Sample records for blotting northern

  1. Northern blotting analysis

    Josefsen, Knud; Nielsen, Henrik


    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...... closing the gap to the more laborious nuclease protection experiments....

  2. Northern blot analysis to investigate the abundance of microorganisms

    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

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

    Larsen, S; Rygaard, K; Asnaes, S; Spang-Thomsen, M


    21-118 h postmortem. Extracted RNA and DNA were examined by Northern and Southern blot analysis using oligo-labelled human DNA probes recognizing gene transcripts of 2-5 kb. The results indicated that, in general, Northern blot analysis was feasible with the applied probes when the tissue was......Fresh biopsy material for molecular biological investigations is not obtainable from all relevant normal human tissues. We studied the feasibility of using RNA and DNA from autopsies for Northern and Southern blot analysis. Tissue samples from seven organs were obtained from 10 autopsies performed...... 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...

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

    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 32P-labeled probe of the pPA8 cDNA clone of BYDV. The distribution of PRC for the F2 population was compared to the distribution of BYD visual symptom scores for 403 F2 plants of a similar F2 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


    LIU Lun-xu; ZHOU Qing-hua; SHI Ying-kang; QIN Yang; SUN Zhi-lin; SUN Ze-fang


    Objective: To investigate the role of nm23 gene expression in human lung cancer. Methods: Forty human lung cancer tissues and 19 non-cancer pulmonary tissues were studied for their nm23-H1 and nm23-H2 mRNA expression with non-radioactive Northern blot hybridization. The correlation of nm23 mRNA expression with clinical features of lung cancer was analyzed. Results: The mRNA expression of nm23-H2 gene in poorly differentiated squamous cell carcinoma was significantly decreased compared to that in moderate-high differentiated squamous cell carcinoma. The mRNA expression of nm23-H1 and nm23-H2 gene in small cell lung cancer was significantly decreased compared to that in squamous cell carcinoma. No significant difference in nm23 mRNA expression was observed between lung cancer with and without lymph node metastasis, nor was there significant difference between tumor stage. Conclusion: The mRNA expression of nm23 gene is correlated with the degree of differentiation of lung cancer, but there is no evidence of metastasis suppression effect by nm23 gene.

  6. The gene encoding vitamin K-dependent anticoagulant protein S is expressed in multiple rabbit organs as demonstrated by northern blotting, in situ hybridization, and immunohistochemistry.

    He, X; Shen, L; Bjartell, A; Dahlbäck, B


    Vitamin K-dependent protein S is an anticoagulant plasma protein that functions as a co-factor to activated protein C in the degradation of coagulation factors Va and VIIIa. We investigated the tissue/cellular distribution of protein S synthesis by Northern blotting, in situ hybridization, and immunohistochemistry. Northern blotting together with in situ hybridization, using specific oligodeoxynucleotide probes, demonstrated protein S mRNA in liver, lung, testis, epididymis, ovary, uterus, and brain. In the reproductive system, protein S mRNA was present in the cytoplasm of Leydig cells, interstitial cells of the ovary, epithelial cells of the epididymis, and in the endometrium, including endometrial mucous glandular membrane in the myometrium. Bronchial epithelial cells and alveolar macrophages were positive in the respiratory system. In the central nervous system, pyramidal neurons in the cerebral cortex and in the hippocampal region, and dentate fascia neurons gave strongly positive signals. Immunohistochemistry with monoclonal antibodies yielded a staining pattern that correlated well with results of in situ hybridization. In conclusion, results from Northern blotting, in situ hybridization, and immunohistochemistry suggested that rabbit protein S is expressed in several extrahepatic tissues. The presence of protein S transcripts in these fully differentiated cells suggests a cell type-specific gene expression which may be related to local anticoagulation or to other as yet unknown protein S functions. PMID:7822769

  7. MicroRNA fate upon targeting with anti-miRNA oligonucleotides as revealed by an improved Northern-blot-based method for miRNA detection

    Torres, Adrian G.; Fabani, Martin M.; Vigorito, Elena; Gait, Michael J.


    MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs) are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such anti-miR ONs include 2′-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs. However, miRNA quanti...

  8. Simultaneous extraction from clinical biopsies of high-molecular-weight DNA and RNA: comparative characterization by biotinylated and 32P-labeled probes on Southern and Northern blots

    A method for efficient simultaneous extraction of high-molecular-weight DNA and RNA from solid mammalian tissues including clinical biopsies is described. It is based on the disruption and subsequent melting of deep frozen tissue in the presence of frozen phenol and nucleic acid extraction buffer; this allows for simultaneous disruption of tissue and inactivation of nucleases. The yield is about 0.7-5.8 mg of DNA and 0.5-8.1 mg of total RNA/g of tissue depending upon the tissue type; this is higher than the yield of other methods tested. Analysis of total RNA by denaturing gel electrophoresis, and of DNA and poly(A)+ RNA by Southern and Northern blot hybridization using 32P and biotinylated probes, indicated that c-Ha-ras gene and its transcripts were undegraded. Biotinylated and 32P probes had approximately the same sensitivity in detecting nucleic acids on Southern and Northern blots. This extraction procedure is simple and, when used with biotinylated probes, is rapid, inexpensive, and nonhazardous. The methodology can be modified for use with other clinical samples and cells grown in culture

  9. MicroRNA fate upon targeting with anti-miRNA oligonucleotides as revealed by an improved Northern-blot-based method for miRNA detection.

    Torres, Adrian G; Fabani, Martin M; Vigorito, Elena; Gait, Michael J


    MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs) are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such anti-miR ONs include 2'-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs. However, miRNA quantification in the presence of anti-miR ONs has proved to be challenging, due to detection artifacts, which has led to poor understanding of miRNA fate upon anti-miR binding. Here we show that anti-miR ON bound to miR-122 can prevent the miRNA from being properly precipitated into the purified RNA fraction using the standard RNA extraction protocol (TRI-Reagent), yielding an RNA extract that does not reflect the real cellular levels of the miRNA. An increase in the numbers of equivalents of isopropanol during the precipitation step leads to full recovery of the targeted miRNA back into the purified RNA extract. Following our improved protocol, we demonstrate by Northern blotting, in conjunction with a PNA decoy strategy and use of high denaturing PAGE, that high-affinity anti-miRs (K-PNA-K3, LNA/DNA, and LNA/OMe) sequester miR-122 without causing miRNA degradation, while miR-122 targeting with a lower-affinity anti-miR (OMe) seems to promote degradation of the miRNA. The technical issues explored in this work will have relevance for other hybridization-based techniques for miRNA quantification in the presence of anti-miR ONs. PMID:21441346

  10. Problem-Solving Test: Southwestern Blotting

    Szeberényi, József


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

  11. Multiplexed Western Blotting Using Microchip Electrophoresis.

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


    Western blotting is a commonly used protein assay that combines the selectivity of electrophoretic separation and immunoassay. The technique is limited by long time, manual operation with mediocre reproducibility, and large sample consumption, typically 10-20 μg per assay. Western blots are also usually used to measure only one protein per assay with an additional housekeeping protein for normalization. Measurement of multiple proteins is possible; however, it requires stripping membranes of antibody and then reprobing with a second antibody. Miniaturized alternatives to Western blot based on microfluidic or capillary electrophoresis have been developed that enable higher-throughput, automation, and greater mass sensitivity. In one approach, proteins are separated by electrophoresis on a microchip that is dragged along a polyvinylidene fluoride membrane so that as proteins exit the chip they are captured on the membrane for immunoassay. In this work, we improve this method to allow multiplexed protein detection. Multiple injections made from the same sample can be deposited in separate tracks so that each is probed with a different antibody. To further enhance multiplexing capability, the electrophoresis channel dimensions were optimized for resolution while keeping separation and blotting times to less than 8 min. Using a 15 μm deep × 50 μm wide × 8.6 cm long channel, it is possible to achieve baseline resolution of proteins that differ by 5% in molecular weight, e.g., ERK1 (44 kDa) from ERK2 (42 kDa). This resolution allows similar proteins detected by cross-reactive antibodies in a single track. We demonstrate detection of 11 proteins from 9 injections from a single Jurkat cell lysate sample consisting of 400 ng of total protein using this procedure. Thus, multiplexed Western blots are possible without cumbersome stripping and reprobing steps. PMID:27270033

  12. Automated design of genomic Southern blot probes

    Komiyama Noboru H


    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

  13. Observation on Plant Leaf Transection by Gelatin Blotting

    TAN Dahai; LI Fuheng; WANG Xiaocen; HUANG Fushan


    Blotting was used to observe cell structures of leaf epidermis cells, and the key method of leaf transaction observation was paraffin section. The concentration, suitable solidification time, melting temperature of gelatin solution and the stain for the gelatin blotting were studied in this research. The results showed that the gelatin blotting could be used to study leaf transaction, it was benefit to make operation easily, and save time, money and so on


    Hubbard, Katherine; Hegarty, Peter


    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. PMID:26924673

  15. HIV-1 western blot assay: What determines an indeterminate status?

    Syed Iqbal


    Full Text Available Background: The Western blot assay is the gold standard for the detection of antibodies to human immunodeficiency virus type1 (HIV-1. However, indeterminate Western blot reactivity to HIV-1 proteins may occur in individuals, who may not be infected with HIV. Aim: This retrospective study was aimed to determine the diagnostic value of the interpretation criteria in relation to commercial kits for HIV -1 diagnosis. Methods and Materials: A total of 556 serum/plasma specimens collected from high-risk population attending our HIV clinic from 2000 - 2004 were tested by three different western blot kits: NEW LAV BLOT I (n=244, HIV BLOT 2.2; (n=112, Genetic Systems HIV-1 (n=237. And the results of western blot strips were analyzed using the various interpretation criteria: WHO/NACO, CDC/ ASTPHLD, ARC, FDA, CRSS and JHU. Some specimens were run on more than one kit. RT-PCR assay was performed on 5 specimens, which were indeterminate with LAV BLOT I. Results: The discrepancy in LAV BLOT I positive results were between 157(64-176(72, and indeterminate results were between 44(18 to 63(25. No such variations were observed in genetic systems. There are some HIV negative (by PCR specimens were indeterminate in LAV BLOT I revealing the kit more sensitive and less effective for diagnostic purpose. Conclusion: The genetic systems kit is superior to other kits we analyzed and its results are concordant with HIV-1 PCR results. To report, the choice of western blot commercial kit is paramount important than the use of particular interpretation criteria for the diagnosis of HIV -1.

  16. Miniaturized fluorescent RNA dot blot method for rapid quantitation of gene expression

    Yadetie Fekadu


    Full Text Available Abstract Background RNA dot blot hybridization is a commonly used technique for gene expression assays. However, membrane based RNA dot/slot blot hybridization is time consuming, requires large amounts of RNA, and is less suited for parallel assays of more than one gene at a time. Here, we describe a glass-slide based miniaturized RNA dot blot (RNA array procedure for rapid and parallel gene expression analysis using fluorescently labeled probes. Results RNA arrays were prepared by simple manual spotting of RNA onto amino-silane coated microarray glass slides, and used for two-color fluorescent hybridization with specific probes labeled with Cy3 and 18S ribosomal RNA house-keeping gene probe labeled with Cy5 fluorescent dyes. After hybridization, arrays were scanned on a fluorescent microarray scanner and images analyzed using microarray image analysis software. We demonstrate that this method gives comparable results to Northern blot analysis, and enables high throughput quantification of transcripts from nanogram quantities of total RNA in hundreds of samples. Conclusion RNA array on glass slide and detection by fluorescently labeled probes can be used for rapid and parallel gene expression analysis. The method is particularly well suited for gene expression assays that involve quantitation of many transcripts in large numbers of samples.

  17. The Design of a Quantitative Western Blot Experiment

    Sean C. Taylor


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


    Proteins of Mycoplasma pneumoniae were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose sheet by blotting. Sera obtained from infected hamsters and immunized rabbits were then incubated with the nitrocellulose strips. Proteins which are capa...

  19. HIV-1 western blot assay: What determines an indeterminate status?

    Syed Iqbal; Balakrishnan P; Solomon Sunil; Murugavel K; Kumarasamy N; Vidya S; Martin S; Thyagarajan S; Mayer Kenneth; Solomon S


    Background: The Western blot assay is the gold standard for the detection of antibodies to human immunodeficiency virus type1 (HIV-1). However, indeterminate Western blot reactivity to HIV-1 proteins may occur in individuals, who may not be infected with HIV. Aim: This retrospective study was aimed to determine the diagnostic value of the interpretation criteria in relation to commercial kits for HIV -1 diagnosis. Methods and Materials: A total of 556 serum/plasma specimens collected from h...

  20. Western Blot of Stained Proteins from Dried Polyacrylamide Gels

    Gruber, Claudia; Stan-Lotter, Helga


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

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

    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 [5'32P]-pCp labeled genomic RNA from a related strain. Hybridization was performed at 520C, 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

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

    Endotoxin (LPS) stimulates a variety of eukaryotic cells. These actions are involved in the pathogenesis of Gram-negative septicemia. The site of action of the LPS toxic moiety, lipid A (LA), is unclear. Their laboratory has previously identified a bioactive LA precursor lipid IV/sub A/, which can be enzymatically labeled with 32P/sub i/ (109 dpm/nmole) and purified (99%). They now show that this ligand binds to specific proteins immobilized on nitrocellulose (NC) from LPS-sensitive RAW 264.7 cultured macrophages. NC blots were incubated with [32P]-IV/sub A/ in a buffer containing BSA, NaCl, polyethylene glycol, and azide. Binding was assessed using autoradiography or scintillation counting. Dot blot binding of the radioligand was inhibited by excess cold IV/sub A/, LA, or ReLPS but not by phosphatidylcholine, cardiolipin, phosphatidylinositol, or phosphatidic acid. Binding was trypsin-sensitive and dependent on protein concentration. Particulate macrophage proteins were subjected to SDS-PAGE and then electroblotted onto NC. Several discrete binding proteins were observed. Identical treatment of fetal bovine serum or molecular weight standards revealed no detectable binding. By avoiding high nonspecific binding of intact membranes, this ligand blotting assay may be useful in elucidating the molecular actions of LPS

  3. Fingerprinting of Natural Product by Eastern Blotting Using Monoclonal Antibodies

    Hiroyuki Tanaka


    Full Text Available We succeeded in developing the fingerprint of natural product by eastern blotting using monoclonal antibodies. After developing and separating them on a TLC plate, solasodine glycosides are oxidized by NaIO4 and reacted with a protein to give conjugates which are recognized with anti-solamargine monoclonal antibody (MAb. Anti-solamargine MAb having wide cross-reactivity can stain and detect all solasodine glycosides by fingerprint. Different sensitivity between solamargine and solasonine was observed. The detection limit was 1.6 ng of solasonine. The hydrolysed products of solamargine were determined by fingerprint of eastern blotting compared to their Rf values depending on the sugar number. Fingerprint by eastern blotting using anti-ginsenoside Rb1 MAb distinguished the formula containing ginseng prescribed in traditional Chinese medicine. By double-staining of ginsenosides it is possible to suggest that the staining color shows the pharmacological activity, such as the purple bands indicate ginsenosides having stimulation activity, and the blue color indicated compound like ginsenosides possessed the depression affect for the central nervous system (CNS, respectively.

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

    A Halajian


    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.


    Texia Gorman


    Full Text Available Se evaluó y caracterizó la respuesta inmune humoral de bovinos naturalmente infectados con Fasciola hepatica frente a un extracto total de antígenos de excreción-secreción (E-S y a una fracción antigénica semipurificada cromatográficamente (Immunodiagnosis of bovine fasciolosis was evaluated in terms of its sensitivity and specificity by ELISA and Western blot using a crude excretory-secretory preparation (C E-S and an E-S partially purified chromatographic fraction of Fasciola hepatica (<30 kDa as antigens. Serum samples from 52 bovines with natural fasciolosis, 18 without the infection and 48 bovines with hydatid cysts, all checked by post mortem examination, were tested as well. The sensitivity and specificity obtained by ELISA using the C E-S antigens were 53% and 100%, respectively. Likewise, when using the partially purified fraction, the sensitivity of ELISA test increased to 90% with a 100% specificity. The C E-S antigens reacted specifically with sera from infected bovines exhibiting bands of 14, 22, 27-39 and 37-38 kDa in Western blots. When the partially purified fraction was tested, the 28-30 kDa band was consistently detected by sera from all infected bovines and was absent in sera from uninfected as well as in sera from those with hydatid infection. These results indicate that F. hepatica presents different antigens, among which the 28-30 kDa fraction represents polypeptides with diagnostic potential that can be further purified and applied in the immunodiagnosis of bovine fasciolosis corroborating previous results recorded in other animal species

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

    Brad L. Ericson


    Full Text Available 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.

  7. Evaluation of an indigenous western blot kit for human immunodeficiency virus

    Lakshmi V; Ponamgi S


    PURPOSE: The Western Blot test is considered a gold standard test for the confirmation of an ELISA and/or rapid assay screened reactive sample in the diagnosis of HIV infection, especially in the low risk population. In this study, an indigenously developed HIV W. Blot kit (J.Mitra & Co., New Delhi, India) was compared for its performance characteristics with a widely used Western Blot kit, HIV Blot 2.2 (Genelabs, Singapore). Antigens of both HIV-1 and the indicator antigen gp36 of HIV...

  8. Evaluation of an indigenous western blot kit for human immunodeficiency virus

    Lakshmi V


    Full Text Available PURPOSE: The Western Blot test is considered a gold standard test for the confirmation of an ELISA and/or rapid assay screened reactive sample in the diagnosis of HIV infection, especially in the low risk population. In this study, an indigenously developed HIV W. Blot kit (J.Mitra & Co., New Delhi, India was compared for its performance characteristics with a widely used Western Blot kit, HIV Blot 2.2 (Genelabs, Singapore. Antigens of both HIV-1 and the indicator antigen gp36 of HIV-2 are included in the strips. METHODS: A panel of 150 clinical serum samples was used in the evaluation. All the sera were tested simultaneously by both the kits. RESULTS: The HIV W. Blot kit had high performance characteristics (100% sensitivity and 100% specificity, like the HIV Blot 2.2. The test procedure was easy to perform. There was clear delineation of the bands. CONCLUSIONS: The interpretation of the results on the HIV W. Blot was less prone to subjective errors. The test gave positive bands at even very high serum dilutions in the test kit. This fact indicates that HIV W. Blot probably has a potential application in early phases of infection, when the antibody concentrations are still very low.

  9. When less is more: a simple Western blotting amendment allowing data acquisition on human single fibers

    Jensen, Thomas Elbenhardt; Richter, Erik A


    This editorial discusses a simple western blotting-amendment allowing rapid data-acquisition on single fibers obtained from freeze-dried human skeletal muscle biopsies.......This editorial discusses a simple western blotting-amendment allowing rapid data-acquisition on single fibers obtained from freeze-dried human skeletal muscle biopsies....


    A western blot assay (WB) was developed and analyzed against the comparable standard, immunoprecipitation of 35[S] methionine/cysteine-labeled ovine progressive pneumonia virus (OPPV) proteins (IP), for its ability to detect anti-OPPV antibodies using endpoint titers. WB is 12-fold more sensitive i...


    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 phosphorylated with ...

  12. Quantitative DNA slot blot analysis: inhibition of DNA binding to membranes by magnesium ions.

    Kube, D M; Srivastava, A.


    Titers of wild-type and recombinant adeno-associated viruses are routinely determined by DNA slot blot analysis. The binding of viral DNA to nylon membranes was found to be inhibited by magnesium ions, which are critical components of the DNase I digestion carried out prior to slot blot analysis. Mg2+ions also interfered with the adsorption of plasmid DNA to nylon and nitrocellulose membranes. These observations yield practical insights into the poorly understood mechanisms by which DNA molec...

  13. Investigation of immunofluorescence cross-reactions against Trichinella spiralis by western blot (immunoblot) analysis.

    Robert, F; Weil, B.; Kassis, N; Dupouy-Camet, J


    Immunofluorescence cross-reactions in Trichinella spiralis serodiagnosis are sometimes difficult to identify. We compared the results of an indirect immunofluorescence assay and the profiles obtained by Western blot (immunoblot) analysis for three groups of patients: 10 T. spiralis-infected patients, 10 patients with autoimmune diseases, and 7 patients with parasitic diseases other than trichinellosis. The degree of immunofluorescence cross-reaction was variable. Western blotting allowed us t...

  14. Identification of immunodiagnostic antigens for cerebrospinal filariasis in horses by western blot analysis

    TAKESUE, Masataka; OSAKA, Yuki; MURANAKA, Masanori; KATAYAMA, Yoshinari; IKADAI, Hiromi


    ABSTRACT In the present study, the serum and cerebrospinal fluid of horses diagnosed with Setaria digitata cerebrospinal filariasis were analyzed by western blot. The results revealed S. digitata protein bands measuring 65, 34, 22, and 18 kDa in molecular weight. In particular, the 18 kDa band is a possible candidate for clinical immunodiagnosis on the basis of western blot findings. PMID:27073332

  15. Northern employment

    Hiring practices and policies and employment opportunities that were available in the Beaufort Sea and MacKenzie Delta project for local residents and for people from southern Canada were dealt with in this chapter. Depending on the source, Northern hiring was a mere token, or a genuine and successful effort on the part of the companies to involve the native population and to share with them the benefits of the project. The fact remains that opening up job opportunities for Northerners was not easily attained, and would never have been realized without the involvement of government and community organizations. Government also played a major role in developing policies and training regimes. By the end of exploration operations, the hiring of Northern residents in the oil and gas industry had become a requirement of drilling applications. Training programs were also created to ensure that Northern residents received the means necessary to take advantage of Northern employment opportunities


    V G Barskova


    Full Text Available There are currently no accepted criteria for positive Western blots in Russian patients with Lyme borreliosis. The purpose of the current study was to develop criteria for a positive IgG westem-blot to aid particularly in the diagnosis of patients with joint manifestation of the disorder. Patients: 97 with Lyme disease, 145 - control subjects. IgG antibody responses were determined to 3 species ofB.burgdorferi sensu lato by Western blotting, using blots prepared by manufacturer. The best discriminatory ability of test criteria was chained by requiring any 3 of 11 IgG bands, a definition that could be used with B. burgdorferi sensu stricto, B.garinii and B.afzelii strains. With these 3 antigen preparation, positive IgG blots were found in 0 to 18% of patients with localized erythema migrans of < 4 weeks duration, 23 to 39% of those with disseminated infection < 20 weeks duration, and in 39 to 46% of those with late arthritis/arthralgia of >6 months duration the specificity was 93 to 99%. Thus, IgG Western blotting may bring greater specificity to serologic testing in Lyme borreliosis, but the sensitivity is limited.

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

    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

  18. HTLV-I/II seroindeterminate Western blot reactivity in a cohort of patients with neurological disease.

    Soldan, S S; Graf, M D; Waziri, A; Flerlage, A N; Robinson, S M; Kawanishi, T; Leist, T P; Lehky, T J; Levin, M C; Jacobson, S


    The human T-cell lymphotropic virus type I (HTLV-I) is associated with a chronic, progressive neurological disease known as HTLV-I-associated myelopathy/tropical spastic paraparesis. Screening for HTLV-I involves the detection of virus-specific serum antibodies by EIA and confirmation by Western blot. HTLV-I/II seroindeterminate Western blot patterns have been described worldwide. However, the significance of this blot pattern is unclear. We identified 8 patients with neurological disease and an HTLV-I/II seroindeterminate Western blot pattern, none of whom demonstrated increased spontaneous proliferation and HTLV-I-specific cytotoxic T lymphocyte activity. However, HTLV-I tax sequence was amplified from the peripheral blood lymphocytes of 4 of them. These data suggest that patients with chronic progressive neurological disease and HTLV-I/II Western blot seroindeterminate reactivity may harbor either defective HTLV-I, novel retrovirus with partial homology to HTLV-I, or HTLV-I in low copy number. PMID:10438355

  19. Optimized semi-quantitative blot analysis in infection assays using the Stain-Free technology.

    Zeitler, Anna F; Gerrer, Katrin H; Haas, Rainer; Jiménez-Soto, Luisa F


    Western blots are a commonly used method for protein detection and quantification in biological samples. Compensation of loading variations is achieved by housekeeping protein (HKP) normalization and/or total protein normalization (TPN). However, under infection conditions, HKP normalization, traditionally used in cell biology for quantification of western blots, can be problematic. Binding of microbes to target cells via specific receptors can induce signal transduction events resulting in drastic changes in the level of expression of HKPs. Additionally, samples collected after infection assays will include cellular and microbial proteins altering the analysis with TPN. Here we demonstrate under experimental infection conditions, how a reliable semi-quantitative analysis of proteins in western blots can be achieved using the Stain-Free technology. PMID:27150675

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

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


    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…

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

    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

  2. A new analytical solution to axisymmetric Blot's consolidation of a finite soil layer


    A new analytical method is presented to study the axisymmetric Blot's consolidation of a finite soil layer. Starting from the governing equations of axisymmetric Blot's consolidation, and based on the property of Laplace transform, the relation of basic variables for a point of a finite soil layer is established between the ground surface (z= 0) and the depth z in the Laplace and Hankel transform domains. Combined with the boundary conditions of the finite soil layer, the analytical solution of any point in the transform domain can be obtained. The actual solution in the physical domain can be obtained by inverse Laplace and Hankel transforms. A numerical analysis for the axisymmetric consolidation of a finite soil layer is carried out.

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

    Carthew, James; Karakesisoglou, Iakowos


    Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (>800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins. PMID:27147045

  4. Western blot analysis of the human antibody response to Campylobacter jejuni cellular antigens during gastrointestinal infection.

    Nachamkin, I; Hart, A. M.


    Western blot analysis was used to identify antigenic components of Campylobacter jejuni whole cells and outer membranes that elicit antibody responses in patients with campylobacter enteritis. Acute- and convalescent-phase sera from eight patients were analyzed for antibody activity against their homologous infecting strains and heterologous clinical isolates. Whole-cell and Sarkosyl-insoluble membrane components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ...

  5. Strategies for laboratory HIV testing: an examination of alternative approaches not requiring Western blot.

    Sato, P. A.; Maskill, W. J.; Tamashiro, H.; Heymann, D L


    Advances in laboratory tests for antibodies to human immunodeficiency virus (HIV) have permitted the development of alternative HIV testing strategies that do not require use of the Western blot approach. Three strategies are proposed. In strategy I, sera are tested for HIV antibody using an enzyme-linked immunosorbent assay (ELISA)/rapid/simple (ERS) test; in strategy II, sera reactive in an initial ERS test are retested using a second ERS test; strategy III involves retesting with a third E...

  6. Detection and Identification of Eight Trichinella Genotypes by Reverse Line Blot Hybridization

    Rombout, Y. B.; Bosch, S.; van der Giessen, J.W.B


    A reverse line blot (RLB) assay was developed to identify different Trichinella genotypes. The RLB assay accomplishes detection and specific identification of the different Trichinella genotypes and relies on hybridization of the amplified 5S ribosomal DNA intergenic spacer regions to specific, membrane-bound oligonucleotide probes. After one single amplification, we were able to detect and genetically identify six sibling species, i.e., T. spiralis, T. britovi, T. nativa, T. murrelli, T. nel...

  7. Evaluation of an adaptive virtual laboratory environment using Western Blotting for diagnosis of disease

    Polly, Patsie; Marcus, Nadine; Maguire, Danni; Belinson, Zack; Gary M Velan


    Background Providing large numbers of undergraduate students in scientific disciplines with engaging, authentic laboratory experiences is important, but challenging. Virtual laboratories (vLABs) are a potential means to enable interactive learning experiences. A vLAB focusing on Western Blotting was developed and implemented in a 3rd year undergraduate Pathology course for science students to facilitate learning of technical molecular laboratory skills that are linked to development of diagno...

  8. Evaluation of western blotting for the diagnosis of enzootic bovine leukemia

    Gonzalez E.T.; Oliva G.A.; Norimine J.; Cid de la Paz V.; Echeverría M.G.


    A western blotting (WB) procedure has been developed for detecting antibodies to bovine leukosis virus (BLV) in cattle sera. Two hundred and thirty three serum samples from naturally infected cattle with BLV virus and serial bleedings from experimentally BLV infected cows were used. An agar gel immunodiffusion test (AGID) was used for comparing with the results obtained by WB. The AGID positive sera showed a different degree of reactivity by WB test against the two most important viral antige...

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

    Yukihiro Shoyama


    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.

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

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


    Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin. PMID:19779698


    刘跃华; 王家璧; 司静懿


    This study differentiated pseudocondyloma of vulva from condyloma acunainata using dot blot hybridization and polymerase chain reaction (PCR). A total of 27 cases o{ pseudocondyloma of vulva and 65 cases of condyloma acuminata were selected for the sttldy. The genital lesions were examined clinically and were biopsled. Each biopsy v-as subjected to histological examination and HPV DNA analysis by dot blot hybridization and PCR. Dot blot analysis detected HPV DNA in 19(82.6%) out of 23 cases of condyloma acuminata and 2(25%) out of 8 cases pseudocondyloma of vulvae(P<0. 05). PCR detected HPV DNA in 51(92.7%) our of 55 cases of eondyloma acuminata, compared with none in 23 cases of pseudocondylorna(P<0. 001). HPV DNA was present in the majority of condyloma acuminata specimens, HPV 6 and 11 were the predominant types. Peudocondyloma is probably not associated with HPV. PCR was the most sensitive and useful techntque for HPV DNA detection.

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

    Tappe D


    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.

  13. Positive IgG Western Blot for Borrelia burgdorferi in Colombia

    Palacios Ricardo


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

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

    de Souza Marcilia MA


    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.

  15. Fluorescent detection of Southern blots and PCR-based genetic typing tests

    Mansfield, E.S.; Worley, J.M. [Molecular Dynamics, Inc., Sunnyvale, CA (United States); Zimmerman, P.A. [Laboratory of Parasitic Diseases, Bethesda, MD (United States)] [and others


    The Southern blot is used to study gene organization, to identify disease-causing genomic rearrangements, or for typing RFLP markers in forensic, paternity, or prenatal diagnostic testing. Fluorescence offers a much greater dynamic range and a more linear response than film used in radioactive or chemiluminescent detection of RFLPs. We therefore investigated using the Fluorimager{trademark} 575 (Molecular Dynamics, Inc.) for analyzing Southern blots. Using a single-locus probe to D2S44 (YNH24) (Promega Corp.), we detect as little as 100 ng (0.05 attomole) genomic DNA. The alkaline phosphatase-labeled probe is detected using AttoPhos (JBL Scientific), and the developed membrane is scanned with the Fluorimager. Biotinylated hybridization probes can also be developed using a streptavidin-alkaline phosphatase conjugate and AttoPhos. The instrument scan parameters can be adjusted to prevent overexposure and accompanying loss of resolution in images of blots, gels, or 96-well microplates. We have used these other sample formats in PCR-based genetic typing assays. We use FluorKit DQS (Molecular Dynamics) to accurately quantify PCR template DNA (1-500 ng) in 96-well microplates scanned using the same instrument. Mutation detection assays run include heteroduplex gels (5% polyacrylamide, 2.7 M urea), short tandem repeat (STR) markers, amplified fragment length polymorphisms (AmpFLP), competitive priming PCR, and allele-specific oligotyping. These assays are run using either 1- or 2-color labeling. We detect unlabeled PCR products, such as the AmpFLP marker D1S80 (Perkin-Elmer) by post-staining gels for 10 minutes with SYBR Green 1 (Molecular Probes) and scanning the wet gel. The Fluorimager scans a 20 x 25 cm sample within three minutes, allowing rapid optimization of fluorescent protocols and high sample throughput.

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

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


    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.


    SATO Neuza Satomi; HIRATA Mário H.; HIRATA Rosário D.C.; Lia Carmen M.S. ZERBINI; Edilene P.R. SILVEIRA; MELO Carmem Silvia de; Ueda, Mirthes


    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-rT...

  18. Reverse Line Blot Assay for Direct Identification of Seven Streptococcus agalactiae Major Surface Protein Antigen Genes

    Zhao, Zuotao; Kong, Fanrong; Gilbert, Gwendolyn L.


    We developed a multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB) to detect the genes encoding members of the family of variable surface-localized proteins of Streptococcus agalactiae (group B streptococcus [GBS]), namely, Bca (Cα), Rib, Epsilon (Epsilon/Alp1/Alp5), Alp2, Alp3, and Alp4, and the immunoglobulin A binding protein, Bac (Cβ). We used the assay to identify these genes in a collection of well-characterized GBS isolates and reference strains. The results showed tha...

  19. Zinc Blotting Assay for Detection of Zinc-Binding Prolamin in Barley (Hordeum vulgare) Grain

    Uddin, Mohammad Nasir; Langkilde, Ane; Vincze, Éva


    -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......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...... zinc blotting method with a zinc-sensing dye, dithizone. Hordeins were extracted from mature barley grain, separated by SDS-PAGE, blotted on a membrane, renatured, overlaid, and probed with zinc; subsequently, zinc-binding specificity of certain proteins was detected either by autoradiography or color...

  20. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting for protein antigen analysis

    Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) has now become a standard tool in most laboratories for protein analysis and purification. Several SDS-PAGE systems have been described but the most widely used one is the discontinuous buffer system introduced for disc gel electrophoresis. Western blotting or the process of transfer of the electrophoretically separated proteins onto immobilizing matrices such as nitrocellulose membrane is an extension of SDS-PAGE system and provides, on the nitrocellulose blot, an identical copy of the electrophoretic separation pattern of the proteins present in the gels. The immobilized proteins can be further reacted with an appropriate probe such as antibody for identification of its corresponding antigen. The protein antigen/antibody complex is then detected by using radioactively labelled or enzyme-linked second antibody probe. The technique is very useful for analysis and characterization of complex protein antigens using immune sera from several sources or vice versa. The protocol given presented here illustrates such a separation by which complex protein antigens of blood stages of Plasmodium vivax obtained from blood of patients with vivax malaria are fractionated by SDS-PAGE and treated with immune sera from patients with acute vivax malaria and the antigen/antibody complex formed are detected by 125I-labelled anti-human immunoglobulins. 5 refs, 2 figs

  1. Monoclonal Antibodies against Small Molecule Natural Products and Their Applications, Eastern Blotting and Knockout Extract

    Yukihiro Shoyama


    Full Text Available To determine the hapten number in hapten-carrier protein conjugate matrix-assisted laser desorption/ionization (MALDI tof mass spectrometry was applied. Highly specific anti-ginsenoside Rb1 and Rg1 monoclonal antibodies (MAbs were prepared. Ginsenosides were developed on thin layer chromatography (TLC plates which were covered by a polyvinylidene difluoride (PVDF membrane resulting in blotting. The membrane was treated with NaIO4 solution to release the aldehyde group on the sugar moiety of the ginsenosides. By treatment of the membrane with a protein solution the ginsenoside-protein conjugation as a Schiff-base occurred, which can function to fix it to the PVDF membrane. A part of the ginsenoside aglycone was reacted with anti-ginsenoside Rb1 MAb, secondary MAb conjugated with enzyme and finally a substrate was added, resulting in a specific and highly sensitive staining that we named Eastern blotting. Furthermore, it makes one-step isolation of ginsenoside Rb1 possible using an immuno-affinity column conjugated with anti-ginsenoside Rb1 MAb. Furthermore, immunoaffinity concentration was carried out allowing high sensitivity analysis of lower concentrations of ginsenoside Rb1 so that several unknown bands could be structurally determined.

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

    N.S. Sato


    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.

  3. Detection of Autophagy in Caenorhabditis elegans by Western Blotting Analysis of LGG-1.

    Palmisano, Nicholas J; Meléndez, Alicia


    A common way to measure the induction of autophagy in yeast and mammalian cells is to compare the amount of Atg8/LC3-I with that of Atg8-PE/LC3-II by using western blot analysis. This is because changes in the amount of LC3-II correlate closely with changes in the number of autophagosomes present in cells. Atg8/LC3 is initially synthesized as an unprocessed form, which is proteolytically processed to form Atg8/LC3-I, and then this is modified into the phosphatidylethanolamine (PE)-conjugated Atg8-PE/LC3-II form. Atg8/LC3-II is membrane bound, whereas Atg8-PE/LC3-I is cytosolic. By associating with both the inner and outer membranes of the autophagosome, Atg8-PE/LC3-II is the only autophagy reporter that is reliably associated with completed autophagosomes. In the nematode Caenorhabditis elegans, the ortholog of Atg8/LC3 is LGG-1. Here, we discuss how changes in the levels of LGG-1-II (and the paralog LGG-2) protein can, with appropriate controls, be used to monitor autophagy activity in nematodes and present a protocol for monitoring changes in the protein levels of different forms of LGG-1 by western blotting. PMID:26832685

  4. Western Blot Detection of Human Anti-Chikungunya Virus Antibody with Recombinant Envelope 2 Protein.

    Yang, Zhaoshou; Lee, Jihoo; Ahn, Hye-Jin; Chong, Chom-Kyu; Dias, Ronaldo F; Nam, Ho-Woo


    Chikungunya virus (CHIKV), a tropical pathogen, has re-emerged and has massive outbreaks abruptly all over the world. Containing many dominant epitopes, the envelope E2 protein of CHIKV has been explored for the vaccination or diagnosis. In the present study, the antigenicity of a recombinant expressed intrinsically disorder domain (IUD) of E2 was tested for the detection of the antibody against CHIKV through western blot method. The gene of the IUD of E2 was inserted into 2 different vectors and expressed as recombinant GST-E2 and recombinant MBP-E2 fusion protein, respectively. Two kinds of fusion proteins were tested with 30 CHIKV patient sera and 30 normal sera, respectively. Both proteins were detected by 25 patients sera (83.3%) and 1 normal serum (3.3%). This test showed a relatively high sensitivity and very high specificity of the recombinant E2 proteins to be used as diagnostic antigens against CHIKV infection. PMID:27180586

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

    Bowlin Gary L


    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.

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

    Sydney Correia Leão


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

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

    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

  8. Development of an improved method for quantitative analysis of skin blotting: Increasing reliability and applicability for skin assessment

    Ogai, Kazuhiro; Matsumoto, Masaru; Minematsu, T; Kitamura, Keiichiro; Kobayashi, M.; Sugama, Junko; Sanada, Hiromi


    Objective A novel skin assessment tool named 'skin blotting' has been recently developed, which can easily predict the skin status to avoid its deterioration. The aim of this study was to propose a normalization method for skin blotting to compensate for individual differences that can hamper the quantitative comparisons and clinical applications. Methods To normalize individual differences, we utilized a total protein as a 'normalizer' with calibration curves. For evaluation, we performed a ...

  9. Application of Western Blotting for the Immunodiagnosis of Fasciola hepatica in Cattle Using Excretory/Secretory Antigens



    In this study, protein bands of excretory/secretory antigens of Fasciola hepatica were determined using SDS-PAGE and Western blotting. Blood and faecal samples were obtained from cattle brought to Kazan slaughterhouse. After examining the organs and faecal samples of these cattle for Fasciola hepatica and other helminths, serum samples were divided into two groups as positive (10 cattle) and negative (5 cattle) for F. hepatica. The sera of these two groups were tested using Western blotting. ...

  10. [Evaluation of IHA, ELISA and Western Blot tests in diagnosis of pulmonary cystic hidatidosis].

    Akisu, Ciler; Bayram Delibaş, Songül; Yuncu, Gökhan; Aksoy, Umit; Ozkoç, Soykan; Biçmen, Can; Sevinç, Serpil; Yaldiz, Sadik


    Pulmonary cystic hidatidosis caused by the larval stages of Echinococcus granulosus is a common parasitic disease in Turkey and throughout the world. In this study IHA, ELISA and Western Blot (WB) tests were performed with a panel of 59 sera from 31 surgically confirmed pulmonary cystic hidatidosis patients, 18 patients with pulmonary disease other than cystic hidatidosis and 10 healthy individual. The overall sensitivity of the IHA, ELISA and WB tests used for the serodiagnosis of pulmonary cystic hidatidosis were found as 96.7%, 87.1%, 100% and the specificities were 82.2%, 89.2% and %85.7, respectively. Using the WB test 8-12 kDa, 24 kDa and 124 kDa bands were detected as valuable for surgically confirmed patients' sera. One or more of these bands were also detected in sera of four patients with other pulmonary diseases false-positively. In conclusion conventional serologic test like IHA and ELISA is valuable in diagnosis of pulmonary cystic hidatidosis, also evaluation of some specific bands in WB would contribute to the diagnosis. PMID:16100652

  11. Analysis of common mitochondrial DNA mutations by allele-specific oligonucleotide and Southern blot hybridization.

    Tang, Sha; Halberg, Michelle C; Floyd, Kristen C; Wang, Jing


    Mitochondrial disorders are clinically and genetically heterogeneous. There are a set of recurrent point mutations in the mitochondrial DNA (mtDNA) that are responsible for common mitochondrial diseases, including MELAS (mitochondrial encephalopathy, lactic acidosis, stroke-like episodes), MERRF (myoclonic epilepsy and ragged red fibers), LHON (Leber's hereditary optic neuropathy), NARP (neuropathy, ataxia, retinitis pigmentosa), and Leigh syndrome. Most of the pathogenic mtDNA point mutations are present in the heteroplasmic state, meaning that the wild-type and mutant-containing mtDNA molecules are coexisting. Clinical heterogeneity may be due to the degree of mutant load (heteroplasmy) and distribution of heteroplasmic mutations in affected tissues. Additionally, Kearns-Sayre syndrome and Pearson syndrome are caused by large mtDNA deletions. In this chapter, we describe a multiplex PCR/allele-specific oligonucleotide (ASO) hybridization method for the screening of 13 common point mutations. This method allows the detection of low percentage of mutant heteroplasmy. In addition, a nonradioactive Southern blot hybridization protocol for the analysis of mtDNA large deletions is also described. PMID:22215554

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

    Parra-Belky, Karlett


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

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

    William H Roldán


    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.

  14. Rapid Detection of Rifampin-resistant Clinical Isolates of Mycobacterium tuberculosis by Reverse Dot Blot Hybridization

    GUO Qian; WAN Kang Lin; YU Yan; ZHU Yan Ling; ZHAO Xiu Qin; LIU Zhi Guang; ZHANG Yuan Yuan; LI Gui Lian; WEI Jian Hao; WU Yi Mou


    Objective A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in‘hot mutation region’ of Mycobacterium tuberculosis (M. tuberculosis). Methods 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. Results The sensitivity and specificity of the RDBH assay were 91.2%(165/181) and 98.3%(117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7%(293/300), 98.2%(164/167), and 97.0%(129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. Conclusion Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.

  15. Evaluation of western blotting for the diagnosis of enzootic bovine leukemia

    Gonzalez E.T.


    Full Text Available A western blotting (WB procedure has been developed for detecting antibodies to bovine leukosis virus (BLV in cattle sera. Two hundred and thirty three serum samples from naturally infected cattle with BLV virus and serial bleedings from experimentally BLV infected cows were used. An agar gel immunodiffusion test (AGID was used for comparing with the results obtained by WB. The AGID positive sera showed a different degree of reactivity by WB test against the two most important viral antigens (gp51 and p24, or against one of them. Other proteins (gp30, p15, p12 and p10 were not detected with any AGID positive sera, being observed occasionally three bands corresponding to the p24 protein. Using sera obtained by BLV experimental inoculation, the antibodies directed to p24 appeared early (between the 2nd and 4th week post inoculation and thereafter antibodies to gp51were detected in some animals. The analysis of field serum samples by AGID as compared to WB showed an agreement of 90.9%. Only 1.7% of sera were negative by AGID and positive by WB and 7.2% that were not conclusive by AGID and were defined by WB (4.2% as positive and 3.0% as negative.

  16. Rapid detection of intestinal pathogens in fecal samples by an improved reverse dot blot method

    Jian-Ming Xing; Su Zhang; Ying Du; Dan Bi; Li-Hui Yao


    AIM:To develop a new, rapid and accurate reverse dot blot (RDB) method for the detection of intestinal pathogens in fecal samples.METHODS:The 12 intestinal pathogens tested were Salmonella spp., Brucella spp., Escherichia coli O157:H7,Clostridium botulinum, Bacillus cereus,Clostridium perfringens, Vibrio parahaemolyticus,Shigella spp., Yersinia enterocolitica, Vibrio cholerae,Listeria monocytogenes and Staphylococcus aureus.The two universal primers were designed to amplify two variable regions of bacterial 16S and 23S rDNA genes from all of the 12 bacterial species tested. Five hundred and forty fecal samples from the diarrhea patients were detected using the improved RDB assay.RESULTS:The methods could identify the 12 intestinal pathogens specifically, and the detection limit was as low as 103 CFUs. The consistent detection rate of the improved RDB assay compared with the traditional culture method was up to 88.75%.CONCLUSION:The hybridization results indicated that the improved RDB assay developed was a reliable method for the detection of intestinal pathogen in fecal samples.

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

    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 32P dCTP and 32P dATP to a specific activity greater then 1.0 x 109 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

  18. Immunodot blot assay to detect Helicobacter pylori using monoclonal antibodies against the 26 kDa protein.

    Amini Najafabadi, Hossein; Paknejad, Maliheh; Farshad, Shohreh; Mohammadian, Taher; Seyyed Ebrahimi, Shadi Sadat; Amini Najafabadi, Azadeh


    Development of a specific immunoassay to detect Helicobacter pylori infection in stool samples requires monoclonal antibody against the specific antigen. The aims of this study were to establish monoclonal antibodies against the 26 kDa protein of H. pylori and develop an immunodot blot for their application to recognize H. pylori infection using stool samples. Mice were immunized intraperitoneally with homogenized gel containing the 26 kDa band of cell surface proteins of H. pylori in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The monoclonal antibodies were produced using the hybridoma technique. Reactivity of monoclonal antibodies was tested with the purified 26 kDa antigen and cell surface proteins from cultured H. pylori by ELISA. Furthermore reactivity of monoclonal antibodies was tested on negative and positive stool samples for H. pylori and suspensions of several major bacteria in stool by immunodot blot assay. Five stable hybridoma monoclones were obtained. The concordant reactivity of the monoclonal antibodies with H. pylori present in the stool samples, which had been tested previously using an ACON ELISA kit for H. pylori stool antigen testing, and unreactivity with several different major fecal bacteria in immunodot blotting indicates high specificity of the immunodot blot based on the reaction of produced monoclonal antibodies with the H. pylori antigen in stools. The findings indicate that the novel immunodot blot developed based on new monoclonal antibodies for stool antigens would be useful as a noninvasive method of diagnosing H. pylori infection. PMID:23244318

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

    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

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

    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 cm2, a spatial resolution of 0.3-1 mm (depending on the field of view) and a sensitivity sufficient to detect 10 fg/mm2 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 104 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/mm2 - 100 pg/mm2. 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 chemiluminescent

  1. Evaluation of western blotting for the diagnosis of enzootic bovine leukemia Avaliação da técnica de western blot no diagnóstico da leucose enzoótica bovina

    E.T. Gonzalez; G.A. Oliva; Norimine, J; V. Cid de la Paz; M. G. Echeverría


    A western blotting (WB) procedure has been developed for detecting antibodies to bovine leukosis virus (BLV) in cattle sera. Two hundred and thirty three serum samples from naturally infected cattle with BLV virus and serial bleedings from experimentally BLV infected cows were used. An agar gel immunodiffusion test (AGID) was used for comparing with the results obtained by WB. The AGID positive sera showed a different degree of reactivity by WB test against the two most important viral antige...

  2. Analysis of cell wall extracts of Candida albicans by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques.

    Ponton, J; J. M. Jones


    Cell walls of intact yeast- and mycelial-phase Candida albicans B311 were extracted with different compounds: dithiothreitol, dithiothreitol with protease, dithiothreitol with lyticase, and dithiothreitol with protease followed by beta-glucuronidase with chitinase. Extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot techniques. Dithiothreitol extracts contained the most satisfactory array of components for study. Analysis of these extracts demo...

  3. Validity of Interpretation Criteria for Standardized Western Blots (Immunoblots) for Serodiagnosis of Lyme Borreliosis Based on Sera Collected throughout Europe

    Hauser, Ulrike; Lehnert, Gisela; Wilske, Bettina


    Western blotting (WB; immunoblotting) is a widely used tool for the serodiagnosis of Lyme borreliosis (LB), but so far, no generally accepted criteria for performance and interpretation have been established in Europe. The current study was preceeded by a detailed analysis of WB with whole-cell lysates of three species of Borrelia burgdorferi sensu lato (U. Hauser, G. Lehnert, R. Lobentanzer, and B. Wilske, J. Clin. Microbiol. 35:1433–1444, 1997). In that study, interpretation criteria for a ...

  4. Simple and Sensitive Detection of HBsAg by Using a Quantum Dots Nanobeads Based Dot-Blot Immunoassay

    Zhang, Pengfei; Lu, Huiqi; Chen, Jia; HAN, HUANXING; MA, Wei


    Simple and sensitive detection of infectious disease at an affordable cost is urgently needed in developing nations. In this regard, the dot blot immunoassay has been used as a common protein detection method for detection of disease markers. However, the traditional signal reporting systems, such as those using enzymes or gold nanoparticles lack sensitivity and thus restrict the application of these methods for disease detection. In this study, we report a simple and sensitive detection meth...

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

    Wiśniewski, Jacek R; Mann, Matthias


    Proteomics and other protein-based analysis methods such as Western blotting all face the challenge of discriminating changes in the levels of proteins of interest from inadvertent changes in the amount loaded for analysis. Mass-spectrometry-based proteomics can now estimate the relative and absolute amounts of thousands of proteins across diverse biological systems. We reasoned that this new technology could prove useful for selection of very stably expressed proteins that could serve as better loading controls than those traditionally employed. Large-scale proteomic analyses of SDS lysates of cultured cells and tissues revealed deglycase DJ-1 as the protein with the lowest variability in abundance among different cell types in human, mouse, and amphibian cells. The protein constitutes 0.069 ± 0.017% of total cellular protein and occurs at a specific concentration of 34.6 ± 8.7 pmol/mg of total protein. Since DJ-1 is ubiquitous and therefore easily detectable with several peptides, it can be helpful in normalization of proteomic data sets. In addition, DJ-1 appears to be an advantageous loading control for Western blot that is superior to those used commonly used, allowing comparisons between tissues and cells originating from evolutionarily distant vertebrate species. Notably, this is not possible by the detection and quantitation of housekeeping proteins, which are often used in the Western blot technique. The approach introduced here can be applied to select the most appropriate loading controls for MS-based proteomics or Western blotting in any biological system. PMID:27297043

  6. Comparison of Crude and Excretory/Secretory Antigens for the Diagnosis of Fasciola hepatica in Sheep by Western Blotting

    GÖNENÇ, Bahadır; SARIMEHMETOĞLU, Hıfsı Oğuz


    Crude and excretory/secretory (E/S) antigens of Fasciola hepatica were subjected to SDS-PAGE and Western blot analysis in order to identify protein bands that would enable the specific and sensitive immunodiagnosis of sheep. Sera from 20 sheep with natural infections of F. hepatica, Dicrocoelium dendriticum, Cyst hydatid, Cysticercus tenuicollis, Trichostrongylidae, Paramphistomum spp., and Trichuris spp. and the same number of sera naturally infected with the same helminths without F. hepati...

  7. Stain Free Total Protein Staining is a Superior Loading Control to β-Actin for Western Blots

    Gilda, Jennifer E.; Aldrin V. Gomes


    Semi-Quantification of proteins using Western blots typically involves normalization against housekeeping genes such as β-actin. More recently, ponceau S and Coomassie blue staining have both been shown to be suitable alternatives to housekeeping genes as loading controls. Stain free total protein staining offers the advantage of no staining or destaining steps. Evaluation of the use of Stain free staining as an alternative to β-actin or the protein stain ponceau S showed that Stain free stai...

  8. Investigation of Anti-Toxocara Antibodies in Epileptic Patients and Comparison of Two Methods: ELISA and Western Blotting

    Mohammad Zibaei


    Full Text Available The relationship between Toxocara infection and epilepsy was previously demonstrated by several case-control studies and case reports. These previous studies were often based on the enzyme-linked immunosorbent assay (ELISA using Toxocara excretory-secretory antigens, which are not specific due to cross-reactivity with other parasitic infections such as ascariasis, trichuriasis, and anisakiasis. An immunoblot analysis is highly specific and can detect low levels of Toxocara antibodies. Therefore, this assay may be useful in the identification of toxocariasis in epileptic patients. We examined patients who had epilepsy and healthy subjects for seropositivity for Toxocara infection by ELISA and Western blotting. Out of 85 epileptic patients, 10 (11.8% and 3 (3.5% persons exhibited Toxocara immunoglobulin G (IgG antibodies responses by ELISA and by both techniques, respectively. Moreover, in the healthy group (, 3 (3.5% persons were positive by ELISA, but none was detected by Western blotting. This study indicates that Toxocara infection is a risk factor for epilepsy in Iran. These findings strongly suggest the need to perform Western blotting immunodiagnosis, as well as the ELISA using Toxocara excretory-secretory antigens, to improve diagnosis of human toxocariasis in patients with epilepsy.

  9. Genetics Home Reference: Northern epilepsy

    ... Understand Genetics Home Health Conditions Northern epilepsy Northern epilepsy Enable Javascript to view the expand/collapse boxes. Download PDF Open All Close All Description Northern epilepsy is a genetic condition that causes recurrent seizures ( ...

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

    Christensen, C B; Theander, T G


    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...

  11. Dot-Blot Hybridization for Detection of Five Cucurbit Viruses by Digoxigenin-Labelled cDNA Probes

    MENG Juan; GU Qin-sheng; LIN Shi-ming; PENG Bin; LIU Li-feng; TIAN Yan-ping; LI Li


    Dot-blot hybridization was applied in this paper to detect five viruses infecting cucurbitaceous crops,Zuccini yellow mosaic virus(ZYMV),Watermelon mosaic virus(WMV),Cucumber mosaic virus(CMV),Papaya ringspot virus watermelon strain(PRSV-W)and Squash mosaic virus(SqMV),as a good alternative assay in seed health test and epidemiological and transgenic research.Digoxigenin-labelled cDNA probes of the five viruses were synthesized by PCR with the specific primers and applied in dot-blot hybridization to detect five viruses in crude extraction of the infected leaves.And three SqMV probes of different lengths(0.55,1.6,and 2.7 kb,respectively)were designed to investigate the effect of hybridization.The results showed that the sensitivity for detecting the crude extraction of infected leaves by ZYMV,WMV,CMV,PRSV-W,and SqMV was down to 1:160,1:160,1:320,1:160,and 1:320,respectively.Three SqMV probes of different length showed no differences on the sensitivity and specificity.The digoxigenin-labelled probes prepared by PCR could be used for accurate and rapid identification of 5 viruses infecting cucurbitaceous crops with good stabilities,sensitivities,specificity,and reproducibilities.

  12. Simple and sensitive detection of HBsAg by using a quantum dots nanobeads based dot-blot immunoassay.

    Zhang, Pengfei; Lu, Huiqi; Chen, Jia; Han, Huanxing; Ma, Wei


    Simple and sensitive detection of infectious disease at an affordable cost is urgently needed in developing nations. In this regard, the dot blot immunoassay has been used as a common protein detection method for detection of disease markers. However, the traditional signal reporting systems, such as those using enzymes or gold nanoparticles lack sensitivity and thus restrict the application of these methods for disease detection. In this study, we report a simple and sensitive detection method for the detection of infectious disease markers that couples the dot-blot immunoassay with quantum dots nanobeads (QDNBs) as a reporter. First, the QDNBs were prepared by an oil-in-water emulsion-evaporation technique. Because of the encapsulation of several QDs in one particle, the fluorescent signal of reporter can be amplified with QDNBs in a one-step test and be read using a UV lamp obviating the need for complicated instruments. Detection of disease-associated markers in complex mixture is possible, which demonstrates the potential of developing QDNBs into a sensitive diagnostic kit. PMID:24505238

  13. Immunohistochemical and Western Blotting Analyses of Ganoine in the Ganoid Scales of Lepisosteus oculatus: an Actinopterygian Fish.

    Sasagawa, Ichiro; Oka, Shunya; Mikami, Masato; Yokosuka, Hiroyuki; Ishiyama, Mikio; Imai, Akane; Shimokawa, Hitoyata; Uchida, Takashi


    In order to compare its characteristics with those of jaw tooth collar enamel, normally developing and experimentally regenerating ganoine from ganoid scales of Lepisosteus oculatus (spotted gar), an actinopterygian fish species, was examined by Western blotting and immunohistochemistry. Amelogenin, a major enamel matrix protein (EMP), is widely found from sarcopterygian fish to mammals. Therefore, we used antimammalian amelogenin antibodies and antisera: an antibody against bovine amelogenin; antiserum against porcine amelogenin; and region-specific antibodies or antiserum against the C-terminus, middle region, or N-terminus of porcine amelogenin in this study. Positive immunoreactivity with the antibody against bovine amelogenin, antiserum against porcine amelogenin, and the middle and C-terminal region-specific antibodies was detected in both normally developing and regenerating ganoine matrix, as well as in granules found within inner ganoine epithelial cells. These immunohistochemical analyses indicated that the Lepisosteus ganoine matrix contains EMP-like proteins with epitopes similar to mammalian amelogenins. In Western blotting analyses of regenerating ganoid scales with the antibovine amelogenin antibody, two protein bands with molecular weights of approximately 78 and 65 kDa were detected, which were similar to those found in Lepisosteus tooth enamel. Our study suggests that in Lepisosteus, EMP-like proteins in the ganoine matrix corresponded to those in tooth enamel. However, it was revealed that the 78 and 65 kDa EMP-like proteins were different from 27 kDa bovine amelogenin. PMID:27139791

  14. Identification of Glioblastoma Phosphotyrosine-Containing Proteins with Two-Dimensional Western Blotting and Tandem Mass Spectrometry

    Tianyao Guo


    Full Text Available To investigate the presence of, and the potential biological roles of, protein tyrosine phosphorylation in the glioblastoma pathogenesis, two-dimensional gel electrophoresis- (2DGE- based Western blotting coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS analysis was used to detect and identify the phosphotyrosine immunoreaction-positive proteins in a glioblastoma tissue. MS/MS and Mascot analyses were used to determine the phosphotyrosine sites of each phosphopeptide. Protein domain and motif analysis and systems pathway analysis were used to determine the protein domains/motifs that contained phosphotyrosine residue and signal pathway networks to clarify the potential biological functions of protein tyrosine phosphorylation. A total of 24 phosphotyrosine-containing proteins were identified. Each phosphotyrosine-containing protein contained at least one tyrosine kinase phosphorylation motif and a certain structural and functional domains. Those phosphotyrosine-containing proteins were involved in the multiple signal pathway systems such as oxidative stress, stress response, and cell migration. Those data show 2DGE-based Western blotting, MS/MS, and bioinformatics are a set of effective approaches to detect and identify glioblastoma tyrosine-phosphorylated proteome and to effectively rationalize the biological roles of tyrosine phosphorylation in the glioblastoma biological systems. It provides novel insights regarding tyrosine phosphorylation and its potential role in the molecular mechanism of a glioblastoma.

  15. Mere end blot pirringer

    Jantzen, Christian; Østergaard, Per


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

  16. Northern Lights Chase Tours : Experiences from Northern Norway

    Bertella, Giovanna


    This study is focused on the development of northern lights chase tourism, a particular type of northern lights tourism consisting in guided tours that have the goal to find good views of the northern lights. The theoretical approach is based on the understanding of the northern lights experience as a visual experience, and on the recognition of the tourism practitioners as the driving force to new product development. The empirical case concerns the recent development of northern lights chas...

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

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


    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. PMID:27329815

  18. Detection of human cytomegalovirus by slot-blot hybridization assay employing oligo-primed 32P-labelled probe

    A 32P-labelled Hind III-0 DNA fragment (nine Kilobases; Kb) from human cytomegalovirus AD-169 (HCMV) was used in slot-blot hybridization assay for the detection of HCMV in clinical samples. The results obtained with DNA hybridization assay (DNA HA) were compared with virus isolation using conventional tube cell culture (CTC) and centrifugation vial culture (CVC), immunofluorescence (IF), and complement fixation test (CFT). Of 15 CTC-positive samples, 13 were positive with DNA HA (sensitivity 86.7%). Also, 14 additional samples were DNA HA-positive but CTC-negative. CVC and/or IF confirmed the diagnosis in nine of 14; the remaining five samples were from three patients who showed fourfold rising antibody titre by CFT. Although DNA HA using 32P-labelled probes is relatively cumbersome and expensive, it is a valuable test for quantitation of viral shedding in patients with HCMV infections who may benefit from antiviral therapy

  19. Detection of enteroinvasive Escherichia coli on a colony blot filter paper by hybridization with a 17 kb probe

    Enteroinvasive Escherichia coli (EIEC) and Shigella cause dysentery by invading epithelial cells of the colon. EIEC are difficult to identify by normal biochemical tests in a routine bacteriology laboratory. The genetics of the virulence of Shigella and EIEC are similar. Both contain plasmids of 120-140 megadalton (mDa) that are necessary for virulence. A 17 kb EcoRI digestion fragment of pWR 100, the 140 MDA plasmid of S. flexneri 5 (M9OT), was shown to be specific in differentiating EIEC from non-EIEC. A radiolabelled probe can be easily prepared and used as a specific probe to identify EIEC in either colony or stool blot sample by the hybridization assay. 9 refs, 2 figs

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

    Luiz Claudio Pereira Ribeiro


    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.

  1. Quantitative Western ligand blotting reveals common patterns and differential features of IGFBP-fingerprints in domestic ruminant breeds and species.

    Wirthgen, Elisa; Höflich, Christine; Spitschak, Marion; Helmer, Carina; Brand, Bodo; Langbein, Jan; Metzger, Friedrich; Hoeflich, Andreas


    The insulin-like growth factor binding proteins (IGFBPs) are determinants of local IGF-effects and thus have an impact on growth and metabolism in vertebrate species. In farm animals, IGFBPs are associated with traits such as growth rate, body composition, milk production, or fertility. It may be assumed, that selective breeding and characteristic phenotypes of breeds are related to differential expression of IGFBPs. Therefore, the aim of the present study was to investigate the effects of selective breeding on blood IGFBP concentrations of farm animals. Breeds of the sheep, goat, and cattle species were investigated. IGFBP-3, -2, and -4 were analyzed with quantitative Western ligand blotting (qWLB), enabling comprehensive monitoring of intact IGFBPs with IGF-binding capacity. We show that in sera of all species and breeds investigated, IGFBP-3, -2, and -4 were simultaneously detectable by qWLB analysis. IGFBP-3 and the total amount of IGFBPs were significantly increased (Pcows had higher levels of IGFBP-4 (P<0.05), if compared to conventional crossbreeds of beef cattle. In Dwarf goats the ratio of IGFBP-3/IGFBP-2 was about 3-fold higher than in other goat breeds (P<0.001). The total IGFBP amount of Toggenburg goats was reduced (P<0.05), compared to the other goat breeds. In conclusion, our data indicate that common and specific features of IGFBP fingerprints are found in different ruminant species and breeds. Our findings may introduce quantitative Western ligand blotting as an attractive tool for biomarker development and molecular phenotyping in farm animal breeds. PMID:26597140

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

    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 125I-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

  3. Serologic immunoreactivity to Neospora caninum antigens in dogs determined by indirect immunofluorescence, western blotting and dot-ELISA.

    Pinheiro, A M; Costa, M F; Paule, B; Vale, V; Ribeiro, M; Nascimento, I; Schaer, R E; Almeida, M A O; Meyer, R; Freire, S M


    Neospora caninum, is a coccidian protozoan known as a major cause of bovine abortion and canine neuropathies. The aim of the present study was to develop a reliable and quick test to detect antibodies to N. caninum in dog sera. Sixty-five serum samples from dogs, including 35 positive and 30 negative for N. caninum antibodies were used for standardization of the test. In parallel, immunoreactivity of the sera to Toxoplasma gondii antigens was investigated using a passive agglutination test. A dot-ELISA test, using soluble extract of N. caninum tachyzoites on nitrocellulose ester membranes, was developed and standardized. SDS-PAGE and complementary analysis of reactivity by Western blotting were used for the characterization of the immunoreactive fractions of all tested sera. The sensitivity and specificity of the dot-ELISA were 94 and 73%, respectively, compared to IFAT at a cut-off of 1:50, and 87 and 100% compared to IFAT at a cut-off of 1:25. Among the sera that tested positively for both IFAT and dot-ELISA, only 8.6% were reactive to T. gondii. The most immunoreactive fractions in Western blots were the 14-, 33-, 42- and 55 kDa bands, with percentages of 42, 60, 42 and 37%, respectively. The 60 kDa band showed a non-specific reaction in 43% of neosporosis-negative animals by both dot-ELISA and IFAT. These results indicate that the dot-ELISA using N. caninum antigen present good sensitivity and specificity, and might be used as a screening test to detect antibodies to N. caninum in dogs. PMID:15893072

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

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


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

  5. New method for simultaneous species-specific identification of equine strongyles (nematoda, strongylida) by reverse line blot hybridization.

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


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

  6. Western blotting using Strongyloides ratti antigen for the detection of IgG antibodies as confirmatory test in human strongyloidiasis

    Luciana Pereira Silva


    Full Text Available The present study was conducted to evaluate the frequency of antigenic components recognized by serum IgG antibodies in Western blotting (WB using a Strongyloides ratti larval extract for the diagnosis of human strongyloidiasis. In addition, the WB results were compared to the enzyme-linked immunosorbent assay (ELISA and the indirect immunofluorescence antibody test (IFAT results. Serum samples of 180 individuals were analyzed (80 with strongyloidiasis, 60 with other intestinal parasitoses, and 40 healthy individuals. S. ratti was obtained from fecal culture of experimentally infected Rattus rattus. For IFAT, S. ratti larvae were used as antigen and S. ratti larval antigenic extracts were employed in WB and ELISA. Eleven S. ratti antigenic components were predominantly recognized by IgG antibodies in sera of patients with strongyloidiasis. There was a positive concordance for the three tests in 87.5% of the cases of strongyloidiasis. The negative concordance in the three tests was 94% and 97.5%, in patients with other intestinal parasitoses and healthy individuals, respectively. In cases of positive ELISA and negative IFAT results, diagnosis could be confirmed by WB. ELISA, IFAT, and WB using S. ratti antigens showed a high rate of sensitivity and specificity. In conclusion, WB using S. ratti larval extract was able to recognize 11 immunodominant antigenic components, showing to be a useful tool to define the diagnosis in cases of equivocal serology.

  7. The use of nitrocellulose blotting for the study of hepatitis B surface antigen electrophoresed in agarose gels

    Nitrocellulose-protein blotting of serum electrophoresed in agarose gels has been adapted for the study of hepatitis B surface antigen (HBsAg). 125I-labeled anti-HBs was used as the antigen probe, and the electrophoretic migration was monitored by autoradiography. The method required 3 μl 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). (Auth)

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

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


    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. PMID:22019182

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

    NUNES Cáris Maroni


    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

  10. "Enzyme-Linked Immunotransfer Blot Analysis of Somatic and Excretory- Secretory Antigens of Fasciola hepatica in Diagnosis of Human Fasciolosis"

    MB Rokni


    Full Text Available The liver fluke Fasciola hepatica causes fascioliasis, a liver disease in most part of the world and particularly in north of Iran. Diagnosis of the diseases is anchored in coprological manner but serological methods are preferable due to some obscurities. In this study, sera obtained from human patients infected with Fasciola hepatica were tested by the enzymelinked immunotrotransfer blot (EITB technique with the parasite s somatic and excretory-secretory (ES antigens in order to evaluate the diagnostic potential of the assay. The study included sera from 40 patients infected with F. hepatica, 20 infected with hydatidosis, 6 with toxocariasis, 10 with strongyloidiasis, 10 with amoebiasis, 5 with malaria and 30 normal controls. By this assay, most pf the serum samples from humans with fascioliasis recognized two antigenic polypeptides of 27 and 29 kDa using both antigens. The sensitivity, specificity, positive and negative predictive values for somatic antigen were 91.0%, 96.2%, 95.2% and 92.7% respectively, while these parameters as for ES antigen were 95.2%, 98.0%, 97.5% and 96.2%, correspondingly. Totally, two cases of reactions for the first antigen and one for the latter were verified. The study suggests that the 27 and 29 kDa bands for two antigens in EITB test could be considered for the immunodiagnosis of human fascioliasis.

  11. The diagnosis of proventricular dilatation disease: use of a Western blot assay to detect antibodies against avian Borna virus.

    Villanueva, Itamar; Gray, Patricia; Mirhosseini, Negin; Payne, Susan; Hoppes, Sharman; Honkavuori, Kirsi S; Briese, Thomas; Turner, Debra; Tizard, Ian


    Avian Borna virus (ABV) has recently been shown to be the causal agent of proventricular dilatation disease (PDD) a lethal neurologic disease of captive psittacines and other birds. An immunoblot assay was used to detect the presence of antibodies against avian Borna virus in the serum of affected birds. A lysate from ABV-infected duck embryo fibroblasts served as a source of antigen. The assay was used to test for the presence of antibodies to ABV in 117 birds. Thirty of these birds had biopsy or necropsy-confirmed proventricular dilatation disease (PDD), while the remaining 87 birds were apparently healthy or were suffering from diseases other than PDD. Sera from 27 of the 30 PDD cases (90%) contained antibodies to ABV. Seventy-three (84%) of the apparently "healthy" birds were seronegative. Additionally, sera from seven macaws and one parrot trapped in the Peruvian Amazon were seronegative. Positive sera recognized the bornaviral nucleoprotein (N-protein). While the presence of antibodies to ABV largely corresponded with the development of clinical PDD, 14 apparently healthy normal birds possessed detectable antibodies to ABV. The existence of a carrier state was confirmed when 13 of 15 apparently healthy cockatiels were shown by PCR to have detectable ABV RNA in their feces. Western blot assays may be of significant assistance in diagnosing proventricular dilatation disease. Many apparently healthy birds may however be seronegative while, at the same time, shedding ABV in their feces. PMID:20036080

  12. 1990 Northern, Iran Images

    National Oceanic and Atmospheric Administration, Department of Commerce — A magnitude 7.7 earthquake occurred in the Gilan Province between the towns of Rudbar and Manjil in northern Iran on Thursday, June 21, 1990 (June 20 at 21:00 GMT)....

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

    Jan Jezbera

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

  14. Evaluation of a Western Blot and ELISA for the detection of anti-Trichinella-IgG in pig sera.

    Nöckler, K; Reckinger, S; Broglia, A; Mayer-Scholl, A; Bahn, P


    Human trichinellosis is a foodborne disease caused by ingestion of infective Trichinella muscle larvae via pork or meat of other food animals which are susceptible to this zoonotic parasite. There are new approaches for a risk-oriented meat inspection for Trichinella in pigs which are accompanied by monitoring programmes on herd level to control freedom from this parasite. For this purpose, testing schemes utilizing serological tests with a high sensitivity and specificity are required. This study aimed at the evaluation of an ELISA and a Western Blot (WB) for the detection of anti-Trichinella-IgG in terms of sensitivity and specificity taking results of artificial digestion as gold standard. For this purpose, 144 field sera from pigs confirmed as Trichinella-free as well as 159 sera from pigs experimentally infected with T. spiralis (123), T. britovi (19) or T. pseudospiralis (17) were examined by ELISA (excretory-secretory antigen) and WB (crude worm extract). Sera from pigs experimentally infected with four other nematode species were included to investigate the cross-reactivity of the antigen used in the WB. For all Trichinella-positive pig sera, band pattern profiles were identified in the WB and results were analysed in relation to ELISA OD% values. Testing of pig sera revealed a sensitivity of 96.8% for the ELISA and 98.1% for the WB whereas the methods showed a specificity of 97.9 and 100%, respectively. WB analysis of Trichinella-positive pig sera revealed five specific band patterns of 43, 47, 61, 66, and 102 kDa of which the 43 kDa protein was identified as the predominant antigen. The frequency of the band pattern profile was irrespective of the dose and the period of infection as well as the Trichinella species investigated. In conclusion, monitoring in swine farms for Trichinella antibodies should be based on screening pig sera by means of ELISA followed by confirmatory testing through WB analysis. PMID:19473770

  15. State-of-the-art housekeeping proteins for quantitative western blotting: Revisiting the first draft of the human proteome.

    Lee, Hyun-Gwan; Jo, Jihoon; Hong, Hyun-Hee; Kim, Kee K; Park, Joong-Ki; Cho, Sung-Jin; Park, Chungoo


    Western blotting (WB) analysis is the most popular and widely used methodology for protein detection and characterization over recent decades. In accordance with the advancement of the technologies for the acquisition of WB signals, a quantitative value is used to present the abundance of target proteins in a complex sample, thereby requiring the use of specific proteins as internal references that represent total proteins. Heretofore, proteins encoded by housekeeping genes such as GAPDH, β-tubulin and β-actin have been commonly used as loading controls without any hesitation because their mRNA expression levels tend to be high and constant in many different cells and tissues. Experimentally, however, some of the housekeeping reference proteins are often displayed with inconsistent expression levels in both homogeneous and heterogeneous tissues, and, in terms of mRNA levels, they have a weak correlation to the abundance of proteins. To estimate accurate, reliable, and reproducible protein quantifications, it is crucial to define appropriate reference controls. For this paper, we explored the recently released large-scale, human proteomic database ProteomicsDB including 16 857 liquid chromatography tandem-mass-spectrometry data from 27 human tissues, and suggest 20 ubiquitously- and constitutively-expressed, putative internal-reference controls for the quantification of differential protein expressions. Intriguingly, the most commonly used, known housekeeping genes were entirely excluded in our newly defined candidates. Although the applications of the candidates under many different biological conditions and in other organisms are yet to be empirically verified, we propose reliable, potential loading controls for a WB analysis in this paper. PMID:27125885

  16. Reduction of telomeric repeats as a possible predictor for development of hepatocellular carcinoma: convenient evaluation by slot-blot analysis.

    Isokawa, O; Suda, T; Aoyagi, Y; Kawai, H; Yokota, T; Takahashi, T; Tsukada, K; Shimizu, T; Mori, S; Abe, Y; Suzuki, Y; Nomoto, M; Mita, Y; Yanagi, M; Igarashi, H; Asakura, H


    Hepatocellular carcinoma (HCC) mainly arises from the liver with chronic inflammation. Because telomere reduction reflects replicative history in somatic cells, we analyzed the possibility that liver tissues surrounding HCC consist of the cells carrying substantial reduction of telomere. We studied 20 HCC and surrounding noncancerous liver tissues (SL) obtained by surgical resection, and 10 laparoscopically obtained needle biopsy specimens of the liver with chronic inflammation including no overt HCC (CI). Five liver tissues without chronic liver diseases (ND) were also examined. Extracted genomic DNAs were blotted on a nylon membrane, and probed at first with radio-labeled d(TTAGGG)(3) and reprobed with radio-labeled d(CCT)(7). The intensity caused by d(TTAGGG)(3) was divided by that of d(CCT)(7). The ratio was defined as telomeric repeats content (TC). Dilution experiments reproducibly revealed almost the same TC. The reduction rate of telomere length through aging estimated by regression analysis of TC was 0.62% per year. Concomitant analyses of TC and average telomere length revealed that both values were significantly correlated (r =.45; P =.009). To compare TC in the liver with respect to chronic inflammation, the value was divided by TC in peripheral blood leukocytes (PBL) from the same donor. The ratio was defined as relative TC (RTC). There was a statistically significant decrease of RTC in CI compared with that in ND (P =.03). Furthermore, RTC in SL was significantly lower than that in CI (P =.0001). These observations suggest that RTC value in liver tissues may digitally indicate a replicative history of hepatocytes under chronic inflammation, and a risk of HCC development. PMID:10421648

  17. Northern pipelines : backgrounder

    Most analysts agree that demand for natural gas in North America will continue to grow. Favourable market conditions created by rising demand and declining production have sparked renewed interest in northern natural gas development. The 2002 Annual Energy Outlook forecasted U.S. consumption to increase at an annual average rate of 2 per cent from 22.8 trillion cubic feet to 33.8 TCF by 2020, mostly due to rapid growth in demand for electric power generation. Natural gas prices are also expected to increase at an annual average rate of 1.6 per cent, reaching $3.26 per thousand cubic feet in 2020. There are currently 3 proposals for pipelines to move northern gas to US markets. They include a stand-alone Mackenzie Delta Project, the Alaska Highway Pipeline Project, and an offshore route that would combine Alaskan and Canadian gas in a pipeline across the floor of the Beaufort Sea. Current market conditions and demand suggest that the projects are not mutually exclusive, but complimentary. The factors that differentiate northern pipeline proposals are reserves, preparedness for market, costs, engineering, and environmental differences. Canada has affirmed its role to provide the regulatory and fiscal certainty needed by industry to make investment decisions. The Government of the Yukon does not believe that the Alaska Highway Project will shut in Mackenzie Delta gas, but will instead pave the way for development of a new northern natural gas industry. The Alaska Highway Pipeline Project will bring significant benefits for the Yukon, the Northwest Territories and the rest of Canada. Unresolved land claims are one of the challenges that has to be addressed for both Yukon and the Northwest Territories, as the proposed Alaska Highway Pipeline will travel through traditional territories of several Yukon first Nations. 1 tab., 4 figs

  18. Detection of lipopolysaccharides in Polyacrylamide gels by transfer to nitrocellulose followed by immunoautoradiography with antibody and 125I-protein A: LPS blotting

    Lipopolysaccharides (LPS), which constitute the somatic (O) antigen of gram-negative bacteria, were used to demonstrate the procedure of LPS blotting involving the electrophoretic transfer of electrophoretically resolved LPS from sodium dodecyl sulfate-polyacrylamide gels to nitrocellulose filters. Immobilized LPS could then be immunoautoradiographically visualized in situ by reaction with specific anti-LPS antibody and subsequent binding of radioiodinated Staphylococcus protein A. LPS blotting is expected to provide an efficient and specific means of investigating the LPS (O) antigens of gram-negative bacteria. 18 references

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

    Timms, Mark; Steel, Rohan; Vine, John


    The recombinant human erythropoietins epoetin alfa (Eprex®), darbepoetin (Aranesp®) and methoxy polyethylene glycol-epoetin beta (Mircera®) were administered to greyhounds for 7, 10 and 14 days respectively. Blood and urine samples were collected and analysed for erythropoietin by ELISA, LC-MS/MS and western blotting. Limits of confirmation in plasma for western blotting and LC-MS/MS methods ranged from a low of 2.5mIU/mL, and closely matched the sensitivity of ELISA screening. PMID:26290355

  20. Evaluation of two sets of immunohistochemical and Western blot confirmatory methods in the detection of typical and atypical BSE cases

    Greenlee Justin J


    Full Text Available Abstract Background Three distinct forms of bovine spongiform encephalopathy (BSE, defined as classical (C-, low (L- or high (H- type, have been detected through ongoing active and passive surveillance systems for the disease. The aim of the present study was to compare the ability of two sets of immunohistochemical (IHC and Western blot (WB BSE confirmatory protocols to detect C- and atypical (L- and H-type BSE forms. Obex samples from cases of United States and Italian C-type BSE, a U.S. H-type and an Italian L-type BSE case were tested in parallel using the two IHC sets and WB methods. Results The two IHC techniques proved equivalent in identifying and differentiating between C-type, L-type and H-type BSE. The IHC protocols appeared consistent in the identification of PrPSc distribution and deposition patterns in relation to the BSE type examined. Both IHC methods evidenced three distinct PrPSc phenotypes for each type of BSE: prevailing granular and linear tracts pattern in the C-type; intraglial and intraneuronal deposits in the H-type; plaques in the L-type. Also, the two techniques gave comparable results for PrPSc staining intensity on the C- and L-type BSE samples, whereas a higher amount of intraglial and intraneuronal PrPSc deposition on the H-type BSE case was revealed by the method based on a stronger demasking step. Both WB methods were consistent in identifying classical and atypical BSE forms and in differentiating the specific PrPSc molecular weight and glycoform ratios of each form. Conclusions The study showed that the IHC and WB BSE confirmatory methods were equally able to recognize C-, L- and H-type BSE forms and to discriminate between their different immunohistochemical and molecular phenotypes. Of note is that for the first time one of the two sets of BSE confirmatory protocols proved effective in identifying the L-type BSE form. This finding helps to validate the suitability of the BSE confirmatory tests for BSE

  1. Detection of H pylori infection by ELISA and Western blot techniques and evaluation of anti CagA seropositivity in adult Turkish dyspeptic patients

    (O)zlem Yilmaz; Nazime (S)en; Ahmet Ali Küpelio(g)lu; (I)lkay (S)im(s)ek


    AIM: To detect H pylori infection and to evaluate the anti CagA seropositivity in adult Turkish dyspeptic patients. METHODS: We evaluated anti-H pylori IgA, IgG and anti-CagA antibodies using commercial enzyme-linked immunoassay (ELISA) and Western blot in dyspeptic Turkish patients. H pylori status was determined by histology and rapid urease testing.RESULTS: Fifty-six patients were entered. Forty-eight (85.7%) out of the 56 patients were positive for H pylori.H pylori IgG seropositivity was 82.1%, IgA seropositivity 48.2%. CagA ELISA showed that IgG was positive in 50% and IgA in 30.4% of those with H pylori infections.Western blot showed that IgG seropositivity was 80.4%and IgA seropositivity 33.9%. Western blot detected IgG antibodies with reactivity to CagA in 50%, VacA in 62.5%, UreB in 87.5%, UreA in 80.4%, and OMP in 57.1%. None of the tests had a sensitivity and specificity above 80%.CONCLUSION: None of these commercial tests seems clinically useful for H pylori detection in adult dyspeptic patients, while Western blot can give seropositivity and determine anti-CagA, VacA virulence factor status of Turkish dyspeptic patients in the Izmir region.

  2. Resolution and identification of major peanut allergens using a combination of fluorescence two-dimensional differential gel electrophoresis, western blotting and Q-TOF mass spectrometry.

    Peanut allergy is triggered by several proteins known as allergens. The matching resolution and identification of major peanut allergens in 2D protein maps, was accomplished by the use of fluorescence two-dimensional differential gel electrophoresis (2D DIGE), Western blotting and quadrupole time-of...

  3. Northern Pintail Telemetry [ds231

    California Department of Resources — Using radio-telemetry, female northern pintail (Anas acuta) survival, distribution, and movements during late August-March in Central California were determined...

  4. Fertilization in northern forests

    Hedwall, Per Ola; Gong, Peichen; Ingerslev, Morten;


    intensive fertilization regimens implying intensive fertilization starting in young forests may, on the other hand, considerably increase the biomass supply and value for the industry. The economic and environmental risks of this type of fertilization may, however, be larger and more research is needed on......Forests of northern ecosystems respond slowly to management activities and the possibilities to increase the growth in a short-term perspective and meet swift increases in society's demand for biomass are small. An exception among the silvicultural measures is fertilization which can be applied in...... combination with present management systems and, almost instantly, enhances forest productivity. There may, however, be both economic and environmental constraints to large-scale applications of fertilizers in forest. Here we review the literature concerning biomass production of forests under different...

  5. Dual infection of chickens with pox and infectious laryngotracheitis (ILT) confirmed with specific pox and ILT DNA dot-blot hybridization assays.

    Fatunmbi, O O; Reed, W M; Schwartz, D L; Tripathy, D N


    Dual infection with fowl pox (FP) and infectious laryngotracheitis (ILT) was diagnosed as the cause of acute mortality in a flock of three age groups of Hy-Line leghorn layers. The affected chickens had not been previously vaccinated against either FP or ILT. The diagnosis was confirmed by virus isolation, histopathology, and the use of specific pox and ILT genomic DNA probes in a dot-blot hybridization assay. FP and ILT vaccinations were recommended to control mortality. The use of FP- and ILT-specific DNA dot-blot hybridization may be used as a routine diagnostic tool to differentiate between the two diseases, especially in atypical cases of either infection or to confirm the existence of the two diseases as a mixed infection in a flock of chickens. PMID:8719232

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

    Nilton Thaumaturgo


    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.

  7. Comparison of immunoperoxidase staining with indirect immunofluorescence, ELISA, and Western blotting assays for detecting anti-HTLV-I antibodies in systemic lupus erythematosus.

    K. Yamaguchi; Matutes, E; Kiyokawa, T.; Nishimura, Y.; Ishii, T.; Takatsuki, K.; Catovsky, D


    Serum antibodies against human T cell leukaemia virus type I (HTLV-I) were investigated in 12 patients by four methods: indirect immunoperoxidase staining, indirect immunofluorescence, enzyme linked immunosorbent assay (ELISA), and strip radioimmunoassay based on the Western blotting assay. Seven patients had systemic lupus erythematosus (SLE) and five various autoimmune diseases with one or more circulating autoantibodies. Serum samples from three patients were found to be HTLV-I-positive by...

  8. Mycoplasma agassizii strain variation and distinct host antibody responses explain differences between enzyme-linked immunosorbent assays and Western blot assays.

    Wendland, Lori D; Klein, Paul A; Jacobson, Elliott R; Brown, Mary B


    The precarious status of desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises has resulted in conservation efforts that now include health assessment as an important component of management decision-making. Mycoplasmal upper respiratory tract disease (URTD) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. In this study, serum samples obtained from eight Gopherus tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with Mycoplasma agassizii as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of mycoplasmal URTD were used to evaluate four distinct clinical isolates of M. agassizii as antigens for ELISA and Western blot analyses. Each animal sample reacted in the Western blot with its homologous M. agassizii strain, but recognition of heterologous M. agassizii strains was variable. Further, individual animals varied significantly with respect to the specific proteins recognized by the humoral immune response. An additional 114 Gopherus serum samples were evaluated using ELISA antigens prepared from the four distinct M. agassizii strains; A₄₀₅ values were significantly correlated (r² goodness of fit range, 0.708 to 0.771; P < 0.0001) for all antigens tested. The results confirm that strain variation is responsible for the observed differences between Western blot binding patterns. Thus, reliance on a single M. agassizii strain as an antigen in Western blot assays may provide false-negative results. This could have adverse consequences for the well-being of these environmentally sensitive hosts if false-negative animals were relocated to sites consisting of true-negative populations. PMID:20810678

  9. Mycoplasma agassizii Strain Variation and Distinct Host Antibody Responses Explain Differences between Enzyme-Linked Immunosorbent Assays and Western Blot Assays ▿

    Wendland, Lori D.; Klein, Paul A.; Jacobson, Elliott R.; Brown, Mary B.


    The precarious status of desert (Gopherus agassizii) and gopher (G. polyphemus) tortoises has resulted in conservation efforts that now include health assessment as an important component of management decision-making. Mycoplasmal upper respiratory tract disease (URTD) is one of very few diseases in chelonians for which comprehensive and rigorously validated diagnostic tests exist. In this study, serum samples obtained from eight Gopherus tortoises documented at necropsy to (i) be enzyme-linked immunosorbent assay (ELISA) seropositive using the PS6 antigen, (ii) be infected with Mycoplasma agassizii as indicated by direct isolation of the pathogen from the respiratory surfaces, and (iii) have histological lesions of mycoplasmal URTD were used to evaluate four distinct clinical isolates of M. agassizii as antigens for ELISA and Western blot analyses. Each animal sample reacted in the Western blot with its homologous M. agassizii strain, but recognition of heterologous M. agassizii strains was variable. Further, individual animals varied significantly with respect to the specific proteins recognized by the humoral immune response. An additional 114 Gopherus serum samples were evaluated using ELISA antigens prepared from the four distinct M. agassizii strains; A405 values were significantly correlated (r2 goodness of fit range, 0.708 to 0.771; P < 0.0001) for all antigens tested. The results confirm that strain variation is responsible for the observed differences between Western blot binding patterns. Thus, reliance on a single M. agassizii strain as an antigen in Western blot assays may provide false-negative results. This could have adverse consequences for the well-being of these environmentally sensitive hosts if false-negative animals were relocated to sites consisting of true-negative populations. PMID:20810678

  10. Western blot analyses of measles virus antibody in normal persons and in patients with multiple sclerosis, subacute sclerosing panencephalitis, or atypical measles.

    Hankins, R W; Black, F L


    A version of the Western blot was developed to detect serum antibodies against measles virus polypeptides. With this technique, a seroepidemiological survey of antibodies to the several measles virus proteins in diverse measles-related conditions was conducted. The sera were obtained from individuals with a recent or long-past history of natural measles, from persons with a history of immunization with live attenuated measles vaccine, and from patients with multiple sclerosis, subacute sclero...

  11. Glycoproteins from the cuticle of the Atlantic shore crab Carcinus maenas: I. Electrophoresis and Western-blot analysis by use of lectins

    Compère, P.; Jaspar-Versali, M.-F.; Goffinet, G.


    The protein and glycoprotein content of four different neutral or acidic solvent extracts (0.5 M KCl, 10% EDTA, 0.1 N HCI, or 2% acetic acid) from the mineralized exoskeleton of a decapod crustacean, the Atlantic shore crab Carcinus maenas, were characterized by quantitative analysis of proteins, SDS-PAGE analysis, and probing with lectins on blots. The lectins used were Conconavalin A, Jacalin, soybean agglutinin, Maackia amurensis agglutinin II, and Sambucus nigra agglutinin. The results sh...

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

    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 (32P) 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 32P 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 H37RV) 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 32P-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 32P-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 32P-oligonucleotide probe is a technique that is rapid and highly specific and sensitive for detection of mutation at codon 315 of

  13. Flight tracks, Northern California TRACON

    National Aeronautics and Space Administration — This dataset contains the records of all the flights in the Northern California TRACON. The data was provided by the aircraft noise abatement office...

  14. Northern Fur Seal Food Habits

    National Oceanic and Atmospheric Administration, Department of Commerce — This data set contains food habits samples, usually scats, collected opportunistically on northern fur seal rookeries and haulouts in Alaska from 1987 to present....

  15. Tornadoes Strike Northern Wisconsin


    A series of tornadoes ripped through the Upper Midwest region of the United States in the evening of June 7, 2007. At least five different tornadoes touched down in Wisconsin, according to the Associated Press, one of which tore through the Bear Paw Resort in northern Wisconsin. Despite dropping as much as fifteen centimeters (six inches) of rain in some places and baseball-size hail in others, authorities were reporting no deaths attributable to the storm system, and only a smattering of injuries, but considerable property damage in some areas. When the MODIS instrument on NASA's Terra satellite observed the area on June 9, 2007, the track torn through the woods by one of the tornadoes stands out quite clearly. This photo-like image uses data collected by MODIS in the normal human vision range to give a familiar natural-looking appearance. The landscape is largely a checkerboard of farms, towns, roads, and cities. The pale land is predominantly farmland where crops have not fully grown in yet. Dark blue shows the winding path of rivers and lakes dotting the landscape. The large blue lake on the east (right) side of the image is Lake Michigan. Towns and cities, including the city of Green Bay, are gray. To the north side, farmland gives way to dark green as land use shifts from agriculture to the Menominee Indian Reservation and Nicolet National Forest. The diagonal slash through the dark green forested land shows the tornado track. Bare land was revealed where the tornado tore down trees or stripped vegetation off the branches. The high-resolution image provided above is at MODIS' full spatial resolution (level of detail) of 250 meters per pixel. The MODIS Rapid Response System provides this image at additional resolutions.

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

    Garth L. Nicolson


    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.’

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

    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 3H-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 35S-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 3H-labeled proteins following blotting to nitrocellulose

  18. Localization and Distribution of 'Candidatus Liberibacter asiaticus’ in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody

    Ding, Fang; Duan, Yongping; Paul, Cristina; Brlansky, Ronald H.; Hartung, John S.


    ‘Candidatus Liberibacter asiaticus’ (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tub...


    Manoj G Tyagi


    Full Text Available Vasopressin, a posterior pituitary hormone is responsible for water reabsorption by the kidneys and maintenance of cardio-vascular homeostasis. Vasopressin receptors are characterized as VR 1 (V1a, VR2 (V2, and VR3 (V1b. VR1, which is abundant in vascular smooth muscles, causes vasoconstriction by increasing intracellular calcium via the phosphatidylinositol bisphosphonate pathway and a positive inotropic effect in cardiac muscle. VR2 has also been shown to be expressed in the heart. There is emerging role for vasopressin receptors in health and disease. This study describes the application of Western blotting to elucidate the importance of vasopressin receptors in the heart cells.

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

    Flora Chávez-Salas; Olenka Vásquez; Hermes Escalante


    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 pacien...

  1. Detection of possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by protein blotting to damaged DNA-fixed membranes

    A novel method for detecting possible DNA repair enzymes on sodium dodecyl sulfate-polyacrylamide gels by blotting them onto a damaged DNA-fixed membrane is presented. To prepare the membrane, highly polymerized calf thymus DNA immobilized on a nylon membrane is damaged chemically. Enzymes, either homogeneous or crude, that are possibly involved in the priming step of DNA repair are fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and are renatured to active form by incubating the gel in an appropriate buffer. The renatured enzyme is then blotted onto the damaged DNA-fixed membrane, a process during which incision and/or excision are introduced to the damaged DNA by the enzymes. The incision and/or excision provide priming sites for repair DNA synthesis in the subsequent step in which the membrane is incubated with DNA polymerase in the presence of alpha-32P-labeled substrate. The site of substrate incorporation on the membrane reflecting the molecular weight of the repair enzyme is finally visualized by autoradiography. The present technique is established using Escherichia coli exonuclease III and a DNA-fixed membrane treated with bleomycin or acid-depurinated. By application of this method, a priming factor (an exonuclease) involved in the initiation of bleomycin-induced DNA repair is detected in the extract of mouse ascites sarcoma cells, and thus the molecular weight of the enzyme is estimated. Some apurinic/apyrimidinic endonucleases of mammals are also detected by the present procedure

  2. Analysis of the insulin receptor gene in noninsulin-dependent diabetes mellitus by denaturing gradient gel blots: A clinical research center study

    Magre, J.; Goldfine, A.B.; Warram, J.H. [Harvard Medical School, Boston, MA (United States)] [and others


    We have used a new technique of denaturing gradient gel blotting to determine the prevalence of alterations in the intracellular domain of the insulin receptor in normal individuals and subjects with non-insulin-dependent diabetes mellitus (NIDDM). This method detects DNA sequence differences as restriction fragment melting polymorphisms (RFMP) and is sensitive to changes in sequence at both restriction sites and within the fragments themselves. Using restriction digests with AluI, HaeIII, HinfI, RsaI, Sau3A, and Sau96, 12 RFMPs were found to localize to the region of the {beta}-subunit of the insulin receptor gene. Using exon-specific probes, these RFMPs could be localized to specific regions surrounding individual exons, including exons, 14, 15, 16, 18, 20, and 22. In general, linkage disequilibrium between polymorphisms was inversely related to their distance in the gene structure, although there was a {open_quotes}hot spot{close_quotes} for recombination between exons 19 and 20. No difference in melting temperatures or allele frequency was observed between NIDDM patients and controls. These data indicate that the region of the insulin receptor gene coding for the intracellular portion of the {beta}-subunit is highly polymorphic and that polymorphisms surrounding specific exons can be identified by denaturing gradient gel blotting, but there is no evidence that variation at this locus contributes to NIDDM susceptibility in most individuals. 36 refs., 3 figs., 3 tabs.

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

    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

  4. 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

    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 32P (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)

  5. Purificación de antígenos de Fasciola hepatica mediante electroelución y su aplicación inmunodiagnóstica mediante Western Blot en la infección animal PURIFICATION OF Fasciola hepatica ANTIGENS BY ELECTROELUTION AND ITS DIAGNOSTIC APPLICATION THROUGH WESTERN BLOT IN ANIMAL INFECTION



    Full Text Available La electroelución implementada en este estudio logró purificar por separado dos polipéptidos, uno de 14 y otro de 29 kDa. La eficiencia diagnóstica de cada uno de estos polipéptidos medida a través de Western blot indicó una sensibilidad de 95% y 97,5% respectivamente y ambos una especificidad de 100%. Además se concluye que son de utilidad en la pesquisa de infecciones prepatentesTwo polypeptides (14 kDa and 29 kDa of adult Fasciola hepatica were purified by electroelution and its diagnostic application through Western blot was evaluated. The value of sensitivity was 95% and 97.5%, respectively and the value of specificity was 100% in both polypeptides. These antigenic components were efficient for the diagnosis of the prepatent stage of the infection

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

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


    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. PMID:26267107

  7. Twenty-two year follow-up of an Indian family with dysferlinopathy-clinical, immunocytochemical, western blotting and genetic features

    Khadilkar Satish


    Full Text Available Long-term observations over a period of 22 years in an Indian family with primary dysferlinopathy are recorded, defining phenotypic variability. In the propositus, the dystrophy began distally in the tibialis anterior muscles, before involving the gastrocnemius. Transient painful calf hypertrophy, followed by calf wasting was observed. The proximal lower and upper limbs weakened after three to four years. The younger sibling presented with the proximo-distal phenotype. Both patients showed very high creatine kinase values early into the illness. Disease progression was slow. The younger sibling lost ambulation 14 years after onset, while the elder one remains ambulatory 22 years into the illness. Muscle biopsy showed dystrophic features and absence of dysferlin. Monocyte western blotting confirmed absence of dysferlin. Genetic analysis detected a heterozygous mutation in Exon 54 [c.6124C>T] in the DYSF gene. This is the first family with a diagnosis of dysferlinopathy supported by genetic data, reported from India.

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

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


    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 is a...... between 18% and 25% to the prokaryotic rRNA pool. The dominant SRB were related to complete oxidizing genera (Desulphococcus, Desulphosarcina and Desulphobacterium), while Desulpho-bacter could not be detected. The vertical profile and quantity of rRNA from SRB was compared with sulphate reduction rates...... (SRR) measured with (SO42-)-S-35 tracer in whole-core incubations. While SRB abundance was highest near the surface, peaking at around 1.5cm, measured sulphate reduction rates were lowest in this region. A second peak of SRB rRNA was observed at the transition zone from oxidized to reduced sediment...

  9. A comparison of antigenic peptides in muscle larvae of several Trichinella species by two-dimensional western-blot analysis with monoclonal antibodies

    Dea-Ayuela M.A.


    Full Text Available The antigens recognised by mAb US5 specific to 53 kDa glycoprotein (gp 53 in T. spiralis L-1 muscle larvae (TSL1 antigens, mAb US9 specific to gp 53 in TSL1 from all encapsulated species and mAb US4 specific to a tyvelose containing tetrasaccharide present in TSL1, were investigated in crude extracts from muscle larvae of T. spiralis, T. nativa and T. britovi by 2D-electrophoresis and western-blot. At least four proteins of different pI were recognised by mAb US5 on T. spiralis antigens. Recognition profile of mAb US9 on T. spiralis antigens exhibited some variation with regard to that of the US5. Polymorphism was apparent in gp 53. High reactivity was shown by the mAb US4 with the three species.

  10. High rate of missed HIV infections in individuals with indeterminate or negative HIV western blots based on current HIV testing algorithm in China.

    Liu, Man-Qing; Zhu, Ze-Rong; Kong, Wen-Hua; Tang, Li; Peng, Jin-Song; Wang, Xia; Xu, Jun; Schilling, Robert F; Cai, Thomas; Zhou, Wang


    It remains unclear if China's current HIV antibody testing algorithm misses a substantial number of HIV infected individuals. Of 196 specimens with indeterminate or negative results on HIV western blot (WB) retrospectively examined by HIV-1 nucleic acid test (NAT), 67.57% (75/111) of indeterminate WB samples, and 16.47% (14/85) of negative WB samples were identified as NAT positive. HIV-1 loads in negative WB samples were significantly higher than those in indeterminate WB samples. Notably, 86.67% (13/15) of samples with negative WB and double positive immunoassay results were NAT positive. The rate of HIV-1 infections missed by China's current HIV testing algorithm is unacceptably high. Thus, China should consider using NAT or integrating fourth generation ELISA into current only antibodies-based HIV confirmation. J. Med. Virol. 88:1462-1466, 2016. © 2016 Wiley Periodicals, Inc. PMID:26856240

  11. Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

    Ding, Fang; Duan, Yongping; Paul, Cristina; Brlansky, Ronald H; Hartung, John S


    'Candidatus Liberibacter asiaticus' (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant. PMID:25946013

  12. Localization and Distribution of 'Candidatus Liberibacter asiaticus’ in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody

    Ding, Fang; Duan, Yongping; Paul, Cristina; Brlansky, Ronald H.; Hartung, John S.


    ‘Candidatus Liberibacter asiaticus’ (CaLas), a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB). Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant. PMID:25946013

  13. Localization and Distribution of 'Candidatus Liberibacter asiaticus' in Citrus and Periwinkle by Direct Tissue Blot Immuno Assay with an Anti-OmpA Polyclonal Antibody.

    Fang Ding

    Full Text Available 'Candidatus Liberibacter asiaticus' (CaLas, a non-cultured member of the α-proteobacteria, is the causal agent of citrus Huanglongbing (HLB. Due to the difficulties of in vitro culture, antibodies against CaLas have not been widely used in studies of this pathogen. We have used an anti-OmpA polyclonal antibody based direct tissue blot immunoassay to localize CaLas in different citrus tissues and in periwinkle leaves. In citrus petioles, CaLas was unevenly distributed in the phloem sieve tubes, and tended to colonize in phloem sieve tubes on the underside of petioles in preference to the upper side of petioles. Both the leaf abscission zone and the junction of the petiole and leaf midrib had fewer CaLas bacteria compared to the main portions of the petiole and the midribs. Colonies of CaLas in phloem sieve tubes were more frequently found in stems with symptomatic leaves than in stems with asymptomatic leaves with an uneven distribution pattern. In serial sections taken from the receptacle to the peduncle, more CaLas were observed in the peduncle sections adjacent to the stem. In seed, CaLas was located in the seed coat. Many fewer CaLas were found in the roots, as compared to the seeds and petioles when samples were collected from trees with obvious foliar symptoms. The direct tissue blot immuno assay was adapted to whole periwinkle leaves infected by CaLas. The pathogen was distributed throughout the lateral veins and the results were correlated with results of qPCR. Our data provide direct spatial and anatomical information for CaLas in planta. This simple and scalable method may facilitate the future research on the interaction of CaLas and host plant.


    A baseline study of the marine mammals of northern Puget Sound and the Strait of Juan de Fuca was undertaken from November 1977 to September 1979 emphasizing certain aspects of the biology of the harbor seal, which is the most abundant marine mammal in this area. The local abunda...

  15. Lyme Disease in Northern California

    Campagna, Joan; Lavoie, Paul E.; Birnbaum, Neal S.; Furman, Deane P.


    Lyme disease is a recently described clinical entity with cutaneous, neurologic, articular and cardiac manifestations. Since the original description of the disease in 1977, more than 500 cases have been reported. Although the vast majority of patients have been from the area near Lyme, Connecticut, we have seen four patients from northern California with various aspects of Lyme disease.

  16. Offshore northern Europe, the challenges

    This paper relates to challenges of the offshore activity in the North Sea. It is appropriate to address these challenges in the context of generating values through efficient management of resources, markets, safety and technology, as the challenges lie therein. The petroleum industry is built to turn natural resources into market value, assuring broad benefits to stake holders and shareholders. In the following, the challenges facing the industry the industry offshore Northern Europe is examined on this background

  17. Carboniferous geology of Northern England

    Waters, Colin N.


    The British Geological Survey (BGS) has produced a wholesale rationalisation of Carboniferous lithostratigraphical nomenclature. This presentation describes the Carboniferous stratigraphy of northern England, illustrated with research carried out as part of recent BGS mapping projects. During the Tournaisian and Visean a phase of north–south rifting resulted in the development of grabens and half-grabens, separated by platforms and tilt-block highs. Visean marine transgressions re...

  18. Viral diseases of northern ungulates

    Frölich, K.


    This paper describes viral diseases reported in northern ungulates and those that are a potential threat to these species. The following diseases are discussed: bovine viral diarrhoea/mucosal disease (BVD/MD), alphaherpesvirus infections, malignant catarrhal fever (MCF), poxvirus infections, parainfluenza type 3 virus infection, Alvsborg disease, foot-and-mouth disease, epizootic haemorrhage disease of deer and bluetongue disease, rabies, respiratory syncytial virus infection, adenovirus infe...

  19. 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

    SATO Neuza Satomi; HIRATA Mário H.; HIRATA Rosário D.C.; Lia Carmen M.S. ZERBINI; Edilene P.R. SILVEIRA; MELO Carmem Silvia de; Ueda, Mirthes


    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-rT...

  20. The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. II. Extraction of spooled DNA and its application to Southern blot hybridization analysis.

    Sato, Y.; Mukai, K.; Matsuno, Y.; Furuya, S.; Kagami, Y.; Miwa, M.; Shimosato, Y.


    In our previous report, we described a new fixation and paraffin-embedding method (the AMeX method) that preserves many of the antigens that are normally destroyed by routine formalin fixation. The current study was conducted to examine the preservation of high-molecular-weight DNA in tissues processed by this method. DNA was extracted from AMeX-processed tissue sections after deparaffinization by the same method as that used to extract DNA from fresh tissues. The total amounts of DNA extracted from 10 mg each in wet weight of AMeX-processed and fresh mouse liver tissues were identical. In tissues of malignant lymphoma, the total amount of spooled DNA extracted from 50 sections, each 20 microns thick, was about 8 micrograms/mm2. The electrophoretic pattern of DNA digested with restriction endonucleases on agarose gel from AMeX-processed tissue sections did not differ from that of fresh materials. Southern blot hybridization analysis also revealed that the mobility of specific DNA fragments was identical for AMeX-processed and fresh tissues. The AMeX method was thus proved to be a versatile multipurpose tissue-processing procedure, which is expected to provide important information regarding the correlation between morphology, phenotypic expression, and gene alteration. Images Figure 3 Figure 4 Figure 5A Figure 5B PMID:2407122

  1. Search for oncogene mutations in X-ray-transformed mouse 10T1/2 cells by denaturing gradient gel electrophoresis blotting

    The authors sought evidence for possible mutations within the c-myc, c-Ha-ras and c-Ki-ras loci of X-ray-transformed mouse C3H10T1/2 cell clones using the denaturing gradient gel electrophoresis (DGGE) blot technique. This method was developed to detect mutations (e.g. single base changes, small deletions) in genomic DNA, by measuring differences in the melting behaviour of short DNA fragments (50-800 bp) obtained by digestion of genomic DNA with several specific 4 bp recognition site restriction enzymes. Genomic DNAs derived from 23 X-ray-transformed clones were digested with several restriction enzymes, electrophorized on denaturing gradient gel and hybridized to c-myc, c-Ha-ras and c-Ki-ras cDNA probes. No alterations in melting patterns were observed for any of these oncogenes as compared with DNA from 18 control, non-irradiated wild-type 10T1/2 cell clones, suggesting that transformation was not associated with mutation of these genes nor with changes in their patterns of methylation. (author)

  2. Inverse PCR and Quantitative PCR as Alternative Methods to Southern Blotting Analysis to Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants.

    Stefano, Biricolti; Patrizia, Bogani; Matteo, Cerboneschi; Massimo, Gori


    One of the major unanswered questions with respect to the commercial use of genetic transformation in woody plants is the stability of the transgene expression over several decades within the same individual. Gene expression is strongly affected by the copy number which has been integrated into the plant genome and by the local DNA features close to the integration sites. Because woody plants cannot be subjected to selfing or backcrossing to modify the transgenic allelic structure without affecting the valuable traits of the cultivar, molecular characterization of the transformation event is therefore crucial. After assessing the transgene copy number of a set of apple transgenic clones with Southern blotting, we describe two alternative methods: the first is based on inverse PCR (i-PCR) and the second on the quantitative PCR (q-PCR). The methods produced comparable results with the exception of the data regarding a high copy number clone, but while the q-PCR-based system is rapid and easily adaptable to high throughput systems, the i-PCR-based method can provide information regarding the transformation event and the characteristics of the sequences flanking the transgenic construct. PMID:26895172

  3. Pteridophyta collected in Northern Nigeria and Northern Cameroon

    Jan kornaś


    Full Text Available 25 species of Pteridophyta were collected in Northern Nigeria (mainly the Lake Chad Basin and the Mandara Mts. and in the neighbouring parts of Cameroon. 11 of them have not been recorded previously from this area: Isoetes schweinfurthii A. Br. in Bak., Selaginella tenerrima A. Br. ex Kuhn, Ophioglossum gomenzianum Welw. ex A. Br., Marsilea coromandeliana Willd., M. distorta A. Br., M. nubica A. Br., M. subterranea Lepr. ex A. Br., Azolla africana Desv., Ceratopteris richardii Brogn., Adiantum capillus-veneris Linn., and Actiniopleris semiflabellata Pic. Ser.

  4. 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

    Hermes Escalante


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

  5. Identification of reference proteins for Western blot analyses in mouse model systems of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD toxicity.

    Stephenie D Prokopec

    Full Text Available Western blotting is a well-established, inexpensive and accurate way of measuring protein content. Because of technical variation between wells, normalization is required for valid interpretation of results across multiple samples. Typically this involves the use of one or more endogenous controls to adjust the measured levels of experimental molecules. Although some endogenous controls are widely used, validation is required for each experimental system. This is critical when studying transcriptional-modulators, such as toxicants like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD.To address this issue, we examined hepatic tissue from 192 mice representing 47 unique combinations of strain, sex, Ahr-genotype, TCDD dose and treatment time. We examined 7 candidate reference proteins in each animal and assessed consistency of protein abundance through: 1 TCDD-induced fold-difference in protein content from basal levels, 2 inter- and intra- animal stability, and 3 the ability of each candidate to reduce instability of the other candidates. Univariate analyses identified HPRT as the most stable protein. Multivariate analysis indicated that stability generally increased with the number of proteins used, but gains from using >3 proteins were small. Lastly, by comparing these new data to our previous studies of mRNA controls on the same animals, we were able to show that the ideal mRNA and protein control-genes are distinct, and use of only 2-3 proteins provides strong stability, unlike in mRNA studies in the same cohort, where larger control-gene batteries were needed.

  6. Evaluation of a New Dot Blot Enzyme Immunoassay (Directigen Flu A+B) for Simultaneous and Differential Detection of Influenza A and B Virus Antigens from Respiratory Samples

    Reina, Jordi; Padilla, Emma; Alonso, Fermin; Ruiz de Gopegui, Enrique; Munar, Maria; Mari, Margarita


    We report a prospective evaluation of a new dot blot enzyme immunoassay (EIA) method for the direct, rapid, qualitative, simultaneous, and differential detection of the influenza A (IA) and B (IB) virus antigen in different respiratory samples. The EIA method was compared with the shell vial culture system (MDCK cell line) used with the same samples. We studied 160 samples from 93 (58.1%) pediatric patients (hospital emergency room) and from 67 (41.9%) adult patients (sentinel network). Seventy-four(46.2%) samples were considered positive; of them, 46 (62.2%) were from pediatric patients and 28 (37.8%) were from an adult group (P < 0.05), with overall positive values of 49.9% and 41.7%, respectively. All 74 (100%) of the positive samples were isolated in cell culture versus the 68.9% that were detected as positive by the new EIA method (P < 0.05). Of the 41 samples positive for the IA virus, the EIA detected 34 (82.9%) positive samples; of the 33 samples positive for the IB virus, the EIA detected 17 (51.5%) positive samples (P < 0.05). No false-positive reaction was detected with the EIA method (specificity and positive predictive value of 100%). The overall results obtained in the comparison between the new EIA and the shell vial culture had a sensibility of 82.9% and predictive negative values of 92.4% for the IA virus and 51.5% and 84.3%, respectively, for the IB virus. This evaluation shows sensitivity and specificity percentages for the new EIA method that is acceptable for routine use in IA virus detection. The results obtained were worse for IB virus detection, but this new EIA method is actually the only one with the capacity to differentiate between the two influenza viruses. PMID:12202608

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



    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.

  8. Discovery of novel poly(ADP-ribose) glycohydrolase inhibitors by a quantitative assay system using dot-blot with anti-poly(ADP-ribose)

    Okita, Naoyuki, E-mail: [Genome and Drug Research Center, Tokyo University of Science (Japan); Ashizawa, Daisuke; Ohta, Ryo [Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022 (Japan); Abe, Hideaki [Genome and Drug Research Center, Tokyo University of Science (Japan); Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022 (Japan); Tanuma, Sei-ichi, E-mail: [Genome and Drug Research Center, Tokyo University of Science (Japan); Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022 (Japan)


    Poly(ADP-ribosyl)ation, which is mainly regulated by poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG), is a unique protein modification involved in cellular responses such as DNA repair and replication. PARG hydrolyzes glycosidic linkages of poly(ADP-ribose) synthesized by PARP and liberates ADP-ribose residues. Recent studies have suggested that inhibitors of PARG are able to be potent anti-cancer drug. In order to discover the potent and specific Inhibitors of PARG, a quantitative and high-throughput screening assay system is required. However, previous PARG assay systems are not appropriate for high-throughput screening because PARG activity is measured by radioactivities of ADP-ribose residues released from radioisotope (RI)-labeled poly(ADP-ribose). In this study, we developed a non-RI and quantitative assay system for PARG activity based on dot-blot assay using anti-poly(ADP-ribose) and nitrocellulose membrane. By our method, the maximum velocity (V{sub max}) and the michaelis constant (k{sub m}) of PARG reaction were 4.46 {mu}M and 128.33 {mu}mol/min/mg, respectively. Furthermore, the IC50 of adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), known as a non-competitive PARG inhibitor, was 0.66 {mu}M. These kinetics values were similar to those obtained by traditional PARG assays. By using our assay system, we discovered two novel PARG inhibitors that have xanthene scaffold. Thus, our quantitative and convenient method is useful for a high-throughput screening of PARG specific inhibitors.

  9. Detection of mutation in embB gene of Mycobacterium tuberculosis from clinical isolates of tuberculous patients in China by means of reverse-dot blot hybridization



    The relationship between embB mutation of Mycobacterium tuberculosis and ethambutol (EMB) resistance of the clinical isolates of tuberculous patients in China was investigated by reversedot blot hybridization (RDBH) in addition to evaluating the clinical value with application of PCR-RD-BH technique to detect EMB resistance. In the present study, the genotypes of the 258 bp fragments of embB genes from 196 clinical isolates of M. tuberculosis were analysed with RDBH and DNA sequencing. It was demonstrated that 60 out of 91 phenotypically EMB-resistant isolates (65.9%) showed 5 types of missense mutations at codon 306 of embB gene, resulting in the replacement of the Met residue of the wild type strain with Val, Ile or Leu residues. In these mutations, the GTP mutation (38/91,41.8% ) and the ATA mutation ( 16/91, 17.6% ) were the most encountered genotypes. The embB mutation at codon 306 could also be found in 69 isolates of phenotypically EMB-sensitive but resistant to other anti-tuberculous drugs, but no such gene mutation could be found in 36 strains of drug-sensitive isolates. Meanwhile, the concordance with the results of DNA sequencing for one wide-type probe and 5 probes for specific mutations was 100%. It was concluded that the EMB-resistance occurring in most M. tuberculosis is due to appearance of embB mutation at codon 306, and the PCR-RDBH assay was proved to be a rapid, simple and reliable method for the detection of gene mutations, which might be a good alternative for the drug-resistance screening.

  10. Northern communities sustainable energy initiative

    Oltman, Ursula; Widmeyer, Scott; Moen, Harlan


    The Circumpolar North may provide the solution to the world's most urgent problems. Combining new technologies with the resources, opportunities and needs of the north, the Arctic region may become instrumental in promoting nature's ability to sequester natural carbons while supplying future energy demands to the world. With the technologies for efficiencies and CCS, the abundant supply of natural gas exists for an efficient northern network of electrical generating facilities in the circumpolar region. A symbiotic relationship between facilities can ensure dependable clean electricity and support East-West distribution of power across international time zones strategically connected to southern grids.

  11. Northern Manitoba, northern Saskatchewan, and the Saskatchewan River Delta: Waterfowl breeding population survey: 1997

    US Fish and Wildlife Service, Department of the Interior — This report summarizes the Waterfowl Breeding Population and Habitat Survey for northern Manitoba, northern Saskatchewan, and the Saskatchewan River Delta during...

  12. Waterfowl production survey: Northern Saskatchewan, northern Manitoba, Saskatchewan River Delta: July 10-22, 1973

    US Fish and Wildlife Service, Department of the Interior — This report summarizes the Waterfowl Production and Habitat Survey for northern Saskatchewan, northern Manitoba, and the Saskatchewan River Delta during 1973. The...

  13. Northern Alaska Landscape/Permafrost GIS Data

    Arctic Landscape Conservation Cooperative — This data set represents an updated Ecological Subsection Map for Northern Alaska. This update includes permafrost mapping to include the following new layers:...

  14. Giant Reed Distribution - Northern California [ds333

    California Department of Resources — The Arundo Distribution layer is a compilation of Arundo donax observations in northern and central California, obtained from several sources, including Arundo...

  15. Inmunoglobulina G Anti Helicobacter Pylori por Elisa y Western-blot en pacientes del Servicio de Gastroenterología del Hospital San Vicente de Paúl, Heredia

    Eugenia Ma Quintana Guzmán


    Full Text Available Justificación y Objetivo: En Costa Rica el cáncer gástrico es una de las malignidades más frecuentes y de la mayor mortalidad y otros estudios han encontrado asociación entre este y el Helicobacter pylori. Esta bacteria es capaz de producir sustancias que alteran la mucosa gástrica, antígenos que han sido reconocidos por medio del Western-blot. El propósito de este trabajo fue realizar, por primera vez en nuestro país, un estudio de anticuerpos IgG-anti H. pylori por medio de Western-blot y lograr identificar los tipos de antígenos contra los cuales se desarrolla la respuesta inmunológica. Para ello se utilizaron sueros y cepas de H. pylori aisladas de pacientes costarricenses. Asimismo, se compararon los resultados obtenidos del Western-blot con una prueba de ELISA para determinar la sensibilidad y especificidad diagnóstica, utilizando como referencia el estudio histológico de biopsia gástrica. Métodos: Se obtuvieron muestras de biopsia gástrica y suero de 83 pacientes referidos al Servicio de Gastroscopía del Hospital San Vicente de Paúl, Heredia. A cada uno se le realizó estudio histológico de biopsia gástrica y de anticuerpos IgG-anti H. pylori, por los métodos de ELISA y Western-blot. Resultados y Conclusiones: Por Western-blot se observaron bandas de proteínas de diferentes pesos moleculares, con un predominio de antígenos de H. pylori de bajo peso molecular. El Western-blot y el ELISA presentaron sensibilidades diagnósticas del 89% y el 88% y especificades diagnósticas del 73% y el 71%, respectivamente, comparados con el estudio histológico.Background and aim: In Costa Rica the gastric cancer is the malignancy that produces more deaths. In other studies their pathology it has been associated with Helicobacter pylori. This bacteria is able to produce substances that alter the gastric mucosa, antigens that have been caracterized by Western-blot. The purpose of this study was to performed for the first time in our

  16. Northern pipelines : challenges and needs

    Dean, D.; Brownie, D. [ProLog Canada Inc., Calgary, AB (Canada); Fafara, R. [TransCanada PipeLines Ltd., Calgary, AB (Canada)


    Working Group 10 presented experiences acquired from the operation of pipeline systems in a northern environment. There are currently 3 pipelines operating north of 60, notably the Shiha gas pipeline near Fort Liard, the Ikhil gas pipeline in Inuvik and the Norman Wells oil pipeline. Each has its unique commissioning, operating and maintenance challenges, as well as specific training and logistical support requirements for the use of in-line inspection tools and other forms of integrity assessment. The effectiveness of cathodic protection systems in a permafrost northern environment was also discussed. It was noted that the delay of the Mackenzie Gas Pipeline Project by two to three years due to joint regulatory review may lead to resource constraints for the project as well as competition for already scarce human resources. The issue of a potential timing conflict with the Alaskan Pipeline Project was also addressed as well as land use issues for routing of supply roads. Integrity monitoring and assessment issues were outlined with reference to pipe soil interaction monitoring in discontinuous permafrost; south facing denuded slope stability; base lining projects; and reclamation issues. It was noted that automatic welding and inspection will increase productivity, while reducing the need for manual labour. In response to anticipated training needs, companies are planning to involve and train Aboriginal labour and will provide camp living conditions that will attract labour. tabs., figs.

  17. Groundwater management in northern Iraq

    Stevanovic, Zoran; Iurkiewicz, Adrian


    Groundwater is vital and the sole resource in most of the studied region of northern Iraq. It has a significant role in agriculture, water supply and health, and the elimination of poverty in rural areas. Although Iraq is currently dramatically disturbed by complex political and socio-economic problems, in its northern part, i.e. the Kurdish-inhabited region, fast urbanization and economic expansion are visible everywhere. Monitoring and water management schemes are necessary to prevent aquifer over-exploitation in the region. Artificial recharge with temporary runoff water, construction of subsurface dams and several other aquifer management and regulation measures have been designed, and some implemented, in order to improve the water situation. Recommendations, presented to the local professionals and decision-makers in water management, include creation of Water Master Plans and Water User Associations, synchronization of drilling programmes, rehabilitation of the existing well fields, opening of new well fields, and the incorporation of new spring intakes in some areas with large groundwater reserves, as well as construction of numerous small-scale schemes for initial in situ water treatment where saline groundwater is present.

  18. Illuminating Northern California's Active Faults

    Prentice, Carol S.; Crosby, Christopher J.; Whitehill, Caroline S.; Arrowsmith, J. Ramón; Furlong, Kevin P.; Phillips, David A.


    Newly acquired light detection and ranging (lidar) topographic data provide a powerful community resource for the study of landforms associated with the plate boundary faults of northern California (Figure 1). In the spring of 2007, GeoEarthScope, a component of the EarthScope Facility construction project funded by the U.S. National Science Foundation, acquired approximately 2000 square kilometers of airborne lidar topographic data along major active fault zones of northern California. These data are now freely available in point cloud (x, y, z coordinate data for every laser return), digital elevation model (DEM), and KMZ (zipped Keyhole Markup Language, for use in Google Earth™ and other similar software) formats through the GEON OpenTopography Portal ( Importantly, vegetation can be digitally removed from lidar data, producing high-resolution images (0.5- or 1.0-meter DEMs) of the ground surface beneath forested regions that reveal landforms typically obscured by vegetation canopy (Figure 2).

  19. Environmental overview of geothermal development: northern Nevada

    Slemmons, D.B.; Stroh, J.M.; Whitney, R.A. (eds.)


    Regional environmental problems and issues associated with geothermal development in northern Nevada are studied to facilitate environmental assessment of potential geothermal resources. The various issues discussed are: environmental geology, seismicity of northern Nevada, hydrology and water quality, air quality, Nevada ecosystems, noise effects, socio-economic impacts, and cultural resources and archeological values. (MHR)

  20. Northern Parkway PTA Makes Health a Habit

    Ferdinand, Marilyn


    Health and fitness have been on the agenda of Northern Parkway Elementary School for quite some time, thanks to the concerted efforts of its involved and active PTA officers and members. For the past five years, the Northern Parkway PTA has held a popular and well-attended Family Fun and Fitness Night and has complemented the activities and…

  1. Earliest Urbanism in Northern Mesopotamia

    Skuldbøl, Tim Boaz Bruun

    This dissertation is based on new evidence from the most recent archaeological fieldwork conducted at Tell Brak in northeastern Syria, one of the most important early cities in Mesopotamia. An analysis of this data underscores the transformative and creative nature of the urbanization process...... and hypotheses about the development of early cities in Mesopotamia, and has important implications for understanding the development of early cities in general, as well as their functional composition. This study primarily draws upon the most recent published reports, and from unpublished data collected from...... fieldwork on the mound of Tell Brak and in its immediate surroundings, as well as the author’s own new comparative ceramic studies, which provide the basis for improvements in chronology of the Late Chalcolithic period in northern Mesopotamia. This extensive empirical material is presented as archaeological...


    B. Theilen-Willige


    Full Text Available Based on LANDSAT ETM and Digital Elevation Model (DEM data derived by the Shuttle Radar Topography Mission (SRTM, 2000 of the coastal areas of Northern Venezuela were investigated in order to detect traces of earlier tsunami events. Digital image processing methods used to enhance LANDSAT ETM imageries and to produce morphometric maps (such as hillshade, slope, minimum and maximum curvature maps based on the SRTM DEM data contribute to the detection of morphologic traces that might be related to catastrophic tsunami events. These maps combined with various geodata such as seismotectonic data in a GIS environment allow the delineation of coastal regions with potential tsunami risk. The LANDSAT ETM imageries merged with digitally processed and enhanced SRTM data clearly indicate areas that might be prone by flooding in case of catastrophic tsunami events.

  3. Contamination from Russian Northern Fleet

    This report, from an environmental group known as Bellona and based in Norway, is highly critical of the Russian navy's apparently short-sighted attitude to nuclear power's longer term economic and environmental consequences. Six main sources of radioactive contamination from the activities of the Russian Northern Fleet are summarised, namely, the still increasing numbers of nuclear-powered submarines in service, naval bases, shipyards (including the storage of solid and liquid radioactive wastes and spent nuclear fuels), safety problems with service ships providing transport for nuclear materials, the whole unaddressed problem of decommissioning nuclear submarines due to go out of service (eighty-eight vessels at present), and accidents involving nuclear submarines. (UK)

  4. Intergroup trust in Northern Ireland.

    Tam, Tania; Hewstone, Miles; Kenworthy, Jared; Cairns, Ed


    Although prominent political agendas have placed a great deal of importance on building trust in postconflict areas, there has been a lack of empirical research on its role in areas of intergroup conflict. The authors conducted two studies to examine the relationship between trust and intergroup behavioral tendencies-and the potential for intergroup contact to build trust in Northern Ireland. Study 1 showed that outgroup trust mediates the impact of intergroup contact on behavioral tendencies toward the outgroup. Study 2 revealed the importance of trusting the outgroup over simply liking the outgroup; establishing outgroup trust is crucial, as trust is a stronger predictor of behavioral tendencies toward the outgroup than positive attitudes are. Results also demonstrated two mechanisms for increasing outgroup trust-through both direct and extended intergroup contact. These studies further our understanding of the psychological mechanisms underlying the formation of intergroup trust and behavior in areas of conflict. PMID:19106077

  5. Northern Security and Global Politics

    This book takes a comprehensive approach to security in the Nordic-Baltic region, studying how this region is affected by developments in the international system. The advent of the new millennium coincided with the return of the High North to the world stage. A number of factors have contributed...... strategic environment. This book will be of much interest to students of Nordic and Baltic politics, international security, foreign policy and IR....... to the increased international interest for the northern part of Europe: climate change resulting in ice melting in Greenland and the Arctic, and new resources and shipping routes opening up across the polar basin foremost among them. The world is no longer "unipolar" and not yet "multipolar," but...

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

    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

  7. Western blot used for detection of syphilis antibodies and its evaluation%免疫印迹法检测梅毒抗体及评价

    曹谊; 金一鸣; 董丽; 江妮娜; 厦伟凤


    目的 建立梅毒抗体免疫印迹法(TP-WB)检测梅毒(TP),并对其效果进行评价.方法 对第1组(13份)用甲苯胺红不加热试验与酶联免疫吸附试验法检测和第2组(28份)对双酶联免疫吸附试验法筛检的可疑样本进行明胶颗粒凝集试验与TP-WB方法确证检测并比较.结果 两组用明胶颗粒凝集试验确证分别为6例阳性、2例可疑、5例阴性和12例阳性、2例可疑、14例阴性,阳性率为46.2%、42.9%;而用TP-WB法检测分别为11例阳性、2例可凝和18例阳性、2例可凝、8例阴性阳性率为84.6%、64.3%.结论 TP-WB法操作简便,结果易观察,无需特殊设备,灵敏度特异性好,更适宜各级实验室梅毒确证检测.%OBJECTIVE To set up western blot method for the detection of syphilis antibodies and evaluate its effect. METHODS Gelatin particle agglutination test was conducted towards the following two groups, 13 shares in Group 1 adopted Toluidine red non-heating test and enzyme-linked immunity and adsorption assay test; 28 shares of spacious samples in Group 2 screened by double enzyme-linked immunity and adsorption assay test performed the gelatin particle agglutination test and confirmatory test and compared the above mentioned test with TP-WB method toward two groups. RESULTS The confirmatory results obtained through gelatin particle agglutination test were 6 cases positive* 2 cases suspicious, 5 cases negative and 12 cases positive, 2 cases suspicious, 14 cases negative; the positive rates were 46. 2% , 42. 9% ; while the results obtained through TP-WB were 11 cases positive, 2 cases suspicious, and 18 cases positive, 2 cases suspicious, 8 cases negative; the positive rates were 78. 4%, 64. 3%. CONCLUSION TP-WB method is simple in operation, easy to observe the results without special equipments, good at sensitivity and specificity, and more suitable for syphilis confirmatory test in all levels of laboratory.

  8. 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

    Neuza Satomi SATO


    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

  9. Case studies: Northern Saskatchewan, Canada

    Northern Saskatchewan comprises an area of about 350 000 km2. In 1951 the population was 11 000 people but by 2003 it was approaching 40 000, of whom about 87% are aboriginal, consisting of either First Nations or Metis people. The first uranium mining area developed in northern Saskatchewan was Uranium City, north of Lake Athabasca. These first mines started production in the early 1950s. Of the 10 producing mines, only Eldorado Nuclear remained in operation after 1965. The development of Uranium City, including better services such as a hospital, drew some aboriginals into the area. There was some aboriginal employment in the early mines but, with few exceptions, these employees only stayed a short time. The mining companies developed training programmes to prepare aboriginals for regular, wage earning jobs. This included lifestyle training such as how to manage personal finances. Further extensive training programmes were required on the job to help these employees become fully contributing members of the workforce, who could advance in their jobs, expand their job opportunities and earnings, and in order to reduce turnover. The question of accommodating mine staff is a complex one, including several options. The first option, a company town, can be developed adjacent to the mine site. It is owned by the company and accommodates everyone who works at the mine and in its service industries. This can result in lower cost accommodation for mine staff with the benefit of no personal capital investment that cannot be recouped after mine closure. The capital cost to the mining company is higher; there is an administrative cost to managing and maintaining many houses, apartments and bunkhouses, and the decommissioning problem at the end of mine life is bigger. Initial developments in northern Saskatchewan were based on the company town concept. At the time there were 25 or more advanced exploration projects in the Uranium City area, 10 of which developed into producing

  10. Bioactive compounds from northern plants.

    Hohtola, Anja


    Northern conditions are characterised by long days with much light and low temperatures during the growing season. It has been chimed that herbs and berries grown in the north are stronger tasting compared to those of southern origin. The compounds imparting aroma and color to berries and herbs are secondary metabolites which in plants mostly act as chemical means of defense. Recently, the production of secondary metabolites using plant cells has been the subject of expanding research. Light intensity, photoperiod and temperature have been reported to influence the biosynthesis of many secondary metabolites. Native wild aromatic and medicinal plant species of different families are being studied to meet the needs of raw material for the expanding industry of e.g., health-promoting food products known as nutraceutics. There are already a large number of known secondary compounds produced by plants, but the recent advances in modern extraction and analysis should enable many more as yet unknown compounds to be found, characterised and utilised. Rose root (Rhodiola rosea) is a perennial herbaceous plant which inhabits mountain regions throughout Europe, Asia and east coastal regions of North America. The extract made from the rhizomes acts as a stimulant like the Ginseng root. Roseroot has been categorized as an adaptogen and is reported to have many pharmacological properties. The biologically active components of the extract are salitroside tyrosol and cinnamic acid glycosides (rosavin, rosarin, rosin). Round-leaved sundew (Drosera rotundifolia L.) has circumboreal distribution. It inhabits nutrient-poor, moist and sunny areas such as peat bogs and wetlands. Sundew leaves are collected from the wild-type for various medicinal preparations and can be utilized in treating e.g., as an important "cough-medicine" for different respiratory diseases. The antimicrobial activity of extracts of aerial parts against various bacteria has been investigated. Drosera produces

  11. Isotope hydrology in northern Chile

    Environmental isotope analyses were done on samples from aquifers in the Pampa del Tamarugal and the Salar de Atacama drainage basin in northern Chile. In the Pampa it is possible to delineate individual groundwater bodies on the basis of their 18O and deuterium contents and, in some cases, to relate these to specific recharge areas. A marked displacement from the meteoric water line indicates that river recharge is an important mechanism for groundwater renewal. Groundwater ages appear high at distance from the Andes and much of the water found in the Pampa may have to be treated as a non-renewable resource. The groundwaters, springs and rivers of the Salar de Atacama drainage basin vary between -6.09 and -8.06%. No difference between the different waters can be recognized and an evaporative isotope enrichment indicates that also here river recharge is an important process. Some groundwaters adjacent to the Salar are very salty but 18O and deuterium data show that these waters are not refluxed brines but simply salty freshwater. The 14C contents in groundwaters and springs are very low but their delta13C values are high. It is concluded that this is probably due to the uptake of volcanic CO2. 14C age dating is thus not possible unless the delta13C values of all possible carbon sources can be defined and the geochemical evolution of the groundwaters is better understood. (author)

  12. Bread ovens in Northern Oretania

    García Huerta, Rosario


    Full Text Available This paper intends to bring to light an unusual type of domestic structure in the northern Oretania, namely the ovens used for the production of bread. The study of their distribution, as well as their dimensions and constructive features, indicates they are more complex structures, with collective or communal characters. At the same time, it gives us some knowledge of the internal organization of the main oritanian oppidas.

    Este artículo pretende dar a conocer un tipo de estructura doméstica poco habitual en la Oretania septentrional, como son los hornos destinados a la producción de pan. El análisis de su distribución, así como sus dimensiones y características constructivas, revela que se trata de estructuras más complejas, de carácter colectivo o comunal, lo que permite aproximarnos al conocimiento de la articulación interna de los principales oppida oretanos.

  13. Transcription factor proteomics: identification by a novel gel mobility shift-three-dimensional electrophoresis method coupled with southwestern blot and high-performance liquid chromatography-electrospray-mass spectrometry analysis.

    Jiang, Daifeng; Jia, Yinshan; Jarrett, Harry W


    Transcription factor (TF) purification and identification is an important step in elucidating gene regulatory mechanisms. In this study, we present two new electrophoretic mobility shift assay (EMSA)-based multi-dimensional electrophoresis approaches to isolate and characterize TFs, using detection with either southwestern or western blotting and HPLC-nanoESI-MS/MS analysis for identification. These new techniques involve several major steps. First, EMSA is performed with agents that diminish non-specific DNA-binding and the DNA-protein complex is separated by native PAGE gel. The gel is then electrotransferred to PVDF membrane and visualized by autoradiography. Next, the DNA-protein complex, which has been transferred onto the blot, is extracted using a detergent-containing elution buffer. Following detergent removal, concentrated extract is separated by SDS-PAGE (EMSA-2DE), followed by in-gel trypsin digestion and HPLC-nanoESI-MS/MS analysis, or the concentrated extract is separated by two-dimensional gel electrophoresis (EMSA-3DE), followed by southwestern or western blot analysis to localize DNA binding proteins on blot which are further identified by on-blot trypsin digestion and HPLC-nanoESI-MS/MS analysis. Finally, the identified DNA binding proteins are further validated by EMSA-immunoblotting or EMSA antibody supershift assay. This approach is used to purify and identify GFP-C/EBP fusion protein from bacterial crude extract, as well as purifying AP1 and CEBP DNA binding proteins from a human embryonic kidney cell line (HEK293) nuclear extract. AP1 components, c-Jun, Jun-D, c-Fos, CREB, ATF1 and ATF2 were successfully identified from 1.5 mg of nuclear extract (equivalent to 3×10(7) HEK293 cells) with AP1 binding activity of 750 fmol. In conclusion, this new strategy of combining EMSA with additional dimensions of electrophoresis and using southwestern blotting for detection proves to be a valuable approach in the identification of transcriptional complexes

  14. Earthquake Damage, Northern Iran, June 21, 1990

    National Oceanic and Atmospheric Administration, Department of Commerce — A magnitude 7.7 earthquake occurred in the Gilan Province between the towns of Rudbar and Manjil in northern Iran on Thursday, June 21, 1990. The event, the largest...

  15. Northern Pintail - Flight Path Telemetry [ds117

    California Department of Resources — North-south flight paths of radio-tagged female northern pintails were monitored in a section of Highway 152 near Los Banos, California during 4 and 11 November and...

  16. Biological processes of the northern Indian Ocean

    Madhupratap, M.; Parulekar, A.H.

    , increases in the abundances of microzooplankton may lead to higher standing stocks of mesozooplankton. Blooms of phytoplankton and swarms of zooplankton are common in the northern Indian Ocean and influence rates of particle fluxes into the ocean interiors...

  17. Northern California 36 arc-second DEM

    National Oceanic and Atmospheric Administration, Department of Commerce — The 36-second Northern California Elevation Grid provides bathymetric data in ASCII raster format of 36-second resolution in geographic coordinates. This grid is...

  18. Northern California 6 arc-second DEM

    National Oceanic and Atmospheric Administration, Department of Commerce — The 6-second Northern California Elevation Grid provides bathymetric data in ASCII raster format of 6-second resolution in geographic coordinates. This grid is...

  19. Permafrost Soils Database for Northern Alaska 2014

    Arctic Landscape Conservation Cooperative — This database contains soil and permafrost stratigraphy for northern Alaska compiled from numerous project data files and reports. The Access Database has main data...

  20. CNMI Northern Island Bottomfish System (NIBS)

    National Oceanic and Atmospheric Administration, Department of Commerce — The Commonwealth of the Northern Mariana Islands (CNMI), Division of Fish and Wildlife (DFW) conducted a market sampling program that existed for a few years back...

  1. Requirements for neonatal cots. Northern Neonatal Network.


    A prospective survey of activity in neonatal nurseries associated with 17 specialist maternity units delivering some 38,700 babies in the Northern region was undertaken during 1991. Data were collected concerning the numbers of babies requiring various forms of neonatal care, using a nursing dependency scale validated by work study. Facilities for prolonged high dependency care are partially decentralised in the Northern region, with a network of five units operating on a flexible and collabo...

  2. Examining Affordability Issues in Northern Ireland

    Brown, L.; S. McGreal; Gibb, K.; Joe Frey; Frey, J.


    The paper explores the relationship between house price growth, income levels, investor activity and the effects on entrants to the housing market. It concentrates on the area of first time buyers to the market and the issues of affordability which are now becoming increasingly apparent in Northern Ireland. House prices in Northern Ireland have experienced unprecedented growth particularly in the last 12 months (37% in 2006 alone according to the University of Ulster Quarterly House Price Ind...

  3. The rock resources of the Northern Emirates

    Mitchell, Clive; Styles, Michael


    The Rock Resources of the Northern Emirates The United Arab Emirates (UAE) has vast resources of limestone and hard rock in the northern Emirates. These are currently exploited by quarrying companies to produce construction aggregate and raw material for the manufacture of cement, with a small amount being used to produce rock wool, dimension stone and mineral filler. The demand by industry for higher value mineral products that could be produced from these resources is mostly met by impor...

  4. Displacing AIDS: therapeutic transitions in Northern Uganda

    Wilhelm-Solomon, M. M.; Alexander, Jocelyn; Daley, Patricia


    This doctoral project, entitled 'Displacing AIDS: Therapeutic Transitions in Northern Uganda' examines the biosocial transitions engendered by the treatment of HIV, focusing on antiretroviral therapy (ART/ARV) interventions, and the ways these are intertwined with the social transitions of conflict, displacement and return. The research involved an inter-disciplinary qualitative study with internally displaced communities living with HIV in northern Uganda, during 10 months fieldwork between ...

  5. Patterns and risks of trichinella infection in humans and pigs in northern Laos.

    James V Conlan

    Full Text Available Several outbreaks of trichinellosis associated with the consumption of raw pork have occurred in Laos since 2004. This cross-sectional study was conducted in four provinces of northern Laos to investigate the seroepidemiology of trichinellosis in the human population and determine the prevalence and species of Trichinella infection in the domestic pig population. Serum samples and questionnaire data were obtained from 1419 individuals. Serum samples were tested for Trichinella antibodies by ELISA using larval excretory-secretory (ES antigens and a subset of 68 positive samples were tested by western blot. The seroprevalence of Trichinella antibodies was 19.1% (95% confidence interval (CI = 17.1-21.1%. The risk of having antibodies detected by ELISA using ES antigens increased with age, being of Lao-Tai ethnicity, living in Oudomxay province and being male. Tongue and diaphragm muscle samples were collected from 728 pigs and tested for Trichinella larvae by the artificial digestion method. Trichinella larvae were isolated from 15 pigs (2.1% of which 13 were identified as T. spiralis by molecular typing; the species of the two remaining isolates could not be determined due to DNA degradation. Trichinella spp. are endemic in the domestic environment of northern Laos and targeted preventative health measures should be initiated to reduce the risk of further outbreaks occurring.

  6. Evaluación diagnóstica de fracciones cromatográficas de Fasciola hepatica mediante Western Blot y ELISA en animales infectados Diagnostic evaluation of chromatographic fractions of Fasciola hepatica by Western Blot and ELISA in infected animals



    Full Text Available La fasciolosis causada por Fasciola hepatica se diagnostica rutinariamente mediante el examen coprológico. Debido a que este examen no es 100% sensible y además es ineficaz en la etapa pre-patente, se realizó este trabajo con el objeto de caracterizar y seleccionar fracciones antigénicas de valor diagnóstico de extractos de excreción-secreción del parásito. Se utilizó cromatografía de exclusión por tamaño molecular (Sephacryl S-300, electroforesis en geles de poliacrilamida en ambiente reductor (SDS-PAGE y posterior western blot (WB, además de un método de "enzyme-linked immuno-sor-bent assay" (ELISA en microplaca. Para evaluar el valor diagnóstico de los antígenos se usaron sueros de las especies ovina, porcina y equina en tres estados (sanos, con otras parasitosis y naturalmente infectados con F. hepatica. Mediante el método cromatográfico se obtuvieron hasta 5 "peaks", que interpolados en una curva patrón representaron polipéptidos de pesos aproximados de 2.000, 400, 150, 29 y menores a 29 kDa. De éstos, los inmunorreactivos específicos para la enfermedad en las tres especies animales, bajo los criterios de SDS-PAGE y posterior WB, fueron los de 400, 150, 29 y The antigenic components of excretory-secretory products of adult F. hepatica, were separated by gel filtration chromatography (Sephacryl S-300 and then analized by polyacrylamide gel electrophoresis (SDS-PAGE, followed by Western Blot (WB. In order to evaluate the sensitivity, specificity and predictive value of the selected fractions an enzyme-linked immunosorbent assay (ELISA was used with sera from sheep, swine and horses infected with F. hepatica, as well as with control sera (uninfected animals. The chromatographic curve presented up to 5 peaks, representing polypeptides with a molecular weight of 2000, 400, 150, 29 and less than 29 kDa, according to the interpolation with a standard curve of commercial polypeptide molecular weights. The results obtained with

  7. Viral diseases of northern ungulates

    K. Frölich


    Full Text Available This paper describes viral diseases reported in northern ungulates and those that are a potential threat to these species. The following diseases are discussed: bovine viral diarrhoea/mucosal disease (BVD/MD, alphaherpesvirus infections, malignant catarrhal fever (MCF, poxvirus infections, parainfluenza type 3 virus infection, Alvsborg disease, foot-and-mouth disease, epizootic haemorrhage disease of deer and bluetongue disease, rabies, respiratory syncytial virus infection, adenovirus infection, hog-cholera, Aujeszky's disease and equine herpesvirus infections. There are no significant differences in antibody prevalence to BVDV among deer in habitats with high, intermediate and low density of cattle. In addition, sequence analysis from the BVDV isolated from roe deer (Capreolus capreolus showed that this strain was unique within BVDV group I. Distinct BVDV strains might circulate in free-ranging roe deer populations in Germany and virus transmission may be independent of domestic livestock. Similar results have been obtained in a serological survey of alpha-herpesviruses in deer in Germany. Malignant catarrhal fever was studied in fallow deer (Cervus dama in Germany: the seroprevalence and positive PCR results detected in sheep originating from the same area as the antibody-positive deer might indicate that sheep are the main reservoir animals. Contagious ecthyma (CE is a common disease in domestic sheep and goats caused by the orf virus. CE has been diagnosed in Rocky Mountain bighorn sheep (Ovis canadensis, mountain goats (Oreamnos americanus, Dall sheep (Ovis dalli, chamois (Rupkapra rupi-capra, muskox {Ovibos moschatus and reindeer (Rangifer tarandus. Most parainfluenza type 3 virus infections are mild or clinically undetectable. Serological surveys in wildlife have been successfully conducted in many species. In 1985, a new disease was identified in Swedish moose (Alces alces, designated as Alvsborg disease. This wasting syndrome probably

  8. Stratigraphy of the Martian northern plains

    Tanaka, K. L.


    The northern plains of Mars are roughly defined as the large continuous region of lowlands that lies below Martian datum, plus higher areas within the region that were built up by volcanism, sedimentation, tectonism, and impacts. These northern lowlands span about 50 x 10(exp 6) km(sup 2) or 35 percent of the planet's surface. The age and origin of the lowlands continue to be debated by proponents of impact and tectonic explanations. Geologic mapping and topical studies indicate that volcanic, fluvial, and eolian deposition have played major roles in the infilling of this vast depression. Periglacial, glacial, fluvial, eolian, tectonic, and impact processes have locally modified the surface. Because of the northern plains' complex history of sedimentation and modification, much of their stratigraphy was obscured. Thus the stratigraphy developed is necessarily vague and provisional: it is based on various clues from within the lowlands as well as from highland areas within and bordering the plains. The results are summarized.

  9. Earthquake hazard analysis in northern Egypt

    Earthquake activity and seismic hazard analysis are important components of the seismic aspects for very essential structures such as major dams and nuclear power plants. Setting of nuclear power plants becomes of increasing important in northern Egypt with the commitment towards promoting nuclear electric generation. Therefore, the annual seismic hazard maps with non-exceedence probability of 80%, 85% and 90% are given. These maps show that northern Egypt is severely affects by earthquakes from potential sources around Sinai peninsula. Three sites (Nile Delta, Cairo, and Ismailia region) have been chosen to estimated the earthquake hazard in more detailes to serve as a basic parameter to the safety factor of different projects in these regions. A seismic safety factor of intensity 8.5 should be considered in designing the vital projects in northern Egypt. (author)

  10. Climate Impacts on Northern Canada: Regional Background

    Prowse, Terry D.; Peters, Daniel L. (Water and Climate Impacts Research Centre, Environment Canada, Dept. of Geography, Univ. of Victoria, Victoria, BC (Canada)). e-mail: terry.prowse@ec.gc.caa; Furgal, Chris (Indigenous Environmental Studies Program, Trent Univ., Peterborough, ON (Canada)); Bonsal, Barrie R. (National Water Research Inst., National Hydrology Research Centre, Environment Canada, Saskatoon, SK (Canada))


    Understanding the implications of climate change on northern Canada requires a background about the size and diversity of its human and biogeophysical systems. Occupying an area of almost 40% of Canada, with one-third of this contained in Arctic islands, Canada's northern territories consist of a diversity of physical environments unrivaled around the circumpolar north. Major ecozones composed of a range of landforms, climate, vegetation, and wildlife include: Arctic, boreal and taiga cordillera; boreal and taiga plains; taiga shield; and northern and southern Arctic. Although generally characterized by a cold climate, there is an enormous range in air temperature with mean annual values being as high as -5 deg C in the south to as low as -20 deg C in the high Arctic islands. A similar contrast characterizes precipitation, which can be >700 mm y-1 in some southern alpine regions to as low as 50 mm y-1 over islands of the high Arctic. Major freshwater resources are found within most northern ecozones, varying from large glaciers or ice caps and lakes to extensive wetlands and peat lands. Most of the North's renewable water, however, is found within its major river networks and originates in more southerly headwaters. Ice covers characterize the freshwater systems for multiple months of the year while permafrost prevails in various forms, dominating the terrestrial landscape. The marine environment, which envelops the Canadian Arctic Archipelago, is dominated by seasonal to multiyear sea ice often several meters thick that plays a key role in the regional climate. Almost two-thirds of northern Canadian communities are located along coastlines with the entire population being just over 100 000. Most recent population growth has been dominated by an expansion of nonaboriginals, primarily the result of resource development and the growth of public administration. The economies of northern communities, however, remain quite mixed with traditional land

  11. Biogeochemical processes in the northern Indian Ocean

    Gaye-Haake, B.; Guptha, M.V.S.; Murty, V.S.N.; Ittekkot, V.

    Indian rivers dominate sedimentation processes in the Bay of Bengal and can reach the 1845–1847 Northern Indian Ocean The majority of contributions deals with results from bilateral programs such as the long-term Indo-German program on ‘‘Particle fluxes... in the northern Indian Ocean’’, and from national research programs of India and Germany con- ducted as a contribution to the international JGOFS Process Study in the Arabian Sea. Addi- tional contributions cover research carried out within India’s Polymetallic...

  12. Rural telemedicine project in northern New Mexico

    Zink, S.; Hahn, H.; Rudnick, J.; Snell, J.; Forslund, D. [Los Alamos National Lab., NM (United States); Martinez, P. [Northern New Mexico Community Coll., Espanola, NM (United States)


    A virtual electronic medical record system is being deployed over the Internet with security in northern New Mexico using TeleMed, a multimedia medical records management system that uses CORBA-based client-server technology and distributed database architecture. The goal of the NNM Rural Telemedicine Project is to implement TeleMed into fifteen rural clinics and two hospitals within a 25,000 square mile area of northern New Mexico. Evaluation of the project consists of three components: job task analysis, audit of immunized children, and time motion studies. Preliminary results of the evaluation components are presented.

  13. Northern Jutland as an Intertextual Location

    Hansen, Kim Toft; Christensen, Jørgen Riber


    With the region of Northern Jutland as a concrete case, the discussion about peripheral areas in Denmark will be contextualized in Augé's theory about anthropological places and non-places, and his theory will be expanded constructively and critically with the concept of intertextual locations. The...... Northern Jutland can be regarded as a location potential of this kind, which is can promote film and media production in the region. An important point in this connection will be that there may be a local and peripheral wish to become an intertextual location because this may lead to regional development...

  14. Seromonitoring of rinderpest in Northern Uganda

    Seromonitoring of rinderpest was carried out in the Northern region of Uganda which covers the sub-regions of West Nile, Northern Uganda, and N.E. Uganda. Three thousand nine hundred ninety two serum samples were collected and tested using the competitive ELISA. The antibody prevalence ranged from 21% to 81%. Although the areas at high risk of rinderpest had low antibody levels their neighbors seem to be establishing immune barrier. Since all the sampled districts were equally supported to carry out vaccination, this survey has emphasized the usefulness of sero monitoring in assessing the success of vaccination programmes. (author). 3 refs, 3 figs, 2 tabs

  15. Northern Land Council v. the Commonwealth

    The Ranger Project Area in the Northern Territory contains deposits of uranium. By section 5 of the Atomic Energy Amendment Act (No. 2) 1980 the assignment of an agreement between the Commonwealth and joint venturers for the conduct of uranium mining was authorised. The Northern Land Council, representing Aboriginal interests, challenged the validity of the section. It was held that the section was valid. The Council also submitted that, although it had entered into an agreement with the Commonwealth in 1978, the agreement was void or voidable

  16. Aisle-truss houses of Northern Jutland

    Eybye, Birgitte Tanderup


    The aisle-truss houses of Northern Jutland were built under hard conditions, such as harsh climate and scarce resources. Hence, the aisle-truss houses display a number of resource-saving and sustainable building principles, including the arcade construction and the use of passive energy strategies......, which make them relevant to research. This paper investigates resource-saving and sustainable principles in the aisle-truss houses of Thy, Northern Jutland. General features as well as three cases of the one-wing dwelling aisle-truss houses are studied. The aim is to improve the understanding of aisle...

  17. Northern and Southern RE Groups Ended Resultlessly


    In September 2002, "Organizing of National Rare EarthEnterprises Group" which was delivered by original NationalEconomy & Trade Committee and original National PlanningCommittee, Ministry of Finance P.R.C, Ministry of Land &Resource P.R.C. and Ministry of Foreign Economy & Trade,was approved by the State Council of P.R.C. to organizeSouthern and Northern Rare Earth Groups. On October 30,2002, China Northern RE Group Co., Ltd Preparation Teamheld the foundation convention in Baotou, which drew thecurtain of ...

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

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


    Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results...... 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...

  19. Regional health library service in northern Ireland.

    Crawford, D S


    The regional medical library service provided to physicians, hospitals, nurses, social workers, and health care administrators throughout Northern Ireland by the Queen's University of Belfast is described. A brief outline of the National Health Service in the United Kingdom is given, and the library service is described in terms of collections, cataloging, interlibrary loan, and reference.

  20. Applied Indigenous Studies at Northern Arizona University.

    Trosper, Ronald L.


    The Applied Indigenous Studies program at Northern Arizona University aims to prepare American Indian students to assume tribal leadership roles. Its location in the College of Ecosystem Science and Management emphasizes its land-oriented and applied focus. The program's development, core courses, and academic requirements for bachelors degrees…

  1. Forest Fire History in the Northern Rockies

    Arno, Stephen F.


    Recent fire-scar studies in the northern Rocky Mountains have documented forest fire history over the past few centuries. They reveal that in some forest types fire maintained many-aged open stands of seral trees. In other types, major fires caused replacement of the stands. Often, however, fires burned at variable intensities, creating a mosaic of stands differing in composition and structure.

  2. Northern Ireland: post-conflict education model?

    Paul Nolan


    Full Text Available Northern Ireland’s Good Friday Agreement of 1998 calledfor “initiatives to facilitate and encourage integratededucation” but progress has been painfully slow. Only 5%of the total school population are in integrated schools(those bringing together students and staff from boththe Protestant and Catholic traditions. Only 1.4% of theadult population has experienced integrated schooling.

  3. The Times and the Northern Ireland Conflict

    Zouhaïr Abassi


    Full Text Available Abstract: In societies in conflict the role of the media is supposed to be neutral and to report conflicts fairly and with balanced analyses. By their public debates on conflicts they are also supposed to take part in pacifying societies and in helping to bring peace. Cottle (1997, for instance, explained that even though some findings related to the British media and its reporting of the Northern Ireland conflict were relevant, he argued that they needed revision. Consequently, he proposed new paradigms of media studies. Elliott (1977 and Curtis(1996 showed that the British media concentrated on violence in general and on republican violence in particular. Moreover, they argued that the British media neglected social and political contexts in their reporting of the conflict. The aim of this paper is then to examine some aspects of how the British media cover the Northern Ireland conflict. We studied the coverage of the Northern Ireland conflict by The (London Times (1990-1995. We used a discourse analysis method to study the paper’s discourse structure in its representation of the Northern Ireland conflict.

  4. Winter cooling in the northern Arabian Sea

    PrasannaKumar, S.; Prasad, T.G.

    forcing that leads to the observed high productivity during winter in the northern Arabian Sea. The weak northerly winds and increased solar insolation during the inter-monsoon period, led to the development of a highly stratified upper layer with warm sea...

  5. Mining and energy in the Northern Territory

    Included in this book is a section on each of the major minerals of present or future importance to the Northern Territory. Brief details of the uranium mining projects at Nabarlek, Ranger, Koongarra and Jabiluka in the Alligator Rivers regions are given. Subjects such as environmental protection, Aboriginal land rights and the geology of the area are also considered

  6. Controlling human oesophagostomiasis in northern Ghana

    Ziem, Juventus Benogle


    This thesis describes aspects of the epidemiology and attempts to control infection and pathology due to the nematode parasite Oesophagostomum bifurcum . In northern Ghana and Togo O. bifurcum is an important parasite of humans; elsewhere it is predominantly seen as a parasite of non-human primates.

  7. Northern oil and gas - annual report 1997

    Oil and gas resources on crown lands north of 60 degrees latitude are managed by the federal government under the Northern Oil and Gas Directorate of the Department of Indian and Northern Affairs and Northern Development. Notable activities in 1997 included the re-introduction in Parliament of the Canada-Yukon Oil and Gas Accord of 1993. The proposed legislation aims to transfer power to the Yukon government by 1998, making it responsible for administration and regulation of onshore oil and gas exploration and development in the territory. In 1997, new gas reserves were uncovered in the southern Northwest Territories. Thirteen new wells were drilled and about $45 million in exploration work was carried out. This included work by Inuvialuit Petroleum Corp., in the Ikhil gas field in the Mackenzie Delta which could end Inuvik's dependency on imported petroleum products. A statistical overview of the oil and gas activities under Northern Oil and Gas was summarized and broken down by region. 11 refs

  8. From Poetry to Music: "Northern Lullaby"

    Cardany, Audrey Berger


    Nancy White Carlstrom's children's book, "Northern Lullaby," conjures through poetry the beauty of the Alaskan landscape in the evening. The book provides an opportunity for music teachers to help their students transform text and visual images to music. The author describes connections for reading comprehension in the general music classroom and…

  9. 76 FR 31299 - Northern New Mexico Resource Advisory Committee


    ... Forest. BILLING CODE 3410-11-P ... Forest Service Northern New Mexico Resource Advisory Committee AGENCY: Forest Service, USDA. ACTION: Correct FR Doc. 2011-12588; Notice of meeting. SUMMARY: The Northern New Mexico Resource...

  10. Discussion on several geological problems and uranium metallogeny on northern border of northern China block (platform)

    According to the informations from the satellite image and the field investigation the following geological events on the northern border of the Northern China Block are recognized and confirmed, duch as suture zones between blocks, folding-reversed fault zones, back-arc collision zones (faulted zones), transitional zone between platform and geosyncline, magmatic are and the double sturcture composed of NNE trend magmatic active belt and fault-depression belts (basins) of Yenshan-Ximalaya age. On thsee bases the following problems, such as the unique structural environment of uranium mineralization related to abyssal magmatic rocks and Yenshan magmatic active zone (including volcanic belt) on the northern border of the Northern China Block, the metallogenetic modes for urnaium deposits of 'magmatic type' and 'neutralized surface type' in fault-depression zone and the classification of uranium metallogenetic belts and the criteria for such classification are studied and discussed. Several uranium deposits are given for illustrations

  11. 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.

    Lamer, S; Jungblut, P R


    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. PMID:11270870

  12. Canada: Native Land Rights and Northern Development. IWGIA Document 26.

    Cumming, Peter A.

    Presenting the critical elements for a new and meaningful relationship between the Inuit and the emerging industrial society of Northern Canada, this publication includes: (1) Canada as a Nation State and Northern Development; (2) Northern Development and Institutions in Decision-Making; (3) The Northwest Territories and Yukon Territory as…

  13. 77 FR 35958 - Northern Natural Gas Company; Notice of Application


    ... Energy Regulatory Commission Northern Natural Gas Company; Notice of Application Take notice that on May 30, 2012, Northern Natural Gas Company (Northern), 1111 South 103rd Street, Omaha, Nebraska 68124... regulations and section 7(b) of the Natural Gas Act, to abandon by sale to DKM Enterprises, LLC (DKM)...

  14. 75 FR 35779 - Northern Natural Gas Company; Notice of Application


    ... Energy Regulatory Commission Northern Natural Gas Company; Notice of Application June 16, 2010. Take notice that on June 2, 2010, Northern Natural Gas Company (Northern), 1111 South 103rd Street, Omaha... Natural Gas Act, for a certificate of public convenience and necessity authorizing the increase...

  15. 78 FR 51716 - Northern Natural Gas Company; Notice of Application


    ... Energy Regulatory Commission Northern Natural Gas Company; Notice of Application Take notice that on August 1, 2013, Northern Natural Gas Company (Northern), 1111 South 103rd Street, Omaha, Nebraska 68124, filed an application pursuant to section 7(c) of the Natural Gas Act and part 157 of the...

  16. Uranium and thorium deposits of Northern Ontario

    This, the second edition of the uranium-thorium deposit inventory, describes briefly the deposits of uranium and/or thorium in northern Ontario, which for the purposes of this circular is defined as that part of Ontario lying north and west of the Grenville Front. The most significant of the deposits described are fossil placers lying at or near the base of the Middle Precambrian Huronian Supergroup. These include the producing and past-producing mines of the Elliot Lake - Agnew Lake area. Also included are the pitchblende veins spatially associated with Late Precambrian (Keweenawan) diabase dikes of the Theano Point - Montreal River area. Miscellaneous Early Precambrian pegmatite, pitchblende-coffinite-sulphide occurrences near the Middle-Early Precambrian unconformity fringing the Lake Superior basin, and disseminations in diabase, granitic rocks, alkalic complexes and breccias scattered throughout northern Ontario make up the rest of the occurrences

  17. Cardiac surgical experience in northern Nigeria.

    Nwiloh, J; Edaigbini, S; Danbauchi, S; Babaniyi, I; Aminu, M; Adamu, Y; Oyati, A


    A pilot study was undertaken to determine the feasibility of establishing a heart surgery programme in northern Nigeria. During three medical missions by a visiting US team, in partnership with local physicians, 18 patients with heart diseases underwent surgery at two referral hospitals in the region. Sixteen (88.9%) patients underwent the planned operative procedure with an observed 30-day mortality of 12.5% (2/16) and 0% morbidity. Late complications were anticoagulant related in mechanical heart valve patients and included a first-trimester abortion one year postoperatively, and a death at two years from haemorrhage during pregnancy. This has prompted us to now consider bioprosthetics as the valve of choice in women of childbearing age in this patient population. This preliminary result has further stimulated the interest of all stakeholders on the urgency to establish open-heart surgery as part of the armamentarium to combat the ravages of heart diseases in northern Nigeria. PMID:22453514

  18. Trace gas fluxes from northern peatlands

    Moore, T. [McGill Univ., Montreal (Canada). Geography Dept.


    Peatlands cover large areas in northern environments: 1.1, 0.1 and 1.7 x 10{sup 4} km{sup 2} in Canada, Finland and the former Soviet Union, respectively. Interest has been generated into the role these extensive areas of peatlands play in controlling the chemistry of the atmosphere. In particular, it has become established that peatlands can be a source of methane (CH{sub 4}) and nitrous oxide (N{sub 2}O), and a sink of carbon dioxide (CO{sub 2}), the latter through the rates of plant production exceeding the rate of decomposition of plant material and peat. In this presentation the recent advances in trace gas flux measurements in northern peatlands are presented. (16 refs.)


    Neil Ferguson


    Full Text Available This article explores the disengagement and deradicalization experiences of Northern Irish loyalist paramilitaries from the Ulster Volunteer Force (UVF and Red Hand Commando (RHC. Interpretative phenomenological analysis was employed to develop an understanding of how the former combatants interpreted and made sense of their disengagement from violence extremism in Northern Ireland after the Belfast Agreement. The analysis of the interviews focusses around push and pull factors which either promote or hinder their ability to move away from violent extremism. The results find a resonance with recent research exploring disengagement and deradicalization processes with terror groupings across the globe and the ideological spectrum. The findings are discussed in relation to a number of topics, including the role of prison, barriers to disengagement, continued commitment and radicalization after desistence from violent extremism, the role of life changes in promoting disengagement and how organizational pressures contain and influence individual disengagement.

  20. Cardiac surgical experience in northern Nigeria

    Nwiloh, J; Edaigbini, S; Danbauchi, S; Aminu, M.; Oyati, A; Babaniyi, I; Adamu, Y.


    Abstract A pilot study was undertaken to determine the feasibility of establishing a heart surgery programme in northern Nigeria. During three medical missions by a visiting US team, in partnership with local physicians, 18 patients with heart diseases underwent surgery at two referral hospitals in the region. Sixteen (88.9%) patients underwent the planned operative procedure with an observed 30-day mortality of 12.5% (2/16) and 0% morbidity. Late complications were anticoagulant related in m...

  1. New geoscience surveys in Northern Ireland

    Young, Mike; Smyth, Dermot


    New regional geochemical and geophysical surveys are in progress in Northern Ireland. These will advance the development of the natural resource industry and provide a baseline of information against which to measure environmental change. Soils, stream-sediments and stream-waters are being sampled, according to the G-BASE standard of the British Geological Survey, and analysed by XRF and ICP. The airborne geophysical survey will be flown by a fixed-wing aircraft equipped with magnetometer, 25...

  2. Lithological control on soil chemistry, Northern Ireland

    Ashton, Nicola; Pattrick, R. A. D.; J. R. Lloyd; B. E. van Dongen; Tye, A.


    The geological diversity of Northern Ireland provides a unique opportunity to investigate the effects of differing lithologies upon the (organic) geochemistry of overlying soils. This will aid the understanding of how this influences the microbial populations within the soil and their role in biochemical cycling. Whilst several factors contribute to the properties of developing soils, source rock is one of the major controls in determining chemistry and formation rate (Chengmin et al., 2...

  3. Ethno-botanical studies from northern Pakistan

    In this research paper efforts have been made to document the ethno-botanical knowledge of important plant species found in Northern Pakistan. It includes Thandiani, Galiat, Kaghan, Swat, Buner, Dir, Chitral and Northern Areas of Pakistan. The area has many climatic and vegetation zones or biomes. Locals residing in mountainous areas belonging to various ethnic groups are traditionally utilizing plants over many generations; these ethnic groups have their distinct life style, belief, traditions and cultural heritage. Plant collection and data regarding traditional uses in various areas of Northern Pakistan has been done periodically in different flowering /fruiting seasons. Locals of old age belonging to various ethnic groups were personally interviewed for establishing uses of plants. Photography is done for easy identification and habitat recognition. Collected plant specimens and seeds were preserved. Plant species were dried, mounted, identified and authenticated. Seventy six species were known to have traditional and ethno botanical uses. Plants have been utilized for many generations. Ethnic groups have distinct life style and have different economic uses for these plants. Due to unsustainable exploitation of natural habitats scarcity of drug plants has occurred. As consequence some species are depleting and may become extinct in near future, e. g. Morchella esculenta, Colchicum lueteum and Viola serpens are just a few of these. Although some sporadic information is available about the flora of this region but very little documented record of the ethno-botanically important plants has been established. It is expected that this research paper will be beneficial for students, researchers, farmers, foresters and general public. On the basis of data obtained it is concluded that ethno-botanical Flora of Northern Pakistan is quite rich and is diverse, due to the difference in altitude, climate and other topographic conditions. (author)

  4. Structural curiosity of the northern Istria

    Ladislav Placer


    In northern Istria certain structural particularities exist that till now were not explained,and some of them not even known. They are all associated with underthrusting of the Adriatic microplate (Istra) under the Dinarides. The Rokava fault of cross-dinaricdirection was discovered that exerts an important influence on hydrographic pattern in this part of Istria. The cross-dinaric sinistral strike-slip faults above Glinščica stream (Rosandra) are of local importance only, since at Kastelec a...

  5. Pneumonic Plague Outbreak, Northern Madagascar, 2011

    Richard, Vincent; Riehm, Julia M; Herindrainy, Perlinot; Soanandrasana, Rahelinirina; Ratsitoharina, Maherisoa; Rakotomanana, Fanjasoa; Andrianalimanana, Samuel; Holger C. Scholz; Rajerison, Minoarisoa


    Yersinia pestis, the causative agent of plague, is endemic to Madagascar, particularly to the central highlands. Although plague has not been previously reported in northern Madagascar, an outbreak of pneumonic plague occurred in this remote area in 2011. Over a 27-day period, 17 suspected, 2 presumptive, and 3 confirmed human cases were identified, and all 15 untreated 20 patients died. Molecular typing of Y. pestis isolated from 2 survivors and 5 Rattus rattus rat samples identified the Mad...

  6. Food irradiation - a Northern Ireland dimension

    Irradiation is a technology which has been exploited in a wide variety of industries ranging from sterilization of medical products and polymer modification to applications with respect to food. Whilst food irradiation has recently become a controversial subject, the process has been studied for many years. Many products could be irradiated to advantage and these need to be thoroughly investigated before final recommendations can be made as to the commercial feasibility and suitability of the processing technology in the Northern Ireland context

  7. Mobile rural youth in northern Ghana

    Gough, Katherine; Birch-Thomsen, Torben


    Young people in northern Ghana are growing up in a very different environment from their southern counterparts. While the south is the locus of the major cities, industries, and most important cash crops, the north is primarily rural with an agricultural base, much of it subsistence. This distinc...... to stop them from moving, their mobility should be seen as a potential source of capital and experience, which those who return can use to invest in farming or establishing an alternative business....


    Barrett, Christopher B.; Chabari, Francis; Bailey, DeeVon; Coppock, D. Layne; Little, Peter D.


    This paper uses detailed, transactions-level data and a structural-heteroskedasticity-in-mean model to identify the determinants of livestock producer prices for pastoralists in the drylands of northern Kenya. The empirical results confirm the importance of animal characteristics, periodic events that predictably shift local demand or supply, and especially rainfall on the prices pastoralists receive for animals. Price risk premia are consistently negative in these livestock markets. The impo...

  9. Early millet use in northern China

    Yang, Xiaoyan; Wan, Zhiwei; Perry, Linda; Lu, Houyuan; Wang, Qiang; Zhao, Chaohong; Li, Jun; XIE, FEI; Yu, Jincheng; Cui, Tianxing; Wang, Tao; Li, Mingqi; Ge, Quansheng


    It is generally understood that foxtail millet and broomcorn millet were initially domesticated in Northern China where they eventually became the dominant plant food crops. The rarity of older archaeological sites and archaeobotanical work in the region, however, renders both the origins of these plants and their processes of domestication poorly understood. Here we present ancient starch grain assemblages recovered from cultural deposits, including carbonized residues adhering to an early p...

  10. Sporotrichosis from the Northern Territory of Australia

    Subedi, Shradha; Kidd, Sarah E.; Baird, Robert W.; Coatsworth, Nicholas; Ralph, Anna P


    We report three cases of lymphocutaneous infection caused by the thermally dimorphic fungus, Sporothrix schenckii from Australia's tropical Northern Territory. Two cases were acquired locally, making them the first to be reported from this region. All three cases presented with ulceration in the limb; however, the classical sporotrichoid spread was present only in the first two cases. Their occurrence within several weeks of each other was suggestive of a common source of environmental contam...