and are coupled with a hydrologic modeling framework. Preliminary results showed that the more complex and processed based algorithms performed better in predicting high sediment flux values and in a basin transferability experiment. However, more testing and validation is required to confirm sediment model skill. This work is carried out in partnership with the Bureau of Reclamation with the goal of evaluating the viability of reservoir sediment yield prediction across the western U.S. using a multi-algorithm approach. Simulations of streamflow and sediment fluxes are validated against observed discharges, as well as a Reservoir Sedimentation Information database that is being developed by the US Army Corps of Engineers. Specific goals of this research include (i) quantifying whether inter-algorithm differences consistently capture observational variability; (ii) identifying whether certain categories of models consistently produce the best results, (iii) assessing the expected sedimentation life-span of several western U.S. reservoirs through long-term simulations.

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

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

International Nuclear Information System (INIS)

Maria Lina R; Andi Yasmon

2011-01-01

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

Pre-bomb marine reservoir ages in the western north Pacific : Preliminary result on Kyoto University collection

NARCIS (Netherlands)

Yoneda, M; Kitagawa, H; van der Plicht, J; Uchida, M; Tanaka, A; Uehiro, T; Shibata, Y; Morita, M; Ohno, T

2000-01-01

The calibration of radiocarbon dates on marine materials involves a global marine calibration with regional corrections. The marine reservoir ages in the Western North Pacific have not been discussed, while it is quite important to determine the timing of palaeo-environmental changes as well as

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.

Taenia solium metacestode preparation in rural areas of sub ...

African Journals Online (AJOL)

Generated crude antigens were applied in a standard western blot assay. Results: 46 out of ... diagnose of T. solium cysticercosis in humans several serological tests ..... tion methods we can only state that there was no rel- evant difference ...

Inactivation of TGF-β signaling in lung cancer results in increased CDK4 activity that can be rescued by ELF

International Nuclear Information System (INIS)

Baek, Hye Jung; Kim, Sang Soo; Silva, Fabio May da; Volpe, Eugene A.; Evans, Stephen; Mishra, Bibhuti; Mishra, Lopa; Blair Marshall, M.

2006-01-01

Escape from TGF-β inhibition of proliferation is a hallmark of multiple cancers including lung cancer. We explored the role of ELF, crucial TGF-β adaptor protein identified from endodermal progenitor cells, in lung carcinogenesis and cell-cycle regulation. Interestingly, elf -/- mice develop multiple defects that include lung, liver, and cardiac abnormalities. Four out of 6 lung cancer and mesothelioma cell lines displayed deficiency of ELF expression with increased CDK4 expression. Immunohistochemistry and Western blot analysis of primary human lung cancers also showed decreased ELF expression and overexpression of CDK4. Moreover, rescue of ELF in ELF-deficient cell lines decreased the expression of CDK4 and resulted in accumulation of G1/S checkpoint arrested cells. These results suggest that disruption in TGF-β signaling mediated by loss of ELF in lung cancer leads to cell-cycle deregulation by modulating CDK4 and ELF highlights a key role of TGF-β adaptor protein in suppressing early lung cancer

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...

Deployment of deer-resistant western redcedar (Thuja plicata)

Science.gov (United States)

John Russell

2008-01-01

Protecting planted western redcedar (Thuja plicata) seedlings from deer browse in the Pacific Northwest and British Columbia is estimated to cost up to CAN$ 25 million annually. Recent studies linking deer browse and needle monoterpenes has resulted in the initiation of a breeding program for deer-resistant western redcedar at Cowichan Lake Research...

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.

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

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

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.

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.

Discrepancy of cytogenetic analysis in Western and eastern Taiwan.

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Chang, Yu-Hsun; Chen, Pui-Yi; Li, Tzu-Ying; Yeh, Chung-Nan; Li, Yi-Shian; Chu, Shao-Yin; Lee, Ming-Liang

2013-06-01

This study aimed at investigating the results of second-trimester amniocyte karyotyping in western and eastern Taiwan, and identifying any regional differences in the prevalence of fetal chromosomal anomalies. From 2004 to 2009, pregnant women who underwent amniocentesis in their second trimester at three hospitals in western Taiwan and at four hospitals in eastern Taiwan were included. All the cytogenetic analyses of cultured amniocytes were performed in the cytogenetics laboratory of the Genetic Counseling Center of Hualien Buddhist Tzu Chi General Hospital. We used the chi-square test, Student t test, and Mann-Whitney U test to evaluate the variants of clinical indications, amniocyte karyotyping results, and prevalence and types of chromosomal anomalies in western and eastern Taiwan. During the study period, 3573 samples, 1990 (55.7%) from western Taiwan and 1583 (44.3%) from eastern Taiwan, were collected and analyzed. The main indication for amniocyte karyotyping was advanced maternal age (69.0% in western Taiwan, 67.1% in eastern Taiwan). The detection rates of chromosomal anomalies by amniocyte karyotyping in eastern Taiwan (45/1582, 2.8%) did not differ significantly from that in western Taiwan (42/1989, 2.1%) (p = 1.58). Mothers who had abnormal ultrasound findings and histories of familial hereditary diseases or chromosomal anomalies had higher detection rates of chromosomal anomalies (9.3% and 7.2%, respectively). The detection rate of autosomal anomalies was higher in eastern Taiwan (93.3% vs. 78.6%, p = 0.046), but the detection rate of sex-linked chromosomal anomalies was higher in western Taiwan (21.4% vs. 6.7%, p = 0.046). We demonstrated regional differences in second-trimester amniocyte karyotyping results and established a database of common chromosomal anomalies that could be useful for genetic counseling, especially in eastern Taiwan. Copyright © 2012. Published by Elsevier B.V.

Study on sensitivity of southern blotting hybridization using a 32P-labeled probe of PCR products in detecting human cytomegalovirus

International Nuclear Information System (INIS)

Bu Hengfu; Chen Juan; Shen Rongsen; Ma Liren; Xu Yongqiang

1996-01-01

Southern blotting hybridization (SBH) using a 32 P-labeled probe is one of the most practical methods for genetic diagnosis of pathogen. On the basis of establishing PCR and nested PCR for detecting human cytomegalovirus (HCMV), a 32 P-labeled probe was prepared with the amplified products of 613 bp PCR outer primers and hybridized with 300 bp inner primer amplified product, resulting in increase in detecting sensitivity from 17 ng (in 1.2% agarose electrophoresis) before SBH to 500 pg (autoradiographed), in other words, increasing the sensitivity of detecting HCMV by 10 2 dilutions after using SBH. The method of PCR and SBH using a 32 P-labeled probe could detect less than 1 gene copy of HCMV, therefore, it is a rapid and reliable diagnosis method for detecting HCMV latent infection

Western Canada Sedimentary Basin competitiveness

International Nuclear Information System (INIS)

Millar, R.H.G.

1996-01-01

Recent dramatic expansion of the natural gas industry in the Western Canada Sedimentary Basin provided ample proof of the potential of this area for further development of natural gas supply. However, the inherent competitive advantages provided by the Western Canada Sedimentary Basin were said to have been offset by low netback prices resulting in poor producer economics when competitiveness is measured by availability of opportunities to find and develop gas supply at costs low enough to ensure attractive returns. Technology was identified as one of the key elements in improving basin competitiveness, but the greatest potential lies in reduced transportation costs and increased access to North American market centres. 8 figs

FIELD ACTIVITIES AND PRELIMINARY RESULTS FROM THE INVESTIGATION OF WESTERN AIRBORNE CONTAMINANTS IN TWO HIGH ELEVATION WATERSHEDS OF ROCKY MOUNTAIN NATIONAL PARK

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The National Park Service initiated the Western Airborne Contaminants Assessment Project (WACAP) in 2002 to determine if airborne contaminants from long-range transport and/or regional sources are having an impact on remote western ecosystems, including AK. Rocky Mountain Nation...

Differential effects of gastric bypass and banding on the ...

African Journals Online (AJOL)

Data collected included changes of body mass index (BMI), blood pressure, fasting blood sugar, fasting serum insulin, insulin resistance (HOMA-IR) and lipid profile. In addition, apo A-IV was determined by the Western blot technique. Results: The results demonstrated a highly significant reduction in body weight as ...

Risk assessment for yellow fever in western and North-Western provinces of Zambia

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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.

Student Performance and Family Socioeconomic Status: Results from a Survey of Compulsory Education in Western China

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Liu, Xiaofei; Lu, Ke

2008-01-01

This study used fifteen-year-old ninth-grade students from rural areas of five provinces in western China as samples to carry out research on the relationship between the socioeconomic status of Chinese families and student academic performance. Based on parents' educational background, occupation, family economic conditions, and other factors,…

Aesthetic Preferences for Eastern and Western Traditional Visual Art: Identity Matters.

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Bao, Yan; Yang, Taoxi; Lin, Xiaoxiong; Fang, Yuan; Wang, Yi; Pöppel, Ernst; Lei, Quan

2016-01-01

Western and Chinese artists have different traditions in representing the world in their paintings. While Western artists start since the Renaissance to represent the world with a central perspective and focus on salient objects in a scene, Chinese artists concentrate on context information in their paintings, mainly before the mid-19th century. We investigated whether the different typical representations influence the aesthetic preference for traditional Chinese and Western paintings in the different cultural groups. Traditional Chinese and Western paintings were presented randomly for an aesthetic evaluation to Chinese and Western participants. Both Chinese and Western paintings included two categories: landscapes and people in different scenes. Results showed a significant interaction between the source of the painting and the cultural group. For Chinese and Western paintings, a reversed pattern of aesthetic preference was observed: while Chinese participants gave higher aesthetic scores to traditional Chinese paintings than to Western paintings, Western participants tended to give higher aesthetic scores to traditional Western paintings than to Chinese paintings. We interpret this observation as indicator that personal identity is supported and enriched within cultural belongingness. Another important finding was that landscapes were more preferable than people in a scene across different cultural groups indicating a universal principle of preferences for landscapes. Thus, our results suggest that, on the one hand, the way that artists represent the world in their paintings influences the way that culturally embedded viewers perceive and appreciate paintings, but on the other hand, independent of the cultural background, anthropological universals are disclosed by the preference of landscapes.

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

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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.

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

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

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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.

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)

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

Internal globalization of Western Balkan

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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.

Western Area Power Administration combined power system financial statements, 30 September 1995 and 1994

Energy Technology Data Exchange (ETDEWEB)

NONE

1995-12-31

The attached report presents the results of the independent certified public accountant`s audit of the Department of Energy`s Western Area Power Administration`s (Western) combined financial statements as of September 30, 1995. The auditors have expressed an unqualified opinion on Western`s 1995 statements. Their reports on Western`s internal control structure and on compliance with laws and regulations are also provided.

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

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

Science.gov (United States)

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

2016-12-01

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

Borrelia burgdorferi antibodies in dogs from Cotia county, São Paulo State, Brazil

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JOPPERT Adriana Marques

2001-01-01

Full Text Available Dogs sera samples collected from Cotia County, São Paulo were tested using indirect immunoenzymatic test (ELISA in order to study Lyme disease serology in dogs. ELISA method was standardized and G39/40 North American strain of Borrelia burgdorferi was used as antigen. Positive results were confirmed employing the Western blotting technique. Because of the possibility of cross-reactions, sera were also tested for different serological strains of Leptospira interrogans and L. biflexa using microscopic sera agglutination test. Twenty-three of 237 (9.7% serum samples were positive in the ELISA; 20 of them (86.9% were confirmed by the Western blotting, what suggests that Cotia may be a risk area for Lyme disease. Although 4 samples (1.7% were positive for Lyme disease and leptospirosis, no correlation was found between the results (X² = 0.725; p = 0.394 what suggests absence of serological cross reactivity.

Mieloencefalite protozoária eqüina (Relato de caso.

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R. C. Gonçalves

2005-03-01

Full Text Available RESUMO :O objetivo deste trabalho foi relatar um caso de mieloencefalite protozoária eqüina no Estado de São Paulo, Brasil. O diagnóstico se baseou nos sinais clínicos, no resultado positivo para anticorpos contra Sarcocystis neurona no soro e no líquor pela técnica de Western blot. Palavras chave: Mieloencefalite, Sarcocystis neurona, eqüinos. SUMMARY: The purpose of this work was to present a case of equine protozoal myeloencephalitis (EPM in the State of São Paulo, Brazil. The diagnosis was based on the clinical sings, the positive results of the serum and cerebrospinal fluid analysis (Western blot for antibodies against Sarcocystis neurona. Keywords: Myeloencephalitis, Sarcocystis neurona,

Actin expression in some Platyhelminthe species.

Science.gov (United States)

Fagotti, A; Panara, F; Di Rosa, I; Simoncelli, F; Gabbiani, G; Pascolini, R

1994-10-01

Actin expression in some Platyhelminthe species was demonstrated by western-blotting and immunocytochemical analysis using two distinct anti-actin antibodies: the anti-total actin that reacts against all actin isoforms of higher vertebrates and the anti-alpha SM-1 that recognizes the alpha-smooth muscle (alpha SM) isotype of endothermic vertebrates (Skalli et al., 1986). Western-blotting experiments showed that all species tested, including some free-living Platyhelminthes (Tricladida and Rhabdocoela) and the parasitic Fasciola hepatica, were stained by anti-total actin antibody while only Dugesidae and Dendrocoelidae showed a positive immunoreactivity against anti-alpha SM-1. These results were confirmed by cytochemical immunolocalization using both avidin biotin conjugated peroxidase reaction on paraffin sections, and immunogold staining on Lowicryl 4KM embedded specimens. Our findings may contribute to the understanding of Platyhelminthes phylogeny.

Hemin offers neuroprotection through inducing exogenous neuroglobin in focal cerebral hypoxic-ischemia in rats

Science.gov (United States)

Song, Xue; Xu, Rui; Xie, Fei; Zhu, Haiyuan; Zhu, Ji; Wang, Xin

2014-01-01

Objective: To investigate the inducible effect of hemin on exogenous neuroglobin (Ngb) in focal cerebral hypoxic-ischemia in rats. Methods: 125 healthy SD rats were randomly divided into five groups: sham-operation control group, operation group, hemin treatment group, exogenous Ngb treatment group, and hemin and exogenous Ngb joint treatment group. Twenty-four hours after focal cerebral hypoxic-ischemia, Ngb expression was evaluated by immunocytochemistry, RT-PCR, and western blot analyses, while the brain water content and infarct volume were examined. Results: Immunocytochemistry, RT-PCR, and western blot analyses showed more pronounced Ngb expression in the hemin and exogenous Ngb joint operation group than in the hemin or exogenous Ngb individual treatment groups, thus producing significant differences in brain water content and infarct volume (p exogenous Ngb. PMID:24966924

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

Science.gov (United States)

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

2009-04-01

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

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)

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

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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.

Teaching the Western.

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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)

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

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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)

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

Evaluations of Extended-Range tropical Cyclone Forecasts in the Western North Pacific by using the Ensemble Reforecasts: Preliminary Results

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Tsai, Hsiao-Chung; Chen, Pang-Cheng; Elsberry, Russell L.

2017-04-01

The objective of this study is to evaluate the predictability of the extended-range forecasts of tropical cyclone (TC) in the western North Pacific using reforecasts from National Centers for Environmental Prediction (NCEP) Global Ensemble Forecast System (GEFS) during 1996-2015, and from the Climate Forecast System (CFS) during 1999-2010. Tsai and Elsberry have demonstrated that an opportunity exists to support hydrological operations by using the extended-range TC formation and track forecasts in the western North Pacific from the ECMWF 32-day ensemble. To demonstrate this potential for the decision-making processes regarding water resource management and hydrological operation in Taiwan reservoir watershed areas, special attention is given to the skill of the NCEP GEFS and CFS models in predicting the TCs affecting the Taiwan area. The first objective of this study is to analyze the skill of NCEP GEFS and CFS TC forecasts and quantify the forecast uncertainties via verifications of categorical binary forecasts and probabilistic forecasts. The second objective is to investigate the relationships among the large-scale environmental factors [e.g., El Niño Southern Oscillation (ENSO), Madden-Julian Oscillation (MJO), etc.] and the model forecast errors by using the reforecasts. Preliminary results are indicating that the skill of the TC activity forecasts based on the raw forecasts can be further improved if the model biases are minimized by utilizing these reforecasts.

Baicalein and U0126 suppress bladder cancer proliferation via ...

African Journals Online (AJOL)

RT-PCR) and western blot. Results: Baicalein and U0126 suppressed bladder cancer cell T24 proliferation by blocking cell cycle in G0~G1 phase. TUNEL and Annexin V/PI detection showed both baicalein and U0126 induced T24 cell ...

Study physico-chemical of the sand of the western ERG (Western South Algeria)

Energy Technology Data Exchange (ETDEWEB)

Allam, M.; Tafraoui, A. [Faculty of sciences and technology, University of Bechar (Algeria)], email: allammessaouda@yahoo.fr

2011-07-01

Silica is gaining increasing importance as it is the base for the production of pure silicon, for which several applications are under development in the electronic and solar energy sectors. The aim of this study is to characterize the sand taken from the Western Erg of Algeria to determine the percentage of silicon it contains. Characterization was done through physical analysis to determine the granulometry of the sand. A chemical analysis was next performed, using diffraction of X-rays and a scanning electron microscope to determine the chemical composition of the sand. Results showed that the sand is mainly made of quartz in the form of rounded and subbarrondis grains and that silicon is prevalent, accounting for 98% of the composition. This study demonstrated that sand from the Western Erg of Algeria is rich in silicon and could be used for silicon production.

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.

Density management and riparian buffer study in Western Oregon: Phase 1 results, launch of phase 2 [brochure

Science.gov (United States)

Rhonda Mazza

2009-01-01

Can we expedite the development of late-successional forest conditions by applying thinning treatments to young forest stands? What effect will these thinning treatments have on headwater ecosystems? These broad questions lie at the foundation of the Density Management and Riparian Buffer Study (DMS) of western Oregon.

Porcine Cysticercosis: Possible Cross-Reactivity of Taenia hydatigena to GP50 Antigen in the Enzyme-Linked Immunoelectrotransfer Blot Assay.

Science.gov (United States)

Muro, Claudio; Gomez-Puerta, Luis A; Flecker, Robert H; Gamboa, Ricardo; Barreto, Percy Vilchez; Dorny, Pierre; Tsang, Victor C W; Gilman, Robert H; Gonzalez, Armando E; Garcia, Hector H; O'Neal, Seth E; For The Cysticercosis Working Group In Peru

2017-12-01

The lentil lectin glycoprotein enzyme-linked immunoelectrotransfer blot (LLGP EITB, reported sensitivity 99% and specificity 100%) is used as a serologic marker of exposure to Taenia solium in pigs. However, only a limited number of parasites have been evaluated for cross reactivity. Pigs may host other related cestode infections, including Taenia hydatigena, which have not been formally evaluated for cross-reactions. We investigated a corral in Tumbes, Peru, a region where a cysticercosis elimination demonstration project was completed in 2012. In this corral, 14/19 (73.7%) 6-8-week-old piglets were reactive to GP50 on LLGP EITB, and all had circulating Taenia sp. antigens. From eight necropsied piglets; four were infected with T. hydatigena metacestodes whereas none had evidence of T. solium infection. Two resident dogs were subsequently confirmed to have T. hydatigena taeniasis. These results suggest GP50 cross-reactivity in T. hydatigena- infected pigs, although controlled experimental infection is needed to confirm this hypothesis.

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

Lagrangian mixed layer modeling of the western equatorial Pacific

Science.gov (United States)

Shinoda, Toshiaki; Lukas, Roger

1995-01-01

Processes that control the upper ocean thermohaline structure in the western equatorial Pacific are examined using a Lagrangian mixed layer model. The one-dimensional bulk mixed layer model of Garwood (1977) is integrated along the trajectories derived from a nonlinear 1 1/2 layer reduced gravity model forced with actual wind fields. The Global Precipitation Climatology Project (GPCP) data are used to estimate surface freshwater fluxes for the mixed layer model. The wind stress data which forced the 1 1/2 layer model are used for the mixed layer model. The model was run for the period 1987-1988. This simple model is able to simulate the isothermal layer below the mixed layer in the western Pacific warm pool and its variation. The subduction mechanism hypothesized by Lukas and Lindstrom (1991) is evident in the model results. During periods of strong South Equatorial Current, the warm and salty mixed layer waters in the central Pacific are subducted below the fresh shallow mixed layer in the western Pacific. However, this subduction mechanism is not evident when upwelling Rossby waves reach the western equatorial Pacific or when a prominent deepening of the mixed layer occurs in the western equatorial Pacific or when a prominent deepening of the mixed layer occurs in the western equatorial Pacific due to episodes of strong wind and light precipitation associated with the El Nino-Southern Oscillation. Comparison of the results between the Lagrangian mixed layer model and a locally forced Eulerian mixed layer model indicated that horizontal advection of salty waters from the central Pacific strongly affects the upper ocean salinity variation in the western Pacific, and that this advection is necessary to maintain the upper ocean thermohaline structure in this region.

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

Directory of Open Access Journals (Sweden)

Maite Sabalza

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

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)

Monoclonal Antibody L1Mab-13 Detected Human PD-L1 in Lung Cancers.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Chang, Yao-Wen; Suzuki, Hiroyoshi; Kaneko, Mika K; Kato, Yukinari

2018-04-01

Programmed cell death ligand-1 (PD-L1) is a type I transmembrane glycoprotein expressed on antigen-presenting cells. It is also expressed in several tumor cells such as melanoma and lung cancer cells. A strong correlation has been reported between human PD-L1 (hPD-L1) expression in tumor cells and negative prognosis in cancer patients. Here, a novel anti-hPD-L1 monoclonal antibody (mAb) L 1 Mab-13 (IgG 1 , kappa) was produced using a cell-based immunization and screening (CBIS) method. We investigated hPD-L1 expression in lung cancer using flow cytometry, Western blot, and immunohistochemical analyses. L 1 Mab-13 specifically reacted hPD-L1 of hPD-L1-overexpressed Chinese hamster ovary (CHO)-K1 cells and endogenous hPD-L1 of KMST-6 (human fibroblast) in flow cytometry and Western blot. Furthermore, L 1 Mab-13 reacted with lung cancer cell lines (EBC-1, Lu65, and Lu99) in flow cytometry and stained lung cancer tissues in a membrane-staining pattern in immunohistochemical analysis. These results indicate that a novel anti-hPD-L1 mAb, L 1 Mab-13, is very useful for detecting hPD-L1 of lung cancers in flow cytometry, Western blot, and immunohistochemical analyses.

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)

Increase of ATP-sensitive potassium (KATP channels in the heart of type-1 diabetic rats

Directory of Open Access Journals (Sweden)

Chen Zhih-Cherng

2012-01-01

Full Text Available Abstract Background An impairment of cardiovascular function in streptozotocin (STZ-diabetic rats has been mentioned within 5 days-to-3 months of induction. ATP-sensitive potassium (KATP channels are expressed on cardiac sarcolemmal membranes. It is highly responsive to metabolic fluctuations and can have effects on cardiac contractility. The present study attempted to clarify the changes of cardiac KATP channels in diabetic disorders. Methods Streptozotocin-induced diabetic rats and neonatal rat cardiomyocytes treated with a high concentration of glucose (a D-glucose concentration of 30 mM was used and cells were cultured for 24 hr were used to examine the effect of hyperglycemia on cardiac function and the expression of KATP channels. KATP channels expression was found to be linked to cardiac tonic dysfunction, and we evaluated the expression levels of KATP channels by Western blot and Northern blot analysis. Results The result shows diazoxide produced a marked reduction of heart rate in control group. Furthermore, the methods of Northern blotting and Western blotting were employed to identify the gene expression of KATP channel. Two subunits of cardiac KATP channel (SUR2A and kir 6.2 were purchased as indicators and showed significantly decreased in both diabetic rats and high glucose treated rat cardiac myocytes. Correction of hyperglycemia by insulin or phlorizin restored the gene expression of cardiac KATP in these diabetic rats. Conclusions Both mRNA and protein expression of cardiac KATP channels are decreased in diabetic rats induced by STZ for 8 weeks. This phenomenon leads to result in desensitization of some KATP channel drugs.

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 ...

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)

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...

Expression of vasoactive proteins in gastric antral mucosa reflects vascular dysfunction in patients with cirrhosis and portal hypertension

DEFF Research Database (Denmark)

Trebicka, Jonel; Wix, Cyrus; von Heydebrand, Matthias

2015-01-01

. Portal pressure gradient was measured in 49 patients with TIPS and in 16 patients without TIPS. Biopsies from the antrum were conserved in formaldehyde for immunohistochemistry or shock-frozen for PCR and Western blot. RESULTS: The mucosal transcription of vascular markers (αSMA, CD31) was higher...

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...

Breviscapine Injection Improves the Therapeutic Effect of Western Medicine on Angina Pectoris Patients.

Science.gov (United States)

Wang, Chuan; Li, Yafeng; Gao, Shoucui; Cheng, Daxin; Zhao, Sihai; Liu, Enqi

2015-01-01

To evaluate the beneficial and adverse effects of breviscapine injection in combination with Western medicine on the treatment of patients with angina pectoris. The Cochrane Central Register of Controlled Trials, Medline, Science Citation Index, EMBASE, the China National Knowledge Infrastructure, the Wanfang Database, the Chongqing VIP Information Database and the China Biomedical Database were searched to identify randomized clinical trials (RCTs) that evaluated the effects of Western medicine compared to breviscapine injection plus Western medicine on angina pectoris patients. The included studies were analyzed using RevMan 5.1.0 software. The literature search yielded 460 studies, wherein 16 studies matched the selection criteria. The results showed that combined therapy using Breviscapine plus Western medicine was superior to Western medicine alone for improving angina pectoris symptoms (OR=3.77, 95% Cl: 2.76~5.15) and also resulted in increased electrocardiogram (ECG) improvement (OR=2.77, 95% Cl: 2.16~3.53). The current evidence suggests that Breviscapine plus Western medicine achieved a superior therapeutic effect compared to Western medicine alone.

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.

Upper mantle flow in the western Mediterranean

Energy Technology Data Exchange (ETDEWEB)

Panza, G F [Dipartimento di Scienze della Terra, Universita degli Studi di Trieste, Trieste (Italy) and Abdus Salam International Centre for Theoretical Physics, Trieste (Italy); Raykova, R [Geophysical Institute of BAS, Sofia (Bulgaria) and Istituto Nazionale di Geofisica e Vulcanologia, Sezione Bologna, Bologna (Italy); Carminati, E; Doglioni, C [Dipartimento di Scienze della Terra, Universita degli Studi di Trieste, Trieste (Italy)

2006-07-15

Two cross-sections of the western Mediterranean Neogene-to-present backarc basin are presented, in which geological and geophysical data of the Transmed project are tied to a new shear-wave tomography. Major results are i) the presence of a well stratified upper mantle beneath the older African continent, with a marked low-velocity layer between 130-200 km of depth; ii) the dilution of this layer within the younger western Mediterranean backarc basin to the north, and iii) the easterly raising of a shallower low-velocity layer from about 140 km to about 30 km in the Tyrrhenian active part of the backarc basin. These findings suggest upper mantle circulation in the western Mediterranean backarc basin, mostly easterly-directed and affecting the boundary between upper asthenosphere (LVZ) and lower asthenosphere, which undulates between about 180 km and 280 km. (author)

Upper mantle flow in the western Mediterranean

International Nuclear Information System (INIS)

Panza, G.F.; Raykova, R.; Carminati, E.; Doglioni, C.

2006-07-01

Two cross-sections of the western Mediterranean Neogene-to-present backarc basin are presented, in which geological and geophysical data of the Transmed project are tied to a new shear-wave tomography. Major results are i) the presence of a well stratified upper mantle beneath the older African continent, with a marked low-velocity layer between 130-200 km of depth; ii) the dilution of this layer within the younger western Mediterranean backarc basin to the north, and iii) the easterly raising of a shallower low-velocity layer from about 140 km to about 30 km in the Tyrrhenian active part of the backarc basin. These findings suggest upper mantle circulation in the western Mediterranean backarc basin, mostly easterly-directed and affecting the boundary between upper asthenosphere (LVZ) and lower asthenosphere, which undulates between about 180 km and 280 km. (author)

Influence of processing on the allergenic properties of pistachio nut assessed in vitro.

Science.gov (United States)

Noorbakhsh, Reihaneh; Mortazavi, Seyed Ali; Sankian, Mojtaba; Shahidi, Fakhri; Maleki, Soheila J; Nasiraii, Leila Roozbeh; Falak, Reza; Sima, Hamid Reza; Varasteh, AbdolReza

2010-09-22

Pistachio (Pistacia vera) is a tree nut that has been reported to cause IgE-mediated allergic reactions. This study was undertaken to investigate the distinctions between different cultivars of pistachio nut and the influence of different processing on the IgE-binding capacity of whole pistachio protein extracts. The influence of different processes on allergenicity was investigated using competitive inhibition ELISA and Western blotting assays. The Western blotting results of extracts from pistachio cultivars showed no marked difference among them. The IgE-binding capacity was significantly lower for the protein extract prepared from steam-roasted than from raw and dry-roasted pistachio nuts. The results of sensory evaluation analysis and hedonic rating proved no significant differences in color, taste, flavor, and overall quality of raw, roasted, and steam-roasted pistachio nut treatments. The most significant finding of the present study was the successful reduction of IgE-binding by pistachio extracts using steam-roast processing without any significant changes in sensory quality of product.

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.

Biogeochemistry of Lakes in Western Papua, Indonesia - First Results of a Pilot Study.

Science.gov (United States)

Kallmeyer, J.; Nomosatryo, S.; Henny, C.; Kopalit, H.

2016-12-01

Despite years of exploration for mineral and hydrocarbon resources, the lakes of Western Papua have received very little attention from a limnogeologic perspective. In some cases not even the maximum water depth of the lakes is published. The only research carried out so far focused on the fish and invertebrate fauna of the lakes, because the macrofauna of Papuan Lakes is significantly different from other islands of western Indonesia. Most lakes harbor numerous endemic species. We carried out a first limnogeologic pilot campaign in spring 2016 to measure water column profiles and take short (max 80 cm long) sediment cores.Lake Sentani is seated in Mesozoic mafic bedrock and consists of four separate basins with maximum water depths of 30 to 40 m. Three basins are connected by shallow sills and one by a natural canal. Although all four basins share almost identical surface water chemistry and exhibit sub- to anoxic bottom waters, each basin has its distinct water column stratification and sediment geochemistry. Despite its coastal location and minimal elevation we could not identify an influx of seawater into the lake. Lake Ayamaru is located further inland on a densely forested karstified carbonate platform. The lake level has dropped significantly in recent years due to water loss into the karst, further reduction of open water surface is caused by massive growth of Pistia. Currently the lake has a maximum depth of around 2 m. Its sediment is mainly composed of carbonate minerals and methane saturated. Due to the carbonate bedrock the lake is highly alkaline (up to 20 meq/L) despite its very low salinity. The initial analyses show that these lakes offer unique biogeochemical conditions that require further in-depth studies.Our research will expand to lakes Anggi Giji and Anggi Gida, which are at almost 2000 m elevation. They have maximum depths of around 200 m and much colder surface waters (12-20°C) compared to the other two lakes that have about 30�

Multiplexed expression and screening for recombinant protein production in mammalian cells

Directory of Open Access Journals (Sweden)

McCafferty John

2006-12-01

Full Text Available Abstract Background A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner. Results A nested PCR approach was used to clone 21 extracellular domains of mouse receptors as CD4 fusions within a mammalian GATEWAY expression vector system. Following transient transfection, HEK-293E cells grown in 2 ml cultures in 24-deep well blocks showed similar growth kinetics, viability and recombinant protein expression profiles, to those grown in 50 ml shake flask cultures as judged by western blotting. Following optimisation, fluorescent dot blot analysis of transfection supernatants was shown to be a rapid method for analysing protein expression yielding similar results as western blot analysis. Addition of urea enhanced the binding of glycoproteins to a nitrocellulose membrane. A good correlation was observed between the results of a plate based small scale transient transfection dot blot pre-screen and successful purification of proteins expressed at the 50 ml scale. Conclusion The combination of small scale multi-well plate culture and dot blotting described here will allow the multiplex analysis of different mammalian expression experiments enabling a faster identification of high yield expression constructs or conditions prior to large scale protein production. The methods for parallel GATEWAY cloning and expression of multiple constructs in cell

Oil and gas competition in Western Europe

International Nuclear Information System (INIS)

Carrie, J.L.

1991-01-01

This paper reports that in Western Europe, the competition between oil and gas began on a large scale during the 1960s. Indeed, natural gas accounted for only 2 percent of Western Europe's energy consumption in 1960 and for already 7 percent in 1970. It now accounts for about 17 percent. Almost all of this increase took place at the detriment of oil products. The competition between those two energy sources has resulted in the development of natural gas supply on one hand, and in several political and economic factors on the other

[RNA interference of HERC4 inhibits proliferation, apoptosis and migration of cervical cancer Hela cells].

Science.gov (United States)

Wei, Min; Zhang, Yan-Ling; Chen, Lan; Cai, Cui-Xia; Wang, Han-Duo

2016-02-20

To explore the effects of silencing HERC4 on the proliferation, apoptosis, and migration of cervical cancer cell line Hela and the possible molecular mechanisms. Three HERC4-specific small interfering RNAs (siRNAs) were transfected into Hela cells, and HERC4 expression in the cells was examined with Western blotting. CCK-8 assay, annexin V-FITC/PI assay, and wound healing assay were used to assess the effect of HERC4 silencing on the proliferation, apoptosis and migration ability of Hela cells. The expression levels of cyclin D1 and Bcl-2 in the cells were detected using Western blotting. Transfection of siRNA-3 resulted in significantly decreased HERC4 protein expression (PHela cells, increased the apoptosis rate (PHela cells in vitro, and the underlying mechanisms may involve the down-regulation of cyclin D1 and Bcl-2.

Effect of recombinant adenovirus encoding human p53 tumor suppressor gene (rAd-p53) on the growth and radiotherapeutic sensitivity of human lymphoma cell lines

International Nuclear Information System (INIS)

Yu Zeyang; Fan Wo; Li Dongqing; Zhu Ran; Wang Yongqing; Wu Jinchang

2008-01-01

Objective: To explore the inhibitory effect and radiation sensitization of recombinant adenovirus encoding human p53 tumor suppressor gene (rAd-p53) on human lymphoma cell lines. Methods: Human lymphoma cell lines Raji and Daudi were treated with rAd-p53, radiation therapy and combined treatment, respectively. The cell growth inhibition was assessed by MTT. The p53 protein expression was detected by Western blotting, and p53 mRNA was detected by BT-PCB. Results: The MTT results showed that the inhibitory effect and radiosensitivity enhancement of rAd-p53 on human lymphoma cell lines were not obvious [Raji: (27.5±4.1)%; Daudi: (28.1±1.6)%]. The results of Western blotting and BT-PCB showed that extrinsic p53 protein and p53 mRNA were expressed to some degree, but not at high-level. In addition, the results didn't demonstrate obvious radiosensitivity enhancement. Conclusions: The role of inhibition and radiosensitivity enhancement of rAd-p53 was not significant on human lymphoma cell lines. (authors)

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.

Western guilt and Third World Development : Part 2

OpenAIRE

Baafi Antwi, Joseph

2011-01-01

This work considered the argument of the opponent of Western guilt and the final verdict was issued. The four thematic areas; colonialism, neo-colonialism, slave trade and trade barriers were used. The work found that these events were of enormous benefits to Third World countries though widely criticized by the proponents of Western guilt. The work also considered factors that have resulted in the underdevelopment of Third World countries. These factors were identified as human resource deve...

For Western girls only? Postfeminism as transnational culture

OpenAIRE

Dosekun, Simidele

2015-01-01

Much of the literature on post-feminism concerns the “Western” world and variously conceptualizes post-feminism as “Western culture.” This article argues that, as a result, feminist cultural scholars have not sufficiently imagined, theorized or empirically researched the possibility of post-feminism in non-Western cultural contexts. By briefly reviewing what has been said in the literature about post-feminism and the non-West, and by putting this in dialogue with transnational feminist cultur...

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.

High DMBT1 concentrations in breast milk correlate with increased risk of infection in preterm and term neonates

DEFF Research Database (Denmark)

Ronellenfitsch, Sebastian; Weiß, Christel; Frommhold, David

2012-01-01

by Western blotting and its concentration was quantified by ELISA in 95 breast milk samples collected from mothers of preterm and term neonates during the first four weeks after delivery. Possible effects of maternal or neonatal parameters were analyzed by different statistical tests. Results: The mean DMBT1...

Apoptosis induced by GanoPoly in human gastric cancer cell line ...

African Journals Online (AJOL)

In order to investigate polysaccharide effect on the cultured human gastric cancer cells (SGC7901), DNA ladder, flow cytometry and western blot were used to examine the morpholog, proliferation and apoptosis of human gastric cancer SGC-7901 cells when they were affected by polysaccharide. Results show that ...

Tetramethylpyrazine attenuates oleic acid-induced acute lung injury ...

African Journals Online (AJOL)

Jane

2011-09-28

Sep 28, 2011 ... exerts potent anti-inflammatory effects in this model. After 4, 8 and 12 ... measured by immunohistochemistry and Western blotting. ... endothelial and infiltrating inflammatory cells results in the ... Electrophoresis apparatus and electric switch slot were ... after tying off the right lung at the main stem bronchus.

TAZ promotes epithelial to mesenchymal transition via the upregulation of connective tissue growth factor expression in neuroblastoma cells.

Science.gov (United States)

Wang, Qiang; Xu, Zhilin; An, Qun; Jiang, Dapeng; Wang, Long; Liang, Bingxue; Li, Zhaozhu

2015-02-01

Neuroblastoma (NB) is a neuroendocrine cancer that occurs most commonly in infants and young children. The Hippo signaling pathway regulates cell proliferation and apoptosis, and its primary downstream effectors are TAZ and yes‑associated protein 1 (YAP). The effect of TAZ on the metastatic progression of neuroblastoma and the underlying mechanisms involved remain elusive. In the current study, it was determined by western blot analysis that the migratory and invasive properties of SK‑N‑BE(2) human neuroblastoma cells are associated with high expression levels of TAZ. Repressed expression of TAZ in SK‑N‑BE(2) cells was shown to result in a reduction in aggressiveness of the cell line, by Transwell migration and invasion assay. In contrast, overexpression of TAZ in SK‑N‑SH human neuroblastoma cells was shown by Transwell migration and invasion assays, and western blot analysis, to result in epithelial‑mesenchymal transition (EMT) and increased invasiveness. Mechanistically, the overexpression of TAZ was demonstrated to upregulate the expression levels of connective tissue growth factor (CTGF), by western blot analysis and chromatin immunoprecipitation assay, while the knockdown of TAZ downregulated it. Furthermore, TAZ was shown by luciferase assay to induce CTGF expression by modulating the activation of the TGF‑β/Smad3 signaling pathway. In conclusion, the present study is, to the best of our knowledge, the first to demonstrate that the overexpression of TAZ induces EMT, increasing the invasive abilities of neuroblastoma cells. This suggests that TAZ may serve as a potential target in the development of novel therapies for the treatment of neuroblastoma.

[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.

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

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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.

Public say food regulatory policies to improve health in Western Australia are important: population survey results

OpenAIRE

Pollard, Christina M; Daly, Alison; Moore, Michael; Binns, Colin W

2013-01-01

Objective To investigate the level of support among Western Australian adults for food control policies to improve diet, reduce obesity and protect the environment. Methods Attitudes towards government food control policies on food labelling, food advertising, and the supply of environmentally friendly food data were pooled from two Nutrition Monitoring Survey Series telephone surveys of 2,147 adults aged 18?64 years collected in 2009 and 2012. Descriptive and logistic regression analyses wer...

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.

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

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.

Exposure to ticks and seroprevalence of [i]Borrelia burgdorferi [/i]among a healthy young population living in the area of southern Podlasie, Poland

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Anna Pańczuk

2014-09-01

Full Text Available [b]Objectives[/b]. The objective of the study was assessment of risk of infection with [i]Borrelia burgdorferi[/i] in the area of southern Podlasie in Poland, near the border with Belarus, by analysis of post-exposure procedure, and evaluation of asymptomatic infection in adolescents bitten by a tick, confirmed by serologic tests. [b]Material and methods[/b]. The study was conducted among 128 healthy individuals aged 16–20 who declared being bitten by a tick. The level of IgM and IgG class antibodies was determined using the immunoenzymatic test (Borrelia 14 kD + OspC IgM ELISA and Borrelia IgG + VlsE ELISA, DRG Diagnostics. Positive and doubtful results were confirmed using the Western blot method (EUROLINE-WB, EUROIMMUN. [b]Results[/b]. In the study group, the largest number of respondents (59.4% declared tick bite in the region of the lower extremities, most often in the knee pit. Among the methods for removing the tick the largest number of respondents indicated removing it with the use of tweezers, with a simple, swift steady movement (29.7%, and pulling it out with the fingers (22.7%. In the ELISA test, a positive or doubtful result in at least one class was observed in 25.0% of respondents (n=32/128: in IgM class – 23.4% (n=30/128, and in IgG class – 4.7% (n=6/128. After verification with the Western blot test, infection was confirmed in 5.5% of respondents (n=7/128: in IgM class – 1.6% (n=2/128, in IgG class – 3.9% (n=5/128. In IgM class antibodies, the Western blot test confirmed positive or doubtful results of the ELISA test in 6.7%, while in IgG class antibodies in 83.3%. [b]Conclusion[/b]. Evaluation of the actual infection with [i]Borrelia spp.[/i] using serologic tests is difficult due to a certain non-specificity of the ELISA test, especially in IgM class antibodies, and difficulties with performance of a wide scope of specific Western blot tests. The variety of methods of tick removal declared by adolescents suggests

Prescriptions of traditional Chinese medicine, western medicine, and integrated Chinese-Western medicine for allergic rhinitis under the National Health Insurance in Taiwan.

Science.gov (United States)

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

2015-09-15

Allergic rhinitis has long been a worldwide health problem with a global growth trend. The use of traditional Chinese medicines alone or integrated Chinese-Western medicines for its treatment is quite common in Taiwan. Respiratory diseases account for the majority of outpatient traditional Chinese medicine treatment, while allergic rhinitis accounts for the majority of respiratory diseases. We hereby conduct a comparative analysis between traditional Chinese medicine treatments and western medicine treatments for allergic rhinitis in Taiwan. The results of the analysis on the prescription difference of traditional Chinese medicine and western medicine treatments would be helpful to clinical guide and health policy decision making of ethnopharmacological therapy. Patients diagnosed as allergic rhinitis with diagnostic code 470-478 (ICD-9-CM) were selected as subjects from 2009-2010 National Health Insurance Research Database based on the claim data from the nationwide National Health Insurance in Taiwan. This retrospective study used Chi-Square test to test the effects of gender and age on visit of traditional Chinese medicine, western medicine, and integrated Chinese-Western medicine treatments. A total of 45,804 patients diagnosed as allergic rhinitis with ICD-9-CM 470-478 were identified from 2009-2010 NHIRD. There were 36,874 subjects for western medicine treatment alone, 5829 subjects for traditional Chinese medicine treatment alone, and 3101 subjects for integrated Chinese-Western medicine treatment. Female patients were more than male in three treatments. 0-9 years children had the highest visit frequency in western medicine and integrated Chinese-Western medicine groups, while 10-19 years young-age rank the highest in traditional Chinese medicine group. The Chi-square test of independence showed that the effects of gender and age on visit of three treatments were significant. The prescription drugs of western medicine treatment alone were almost for

Western Option - Disarmament of Russian Weapon Plutonium

International Nuclear Information System (INIS)

Tveiten, B.; Petroll, M.R.

2002-01-01

The Western Option concept describes an approach to the conversion of weapon-grade plutonium from Russian nuclear warheads under the special aspects of meeting the criteria of irreversible utilization. Putting this concept of plutonium conversion into non-weapon-grade material into effect would make a major contribution to improving security worldwide. This study is based on an agreement between the Russian Federation and the United States of America concluded in September 2000. It provides for the conversion of 34 t of weapon-grade plutonium in each of the two states. This goal is also supported by other G8 countries. While the United States performs its part of the agreement under its sole national responsibility, the Russian program needs financial support by Western states. Expert groups have pointed out several options as a so-called basic scenario. The funds of approx. US Dollar 2 billion required to put them into effect have not so far been raised. The Western Option approach described in this contribution combines results of the basic scenario with other existing experience and with technical solutions available for plutonium conversion. One of the attractions of the Western Option lies in its financial advantages, which are estimated to amount to approx. US Dollar 1 billion. (orig.) [de

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.

Targeting and Therapy of Glioblastoma in a Mouse Model Using Exosomes Derived From Natural Killer Cells

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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

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

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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...

Ameliorative potential of Artemisia Capillaris Formula on ...

African Journals Online (AJOL)

Hepatic changes were observed through pathological examination of Hematoxylin and eosin-stained tissues, quantification of lipid metabolites from sera (ALT, AST, ALP activity and TG, TC, HDL-C, LDL-C), and quantification of related gene and protein expression levels by RT-PCR and Western blotting. Results: A high-fat ...

Vascular and Skeletal Muscle Function in Gulf War Veterans Illness

Science.gov (United States)

2017-07-01

DNA, RNA, and protein from muscle tissue samples. Prepare cDNA from RNA samples. 4-28 Pending Complete qPCR and Western Blot to assess genes and...Lab from cDNA samples 24-30 Pending Interpret results and identify candidate genes related to Gulf War Illness 24-30 Pending Milestone: Complete

Native human serum amyloid P component is a single pentamer

DEFF Research Database (Denmark)

Sørensen, Inge Juul; Andersen, Ove; Nielsen, EH

1995-01-01

and ELISA, and SAP peak fractions were analysed by use of SDS-PAGE, Western blotting, and electron microscopy. The results indicate that native SAP circulates as a single pentamer, a part of which forms complexes with C4b-binding protein. The properties of SAP changed during purification as indicated...

Prevalence of intermittent preventive treatment with sulphadoxine ...

African Journals Online (AJOL)

by the national diagnostic algorithm of two rapid antibody test and western blot confirmation. Results: Of ... problem, affecting nearly half of all pregnant women .... bottle and temporarily stored in an ice chest and were ... copy of the concentrated sediments in urine. ..... cacy and cost effectiveness of IPTp-SP, SP drug resist-.

Wortmannin as Targeted Therapeutic Agent for the Treatment of ...

African Journals Online (AJOL)

(7 mg/kg) in DMSO daily intraperitoneally whereas the animals in control groups received an equal volume of DMSO for 21 days after the cancer attained palpable stage. Western blot analysis was carried out using enhanced chemiluminescence reagent while Protean IEF cell unit was used for 1-D electrophoresis. Results: ...

[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

Radiation-induced apoptosis in developing rats and kainic acid-induced excitotoxicity in adult rats are associated with distinctive morphological and biochemical c-Jun/AP-1 (N) expression

Energy Technology Data Exchange (ETDEWEB)

Pozas, E. [Unitat de Neuropatologia, Servei d' Anatomia Patologica, Hospital Princeps d' Espanya, Universitat de Barcelona, 08907 Hospitalet de Llobregat (Spain); Planas, A.M. [Departament de Farmacologia i Toxicologia, IIBB, CSIC Barcelona (Spain); Ferrer, I. [Unitat de Neuropatologia, Servei d' Anatomia Patologica, Hospital Princeps d' Espanya, Universitat de Barcelona, 08907 Hospitalet de Llobregat (Spain)

1997-07-14

Ionizing radiation produces apoptosis in the developing rat brain. Strong c-Jun immunoreactivity, as revealed with the antibody c-Jun/AP-1 (N) which is raised against the amino acids 91-105 mapping with the amino terminal domain of mouse c-Jun p39, is simultaneously observed in the nucleus and cytoplasm of apoptotic cells. Western blotting of total brain homogenates, using the same antibody, shows a p39 band in control rats which is accompanied by a strong, phosphorylated p62 double-band in irradiated animals. In addition, increased c-Jun N-terminal kinase 1 expression, as found on western blots, is found in irradiated rats when compared with controls. Intraperitoneal injection of kainic acid at convulsant doses to the adult rat produces cell death with morphological features of necrosis, together with the appearance of cells with fine granular chromatin degeneration and small numbers of apoptotic-like cells, in the entorhinal and piriform cortices, basal amygdala, certain thalamic nuclei, and CA1 region of the hippocampus. c-Jun expression in kainic acid-treated rats, as revealed with the c-Jun/AP-1 (N) antibody, is found in the nuclei of a minority of cells in the same areas. The vast majority of c-Jun-immunoreactive cells have normal nuclear morphology, whereas necrotic cells are negative and only a few cells with fine granular chromatin condensation and apoptotic cells following kainic acid injection are stained with c-Jun antibodies. Western blotting, using the same antibody, shows a p39 band in control rats, which is accompanied by a band at about p26 from 6 h onwards following kainic acid injection. Decreased c-Jun N-terminal kinase 1 expression, as revealed on western blots, is observed in kainic acid-treated rats.These results show that the antibody c-Jun/AP-1 (N) recognizes three different forms of c-Jun-related immunoreactivity in normal and pathological states, which are associated with the different outcome of cells. These results stress the necessity

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

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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

Determination of protein-carbonyls and ubiquitin-mediated proteolysis as biomarkers of oxidative-stress in bivalvia and anthozoa

International Nuclear Information System (INIS)

Walker, Stephen Thomas

2002-01-01

This study describes the investigation of biomarkers of oxidative-stress in the bivalves Mytilus edulis and Dosinia lupinus, together with host and symbiont tissues of the scleractinian Anthozoa Agaricia agaricites. The biomarkers used were assay of total (via spectrophotometry) and individual (via Western blotting; Oxyblot kit) protein-carbonyls (PC=Os) and content of ubiquitin protein conjugates (UPC) via Western blotting (Bivalvia and Anthozoa) and immunohistochemistry (Anthozoa only). Additional assays for Bivalvia were Trolox equivalent antioxidant capacity (TEAC); and post γ-irradiation survival rates. Experimental stressors for Bivalvia were increased seawater temperature, H 2 O 2 and 60 Co γ-radiation (latter two were used in vivo and in vitro). Comparisons of clean and polluted marine sites are included. Stressors used for Anthozoa were increased solar irradiation concomitant with elevated seawater temperature. Results and conclusions were as follows: individual samples showed considerable variation, pooling of samples improved consistency. Controls for both biomarkers had detectable background levels in each phylum, against which relatively small differences were assessed. In M. edulis, no measurable differences in PC=Os could be determined when elevated seawater temperature or dilute H 2 O 2 ( 2 O 2 (30% v/v) produced a small difference. 60 Co γ-radiation produced clearer differences via Oxyblot and spectrophotometric assays. Comparison of four different tissues from the two bivalves found considerable species-specific and tissue-specific differences. Post-irradiation mortality between species was significantly different (<0.001), D. lupinus was more susceptible than M. edulis. TEAC values generally showed a decrease following irradiation (except for digestive gland). UPCs were clearly different between tissues and between species. PC=Os can be detected by DNPH-reactivity/Western blotting assay in host A. agaricites. UPCs can be assayed via Western

Proposed framework for the Western Area Power Administration Environmental Risk Management Program

Energy Technology Data Exchange (ETDEWEB)

Glantz, C.S.; DiMassa, F.V.; Pelto, P.J.; Brothers, A.J. [Pacific Northwest Lab., Richland, WA (United States); Roybal, A.L. [Western Area Power Administration, Golden, CO (United States)

1994-12-01

The Western Area Power Administration (Western) views environmental protection and compliance as a top priority as it manages the construction, operation, and maintenance of its vast network of transmission lines, substations, and other facilities. A recent Department of Energy audit of Western`s environmental management activities recommends that Western adopt a formal environmental risk program. To accomplish this goal, Western, in conjunction with Pacific Northwest Laboratory, is in the process of developing a centrally coordinated environmental risk program. This report presents the results of this design effort, and indicates the direction in which Western`s environmental risk program is heading. Western`s environmental risk program will consist of three main components: risk communication, risk assessment, and risk management/decision making. Risk communication is defined as an exchange of information on the potential for threats to human health, public safety, or the environment. This information exchange provides a mechanism for public involvement, and also for the participation in the risk assessment and management process by diverse groups or offices within Western. The objective of risk assessment is to evaluate and rank the relative magnitude of risks associated with specific environmental issues that are facing Western. The evaluation and ranking is based on the best available scientific information and judgment and serves as input to the risk management process. Risk management takes risk information and combines it with relevant non-risk factors (e.g., legal mandates, public opinion, costs) to generate risk management options. A risk management tool, such as decision analysis, can be used to help make risk management choices.

Health behaviour among non-Western immigrants with Danish citizenship

DEFF Research Database (Denmark)

Hansen, Anne R; Ekholm, Ola; Kjøller, Mette

2008-01-01

AIMS: To compare belief in own effort to stay healthy, health behaviour and body mass index (BMI) among non-Western immigrants with Danish citizenship and citizens with Danish background. METHODS: Based on the National Health Interview Survey 2005, logistic regression analyses were used to examine...... differences in belief in own effort to stay healthy, in health behaviour and in BMI between 136 non-Western immigrants with Danish citizenship and 9,901 citizens with Danish background in the age group 25-64 years. RESULTS: Non-Western immigrants had lower odds for reporting that own effort is very important...... to maintain good health (odds ratio (OR) 0.45; 95% confidence interval (CI) 0.32-0.62) and for reporting consuming more alcohol on a weekly basis than recommended by the Danish National Board of Health (OR 0.21, 95% CI 0.09-0.51). The odds were higher for non-Western immigrants for than citizens with Danish...

Further evidence for increased macrophage migration inhibitory factor expression in prostate cancer

Directory of Open Access Journals (Sweden)

Iczkowski Kenneth A

2005-07-01

Full Text Available Abstract Background Macrophage migration inhibitory factor (MIF is a cytokine associated with prostate cancer, based on histologic evidence and circulating (serum levels. Recent studies from another laboratory failed to document these results. This study's aims were to extend and confirm our previous data, as well as to define possible mechanisms for the discrepant results. Additional aims were to examine MIF expression, as well as the location of MIF's receptor, CD74, in human prostatic adenocarcinoma compared to matched benign prostate. Methods MIF amounts were determined in random serum samples remaining following routine PSA screening by ELISA. Native, denaturing and reducing polyacrylamide gels and Western blot analyses determined the MIF form in serum. Prostate tissue arrays were processed for MIF in situ hybridization and immunohistochemistry for MIF and CD74. MIF released into culture medium from normal epithelial, LNCaP and PC-3 cells was detected by Western blot analysis. Results Median serum MIF amounts were significantly elevated in prostate cancer patients (5.87 ± 3.91 ng/ml; ± interquartile range; n = 115 compared with patients with no documented diagnosis of prostate cancer (2.19 ± 2.65 ng/ml; n = 158. ELISA diluent reagents that included bovine serum albumin (BSA significantly reduced MIF serum detection (p Conclusion Increased serum MIF was associated with prostate cancer. Diluent reagents that included BSA resulted in MIF serum immunoassay interference. In addition, significant amounts of complexed MIF (180 kDa under denaturing conditions by Western blot found in the serum do not bind to the MIF capture antibody. Increased MIF mRNA expression was observed in prostatic adenocarcinoma compared to benign tissue from matched samples, supporting our earlier finding of increased MIF gene expression in prostate cancer.

Disruption of FGF5 in Cashmere Goats Using CRISPR/Cas9 Results in More Secondary Hair Follicles and Longer Fibers

Science.gov (United States)

Zhu, Haijing; Niu, Yiyuan; Ma, Baohua; Yu, Honghao; Lei, Anmin; Yan, Hailong; Shen, Qiaoyan; Shi, Lei; Zhao, Xiaoe; Hua, Jinlian; Huang, Xingxu; Qu, Lei; Chen, Yulin

2016-01-01

Precision genetic engineering accelerates the genetic improvement of livestock for agriculture and biomedicine. We have recently reported our success in producing gene-modified goats using the CRISPR/Cas9 system through microinjection of Cas9 mRNA and sgRNAs targeting the MSTN and FGF5 genes in goat embryos. By investigating the influence of gene modification on the phenotypes of Cas9-mediated goats, we herein demonstrate that the utility of this approach involving the disruption of FGF5 results in increased number of second hair follicles and enhanced fiber length in Cas9-mediated goats, suggesting more cashmere will be produced. The effects of genome modifications were characterized using H&E and immunohistochemistry staining, quantitative PCR, and western blotting techniques. These results indicated that the gene modifications induced by the disruption of FGF5 had occurred at the morphological and genetic levels. We further show that the knockout alleles were likely capable of germline transmission, which is essential for goat population expansion. These results provide sufficient evidences of the merit of using the CRISPR/Cas9 approach for the generation of gene-modified goats displaying the corresponding mutant phenotypes. PMID:27755602

Enhanced production and purification of recombinant surface array protein (Sap) for use in detection of Bacillus anthracis.

Science.gov (United States)

Puranik, Nidhi; Tripathi, N K; Pal, V; Goel, Ajay Kumar

2018-05-01

Surface array protein (Sap) can be an important biomarker for specific detection of Bacillus anthracis , which is released by the bacterium during its growth in culture broth. In the present work, we have cloned and expressed Sap in Escherichia coli . The culture conditions and cultivation media were optimized and used in batch fermentation process for scale up of Sap in soluble form. The recombinant Sap was purified employing affinity chromatography followed by diafiltration. The final yield of purified protein was 20 and 46 mg/l of culture during shake flasks and batch fermentation, respectively. The protein purity and its reactivity were confirmed employing SDS-PAGE and Western blot, respectively. The antibodies raised against purified Sap were evaluated by Western blotting for detection of Sap released by B. anthracis . Our results showed that the Sap could be a novel marker for detection and confirmation of B. anthracis .

Generation and characterization of polyclonal antibodies specific to N-terminal extension of p85 isoform of ribosomal protein S6 kinase 1 (p85 S6K1

Directory of Open Access Journals (Sweden)

Savinska L. O.

2015-08-01

Full Text Available Aim. Generation of polyclonal antibodies specific to the ribosomal protein S6 kinase isoform – p85S6K1 and directed to the N-terminal (1–23 aa extension of p85S6K1. Methods. Animal immunization with synthetic (1–23 aa peptide, ELISA, Western blot, Immunoprecipitation, immunofluorescent analysis. Results. Polyclonal antibodies have been generated, which specifically recognize only p85 but not p70 isoform of S6K1 in western blot, immunoprecipitation and immunofluorescence analysis. Conclusions. The obtained antibodies can be recommended for studies on the p85S6K1 and other S6K1 isoforms possessing the N-terminal extension – the identification of binding protein partners, analysis of subcellular localization under different physiological conditions, elucidation of the signal transduction pathways involving different S6K1 isoforms.

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

Post-early cretaceous landform evolution along the western margin of the banca~nnia trough, western nsw

Science.gov (United States)

Gibson, D.L.

2000-01-01

Previously undated post-Devonian sediments outcropping north of Fowlers Gap station near the western margin of the Bancannia Trough are shown by plant macro- and microfossil determinations to be of Early Cretaceous (most likely Neocomian and/or Aptian) age, and thus part of the Eromanga Basin. They are assigned to the previously defined Telephone Creek Formation. Study of the structural configuration of this unit and the unconformably underlying Devonian rocks suggests that the gross landscape architecture of the area results from post-Early Cretaceous monoclinal folding along blind faults at the western margin of the trough, combined with the effects of differential erosion. This study shows that, while landscape evolution in the area has been dynamic, the major changes that have occurred are on a geological rather than human timescale.

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...

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...

LyGDI expression in HeLa cells increased its sensitivity to radiation-induced apoptosis

International Nuclear Information System (INIS)

Zhou Xinwen; Xu Yaxiang

2006-01-01

Objective: In order to confirm whether LyGDI has apoptotic signal transduction function and can increase the apoptotic rate of radiation-induced cell death, the lyGDI and mutant D19lyGDI gene, which constructed with the pCDNA3. 1 His A, were transfected into no-endogenous lyGDI HeLa cells. Methods Transient expressions of lyGDI and D19lyGDI in HeLa cells were analyzed by Western blot using anti-mono antibody of LyGDI and Xpress tag. Cell apoptosis was assayed with Annexin V-FITC apoptosis kit. To select stable clone, the transferred HeLa cells had been maintained in G418 medium for 3 weeks, then a cell line, which stably expressed LyGDI and mutant D19lyGDI, was selected. The selected cell line was irradiated with 12 Gy 60 Co y-rays. Caspase-3 activity of the cells was determined by Western blot and cell viability by clone-forming assay after 48 hours post-irradiation culture. Results: Western blot and Annexin V-FITC apoptotic analysis revealed that lyGDI and D19lyGDI transient expressions in HeLa cells induced apoptosis; Caspase-3 activity measurement and clone-forming assay showed that lyGDI increased sensitivity to radiation-induced cell apoptosis. Conclusions: lyGDI performs function in apoptosis signal transduction, its expression in HeLa cells can increase the sensitivity to radiation-induced cell apoptosis. (authors)

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.

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.

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.

Construction of a fusion gene containing hepatitis B virus L gene ...

African Journals Online (AJOL)

The results of SDS-PAGE and Western blot showed that the recombinant protein was induced by methanol and stably expressed in P. pastoris, while it has specific reaction with the serum containing anti-HbsAg or anti-Ag85B. However, the successful construction of a recombinant yeast expression vector containing gene ...

The expression of GFP under the control of fibroin promotor in ...

Indian Academy of Sciences (India)

... mediated rapid amplification of cDNA ends (RLM-RACE). The expression vector (pGFP-N2/Fib) was constructed by use of replacing the CMV promoter with the fibroin promoter. The results of visual screening under a fluorescent inverted microscope and Western blot analysis indicated that the GFP gene was expressed in ...

OCT4 and downstream factors are expressed in human somatic urogenital epithelia and in culture of epididymal spheres

DEFF Research Database (Denmark)

Audouze, Karine Marie Laure; Brunak, Søren; Kristensen, DM

2010-01-01

and microdissected tissues, in situ hybridisation, immunohistochemistry, and Western blotting to show that OCT4 and SOX2 together with downstream targets, UTF1 and REX1, are expressed in the human male urogenital tract. We further supported these results by analysis of DNA methylation of a region in the OCT4...

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...

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...

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...

Effects of the Maillard Reaction on the Immunoreactivity of Amandin in Food Matrices.

Science.gov (United States)

Chhabra, Guneet S; Liu, Changqi; Su, Mengna; Venkatachalam, Mahesh; Roux, Kenneth H; Sathe, Shridhar K

2017-10-01

Amandin is the major storage protein and allergen in almond seeds. Foods, containing almonds, subjected to thermal processing typically experience Maillard browning reaction. The resulting destruction of amino groups, protein glycation, and/or denaturation may alter amandin immunoreactivity. Amandin immunoreactivity of variously processed almond containing foods was therefore the focus of the current investigation. Commercial and laboratory prepared foods, including those likely to have been subjected to Maillard browning, were objectively assessed by determining Hunter L * , a * , b * values. The L * values for the tested samples were in the range of 31.75 to 85.28 consistent with Maillard browning. Three murine monoclonal antibodies, 4C10, 4F10, and 2A3, were used to determine the immunoreactivity of the targeted samples using immunoassays (ELISA, Western blot, dot blot). The tested foods did not exhibit cross-reactivity indicating that the immunoassays were amandin specific. For sandwich ELISAs, ratio (R) of sample immunoreactivity to reference immunoreactivity was calculated. The ranges of R values were 0.67 to 15.19 (4C10), 1.00 to 11.83 (4F10), and 0.77 to 23.30 (2A3). The results of dot blot and Western blot were consistent with those of ELISAs. Results of these investigations demonstrate that amandin is a stable marker protein for almond detection regardless of the degree of amandin denaturation and/or destruction as a consequence of Maillard reaction encountered under the tested processing conditions. Foods containing almond are often subjected to processing prior to consumption. Amandin, the major allergen in almond, may experience Maillard reaction. Understanding the change in amandin immunoreactivity as a result of Maillard reaction is important for amandin detection and production of hypoallergenic food products. © 2017 Institute of Food Technologists®.

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

Attachment in cultural context : Differences in attachment between Eastern and Western Europeans

NARCIS (Netherlands)

Polek, Elzbieta

2008-01-01

Political changes after 1989 and resulting from the European Union enlargement increased emigration from Eastern to Western Europe. In the introductory Chapter 1 the data about migration from the Eastern to Western Europe are presented, as well as the data about cultural, social and economic

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

From Marshes to the Continental Shelf: Results of the Western Component of the US EPA National Coastal Assessment

Science.gov (United States)

W. G. Nelson; H. II Lee; J. O. Lamberson

2006-01-01

The National Coastal Assessment of the US EPA began field work in the Western US in 1999-2000. Probabilistic sampling for biotic and abiotic condition indicators was conducted at 381 stations within estuaries and coastal embayments of Washington, Oregon and California. In 2002, intertidal and low salt marsh habitats were sampled at an additional 190 stations. As part...

Expression of c-fos and c-jun proteins in the marginal division of the rat striatum during learning and memory training

Institute of Scientific and Technical Information of China (English)

BAO Xin-min; SHU Si-yun; WANG Hong

2005-01-01

Background A new brain region, the marginal division (MrD), was discovered at the caudal margin of the neostriatum. The MrD was shown to be involved in learning and memory in the rat. The aim of this study was to investigate the expression of the immediate-early genes c-fos and c-jun in the MrD of the striatum during learning and memory processes in the rat, immunocytochemical and Western blot methods were used to examine Y-maze trained rats.Methods The rats were divided into three groups, namely the training, pseudotraining, and control groups. After Y-maze training, the expression of the immediate-early genes c-fos and c-jun in the MrD of the rats was investigated using immunocytochemical and Western blot methods. Results After one hour of Y-maze training, the expression of c-jun and c-fos proteins was significantly enhanced in the MrD; the c-jun protein, in particular, was more intensely expressed in this region than in other parts of the striatum. The expression of these two proteins in the training group was significantly higher than in the pseudotraining and control groups. In addition, positive expression was also found in the hippocampus, cingulum cortex, thalamus, and in other areas. Western blot disclosed two immunoreactive bands for the anti-c-fos antibody (47 kD and 54 kD) and two immunoreactive bands for the anti-c-jun antibody (39 kD and 54 kD). Conclusions These results indicate that the immediate-early genes c-fos and c-jun participate in signal transduction during the learning and memory processes associated with Y-maze training in rats.

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.

Novel levamisole derivative induces extrinsic pathway of apoptosis in cancer cells and inhibits tumor progression in mice.

Directory of Open Access Journals (Sweden)

Mahesh Hegde

Full Text Available BACKGROUND: Levamisole, an imidazo(2,1-bthiazole derivative, has been reported to be a potential antitumor agent. In the present study, we have investigated the mechanism of action of one of the recently identified analogues, 4a (2-benzyl-6-(4'-fluorophenyl-5-thiocyanato-imidazo[2,1-b][1], [3], [4]thiadiazole. MATERIALS AND METHODS: ROS production and expression of various apoptotic proteins were measured following 4a treatment in leukemia cell lines. Tumor animal models were used to evaluate the effect of 4a in comparison with Levamisole on progression of breast adenocarcinoma and survival. Immunohistochemistry and western blotting studies were performed to understand the mechanism of 4a action both ex vivo and in vivo. RESULTS: We have determined the IC(50 value of 4a in many leukemic and breast cancer cell lines and found CEM cells most sensitive (IC(50 5 µM. Results showed that 4a treatment leads to the accumulation of ROS. Western blot analysis showed upregulation of pro-apoptotic proteins t-BID and BAX, upon treatment with 4a. Besides, dose-dependent activation of p53 along with FAS, FAS-L, and cleavage of CASPASE-8 suggest that it induces death receptor mediated apoptotic pathway in CEM cells. More importantly, we observed a reduction in tumor growth and significant increase in survival upon oral administration of 4a (20 mg/kg, six doses in mice. In comparison, 4a was found to be more potent than its parental analogue Levamisole based on both ex vivo and in vivo studies. Further, immunohistochemistry and western blotting studies indicate that 4a treatment led to abrogation of tumor cell proliferation and activation of apoptosis by the extrinsic pathway even in animal models. CONCLUSION: Thus, our results suggest that 4a could be used as a potent chemotherapeutic agent.

Anti-tumor effects of ephedrine and anisodamine on SKBR3 human ...

African Journals Online (AJOL)

Western blot was used to measure PKA. Results: Ephedrine and anisodamine inhibited cell proliferation and arrested SKBR3 cells at G0/G1 phases. Ephedrine and anisodamine increased the level of CD1 in SKBR3 cells. Furthermore, significant change in intracellular cAMP concentration was found in SKBR3 cells treated ...

Soluble expression and purifiation of hepatitis B core antigen (HBcAg subgenotype B3 in Escherichia coli using thioredoxin fusion tag

Directory of Open Access Journals (Sweden)

Rahmah Waty

2017-08-01

Full Text Available Objective: To express HBcAg protein (hepatitis B virus subgenotype B3 in Escherichia coli in soluble form. Methods: HBcAg sequence of hepatitis B virus subgenotype B3 was cloned into plasmid pET32a and introduced to E. coli BL21 (DE3. The E. coli was grown in Luria-Bertani (LB medium supplemented with ampicillin with agitation. Protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG at concentrations of 0.1 mmol/L, 0.3 mmol/L, and 0.5 mmol/L at room temperature (28 °C. The bacteria were dissolved in lysis buffer and lysed by freeze-thawing method then sonication. The fusion protein [thioredoxin A-(His6tag-HBcAg] was purified using immobilized metal affinity chromatography. The protein expression was analyzed by SDS-PAGE, dot blot, and western blot. Results: This research showed that DNA sequence of HBcAg could be propagated in pET32a and soluble protein was successfully expressed in E. coli. Induction with 0.3 mmol/L IPTG and 4-hour incubation was the best condition to express the HBcAg protein. SDS-PAGE and dot blot analysis showed that HBcAg protein could be expressed in E. coli. Western blot analysis showed that molecular weight of HBcAg fusion protein was about 38.5 kDa. Conclusions: This study confirmed that HBcAg protein could be expressed in soluble form in E. coli.

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.

Wet and wild: results from a pilot study assessing injuries among recreational water users in Western Australia.

Science.gov (United States)

Pikora, T J; Braham, R; Hill, C; Mills, C

2011-06-01

To identify, describe and compare injuries among three water sport activities: kite surfing (KS), personal watercraft (PWC) and towed water sports (TWS). The study was a cross sectional, online survey. The setting was on Perth, Western Australia's popular beaches and riverbanks. Main outcome measures were number of injuries and level of severity; level of exposure and protection measures. Overall, 43% reported at least one injury in the past 12 months, a rate of 22.3 injuries per 100 h. Kite surfers were more likely to report an injury than PWC or TWS. One-half of injuries occurred while on the water. Most injuries were caused by landing awkwardly (56%) and/or trying new tricks (41%). Despite 90% of respondents having used at least one personal protective equipment (PPE) item, half (49%) reported always using a personal floatation device. This study provided information on KS, PWC and TWS injuries as well as a range of safety behaviours. It is recommended that these results form the basis of further research to reduce injury rates and encourage the use of PPE items.

Inhibiting effects of rhynchophylline on methamphetamine-dependent zebrafish are related with the expression of tyrosine hydroxylase (TH).

Science.gov (United States)

Zhu, Chen; Liu, Wei; Luo, Chaohua; Liu, Yi; Li, Chan; Fang, Miao; Lin, Yingbo; Ou, Jinying; Chen, Minting; Zhu, Daoqi; Yung, Ken Kin-Lam; Mo, Zhixian

2017-03-01

In this study, to study the effect of rhynchophylline on TH in midbrain of methamphetamine-induced conditioned place preference (CPP) adult zebrafish, place preference adult zebrafish models were established by methamphetamine (40μg/g) and the expression of TH was observed by immunohistochemistry technique and Western blot. Ketamine (150μg/g), high dose of rhynchophylline (100μg/g) group can significantly reduce the place preference; immunohistochemistry results showed that the number of TH-positive neurons in midbrain was increased in the methamphetamine model group, whereas less TH-positive neurons were found in the ketamine group and high dosage rhynchophylline group. Western blot results showed that the expression of TH protein was significantly increased in the model group, whereas less expression was found in the ketamine group, high dosage rhynchophylline group. Our data pointed out that TH plays an important role in the formation of methamphetamine-induced place preference in adult zebrafish. Rhynchophylline reversed the expression of TH in the midbrain demonstrates the potential effect of mediates methamphetamine induced rewarding effect. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of Sirtuin-1 on Synaptic Plasticity in Nucleus Accumbens in a Rat Model of Heroin Addiction.

Science.gov (United States)

Xia, Baijuan; Li, Yixin; Li, Rongrong; Yin, Dan; Chen, Xingqiang; Li, Jie; Liang, Wenmei

2018-06-05

BACKGROUND Synaptic plasticity plays an important role in the process of addiction. This study investigated the relationship between synaptic plasticity and changes in addictive behavior and examined the expression of synaptic plasticity-associated proteins and genes in the nucleus accumbens (NAc) region in different rat models. MATERIAL AND METHODS Heroin addiction, SIRT1-overexpression, and SIRT1-silenced rat models were established. Polymerase chain reaction gene chip technology, immunohistochemistry, Western blotting, and transmission electron microscopy were used to detect changes in synaptic plasticity-related gene and protein expression, and changes in the ultrastructure of synapses, in the NAc. RESULTS Naloxone withdrawal symptoms appeared in the SIRT1-overexpression group. In the SIRT1-silenced group the symptoms were reduced. Immunohistochemistry and Western blotting results showed that FOXO1 expression decreased in the heroin addiction (HA) group but increased in the SIRT1-silenced group (paddiction. SIRT1 overexpression can increase behavioral sensitization in the NAc of rats, and SIRT1 silencing might ease withdrawal symptoms and reduce conditioned place preferences.

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

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.

SELECTED MACROECONOMIC FACTORS OBSERVATION IMPACT ON THE ECONOMY OF THE REPUBLIC OF SERBIA AND THE WESTERN BALKANS IN THE OBSERVATION PERIOD 2000-2012, SHOWING RESULTS FROM 2008 TO 2012.

Directory of Open Access Journals (Sweden)

BOGDAN LABAN

2017-12-01

Full Text Available The application of the neoliberal concept in the Western Balkans has had effects on the economies of those countries that can say das resulted in unsustainable economic growth and macroeconomic instability. Besides the application of the same given the developments that had features of weakness especially since 2008., from the onset of the global economic crisis. The consequences of the liberalization of international economic developments have enabled the countries of the Western Balkans obtain foreign investment in the form of foreign direct investment, but their economies have done even more dependent on foreign capital, which is increasingly going in economic activities that have a faster way could fertilize the invested capital.

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.

Small, round-structured viruses (SRSVs) associated with acute gastroenteritis outbreaks in Gifu, Japan.

Science.gov (United States)

Kawamoto, H; Hasegawa, S; Sawatari, S; Miwa, C; Morita, O; Hosokawa, T; Tanaka, H

1993-01-01

Two outbreaks of non-bacterial gastroenteritis occurred in Gifu prefecture in January 1989 and in January 1991. Both outbreaks were closely related to the consumption of raw oysters, and showed similar clinical features. Small, round-structured virus particles were found in patient stools in both outbreaks by electron microscopy. The role of these particles as the causative agents of the outbreaks were strongly suggested by immune electron microscopy and/or western-blotting immunoassay. When compared with SRSV-9 (Tokyo/SRSV/86-510) reported previously (Hayashi et al, J. Clin. Microbiol., 27: 1728-1733, 1989), it was found that these viral particles were antigenically similar to SRSV-9, and had a major structural protein of 63 kilodaltons (kDa). Further, the prevalence of this agent in Gifu area was examined by western blot antibody assay using 67 serum samples collected from the inhabitants in 1991. The results indicated the circulation of the same or antigenically similar agent in this area.

Proteomic analysis of pancreas derived from adult cloned pig

International Nuclear Information System (INIS)

Chae, Jung-Il; Cho, Young Keun; Cho, Seong-Keun; Kim, Jin-Hoi; Han, Yong-Mahn; Koo, Deog-Bon; Lee, Kyung-Kwang

2008-01-01

The potential medical applications of animal cloning include xenotransplantation, but the complex molecular cascades that control porcine organ development are not fully understood. Still, it has become apparent that organs derived from cloned pigs may be suitable for transplantation into humans. In this study, we examined the pancreas of an adult cloned pig developed through somatic cell nuclear transfer (SCNT) using two-dimensional electrophoresis (2-DE) and Western blotting. Proteomic analysis revealed 69 differentially regulated proteins, including such apoptosis-related species as annexins, lamins, and heat shock proteins, which were unanimously upregulated in the SCNT sample. Among the downregulated proteins in SCNT pancreas were peroxiredoxins and catalase. Western blot results indicate that several antioxidant enzymes and the anti-apoptotic protein were downregulated in SCNT pancreas, whereas several caspases were upregulated. Together, these data suggest that the accumulation of reactive oxygen species (ROS) in the pancreas of an adult cloned pig leads to apoptosis

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

[Exploration of microcosmic Chinese medicine used by western medicine].

Science.gov (United States)

Zheng, Zhi-jing

2015-02-01

"Microcosmic syndrome", "treatment based on syndrome differentiation", and "combination of disease identification and syndrome differentiation" generally refer to a mode: following the syndrome if with no disease identified, following the disease if with no syndrome type differentiated. For example, Chinese medical treatment of hypertension, high blood lipids, increased transaminase, and so on candirectly use Chinese recipes, but no longer with syndrome differentiation. Clinical application of Chinese patent medicine can also obtain favorable clinical. Western doctors need not follow syndrome differentiation. The invention of artemisinin was screened from more than 40 000 kinds of compounds and herbs, but with no reference of any traditional Chinese medical theory. A lot of folk remedy and empirical recipes have obtained effective efficacy but unnecessarily with profound Chinese medical theories. Various evidences showed that disease can also be cured without syndrome differentiation. I held that it might be associated with the same mechanism of Chinese medicine and Western medicine. Any disease can be cured or alleviated by Chinese medicine is a result from its modern pharmacological effect, which is achieved by improving etiologies, and pathogeneses. I was inspired by whether we can directly use traditional Chinese medicine with modern pharmacological effects to treat symptomatic disease. So I raised an idea of microcosmic Chinese medicine used by Western medicine, i.e., we find and use Chinese herbs with relatively effective modern pharmacological effect to treat diseases targeting at patients' clinical symptoms and signs, as well as various positive laboratory results (collectively called as microscopic dialectical indicators). More Western doctors would use it to treat disease due to omission of complicated and mysterious syndrome differentiation. This will promote extensive application and expansion of Chi- nese medicine and pharmacy, enlarge the team of

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.

Polyvalent horse F(Ab`) 2 snake antivenom: Development of ...

African Journals Online (AJOL)

F(ab´)2 fragments were further purified by Q-Fast Flow chromatography, concentrated by molecular ultrafiltration and sterilized by filtration through 0.22 m membranes. The resulting F(ab´)2 preparations were rich in intact L and in pieces of H IgG(T) chains, as demonstrated by electrophoresis and Western blot and exhibited ...

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

Irradiation With Carbon Ion Beams Induces Apoptosis, Autophagy, and Cellular Senescence in a Human Glioma-Derived Cell Line

International Nuclear Information System (INIS)

Jinno-Oue, Atsushi; Shimizu, Nobuaki; Hamada, Nobuyuki; Wada, Seiichi; Tanaka, Atsushi; Shinagawa, Masahiko; Ohtsuki, Takahiro; Mori, Takahisa; Saha, Manujendra N.; Hoque, Ariful S.; Islam, Salequl; Kogure, Kimitaka; Funayama, Tomoo; Kobayashi, Yasuhiko

2010-01-01

Purpose: We examined biological responses of human glioma cells to irradiation with carbon ion beams (C-ions). Methods and Materials: A human glioma-derived cell line, NP-2, was irradiated with C-ions. Apoptotic cell nuclei were stained with Hoechst 33342. Induction of autophagy was examined either by staining cells with monodansylcadaverine (MDC) or by Western blotting to detect conversion of microtuble-associated protein light chain 3 (MAP-LC3) (LC3-I) to the membrane-bound form (LC3-II). Cellular senescence markers including induction of senescence-associated β-galactosidase (SA-β-gal) were examined. The mean telomere length of irradiated cells was determined by Southern blot hybridization. Expression of tumor suppressor p53 and cyclin/cyclin-dependent kinase inhibitor p21 WAF1/CIP1 in the irradiated cells was analyzed by Western blotting. Results: When NP-2 cells were irradiated with C-ions at 6 Gy, the major population of the cells died of apoptosis and autophagy. The residual fraction of attached cells ( WAF1/CIP1 was induced in NP-2 cells after irradiation. Furthermore, we found that irradiation with C-ions induced cellular senescence in a human glioma cell line lacking functional p53. Conclusions: Irradiation with C-ions induced apoptosis, autophagy, and cellular senescence in human glioma cells.

Hepatocyte-specific deletion of Cdc42 results in delayed liver regeneration after partial hepatectomy in mice

DEFF Research Database (Denmark)

Yuan, Haixin; Zhang, Hong; Wu, Xunwei

2009-01-01

Cdc42, a member of the Rho guanosine triphosphatase (GTPase) family, plays important roles in the regulation of the cytoskeleton, cell proliferation, cell polarity, and cellular transport, but little is known about its specific function in mammalian liver. We investigated the function of Cdc42...... in regulating liver regeneration. Using a mouse model with liver-specific knockout of Cdc42 (Cdc42LK), we studied liver regeneration after partial hepatectomy. Histological analysis, immunostaining, and western blot analysis were performed to characterize Cdc42LK livers and to explore the role of Cdc42 in liver...... regeneration. In control mouse livers, Cdc42 became activated between 3 and 24 hours after partial hepatectomy. Loss of Cdc42 led to a significant delay of liver recovery after partial hepatectomy, which was associated with reduced and delayed DNA synthesis indicated by 5-bromo-2'-deoxyuridine staining...

Protein and antigen profiles of Leptospira interrogans serovar Hardjo Perfil proteico e antigênico da Leptospira interrogans sorovariedade Hardjo

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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.

Lagrangian Observations of the Deep Western Boundary Currents in the North Pacific During KERE

National Research Council Canada - National Science Library

Riser, Stephen

1997-01-01

.... This study resulted from the observation that numerical model solutions of upper ocean flows in the western Pacific often appear to depend on the state of the deep ocean flow near the western boundary...

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).

The Correlation Of Islamic Civilization In Sciences With Western World ( Eastern Impact Through Sciences On Western World

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Mohamed Mohamed Tolba Said

2018-01-01

Full Text Available The nature of scientific verification of knowledge distinguishes it from mystical knowledge in empirical sciences. Islam is a religion and a civilization, historically connecting various stages of human history for more than fourteen centuries. The Islamic ethics and law “Sharia’h” are coherent legal system to protect private property within a comprehensive and rational system. Capitalism and the industrial revolution of western world dramatically transformed resulting in a socio-economic schism consequently emerged as a domineer for existence and affected the Islamic world. The secular and rationalized legal framework needed capitalism, which is incompatible with the nature of Islamic law. The western science in this civilization is also separated from morality and noble values because it has adopted materialistic philosophies and ideologies, such as Pragmatism, Darwinism, Existentialism and any other philosophy that is against the Islamic religion.

Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli

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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.

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.

Lime stabilization of fine-grained sediments in western Greenland

DEFF Research Database (Denmark)

Jørgensen, Anders Stuhr; Ingeman-Nielsen, Thomas; Skels, Peteris

2011-01-01

Thick deposits of fine-grained marine sediments exist in large areas of western Greenland. Many places these sediments are located above sea-level, and now complicate construction projects in urban areas. The mineralogy of the fine-grained sediments is very different from European sediments, mainly...... due to the cold climate, and it is therefore of great interest to study possible methods to improve the stability and strength properties. This project includes laboratory studies of lime stabilization on fine-grained marine sediments from Kangerlussuaq, western Greenland. The results have included...

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...

Integrated ecosystem assessment for western development of China

Institute of Scientific and Technical Information of China (English)

无

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.

Human transmissible spongiform encephalopathy: Case report

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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.

Approach to valuing visual pollution from Western Electricity Production. [For Western Systems Coordinating Council area

Energy Technology Data Exchange (ETDEWEB)

Erickson, L.E.

1977-02-01

This paper outlines an approach to valuing visual pollution from electric power plants. The differences between public-good pollution externalities, such as these aesthetic damages, and other market failures are discussed. Approaches generally used to value externalities are briefly described. The approach used relies heavily on an earlier application of bidding games to estimate people's willingness to pay for abatement of emissions from the Four Corners fossil-fuel power plant in northwestern New Mexico. The results of these surveys were used here to estimate the value of visual pollution from electric power plants for residents of and visitors to the Four Corners Air Quality Control Region, as a function of power plant emissions in that region. The approach presented here for the Four Corners region is structured so that replication for other air quality control regions is relatively easy. Preliminary results of this procedure for all of the air quality control regions in the Western Systems Coordinating Council area are presented. Visual pollution damages from electric power plants to residents of and recreational visitors to these western regions are estimated to total more than $100 million annually by 1985. These damages are expected to occur unless additional pollution controls are implemented, even if these subject populations do not increase.

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...

Demethoxycurcumin Suppresses Migration and Invasion of Human Cervical Cancer HeLa Cells via Inhibition of NF-κB Pathways.

Science.gov (United States)

Lin, Chin-Chung; Kuo, Chao-Lin; Huang, Yi-Ping; Chen, Cheng-Yen; Hsu, Ming-Jie; Chu, Yung Lin; Chueh, Fu-Shin; Chung, Jing-Gung

2018-05-01

Demethoxycurcumin (DMC), one of the curcuminoids present in turmeric, has been shown to induce cell death in many human cancer cell lines, however, there has not been any investigation on whether DMC inhibits metastatic activity in human cervical cancer cells in vitro. In the present study, DMC at 2.5-15 μM decreased cell number, thus, we used IC 20 (7.5 μM) for further investigation of its anti-metastatic activity in human cervical cancer HeLa cells. The wound healing, migration, invasion, zymography, and western blotting assays were used to investigate the effects of DMC on HeLa cells. The wound healing assay was used to show that DMC suppressed cell movement of HeLa cells. Furthermore, the trans-well chamber assay was used to show that DMC suppressed HeLa cell migration and invasion. Gelatin zymography assay did not show any significant effects of DMC on the gelatinolytic activity (MMP-2 and -9) in conditioned media of HeLa cells treated by DMC. Western blotting showed that DMC significantly reduced protein levels of GRB2, MMP-2, ERK1/2, N-cadherin and Ras but increased the levels of E-cadherin and NF-κB in HeLa cells. Confocal laser microscopy indicated that DMC increased NF-κB in HeLa cells confirming the results from Western blotting. DMC may be used as a novel anti-metastatic agent for the treatment of human cervical cancer in the future. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

A hybrid of coumarin and phenylsulfonylfuroxan induces caspase-dependent apoptosis and cytoprotective autophagy in lung adenocarcinoma cells.

Science.gov (United States)

Wang, Qian; Guo, Yalan; Jiang, Shanshan; Dong, Mengxue; Kuerban, Kudelaidi; Li, Jiyang; Feng, Meiqing; Chen, Ying; Ye, Li

2018-01-15

Lung adenocarcinoma is the most primary histologic subtype of non-small cell lung cancer (NSCLC). Compound 8b, a novel coumarin derivative with phenylsulfonylfuroxan group, shows significant antiproliferation activity against lung adenocarcinoma cell with low toxicity. This study aims to uncover the potential of compound 8b in relation to apoptosis as well as autophagy induction in lung adenocarcinoma cells. The cytotoxicity and apoptosis of A549 and H1299 cells induced by compound 8b were detected by MTT, microscope and western blot analysis. Autophagy was determined by TEM, confocal microscopy and western blot analysis. Akt/mTOR and Erk signaling pathway were also examined by western blot analysis. First, significant growth inhibition and caspase-dependent apoptosis were observed in compound 8b-treated A549 and H1299 cells. Then, we confirmed compound 8b-induced autophagy by autophagosomes formation, upregulated expression of autophagy-related protein LC3-II and autophagic flux. Importantly, abolishing autophagy using inhibitors and ATG5 siRNA enhanced the cytotoxicity of compound 8b, indicating the cytoprotective role of autophagy in lung adenocarcinoma. Further mechanistic investigations suggested that Akt/mTOR and Erk signaling pathways contributed to autophagy induction by compound 8b. This results demonstrate that compound 8b induces caspase-dependent apoptosis as well as cytoprotective autophagy in lung adenocarcinoma cells, which may provide scientific evidence for developing this furoxan-based NO-releasing coumarin derivative as a potential anti-lung adenocarcinoma therapeutic agents. Copyright © 2017 Elsevier GmbH. All rights reserved.

Cloning and Expression of 31kDa Outer Membrane Protein of Brucella melitansis in E.coli

Directory of Open Access Journals (Sweden)

Sayeneh Khodadadi

2012-04-01

Full Text Available Background & Objectives: The identification of Brucella spp. antigens with the capacity to elicit a protective immune response is of the great interest for the researchers. So, characterization and assessment of diverse antigens of Brucella need to be evaluated. In this study, we report the cloning and expression of the gene coding for 31 KDa OMP (OMP31 of Brucella melitensis 16M.   Methods: Brucella melitensis Omp31 gene was amplified with specific primers, cloned into pJET1/2 and subsequently subcloned in pET28a (+ vector. Both these recombinant plasmids were sequenced and then after, expression of recombinant protein was induced by 1mM IPTG. Western blot analysis was also performed by polyclonal rabbit antiserum.   Results: Omp31 successfully was cloned in both plasmid vectors. The recombinant Omp31 was expressed in E.coli host and purified with significant yield. Western blot results along with those of sequencing ensured accurate production of recombinant omp31 and retaining of its partial epitopes.   Conclusion: Our results show that, an expression host such as E. coli is suitable for omp31 production.

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

Odour reduction strategies for biosolids produced from a Western Australian wastewater treatment plant: results from Phase I laboratory trials.

Science.gov (United States)

Gruchlik, Yolanta; Heitz, Anna; Joll, Cynthia; Driessen, Hanna; Fouché, Lise; Penney, Nancy; Charrois, Jeffrey W A

2013-01-01

This study investigated sources of odours from biosolids produced from a Western Australian wastewater treatment plant and examined possible strategies for odour reduction, specifically chemical additions and reduction of centrifuge speed on a laboratory scale. To identify the odorous compounds and assess the effectiveness of the odour reduction measures trialled in this study, headspace solid-phase microextraction gas chromatography-mass spectrometry (HS SPME-GC-MS) methods were developed. The target odour compounds included volatile sulphur compounds (e.g. dimethyl sulphide, dimethyl disulphide and dimethyl trisulphide) and other volatile organic compounds (e.g. toluene, ethylbenzene, styrene, p-cresol, indole and skatole). In our laboratory trials, aluminium sulphate added to anaerobically digested sludge prior to dewatering offered the best odour reduction strategy amongst the options that were investigated, resulting in approximately 40% reduction in the maximum concentration of the total volatile organic sulphur compounds, relative to control.

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.

Empathy and Emotional Intelligence among Eastern and Western Counsellor Trainees: A Preliminary Study

Science.gov (United States)

Young Kaelber, Kara A.; Schwartz, Robert C.

2014-01-01

This study explored degree of empathy and emotional intelligence among Thai (n?=?48) and American (n?=?53) counsellor trainees to determine if differences in Eastern and Western cultural orientations (e.g., interdependent versus independent self-construals) affect foundational counselling skills. Results indicated that Western trainees showed…

Yanai waves in the western equatorial Indian Ocean

Digital Repository Service at National Institute of Oceanography (India)

Chatterjee, A.; Shankar, D.; McCreary, J.P.; Vinayachandran, P.N.

; interference between the interior and boundary responses results in a complex surface pattern that propagates eastward and has nodes. Yanai waves are also forced by instabilities primarily during June/July in a region offshore from the western boundary (52...

Deletion of the AcMNPV core gene ac109 results in budded virions that are non-infectious

International Nuclear Information System (INIS)

Fang Minggang; Nie, Yingchao; Theilmann, David A.

2009-01-01

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac109 is a core gene and its function in the virus life cycle is unknown. To determine its role in the baculovirus life cycle, we used the AcMNPV bacmid system to generate an ac109 deletion virus (vAc 109KO ). Fluorescence and light microscopy showed that transfection of vAc 109KO results in a single-cell infection phenotype. Viral DNA replication is unaffected and the development of occlusion bodies in vAc 109KO -transfected cells evidenced progression to the very late phases of viral infection. Western blot and confocal immunofluorescence analysis showed that AC109 is expressed in the cytoplasm and nucleus throughout infection. In addition, AC109 is a structural protein as it was detected in both budded virus (BV) and occlusion derived virus in both the envelope and nucleocapsid fractions. Titration assays by qPCR and TCID 50 showed that vAc 109KO produced BV but the virions are non-infectious. The vAc 109KO BV were indistinguishable from the BV of repaired and wild type control viruses as determined by negative staining and electron microscopy.

Trial use of the Personal Qualities Assessment (PQA) in the entrance examination of a Japanese medical university: similarities to the results in western countries.

Science.gov (United States)

Fukui, Yuriko; Noda, Saeko; Okada, Midori; Mihara, Nakako; Kawakami, Yoriko; Bore, Miles; Munro, Don; Powis, David

2014-01-01

The Personal Qualities Assessment (PQA), developed by the University of Newcastle, Australia to assess the aptitude of future medical professionals, has been used in Western countries. The objective was to investigate whether the PQA is appropriate for Japanese medical school applicants. Two of the PQA tests, Libertarian-Dual-Communitarian moral orientations (Mojac) and Narcissism, Aloofness, Confidence, and Empathy (NACE), were translated into Japanese, and administered at the Tokyo Women's Medical University entrance examinations from 2007 to 2009. The distributions of the applicants' Mojac and NACE scores were close to the normal distribution, and the mean scores did not exhibit a large difference from those in Western countries. The only significant difference was that the mean score of the NACE test was slightly lower than the Western norm. The translated PQA tests may be appropriate for use with Japanese applicants, though further research considering cultural differences is required.

Western Wind and Solar Integration Study: Phase 2 (Presentation)

Energy Technology Data Exchange (ETDEWEB)

Lew, D.; Brinkman, G.; Ibanez, E.; Lefton, S.; Kumar, N.; Venkataraman, S.; Jordan, G.

2013-09-01

This presentation summarizes the scope and results of the Western Wind and Solar Integration Study Phase 2, which examined operational impacts of high penetrations of variable renewable generation in the West.

Estimation of North American population doses resulting from radon-222 release in western United States: methodology

International Nuclear Information System (INIS)

Fields, D.E.; Travis, C.C.; Watson, A.P.; McDowell-Boyer, L.M.

1979-12-01

The report represents a compilation of computer codes used to estimate potential human exposures and inhalation doses due to unit releases of 222 Rn from uranium milling sites in western United States. The populations considered for potential exposure to risk from 222 Rn and associated daughters are the inhabitants of North America between 20 0 and 60 0 North latitude. The primary function of these codes is to integrate spatially atmospheric radionuclide concentrations with current population data for the geographic area under consideration. It is expected that these codes will be of assistance to anyone interested in assessing nuclear or nonnuclear population exposures over large geographic areas

FLORA LICHEN WESTERN MOUNTAINS VRANJE PLACE

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Gordana Bogdanović

2016-07-01

Full Text Available Lichen is a symbiotic plant built by the cells of algae and fungi hyphae. Algae are usually presented - green (Chlorophyta or blue green (Cyanophyta, a mushroom commonly found is ascomycetae and sometimes basidiomycetae. Mushrooms receive oxygen and carbohydrates from algae, and they in turn provide water, CO2 and mineral salts. Lichens are often found on trees and rocks in unpolluted environments and can be used as a bioindicator species. In during 2015-2016. was realized a survey of epiphytic lichen flora of the western mountains in environment of Vranje. Sampling was carried out at 4 locations: Borino brdo, Krstilovica, Markovo Kale and Pljačkovica. Based on the collected and determined samples can be concluded that the study implemented of the area of 25 species of lichens of which: 8 as crust, leafy 12 and 5 shrub. The research results indicate that the lichen flora of the western mountains environments Vranje of a rich and diverse as a result of favorable geographic position, geological and soil composition, climate and plant cover that provide opportunities for the development and survival of lichens.

Prevalencia de anticuerpos contra Trypanosoma cruzi en donadores de sangre del IMSS, Orizaba, Veracruz, México Prevalence of antibodies against Trypanossoma cruzi in blood bank donors from the IMSS General Hospital in Onizaba, Veracruz, Mexico

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Angel Ramos-Ligonio

2006-02-01

Full Text Available OBJETIVO: Determinar la prevalencia de anticuerpos contra Trypanosoma cruzi en donadores del Hospital General Regional del Instituto Mexicano del Seguro Social (IMSS en la ciudad de Orizaba, Veracruz. MATERIAL Y MÉTODOS: Se examinaron muestras de donadores del banco de sangre del Hospital General Regional (HGRO del IMSS para la búsqueda de antiT. cruzi por ELISA, Western blot e IFI, utilizando una proteína recombinante (MBP::Hsp70 y un extracto crudo de epimastigotes. Las muestras fueron obtenidas entre los meses de octubre de 2001 a enero de 2002. RESULTADOS: Los 420 donadores de sangre analizados fueron seronegativos para HBV, HCV, BrA, VDRL y HIV. Después del tamizaje de los 420 donadores, se identificaron dos individuos seropositivos por las pruebas de ELISA, Western blot e IFI, con una seroprevalencia de 0.48%. CONCLUSIONES: En este estudio se muestran evidencias de seropositividad para T. cruzi en donadores de sangre del HGRO, lo que sugiere la existencia de riesgo de contaminación por transfusión sanguínea. Por tal motivo, es necesario aplicar programas para el tamizaje serológico a través de técnicas inmunológicas con alta sensibilidad y especificidad.OBJECTIVE: To estimate the prevalence of antibodies against Trypanosoma cruzi in blood donors from Hospital General Regional (HGRO of the Mexican Institute of Social Security (IMSS per its abbreviation in Spanish. MATERIAL AND METHODS: Between October 2001 and January 2002, blood samples were collected from voluntary donors at the blood bank of the Hospital General Regional of IMSS in Orizaba; Veracruz, Mexico. The samples were assayed for anti-T. cruzi by ELISA, Western blot and IFI, using a recombinant protein (MBP::Hsp70, and crude extract from epimastigotes. RESULTS: A total of 420 blood donors were studied; two of them were seropositive for ELISA, Western blot and IFI, with a seroprevalence of 0.48%. CONCLUSIONS: Some blood donors at the HGRO hospital were seropositive for T

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

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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

Determination of protein-carbonyls and ubiquitin-mediated proteolysis as biomarkers of oxidative-stress in bivalvia and anthozoa

Energy Technology Data Exchange (ETDEWEB)

Walker, Stephen Thomas

2002-07-01

This study describes the investigation of biomarkers of oxidative-stress in the bivalves Mytilus edulis and Dosinia lupinus, together with host and symbiont tissues of the scleractinian Anthozoa Agaricia agaricites. The biomarkers used were assay of total (via spectrophotometry) and individual (via Western blotting; Oxyblot kit) protein-carbonyls (PC=Os) and content of ubiquitin protein conjugates (UPC) via Western blotting (Bivalvia and Anthozoa) and immunohistochemistry (Anthozoa only). Additional assays for Bivalvia were Trolox equivalent antioxidant capacity (TEAC); and post {gamma}-irradiation survival rates. Experimental stressors for Bivalvia were increased seawater temperature, H{sub 2}O{sub 2} and {sup 60}Co {gamma}-radiation (latter two were used in vivo and in vitro). Comparisons of clean and polluted marine sites are included. Stressors used for Anthozoa were increased solar irradiation concomitant with elevated seawater temperature. Results and conclusions were as follows: individual samples showed considerable variation, pooling of samples improved consistency. Controls for both biomarkers had detectable background levels in each phylum, against which relatively small differences were assessed. In M. edulis, no measurable differences in PC=Os could be determined when elevated seawater temperature or dilute H{sub 2}O{sub 2} (<30% v/v) stressors were used, nor with between-site comparisons. Concentrated H{sub 2}O{sub 2} (30% v/v) produced a small difference. {sup 60}Co {gamma}-radiation produced clearer differences via Oxyblot and spectrophotometric assays. Comparison of four different tissues from the two bivalves found considerable species-specific and tissue-specific differences. Post-irradiation mortality between species was significantly different (<0.001), D. lupinus was more susceptible than M. edulis. TEAC values generally showed a decrease following irradiation (except for digestive gland). UPCs were clearly different between tissues and

Respuesta IgE específica anti-Blomia tropicalis en niños asmáticos residentes en Santa Marta, una ciudad del Caribe colombiano

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Dary Luz Mendoza Meza

2013-10-01

Full Text Available ResumenBlomia tropicalis es un ácaro intradomiciliario predominante en Colombia. Sin embargo, existe poca información disponible sobre la sensibilización a este y otros ácaros intradomiciliarios en pacientes asmáticos de Santa Marta. El propósito de este estudio fue establecer la respuesta IgE específica contra el ácaro B. tropicalis en pacientes pediátricos asmáticos de Santa Marta. La respuesta IgE específica se determinó por ELISA indirecto en 77 niños con diagnóstico de asma bronquial alérgica. Las fracciones alergénicas mayoritarias de B. tropicalis se identificaron por Western Blotting usando un extracto de proteínas de B. tropicalis. Setenta y seis (98.7% niños presentaron niveles elevados de IgE total, 48 (88% tenían IgE anti- B. tropicalis positiva. El Western Blot identificó 16 fracciones alergénicas con rango entre 80- 21 kDa. Estos resultados indican que los alérgenos del ácaro B. tropicalis deben incluirse en el diagnóstico exacto de la sensibilización a los ácaros del polvo doméstico en Santa Marta, Colombia. (DUAZARY 2011, 9 - 16AbstractBlomia tropicalis is the predominant indoor mite in Colombia, nevertheless there is not enough information about sensitization to this and other indoor mites, in the asthmatic patients living in Santa Marta. The purpose of this study was to establish the specific IgE responses against to B. tropicalis in pediatric asthmatic patients in Santa Marta. Specific IgE responses were determined by indirect ELISA in 77 children with bronchial asthma. The B. tropicalis major allergenic fractions were identified by Western Blotting using a B. tropicalis protein extract. Seventy six (98.7% children showed high levels of total IgE, 48 (88% had IgE anti-B. tropicalis positive. Sixteen allergenic fractions, between 80 – 21 kDa, were identified by Western Blotting. These results indicate that the B. tropicalisallergens must be included in the precise diagnosis of sensitization to house

Acetate supplementation reduces microglia activation and brain interleukin-1β levels in a rat model of Lyme neuroborreliosis

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Brissette Catherine A

2012-11-01

Full Text Available Abstract Background We have found that acetate supplementation significantly reduces neuroglia activation and pro-inflammatory cytokine release in a rat model of neuroinflammation induced with lipopolysaccharide. To test if the anti-inflammatory effect of acetate supplementation is specific to a TLR4-mediated injury, we measured markers of neuroglia activation in rats subjected to B. burgdorferi-induced neuroborreliosis that is mediated in large part by a TLR2-type mechanism. Methods In this study, rats were subjected to Lyme neuroborreliosis following an intravenous infusion of B. burgdorferi (B31-MI-16. Acetate supplementation was induced using glyceryl triacetate (6g/kg by oral gavage. Immunohistochemistry, qPCR, and western blot analyses were used to measure bacterial invasion into the brain, neuroglial activation, and brain and circulating levels of interleukin 1β. Statistical analysis was performed using one-way analysis of variance (ANOVA followed by a Tukey’s post hoc tests or using a Student’s t test assuming unequal variances when appropriate. Results We found that acetate supplementation significantly reduced microglia activation by 2-fold as determined by immunohistochemical and western blot analysis. Further, acetate supplementation also reduced the expression of the pro-inflammatory cytokine IL-1β by 2-fold as compared to controls. On the other hand, the inoculation of rats with B. burgdorferi had no effect on astroglial activation as determined by immunocytochemistry and western blot analysis despite significant increases in circulation levels of antigen toward B. burgdorferi and presence of the bacteria in the central nervous system. Conclusions These results suggest that microglial activation is an essential component to neuroborreliosis and that acetate supplementation may be an effective treatment to reduce injury phenotype and possibly injury progression in Lyme neuroborreliosis.

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.

A Case of Apparent Contact Dermatitis Caused by Toxocara Infection

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Rosanna Qualizza

2014-01-01

Full Text Available Infection from Toxocara species may give rise to a large array of clinical symptoms, including apparent manifestations of allergy such as asthma, urticaria/angioedema, and dermatitis. We report a case, thus far not described, of contact dermatitis attributed to nickel allergy but caused by Toxocara infection. The patient was a 53-year-old woman presenting from 10 years a dermatitis affecting head, neck, and thorax. Patch tests initially performed gave a positive result to nickel, but avoidance of contact with nickel did not result in recovery. The patient referred to our Allergy Service in 2010 because of dermatitis to feet. Patch testing confirmed the positive result for nickel, but expanding the investigation a positive result for IgG antibodies to Toxocara was detected by Western blotting and ELISA. Treatment with mebendazole achieved immediate efficacy on feet dermatitis. Then, two courses of treatment with albendazole resulted in complete regression of dermatitis accompanied by development of negative ELISA and Western blotting for Toxocara antibodies. This report adds another misleading presentation of Toxocara infection as apparent contact dermatitis caused by nickel and suggests bearing in mind, in cases of contact dermatitis not responding to avoidance of the responsible hapten and to medical treatment, the possible causative role of Toxocara.

2-tiered antibody testing for early and late Lyme disease using only an immunoglobulin G blot with the addition of a VlsE band as the second-tier test.

Science.gov (United States)

Branda, John A; Aguero-Rosenfeld, Maria E; Ferraro, Mary Jane; Johnson, Barbara J B; Wormser, Gary P; Steere, Allen C

2010-01-01

Standard 2-tiered immunoglobulin G (IgG) testing has performed well in late Lyme disease (LD), but IgM testing early in the illness has been problematic. IgG VlsE antibody testing, by itself, improves early sensitivity, but may lower specificity. We studied whether elements of the 2 approaches could be combined to produce a second-tier IgG blot that performs well throughout the infection. Separate serum sets from LD patients and control subjects were tested independently at 2 medical centers using whole-cell enzyme immunoassays and IgM and IgG immunoblots, with recombinant VlsE added to the IgG blots. The results from both centers were combined, and a new second-tier IgG algorithm was developed. With standard 2-tiered IgM and IgG testing, 31% of patients with active erythema migrans (stage 1), 63% of those with acute neuroborreliosis or carditis (stage 2), and 100% of those with arthritis or late neurologic involvement (stage 3) had positive results. Using new IgG criteria, in which only the VlsE band was scored as a second-tier test among patients with early LD (stage 1 or 2) and 5 of 11 IgG bands were required in those with stage 3 LD, 34% of patients with stage 1, 96% of those with stage 2, and 100% of those with stage 3 infection had positive responses. Both new and standard testing achieved 100% specificity. Compared with standard IgM and IgG testing, the new IgG algorithm (with VlsE band) eliminates the need for IgM testing; it provides comparable or better sensitivity, and it maintains high specificity.

Traditional African economies in conflict with western capitalism

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C.M. Pauw

1996-04-01

Full Text Available Traditional Mrican economies� in conflict with western capitalism Some of� the fundamental differences between two� economic systems which, by� and large,� have� come� into� conflict with� one another� in Africa south� of the� Sahara� are� analised,� i e� traditional African� economies� and western, capitalist oriented� economies.� The� dire� economic� conditions prevailing� in Africa are the result,� to a� large extent,� of� a� long history of� exploitation and economic� disempowerment particularly� by� western� powers.� Not� all� the strategies and programs to� counter this poverty are equally appropriate or acceptable.� In� the� meantime� a� unique� coping� mechanism� is� developing, particularly within African Independent Churches which may provide some answers.

Genotyping of Canine parvovirus in western Mexico.

Science.gov (United States)

Pedroza-Roldán, César; Páez-Magallan, Varinia; Charles-Niño, Claudia; Elizondo-Quiroga, Darwin; De Cervantes-Mireles, Raúl Leonel; López-Amezcua, Mario Alberto

2015-01-01

Canine parvovirus (CPV) is one of the most common infectious agents related to high morbidity rates in dogs. In addition, the virus is associated with severe gastroenteritis, diarrhea, and vomiting, resulting in high death rates, especially in puppies and nonvaccinated dogs. To date, there are 3 variants of the virus (CPV-2a, CPV-2b, and CPV-2c) circulating worldwide. In Mexico, reports describing the viral variants circulating in dog populations are lacking. In response to this deficiency, a total of 41 fecal samples of suspected dogs were collected from October 2013 through April 2014 in the Veterinary Hospital of the University of Guadalajara in western Mexico. From these, 24 samples resulted positive by polymerase chain reaction, and the viral variant was determined by restriction fragment length polymorphism. Five positive diagnosed samples were selected for partial sequencing of the vp2 gene and codon analysis. The results demonstrated that the current dominant viral variant in Mexico is CPV-2c. The current study describes the genotyping of CPV strains, providing valuable evidence of the dominant frequency of this virus in a dog population from western Mexico. © 2014 The Author(s).

Different collagen types define two types of idiopathic epiretinal membranes

OpenAIRE

Kritzenberger , Michaela; Junglas , Benjamin; Framme , Carsten; Helbig , Horst; Gabel , Veit-Peter; Fuchshofer , Rudolf; Tamm , Ernst R; Hillenkamp , Jost

2011-01-01

Abstract Aims: To identify differences in extracellular matrix contents between idiopathic epiretinal membranes (IEM) of cellophane macular reflex (CMRM) or preretinal macular fibrosis (PMFM) type. Methods and results: IEM were analyzed by light and quantitative transmission electron microscopy, immunohistochemistry, and Western blotting. Substantial differences between CMRM and PMFM were observed regarding the nature of extracellular fibrils. In CMRM, the fibrils were thin with...

Differential aetiology and impact of phosphoinositide 3-kinase (PI3K) and Akt signalling in skeletal muscle on in vivo insulin action

DEFF Research Database (Denmark)

Friedrichsen, Martin; Poulsen, P.; Richter, Erik

2010-01-01

signalling was evaluated at three key levels, i.e. the insulin receptor, IRS-1 and V-akt murine thymoma viral oncogene (Akt) levels, employing kinase assays and phospho-specific western blotting. RESULTS: Proximal insulin signalling was not associated with obesity, age or sex. However, birthweight...... for most measures of insulin signalling activity. Glucose disposal was positively associated with Akt-308 phosphorylation (p

Studies on antigenic cross-reactivity of Trichuris ovis with host mucosal antigens in goat

OpenAIRE

Gautam Patra; Seikh Sahanawaz Alam; Sonjoy Kumar Borthakur; Hridayesh Prasad

2015-01-01

Objective: To ascertain whether immunodominant antigens of Trichuris ovis might share and cross react with host molecule. Methods: Two crude protein preparations from anterior and posterior parts of Trichuris ovis were characterized along with host mucosal antigen by double immunodiffusion, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting technique. Conventional scanning electron microscopy was performed as per standard procedure. Results: Sharp...

Determinants of Primary School Non-Enrollment and Absenteeism: Results from a Retrospective, Convergent Mixed Methods, Cohort Study in Rural Western Kenya.

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Nia King

Full Text Available Education is a key element in the socioeconomic development required to improve quality of life in Kenya. Despite the introduction of free primary education, primary school enrollment and attendance levels remain low. Drawing on qualitative and quantitative data, this study explores the determinants of non-enrollment and absenteeism in rural western Kenya and potential mitigation strategies to address these issues.The study was conducted in Bwaliro village in rural western Kenya. A random sample of 64 students was obtained by blocking the village primary school's student population according to grade level, gender, and orphan status. Qualitative and quantitative data were collected through interviews with parents, guardians, and key informants, and focus group discussions with students. Quantitative data were compared using chi-square tests, Student's T-test, and Poisson regressions. Qualitative data were analyzed using thematic content analysis.Malaria, menstruation, and lack of money were among the most notable determinants of primary school dropout and absenteeism, and these factors disproportionately impacted orphans and female students. Potential mitigation strategies suggested by the community included provision of malaria treatment or prevention, reduction in education costs, expansion of the established school-feeding program, and provision of sanitary pads.Despite free primary education, numerous factors continue to prevent children in rural western Kenya from attending primary school. The findings suggest that interventions should primarily target orphaned and female students. Prior to implementation, suggested mitigation strategies should be assessed for cost-effectiveness.

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.

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.

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.

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…

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

Stumpage market integration in western national forests

Science.gov (United States)

Jean M. Daniels

2011-01-01

This study presents results of statistical tests for stumpage market integration on 62 national forests in the Western United States. Quarterly stumpage prices from 1984 to 2007 obtained from cut and sold reports for USDA Forest Service Regions 1, 4, 5, and 6 (Northern, Intermountain, Pacific Southwest, and Pacific Northwest, respectively) were analyzed to establish...

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

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.

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...

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)

The study for the Spatial Distribution Pattern of NDVI in the Western of Jilin Province

Science.gov (United States)

Yang, Shu-jie; Li, Xiao-dong; Yan, Shou-gang

2018-02-01

Using methods of spatial autocorrelation analysis and trend analysis, the paper studies the spatial distribution pattern of NDVI based on the GIMMS NDVI dataset (1998-2008), in Western Jilin. The maximum value for 15d is got through the method of MAX processing. Results show that: the NDVI in growing season shows a rising trend in western Jilin in 1998-2008. In the study area, the NDVI in Western Jilin shows positive spatial autocorrelation in the whole region, but the partial NDVI is apt to scattered distribution, which means the vegetation cover of Western Jilin is generally fragmental.

Peabody Western brings its old mines into new competitive era

Energy Technology Data Exchange (ETDEWEB)

Sprouls, M.W.

1994-10-01

Peabody Western Coal Co., manages four surface mines that mark Peabody's expansion from the Midwest to the West. The mines began operating between 1964 and 1973, before the surge of western coal mine start-ups brought about by the oil price shock of 1973 and 1974 and the subsequent investments in coal by American oil companies. Despite good demand for western low-sulfur coals, the prices are low due to production overcapacity that was built in the 1970s. The result is that older mines have had to be creative to reduce costs and retain customers amid stiff competition. The mining units - Black Mesa and Kayenta in Arizona, Big Sky in Montana and Seneca in Colorado - now must handle coal supply administration, financial analysis, engineering, environmental compliance, labor relations and community relations at their sites. Peabody Western's Flagstaff, Ariz., headquarters staff will be reduced by about 30 people, although many of them will be assigned to mining units. All four surface mines use draglines, and each supplies a primary customer, but the mines have as many differences as similarities. Altogether, the mines produce about 17 million tons annually and employ 1,100 people.

Spectra of molecular changes induced in DNA of Drosophila spermatozoa by 1-ethyl-1-nitrosourea and X-rays

International Nuclear Information System (INIS)

Batzer, M.A.; Fossett, N.G.; Lee, W.R.; Louisiana State Univ., Baton Rouge; Tedeschi, B.; Tucker, A.; Kilroy, G.; Arbour, P.

1988-01-01

Mutations induced in Drosophila spermatozoa at the alcohol dehydrogenase Adh locus by 1-ethyl-1-nitrosourea (ENU) were compared to X-ray-induced mutations using genetic tests for complementation, southern blotting, western blotting and northern blotting. 8 of 10 ENU-induced mutations complemented all known adjacent loci and were presumed to be intragenic. In contrast, 8 of 30 X-ray-induced mutations were intragenic. The interpretation of these results is that in spermatozoa X-rays induce primarily deletions that either produce deficiencies of the Adh locus or nonsense mutations within the locus, whereas ENU induces primarily missense mutations. This forward mutation assay based on loss of enzymatic activity efficiently recovered a broad spectrum of mutations ranging from missense to intragenic deletions and multi-locus deficiencies. Only 3 of these 40 mutations produced a polypeptide detectable as an electrophoretic variant. 37 refs.; 6 figs.; 2 tabs

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.

Spatio-temporal Variations of Nitrogen Dioxide over Western China from Satellite Observations during 2005-2013

Science.gov (United States)

Cui, Y.; Lin, J.; Huang, B.; Song, C.

2015-12-01

Western China has experienced rapid urbanization and industrialization since the implementation of National Western Development Strategy by Chinese Government. Most resource-intensive industries and high-pollution factories had been moved from the east coast to Western China after 2000. In this research, the spatial and temporal variations of tropospheric NO2 concentration in 2005 - 2013 is analyzed based on the satellite observations by Ozone Measurement Instrument (OMI). The annual trends and seasonality of tropospheric NO2 over Western China are calculated. The results show that large increases are observed in urban areas and the polluted regions are expanding. Additionally, the seasonal patterns of some regions over Western China are changing significantly and more clean areas tend to changing from the characteristics of natural emissions to those of anthropogenic emissions. The spatial and temporal variations of NO2 concentrations are well responded to the rapid urbanization and industrialization over Western China.

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

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.

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.

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...

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.

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

International Nuclear Information System (INIS)

Margolin, A.B.

1986-01-01

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

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

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-.

Radiogeochronologic data on rocks of crystalline massifs of Western Carpathians and their interpretation in radiogeochronologic dating

International Nuclear Information System (INIS)

Cambel, B.

1979-01-01

The results are summed up and evaluated of the measurement of the age of various crystalline rocks in the Western Carpathians using the method of isotope dating. The obtained results show that the rocks in the Western Carpathians were formed in the period of the Variscian and Alpine orogenesis, ie., 90 to 550 million years ago. A list of problems is given which must be resolved using radiogeochronological methods for age determination of geological processes and for specifying the stratigraphy of crystalline rocks in the Western Carpathians. (Ha)

Cultural Novelty and Adjustment: Western Business Expatriates in China

DEFF Research Database (Denmark)

Selmer, Jan

2006-01-01

Western business expatriates in China. Three sociocultural adjustment variables were examined; general, interaction and work adjustment. Although a negative relationship was hypothesized between cultural novelty and the three adjustment variables, results of the hierarchical multiple regression analysis...

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

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.

CEPF Western Ghats Special Series: An overview of Asian Elephants in the Western Ghats, southern India: implications for the conservation of Western Ghats ecology

Directory of Open Access Journals (Sweden)

N. Baskaran

2013-10-01

Full Text Available The Western Ghats region is a global biodiversity hotspot and the source of all the major rivers of peninsular India. The conservation of this region is important for the biodiversity it harbours, and for ecological functions that include climate stability, erosion control, clean water and air, which are essential to safeguard economic growth, social stability and quality of life for the people of peninsular India. Possessing a unique diversity in topography, climate, vegetation, faunal communities, endemism and human communities, the Western Ghats is also known for its spectacular assemblage of larger mammals, including 25% of the global population of Asian Elephants. There are four major landscapes in the Western Ghats: (1 Uttara Kannada, (2 Brahmagiri-Nilgiris, (3 Anamalai-Nelliyampathy-High Range, and (4 Periyar-Agasthyamalai, spread across 30,000km2, harbouring a minimum 10,000 elephants in six different populations with signs of an increasing trend in some populations. The second landscape (Brahmagiri-Nilgiris with over 50% of the Ghats elephant population, along with its contiguity to the Eastern Ghats elephant landscape, forms the single largest global population of Asian Elephants. However, major threats to the long-term conservation of the elephant include further fragmentation of habitat, continued poaching of bulls for ivory, and escalation in human-elephant conflicts resulting in public antagonism toward the species. The goals of management should thus be to: (1 consolidate habitats and preserve corridors to avoid further fragmentation; (2 take steps through integrated land use planning at the landscape level to reduce human-elephant conflicts; and (3 build up a demographically and genetically viable elephant population by protecting the tusked males from ivory poaching. Being a wide-ranging umbrella species, ensuring the long-term conservation of Asian Elephants in the Ghats implies protecting its biodiversity and ecological

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.

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.

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

Three major nucleolar proteins migrate from nucleolus to nucleoplasm and cytoplasm in root tip cells of Vicia faba L. exposed to aluminum.

Science.gov (United States)

Qin, Rong; Zhang, Huaning; Li, Shaoshan; Jiang, Wusheng; Liu, Donghua

2014-09-01

Results from our previous investigation indicated that Al could affect the nucleolus and induce extrusion of silver-staining nucleolar particles containing argyrophilic proteins from the nucleolus into the cytoplasm in root tip cells of Vicia faba L. So far, the nucleolar proteins involved have not been identified. It is well known that nucleophosmin (B23), nucleolin (C23), and fibrillarin are three major and multifunctional nucleolar proteins. Therefore, effects of Al on B23, C23, and fibrillarin in root tip cells of V. faba exposed to 100 μM Al for 48 h were observed and analyzed using indirect immunofluorescence microscopy and Western blotting. The results from this work demonstrated that after 100 μM of Al treatment for 48 h, B23 and C23 migrated from the nucleolus to the cytoplasm and fibrillarin from the nucleolus to the nucleoplasm. In some cells, fibrillarin was present only in the cytoplasm. Western blotting data revealed higher expression of the three major nucleolar proteins in Al-treated roots compared with the control and that the B23 content increased markedly. These findings confirmed our previous observations.

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.

Obesity and nutrition behaviours in Western and Palestinian outpatients with severe mental illness

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Aljeesh Yousef

2011-10-01

Full Text Available Abstract Background While people with severe mental illness have been found to be more overweight and obese in Western nations, it is unknown to what extent this occurs in Middle Eastern nations and which eating behaviours contribute to obesity in Middle Eastern nations. Method A total of 665 responses were obtained from patients with serious mental illness attending out-patient clinics in Western developed countries (Germany, UK and Australia; n = 518 and Palestine (n = 147. Patients were evaluated by ICD-10 clinical diagnosis, anthropometric measurements and completed a self-report measure of frequencies of consuming different food items and reasons for eating. Nutritional habits were compared against a Western normative group. Results More participants from Palestine were overweight or obese (62% compared to Western countries (47%. In the Western sample, obese patients reported consuming more low-fat products (OR 2.54, 95% CI 1.02-6.33 but also greater eating due to negative emotions (OR 1.84, 95% CI 1.31-2.60 than patients with a healthy body-mass index. In contrast, obese patients from Palestine reported increased consumption of unhealthy snacks (OR 3.73 95% CI 1.16-12.00. Conclusion Patients with mental illness have poorer nutritional habits than the general population, particularly in Western nations. Separate interventions to improve nutritional habits and reduce obesity are warranted between Western nations and Palestine.

Establishment of H2Mab-119, an Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody, Against Pancreatic Cancer.

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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.

H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer.

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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.

The Greek evil eye, African witchcraft, and Western ethnocentrism

African Journals Online (AJOL)

p1243322

often projected onto non-Western cultures resulting in the problem of ethnocentrism. ..... were most susceptible to the evil eye, as were children, work places and animals .... The priest then holds the candidate upright, looks to the east and.

Western Area Power Administration combined power system financial statements September 30, 1994 and 1993 and management overview and performance measurements

Energy Technology Data Exchange (ETDEWEB)

Marwick, P.

1994-12-31

The attached report presents the results of the independent certified public accountant`s audit of the Department of Energy`s Western Area Power Administration`s (Western) combined financial statements as of September 30, 1994. The auditors have expressed an unqualified opinion on Western`s 1994 statements. Their reports on Western`s internal control structure and on compliance with laws and regulations are also provided. Western was established in December 1977, and has the responsibility for the Federal electric power marketing and transmission functions in 15 central and western states. Western markets power, as required by existing law, at the lowest possible rates consistent with sound business principles to recover the costs of operation and capital invested in power facilities.

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

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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.

Investigating adolescents' sweetened beverage consumption and Western fast food restaurant visits in China, 2006-2011.

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Lee, Yen-Han; Chiang, Timothy C; Liu, Ching-Ti; Chang, Yen-Chang

2018-05-25

Background China has undergone rapid Westernization and established dramatic social reforms since the early 21st century. However, health issues led to challenges in the lives of the Chinese residents. Western fast food and sweetened beverages, two food options associated with chronic diseases and obesity, have played key roles to alter adolescents' dietary patterns. This study aims to examine the association between adolescents' visits to Western fast food restaurants and sweetened beverage consumption. Methods Applying three waves of the China Health and Nutrition Study (CHNS) between 2006 and 2011 (n = 1063), we used generalized Poisson regression (GPR) to investigate the association between adolescents' Western fast food restaurant visits and sweetened beverage consumption, as the popularity of fast food and sweetened beverages has skyrocketed among adolescents in contemporary China. A linear-by-linear association test was used as a trend test to study general patterns between sweetened beverage consumption and Western fast food restaurant visits. We adjusted all models with sweetened beverage consumption frequency, four food preferences (fast food, salty snacks, fruits and vegetables), school status, gross household income, provinces, rural/urban regions, age and gender. Results From the results of the trend test, frequent sweetened beverage consumption was highly associated with more Western fast food restaurant visits among Chinese adolescents in the three waves (p beverage consumption or did not drink them at all, had much less likelihood of visiting Western fast food restaurants (p beverage consumption was highly associated with Western fast food restaurant visits in contemporary China. Further actions are needed from the Chinese central government to create a healthier dietary environment for adolescents.

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...

Predominance of Trypanosoma rangeli infection in children from a Chagas disease endemic area in the west-shore of the Panama canal

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Azael Saldaña

2005-11-01

Full Text Available A total of 206 serum samples from children (3-14 years old living in the Amador County (La Chorrera District, Province of Panama were screened by indirect immunofluorescence antibody test (IFAT for the presence of antibodies against Trypanosoma cruzi. Positive sera were confirmed by recombinant enzyme-linked immunosorbent assay (ELISA and Western blot analysis. The presence of blood trypanosomes was investigated by hemoculture and subsequently identify by a duplex polymerase chain reaction (PCR followed by dot blot hybridization. The results indicated a prevalence of 9.7% for trypanosome infections, a seroprevalence of 2.9% against T. cruzi and a predominance of T. rangeli infection (6.8%. The immunological and clinical implications of these findings are discussed.

Rethinking Education Research methodologies-Dominance of Western perspectives?

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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.

Cytokine Gene Expression in Response to SnSAG1 in Horses with Equine Protozoal Myeloencephalitis

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Spencer, Jennifer A.; Deinnocentes, Patricia; Moyana, Edith M.; Guarino, Anthony J.; Ellison, Siobhan E.; Bird, R. Curtis; Blagburn, Byron L.

2005-01-01

Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome seen in horses from the Americas and is mainly caused by Sarcocystis neurona. Recently, a 29-kDa surface antigen from S. neurona merozoites was identified as being highly immunodominant on a Western blot. This antigen has been sequenced and cloned, and the expressed protein has been named SnSAG1. In a previous study, cell-mediated immune responses to SnSAG1 were shown to be statistically significantly reduced in horses with EPM in comparison to EPM-negative control horses. It therefore appears as though the parasite is able to induce immunosuppression towards parasite-derived antigens as parasite-specific responses are decreased. Isolated peripheral blood lymphocytes from 21 EPM (cerebrospinal fluid [CSF] Western blot)-negative horses with no clinical signs and 21 horses with clinical signs of EPM (CSF Western blot positive) were cocultured with SnSAG1 for 48 and 72 h, and the effect on cytokine production was investigated by means of reverse transcriptase PCR. Cytokines assayed include gamma interferon (IFN-γ), tumor necrosis factor alpha, interleukin (IL)-2, IL-4, and IL-6. β-Actin was used as the housekeeping gene. A Wilcoxon signed-rank test of the findings indicated that there was a statistically significant decrease in IFN-γ production after 48 h in culture for samples from horses with clinical disease. There was also a statistically significant increase in IL-4 production after 72 h in culture for samples from horses with EPM. These results further support the notion that this parasite is able to subvert the immune system in horses with clinical disease. PMID:15879026

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

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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.

Proportion of collagen type II in the extracellular matrix promotes the differentiation of human adipose-derived mesenchymal stem cells into nucleus pulposus cells.

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Tao, Yiqing; Zhou, Xiaopeng; Liu, Dongyu; Li, Hao; Liang, Chengzhen; Li, Fangcai; Chen, Qixin

2016-01-01

During degeneration process, the catabolism of collagen type II and anabolism of collagen type I in nucleus pulposus (NP) may influence the bioactivity of transplanted cells. Human adipose-derived mesenchymal stem cells (hADMSCs) were cultured as a micromass or in a series of gradual proportion hydrogels of a mix of collagen types I and II. Cell proliferation and cytotoxicity were detected using CCK-8 and LDH assays respectively. The expression of differentiation-related genes and proteins, including SOX9, aggrecan, collagen type I, and collagen type II, was examined using RT-qPCR and Western blotting. Novel phenotypic genes were also detected by RT-qPCR and western blotting. Alcian blue and dimethylmethylene blue assays were used to investigate sulfate proteoglycan expression, and PI3K/AKT, MAPK/ERK, and Smad signaling pathways were examined by Western blotting. The results showed collagen hydrogels have good biocompatibility, and cell proliferation increased after collagen type II treatment. Expressions of SOX9, aggrecan, and collagen type II were increased in a collagen type II dependent manner. Sulfate proteoglycan synthesis increased in proportion to collagen type II concentration. Only hADMSCs highly expressed NP cell marker KRT19 in collagen type II culture. Additionally, phosphorylated Smad3, which is associated with phosphorylated ERK, was increased after collagen type II-stimulation. The concentration and type of collagen affect hADMSC differentiation into NP cells. Collagen type II significantly ameliorates hADMSC differentiation into NP cells and promotes extracellular matrix synthesis. Therefore, anabolism of collagen type I and catabolism of type II may attenuate the differentiation and biosynthesis of transplanted stem cells. © 2016 International Union of Biochemistry and Molecular Biology.

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

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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.

TGF-β1 is Involved in Vitamin D-Induced Chondrogenic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells by Regulating the ERK/JNK Pathway

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Xiaorui Jiang

2017-08-01

Full Text Available Background/Aims: Osteoarthritis (OA is characterized by degradation of cartilage, sole cell type of which is chondrocytes. Bone marrow-derived mesenchymal stem cells (BMSCs possess multipotency and can be directionally differentiated into chondrocytes under stimulation. This study was aimed to explore the possible roles of vitamin D and transforming growth factor-β1 (TGF-β1 in the chondrogenic differentiation of BMSCs. Methods: BMSCs were isolated from femurs and tibias of rats and characterized by flow cytometry. After stimulation with vitamin D, BMSC proliferation and migration were measured by Cell Counting Kit-8 (CCK-8 and Transwell assays, respectively. Chondrogenic differentiation was estimated through expression levels of specific markers by qRT-PCR and Western blot analysis. After stable transfection, the effects of aberrantly expressed TGF-β1 on vitamin D-induced alterations, including BMSC viability, migration and chondrogenic differentiation, were all evaluated utilizing CCK-8 assay, Transwell assay, qRT-PCR and Western blot analysis. Finally, the phosphorylation levels of key kinases in the extracellular signal-regulated kinase (ERK and c-Jun N-terminal kinase (JNK pathways were determined by Western blot analysis. Results: Vitamin D remarkably promoted BMSC viability, migration and chondrogenic differentiation. These alterations of BMSCs induced by vitamin D were reinforced by TGF-β1 overexpression while were reversed by TGF-β1 silencing. Additionally, the phosphorylation levels of ERK, JNK and c-Jun were enhanced by TGF-β1 overexpression but were reduced by TGF-β1 knockdown. Conclusion: Vitamin D promoted BMSC proliferation, migration and chondrogenic differentiation. TGF-β1 might be implicated in the vitamin D-induced alterations of BMSCs through regulating ERK/JNK pathway.

[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.

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

Science.gov (United States)

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

2010-12-15

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

Fathers in situational crisis: a comparison of Asian and Western cultures.