Sample records for antiserum

  1. Production of a Mouse Antiserum to Bacteroides fragilis Enterotoxin Using a Recombinant Enterotoxin Precursor


    Sandini, Silvia; d'Abusco, Anna Scotto; La Valle, Roberto; Pantosti, Annalisa


    The precursor of the Bacteroides fragilis metalloprotease enterotoxin was cloned and expressed in Escherichia coli, which was not able to process the precursor into the biologically active enterotoxin. Mouse antiserum elicited to the recombinant precursor reacted with the purified enterotoxin and with a crude enterotoxin preparation from an enterotoxigenic strain. The antiserum neutralized the cytotoxic activity of the enterotoxin in HT-29 cells.

  2. Neonatal Treatment with Antiserum to Prolactin Lowers Blood Pressure in Rats (United States)

    Mills, David E.; Buckman, Maire T.; Peake, Glenn T.


    Prolactin administration reportedly increases blood pressure in rats and rabbits. To study the effects of prolactiin deficiency on blood pressure, rats were given saline, normal rabbit serum, or rabbit antiserum to rat prolactin on postnatal days 2 to 5. Both males and females given antiserum had significantly lower blood pressure at 14 weeks than rats given saline or normal rabbit serum. Blood pressure differences between females given antiserum and females given saline disappeared during and following pregnancy. The antiserum also lowered the concentration of prolactin in plasma 49 percent in males and decreased the prolactin response to ether stress in both sexes. These results suggest that endogenous prolactin is involved in blood pressure regulation.

  3. Neutralization of glucagon by antiserum as a tool in glucagon physiology. Lack of depression of basal blood glucose after antiserum treatment in rats

    DEFF Research Database (Denmark)

    Holst, J J; Galbo, H; Richter, Erik


    to more than one-third of the total glucagon content in the rat pancreas. That rapid, extensive, and lasting neutralization of glucagon had taken place after antiserum treatment was indicated by the following findings: When examined more than 1 h after the injection and after 60 min of exercise...

  4. Use of antiserum to neurotensin reveals a physiological role for the peptide in rat prolactin release. (United States)

    Vijayan, E; Carraway, R; Leeman, S E; McCann, S M


    Previous studies have indicated that the brain peptide neurotensin can stimulate prolactin release by direct action on the pituitary gland, whereas its action within the hypothalamus is inhibitory. The inhibitory action is mediated by the release of dopamine into the hypophyseal portal veins, which deliver the neurotransmitter to the anterior pituitary gland to inhibit prolactin release. Our experiments were done to evaluate the physiologic significance of these neurotensin actions by injecting the globulin fraction of highly specific neurotensin antiserum either intravenously or intraventricularly. Injection into the third ventricle of either 1 or 3 microliter of neurotensin antiserum significantly increased plasma prolactin concentrations in (i) ovariectomized and (ii) ovariectomized estrogen- and progesterone-primed rats within 1 hr of injection. The response was more pronounced in the ovariectomized than in the ovariectomized estrogen- and progesterone-treated animals and was dose related. Intraventricular injection of these doses of neurotensin antiserum also evoked elevations in plasma prolactin in intact males, which were significant but smaller in magnitude than those seen in female rats. To evaluate the effect of the antiserum on the pituitary directly, the antiserum was injected intravenously at a dose of 40 microliter, which was sufficient to block the blood pressure-lowering effect of neurotensin. After the intravenous injection of antiserum, a highly significant suppression of plasma prolactin occurred, detectable when first measured at 1 hr after injection in both ovariectomized and ovariectomized estrogen- and progesterone-treated animals; however, the intravenous injection of antiserum had no significant effect on the prolactin release in males. These data indicate the physiological significance of the hypothalamic inhibitory actions of neurotensin on prolactin release, which are probably mediated by its stimulation of dopamine release that in turn


    Institute of Scientific and Technical Information of China (English)

    聂新民; 周鸣; 桂嵘; 李小玲; 张必成; 李伟芳; 王蓉; 曹利; 李桂源


    Objective: To express nitroreductase gene NOR1 in Escherichia coli and to purify the expressed protein in order to get the polyclonal antibody of NOR1. Methods: The full length of NOR1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested with BamHI and XhoI restriction endonucleases.The plasmid pGEX-4T-2 was also digested with BamHI and XhoI, then the NOR1 gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR1 was identified by sequencing and restriction enzymes digestion. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express the GST fusion protein. The purified targeted protein obtained by affinity chromatography was used to immunize New Zealand rabbits to acquire antiserum.Antiserum was analyzed with immunoblot. Results: The1.25 kb NOR1 gene was successfully isolated. After induction,a new anticipated protein of 74 kDa appeared on sodiumdodecylsulfate polyacrylamide (SDS-PAGE). The result was confirmed by Western blot analysis, and the purified targeted protein was obtained by affinity chromatography.The titer of antiserum was 1:8. Conclusion: A high level of expression of GST-NOR1 is obtained in JM 105, and its antiserum can be prepared successfully.

  6. Isolation of labile Fcgamma-receptors from human peripheral blood lymphocytes and production of an antiserum. (United States)

    Sandilands, G P; Peel, M G; Froebel, K S; Belch, J J; MacSween, R N


    In this study, we have isolated membranelabile Fcgamma-receptors (i.e. FcgammaR I) from normal human peripheral blood lymphocytes and have produced a rabbit antiserum to this protein. Using this antiserum, we have shown that membrane-labile and membrane-stable (i.e. FcgammaR II) Fcgamma-receptors are antigenically distinct and that these two forms of the receptors probably coexist on the same lymphocyte subpopulation. Moreover, it was apparent that lymphocyte FcgammaR Is are distinct from FcgammaRs expressed on other cell types (e.g. monocytes, polymorphs and spermatozoa). Preliminary evidence does suggest, however, that human platelets express an FcgammaR which is antigenically similar to human lymphocyte FcgammaR I.

  7. Beneficial effects of anti-growth hormone antiserum in avian muscular dystrophy. (United States)

    Kurtenbach, E; Moraes, S S; Trocado, M T; Lôbo, G F; Nascimento, P S; Verjovski-Almeida, S


    Human subjects and mice have been found to have a milder progression of muscular dystrophy when the disease is associated with genotypically determined dwarfism. In this paper we describe an experimental test for reducing growth hormone in dystrophic chickens that uses rabbit anti-chicken growth hormone anti-serum (anti-cGH). Antiserum was injected daily into dystrophic (line 413) male chickens from day 1 to day 8 after hatching. Dystrophic chickens injected with anti-cGH maintained a significantly higher score in the standardized test for righting ability (P less than 0.001-0.051) from 3 to 9 1/2 wk after hatching when compared with dystrophic controls. The observed prolongation of the functional ability of injected dystrophic animals suggests that growth hormone plays a role in potentiating the symptoms of dystrophy in chickens.

  8. Cloning, Expression of Crocus sativus Phytoene Desaturase Gene and Preparation of Antiserum against It

    Institute of Scientific and Technical Information of China (English)

    Bai Jie; Miao Chen; Xu Ying; Tang Lin; Wang Zhi-tao; Chen Fang


    A 2 149 bp full length phytoene desaturase (PDS) cDNA was first cloned from saffron (Crocus sativus L.) stigma using RT-PCR technique and a rapid amplification of cDNA end (RACE) strategy. The cDNA has an open reading frame of 1 697 bp, which encodes a polypeptide of 565 amino acids. The coding region of the cDNA was inserted into a prokaryotic expression vector pET -21a(+) and overexpressed in E. coli BL21 (DE3). The fusion proteins were found largely in an insoluble inclusion bodies. The purified fusion protein was used to immunize rabbits to obtain polyclonal antiserum with titer of 1×105. Western blot analysis by using this particular antiserum showed that the higher expression level of PDS in mature stigma than in leaves and stamen, and the higher expression level of PDS in mature stigma than in young stigma.

  9. Expression of Recombinant Human FADD, Preparation of Its Polyclonal Antiserum and the Application in Immunoassays

    Institute of Scientific and Technical Information of China (English)

    Faiz MMT Marikar; Dingyuan Ma; Jianqiang Ye; Bo Tang; Weijuan Zheng; Jing Zhang; Min Lu; Zichun Hua


    The wild-type human Fas-associated death domain (FADD) protein was expressed as a His-tag fusion protein in Escherichla coli. Recombinant FADD proteins were purified under the denatured condition. After denatured protein purification, it was refoided and obtained at a yield of about 23 mg/L. Purified FADD exhibited as a homogenous band corresponding to the molecular weight of 31 kDa. Immunization of rabbits against the refolded FADD不得 protein was allowed the production of high titre polycional antiserum. This new polyclonal antibody could recognize recombinant FADD protein in Western blot. Immunoreactivity was also observed in immunofluorescence assay. The low cost polyclonal antiserum was applicable to extensive detection of FADD in various immunoassays.Cellular & Molecular Immunology. 2008;5(6):471-474.

  10. Evaluation of commercial chromatographic adsorbents for the direct capture of polyclonal rabbit antibodies from clarified antiserum

    DEFF Research Database (Denmark)

    Bak, Hanne; Thomas, O.R.T.


    We have carried out a rigorous evaluation of eight commercially available packed bed chromatography adsorbents for direct capture and purification of immumoglobulins from clarified rabbit antiserum. Three of these materials featured rProtein A (rProtein A Sepharose Fast Flow, Mabselect, Prosep rP...... evaluated on the basis of dynamic binding capacity, recovery, and purity) were obtained, which allowed clear recommendations concerning the choice of adsorbents best suited for antibody capture from rabbit antisera, to be made....

  11. Transcriptomic basis for an antiserum against Micrurus corallinus (coral snake venom

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    Ho Paulo L


    Full Text Available Abstract Background Micrurus corallinus (coral snake is a tropical forest snake belonging to the family Elapidae. Its venom shows a high neurotoxicity associated with pre- and post-synaptic toxins, causing diaphragm paralysis, which may result in death. In spite of a relatively small incidence of accidents, serum therapy is crucial for those bitten. However, the adequate production of antiserum is hampered by the difficulty in obtaining sufficient amounts of venom from a small snake with demanding breeding conditions. In order to elucidate the molecular basis of this venom and to uncover possible immunogens for an antiserum, we generated expressed sequences tags (ESTs from its venom glands and analyzed the transcriptomic profile. In addition, their immunogenicity was tested using DNA immunization. Results A total of 1438 ESTs were generated and grouped into 611 clusters. Toxin transcripts represented 46% of the total ESTs. The two main toxin classes consisted of three-finger toxins (3FTx (24% and phospholipases A2 (PLA2s (15%. However, 8 other classes of toxins were present, including C-type lectins, natriuretic peptide precursors and even high-molecular mass components such as metalloproteases and L-amino acid oxidases. Each class included an assortment of isoforms, some showing evidence of alternative splicing and domain deletions. Five antigenic candidates were selected (four 3FTx and one PLA2 and used for a preliminary study of DNA immunization. The immunological response showed that the sera from the immunized animals were able to recognize the recombinant antigens. Conclusion Besides an improvement in our knowledge of the composition of coral snake venoms, which are very poorly known when compared to Old World elapids, the expression profile suggests abundant and diversified components that may be used in future antiserum formulation. As recombinant production of venom antigens frequently fails due to complex disulfide arrangements, DNA

  12. [Prokaryotic expression of vp3 gene of Muscovy duck parvovirus, and its antiserum preparation for detection of virus multiplication]. (United States)

    Huang, Yu; Zhu, Yumin; Dong, Shijuan; Yu, Ruisong; Zhang, Yuanshu; Li, Zhen


    New epidemic broke out in recent year which was suspected to be caused by variant Muscovy duck parvovirus (MDPV). For this reason, new MDPV detection methods are needed for the new virus strains. In this study, a pair of primers were designed according to the full-length genome of MDPV strain SAAS-SHNH, which were identified in 2012, and were used to amplify the vp3 gene of MDPV by polymerase chain reaction. After being sequenced, the vp3 gene was subcloned into the prokaryotic expression vector PET28a. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. SDS-PAGE and Western blotting analysis showed the MDPV vp3 gene was successfully expressed. After being purified by Ni2+ affinity chromatography system, the recombinant protein was used as antigen to immunize rabbits to obtain antiserum. Western blotting analysis showed that the acquired antiserum could react specifically with VP3 protein of J3D6 strain and MDPV vaccine strain. The antiserum could also be used for detection of cultured MDPV from primary duck embryo fibroblasts by immune fluorescence assay (IFA). It could be concluded that the VP3 protein and its antibody prepared in the research could be used for detection of VP3 antiserum and antigen respectively.

  13. Inhibition of dye-coupling in Patella (mollusca) embryos by microinjection of antiserum against Nephrops (arthropoda) gap junctions

    NARCIS (Netherlands)

    Serras, F.; Buultjens, T.E.J.; Finbow, M.E.


    Antiserum raised against Nephrops gap junctions was injected into single cells of the 2-, 4-, 8-, 16-, and 32-cell stage of the Patella vulgata embryos. The pattern of junctional communication by iontophoresis of Lucifer Yellow CH was tested at the 32-cell stage. The results show that the normal pat

  14. Production of polyclonal antiserum specific to the 27.5 kDa envelope protein of white spot syndrome virus

    NARCIS (Netherlands)

    You, Z.O.; Nadala, E.C.B.; Yang, J.S.; Hulten, van M.C.W.; Loh, P.C.


    A truncated version of the white spot syndrome virus (WSSV) 27.5 kDa envelope protein was expressed as a histidine tag fusion protein in Escherichia coli. The bacterial expression system allowed the production of up to 10 mg of purified recombinant protein per liter of bacterial culture. Antiserum f

  15. Failure of zinc to prevent dysmorphogenesis of cultured rat conceptuses by anti-yolk sac antiserum

    Energy Technology Data Exchange (ETDEWEB)

    Marlow, R.; Freeman, S.J.


    Day 10 rat conceptuses were cultured for 48h in the presence of either cadmium or anti-vesceral yolk sac antiserum (AVYS). Cadmium was embryotoxic at concentrations exceeding 0.25 ug/ml while AVYS caused embryonic dysmorphogenesis, particularly affecting the optic vesicles, at concentrations of 2 ul/ml and above. The effect of pretreatment with zinc on embryotoxicity caused by cadmium or AVYS was studied. Zinc ameliorated the effects of cadmium but had no effect on AVYS-induced embryonic abnormalities. In a second set of experiments inhibition of /sup 125/I-labelled PVP uptake by the yolk sac of cultured whole conceptuses was studied. Cadmium and AVYS both inhibited uptake compared to control cultures. Zinc again ameliorated the effect of cadmium but had no action against AVYS-induced inhibition. These results are in contrast to their previous findings using isolated cultured yolk sacs in which zinc ameliorated the inhibitory effects on /sup 125/I-labelled PVP uptake of both cadmium and AVYS. These data show that in experiments using the isolated cultured yolk sac and the intact cultured conceptus, a qualitatively different response in yolk sac behavior is observed under similar experimental conditions.

  16. Antiserum against Raoultella terrigena ATCC 33257 identifies a large number of Raoultella and Klebsiella clinical isolates as serotype O12. (United States)

    Mertens, Katja; Müller-Loennies, Sven; Stengel, Petra; Podschun, Rainer; Hansen, Dennis S; Mamat, Uwe


    Raoultella terrigena ATCC 33257, recently reclassified from the genus Klebsiella, is a drinking water isolate and belongs to a large group of non-typeable Klebsiella and Raoultella strains. Using an O-antiserum against a capsule-deficient mutant of this strain, we could show a high prevalence (10.5%) of the R. terrigena O-serotype among non-typeable, clinical Klebsiella and Raoultella isolates. We observed a strong serological cross-reaction with the K. pneumoniae O12 reference strain, indicating that a large percentage of these non-typeable strains may belong to the O12 serotype, although these are currently not detectable by the K. pneumoniae O12 reference antiserum in use. Therefore, we analyzed the O-polysaccharide (O-PS) structure and genetic organization of the wb gene cluster of R. terrigena ATCC 33257, and both confirmed a close relation of R. terrigena and K. pneumoniae O12. The two strains possess an identical O-PS, lipopolysaccharide core structure, and genetic organization of the wb gene cluster. Heterologous expression of the R. terrigena wb gene cluster in Escherichia coli K-12 resulted in the WecA-dependent synthesis of an O-PS reactive with the K. pneumoniae O12 antiserum. The serological data presented here suggest a higher prevalence of the O12-serotype among Klebsiella and Raoultella isolates than generally assumed.

  17. Passive immunization with LINGO-1 polyclonal antiserum afforded neuroprotection and promoted functional recovery in a rat model of spinal cord injury. (United States)

    Lv, Jun; Xu, Ru-xiang; Jiang, Xiao-dan; Lu, Xin; Ke, Yi-quan; Cai, Ying-qian; Du, Mou-xuan; Hu, Changchen; Zou, Yu-xi; Qin, Ling-sha; Zeng, Yan-jun


    LINGO-1 (leucine-rich repeat and Ig domain-containing, Nogo receptor-interacting protein) is an important component of the NgR receptor complex involved in RhoA activation and axon regeneration. The authors report on passive immunization with LINGO-1 polyclonal antiserum, a therapeutic approach to overcome NgR-mediated growth inhibition after spinal cord injury (SCI). The intrathecally administered high-titer rabbit-derived antiserum can be detected around the injury site within a wide time window; it blocks LINGO-1 in vivo with high molecular specificity. In this animal model, passive immunization with LINGO-1 antiserum significantly decreased RhoA activation and increased neuronal survival. Adult rats immunized in this manner show recovery of certain hindlimb motor functions after dorsal hemisection of the spinal cord. Thus, passive immunotherapy with LINGO-1 polyclonal antiserum may represent a promising repair strategy following acute SCI.

  18. Antigen-induced and non-antigen-induced histamine release from rat mast cells sensitized with mouse antiserum.

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    Full Text Available Marked IgE-mediated histamine release from rat mast cells sensitized in vitro with mouse antiserum occurs in the presence of added Ca++ and phosphatidylserine (PS, although a considerable degree of antigen-induced histamine release which may utilize intracellular or cell-bound calcium is also observed. The decay in the responsiveness to Ca++ of the sensitized cells stimulated by antigen in Ca++-free medium in the presence of PS is relatively slow, and maximum release is produced by Ca++ added 1 min after antigen. Histamine release also occurs when Ca++ is added after PS in the absence of antigen to the sensitized cells suspended in Ca++-free medium. Unlike the antigen-induced release, the intensity of this non-antigen-induced release varies depending on both mast-cell and antiserum pools. A heat-labile factor(s, which is different from antigen-specific IgE antibody and is also contained in normal mouse serum, is involved in this reaction. In the antigen-nondependent (PS + Ca++-induced release, no decay in the responsiveness to Ca++ is observed after PS addition. Both the antigen-induced and non-antigen-induced release are completed fairly rapidly and are dependent of temperature, pH and energy.

  19. A Simple and Novel Strategy for the Production of a Pan-specific Antiserum against Elapid Snakes of Asia (United States)

    Ratanabanangkoon, Kavi; Tan, Kae Yi; Eursakun, Sukanya; Tan, Choo Hock; Simsiriwong, Pavinee; Pamornsakda, Teeraporn; Wiriyarat, Witthawat; Klinpayom, Chaiya; Tan, Nget Hong


    Snakebite envenomation is a serious medical problem in many tropical developing countries and was considered by WHO as a neglected tropical disease. Antivenom (AV), the rational and most effective treatment modality, is either unaffordable and/or unavailable in many affected countries. Moreover, each AV is specific to only one (monospecific) or a few (polyspecific) snake venoms. This demands that each country to prepare AV against its local snake venoms, which is often not feasible. Preparation of a ‘pan-specific’ AV against many snakes over a wide geographical area in some countries/regions has not been possible. If a ‘pan-specific’ AV effective against a variety of snakes from many countries could be prepared, it could be produced economically in large volume for use in many countries and save many lives. The aim of this study was to produce a pan-specific antiserum effective against major medically important elapids in Asia. The strategy was to use toxin fractions (TFs) of the venoms in place of crude venoms in order to reduce the number of antigens the horses were exposed to. This enabled inclusion of a greater variety of elapid venoms in the immunogen mix, thus exposing the horse immune system to a diverse repertoire of toxin epitopes, and gave rise to antiserum with wide paraspecificity against elapid venoms. Twelve venom samples from six medically important elapid snakes (4 Naja spp. and 2 Bungarus spp.) were collected from 12 regions/countries in Asia. Nine of these 12 venoms were ultra-filtered to remove high molecular weight, non-toxic and highly immunogenic proteins. The remaining 3 venoms were not ultra-filtered due to limited amounts available. The 9 toxin fractions (TFs) together with the 3 crude venoms were emulsified in complete Freund’s adjuvant and used to immunize 3 horses using a low dose, low volume, multisite immunization protocol. The horse antisera were assayed by ELISA and by in vivo lethality neutralization in mice. The findings

  20. A Simple and Novel Strategy for the Production of a Pan-specific Antiserum against Elapid Snakes of Asia.

    Directory of Open Access Journals (Sweden)

    Kavi Ratanabanangkoon


    Full Text Available Snakebite envenomation is a serious medical problem in many tropical developing countries and was considered by WHO as a neglected tropical disease. Antivenom (AV, the rational and most effective treatment modality, is either unaffordable and/or unavailable in many affected countries. Moreover, each AV is specific to only one (monospecific or a few (polyspecific snake venoms. This demands that each country to prepare AV against its local snake venoms, which is often not feasible. Preparation of a 'pan-specific' AV against many snakes over a wide geographical area in some countries/regions has not been possible. If a 'pan-specific' AV effective against a variety of snakes from many countries could be prepared, it could be produced economically in large volume for use in many countries and save many lives. The aim of this study was to produce a pan-specific antiserum effective against major medically important elapids in Asia. The strategy was to use toxin fractions (TFs of the venoms in place of crude venoms in order to reduce the number of antigens the horses were exposed to. This enabled inclusion of a greater variety of elapid venoms in the immunogen mix, thus exposing the horse immune system to a diverse repertoire of toxin epitopes, and gave rise to antiserum with wide paraspecificity against elapid venoms. Twelve venom samples from six medically important elapid snakes (4 Naja spp. and 2 Bungarus spp. were collected from 12 regions/countries in Asia. Nine of these 12 venoms were ultra-filtered to remove high molecular weight, non-toxic and highly immunogenic proteins. The remaining 3 venoms were not ultra-filtered due to limited amounts available. The 9 toxin fractions (TFs together with the 3 crude venoms were emulsified in complete Freund's adjuvant and used to immunize 3 horses using a low dose, low volume, multisite immunization protocol. The horse antisera were assayed by ELISA and by in vivo lethality neutralization in mice. The

  1. A Simple and Novel Strategy for the Production of a Pan-specific Antiserum against Elapid Snakes of Asia. (United States)

    Ratanabanangkoon, Kavi; Tan, Kae Yi; Eursakun, Sukanya; Tan, Choo Hock; Simsiriwong, Pavinee; Pamornsakda, Teeraporn; Wiriyarat, Witthawat; Klinpayom, Chaiya; Tan, Nget Hong


    Snakebite envenomation is a serious medical problem in many tropical developing countries and was considered by WHO as a neglected tropical disease. Antivenom (AV), the rational and most effective treatment modality, is either unaffordable and/or unavailable in many affected countries. Moreover, each AV is specific to only one (monospecific) or a few (polyspecific) snake venoms. This demands that each country to prepare AV against its local snake venoms, which is often not feasible. Preparation of a 'pan-specific' AV against many snakes over a wide geographical area in some countries/regions has not been possible. If a 'pan-specific' AV effective against a variety of snakes from many countries could be prepared, it could be produced economically in large volume for use in many countries and save many lives. The aim of this study was to produce a pan-specific antiserum effective against major medically important elapids in Asia. The strategy was to use toxin fractions (TFs) of the venoms in place of crude venoms in order to reduce the number of antigens the horses were exposed to. This enabled inclusion of a greater variety of elapid venoms in the immunogen mix, thus exposing the horse immune system to a diverse repertoire of toxin epitopes, and gave rise to antiserum with wide paraspecificity against elapid venoms. Twelve venom samples from six medically important elapid snakes (4 Naja spp. and 2 Bungarus spp.) were collected from 12 regions/countries in Asia. Nine of these 12 venoms were ultra-filtered to remove high molecular weight, non-toxic and highly immunogenic proteins. The remaining 3 venoms were not ultra-filtered due to limited amounts available. The 9 toxin fractions (TFs) together with the 3 crude venoms were emulsified in complete Freund's adjuvant and used to immunize 3 horses using a low dose, low volume, multisite immunization protocol. The horse antisera were assayed by ELISA and by in vivo lethality neutralization in mice. The findings were: a

  2. Production of antiserum to a non-structural potyviral protein and its use to detect narcissus yellow stripe and other potyviruses. (United States)

    Mowat, W P; Dawson, S; Duncan, G H


    A protein, of apparent molecular weight 72,000, was purified from experimentally infected narcissus plants with yellow stripe symptoms utilising SDS-polyacrylamide gel electrophoresis. This protein was excised from the gels and used to prepare antiserum, which reacted specifically with cytoplasmic cylindrical inclusions in ultra-thin sections of virus-infected cells and, in immunoblots, with the 72 kDa protein in preparations containing cytoplasmic inclusions. The antiserum reacted in ELISA with leaf extracts from yellow stripe diseased plants of four narcissus cultivars but not with extracts from comparable symptomless plants. In tests with extracts of plants infected with seven definitive potyviruses, reactions were obtained with bean yellow mosaic and iris mild mosaic viruses. Virus-specific reactions in dot-blot ELISA were dependent on the presence of Tween 20 in the extraction buffer. In contrast, an antiserum to the putative cytoplasmic inclusion protein of alstroemeria mosaic virus reacted only with SDS-treated leaf extracts of infected plants. In limited tests, the method of purifying cytoplasmic inclusion protein was successfully applied to four definitive potyviruses, suggesting that it may be generally applicable to potyviruses and of use for preparing antisera when purification of virus particles is difficult.

  3. Antiserum development for chicken infectious bursal disease virus%鸡传染性法氏囊病病毒抗血清的研制

    Institute of Scientific and Technical Information of China (English)

    王占伟; 邵国青; 刘茂军; 冯志新; 王丽; 高云飞; 周开华; 尹秀凤


    [Objective]This research developed high performing and safe chicken infectious bursal disease (IBD) prevention and treatment products to ensure the high efficiency of chicken infectious bursal disease virus (IBDV) antiserum and to solve the safety and immunological stress problems at the same time. [ Method ] Using SPF chicken as the immune animal, the corresponding IBDV antiserum was prepared according to the requirements from《Veterinary Biological Products Technical Guidance and Principle Experimental Studies 》 and 《Veterinary Pharmacopoeia of P.R. China》(2010 Ed). After detoxification treatment, the semi-finished and finished antiserums underwent production inspections. [Result] For the antiserums obtained from IBDV NJ immunized SPF chickens at 4-weeks old, the serum titer before detoxification treatment was 5≥1:64, while the serum titer after detoxification treatment was 5≥1:32. From the test results of semi-finished and finished serums, the antiserum products after detoxification treatment were orange-hued, sterile, without mycoplas-ma, and without exogenous virus; the formaldehyde and thimerosal residues also did not exceed the prescribed standards, which were safe to use in mice, guinea pigs, and SPF chickens. [Conclusion]The IBDV antiserum products, developed according to the standards of (Veterinary Pharmacopoeia of P. R. China), could be used for emergency IBD prevention and early IBD treatment. The antiserum could also be listed as a formal sales product in the future to meet market demands.%[目的]确保鸡传染性法氏囊病病毒(IBDV)抗血清高效力的同时又彻底解决安全性和免疫应激问题,旨在研制出一种高效、安全的鸡传染性法氏囊病(IBD)预防和治疗制品.[方法]以SPF鸡为免疫动物,按照《兽用生物制品试验研究指导技术原则》和《中华人民共和国兽药典》(2010年版)的要求研制IBDV抗血清,经脱毒处理后,对其半成品及

  4. Passive immunization with a polyclonal antiserum to the hemoglobin receptor of Haemophilus ducreyi confers protection against a homologous challenge in the experimental swine model of chancroid. (United States)

    Leduc, Isabelle; Fusco, William G; Choudhary, Neelima; Routh, Patty A; Cholon, Deborah M; Hobbs, Marcia M; Almond, Glen W; Orndorff, Paul E; Elkins, Christopher


    Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme.

  5. Over-expression of 72 ku protein of wheat yellow mosaic virus in E.coli and preparation of its antiserum

    Institute of Scientific and Technical Information of China (English)


    By reverse transcription-polymerase chain reaction (RT-PCR),cDNA fragment of wheat yellow mosaic virus (WYMV) RNA2 encoding 72 ku protein has been synthesized and cloned into plasmid pET30a(+) for overexpression in prokaryotic cells.BL21(DE3) pLys S of E.coli transformed with the recombinant plasmid pETP72 containing the fragment has been induced to express the 72 ku protein on high level.The produced protein has been purified from sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for its antiserum preparation.In Western-blotting analysis,the antibodies reacted with the 72 ku protein expressed in E.coli.

  6. Serological survey of Seewis virus antibodies in patients suspected for hantavirus infection in Finland; a cross-reaction between Puumala virus antiserum with Seewis virus N protein? (United States)

    Ling, Jiaxin; Vaheri, Antti; Hepojoki, Satu; Levanov, Lev; Jääskeläinen, Anne; Henttonen, Heikki; Vapalahti, Olli; Sironen, Tarja; Hepojoki, Jussi


    Puumala virus (PUUV, carried by Myodes glareolus) co-circulates with Seewis virus (SWSV, carried by Sorex araneus) in Finland. While PUUV causes 1000-3000 nephropathia epidemica (NE) cases annually, the pathogenicity of SWSV to man is unknown. To study the prevalence of SWSV antibodies in hantavirus fever-like patients' sera, we used recombinant SWSV nucleocapsid (N) protein as the antigen in ELISA, immunofluorescence assay (IFA) and immunoblotting. While characterizing the recombinant SWSV N protein, we observed that a polyclonal rabbit antiserum against PUUV N protein cross-reacted with SWSV N protein and vice versa. We initially screened 486 (450 PUUV-seronegative and 36 PUUV-seropositive) samples sent to Helsinki University Hospital Laboratory for PUUV serodiagnosis during 2002 and 2007 in an SWSV N protein IgG ELISA. In total, 4.2 % (19/450) of the PUUV-seronegative samples were reactive in the SWSV N protein IgG ELISA and none of the tested samples [43 PUUV-seronegative (weakly reactive in the SWSV IgG ELISA) and 15 random] were reactive in the SWSV N protein IgM ELISA. None of the IgG reactions could be confirmed by IFA or immunoblotting. Furthermore, among the 36 PUUV-seropositive samples three were reactive in SWSV N protein IgG and ten in SWSV N protein IgM ELISA. One PUUV-seropositive sample reacted with SWSV N protein in IFA and four in immunoblotting. Finally, we applied competitive ELISA to confirm that the observed reactivity was due to cross-reactivity rather than a true SWSV response. In conclusion, no evidence of SWSV infection was found among the 486 samples studied; however, we did demonstrate that PUUV antiserum cross-reacted with shrew-borne hantavirus N protein.

  7. Prokaryotic Expression and the Antiserum Preparation of Duck Enteritis Virus US10 Gene%鸭肠炎病毒US10基因的原核表达及抗血清的制备

    Institute of Scientific and Technical Information of China (English)

    张蕾; 董井泉; 母晓宇; 马波; 王君伟


    The US10 gene was expressed in pET-32a( + )/Rosetta(DE3) pLys system using the DNA of Duck enteritis virus (DEV) C-KCE as temple, US10 gene ORF was amplified by polymerase chain reaction (PCR). Then the recombinant protein of US 10 gene induction with IPTG by SDS - PAGE analysis showed that the object protein is mainly expressed as inclusion bodies. Using the Ni-NTA Purification System, the recombinant protein immunized to rabbits was purified to make the antiserum. The titer of the antiserum tested by agar diffusion reaction was 1:4. The titer of the antiserum tested by ELISA was 1:102 400. The prepared antiserum has a high specificity with the western-blot analysis, and the indirect immunofluorescence (IIF) test proved that the antiserum could react with DEV US10 encoded product, which lays a foundation for further research on molecular structure and function.%以鸭肠炎病毒(DEV) C-KCE株基因组DNA为模板,PCR扩增获得US10基因.构建了其原核表达载体pET-32a-US10,经1.0 mmol/L IPTG诱导,目的蛋白在Rosetta(DE3) pLys宿主菌中获得了表达.SDS-PAGE 电泳结果显示目的蛋白以包涵体形式表达,与预期大小相符.利用亲和层析法纯化目的蛋白,以纯化的蛋白为免疫原制备其抗血清,琼脂扩散法测得抗体效价为1:4,间接ELISA法测定抗体效价为1∶102 400.间接免疫荧光试验证实制备的抗血清可特异性识别鸭肠炎病毒的US10基因编码产物.

  8. An Observation in Vitro of Recombinant Ferritin Protein Antiserum Impaired the Protoscolex of Echinococcus Granulosus%Eg.ferritin抗血清介导的体外杀伤细粒棘绦虫原头节作用的观察

    Institute of Scientific and Technical Information of China (English)

    王娅娜; 张炎; 朱佳


    目的 体外观察细粒棘球绦虫重组铁蛋白(Eg.ferritin)抗血清对细粒棘球蚴原头节(PSC)的杀伤作用.方法 用纯化的Eg.ferritin免疫新西兰家兔制备抗血清,以不同浓度的抗血清与细粒棘球绦虫原头蚴体外共培养,光镜观察培养不同时间点细粒棘球原头节的变化,透射电子显微镜观察原头节超微结构的改变.结果 抗血清组的原头蚴的死亡率及破坏程度与抗血清的浓度呈正相关,与对照组相比,抗血清对原头节有杀伤作用(P<0.01).在20%抗血清组中,原头节内部被大量空泡及髓样结构代替或无结构.结论 细粒棘球蚴重组铁蛋白抗体对细粒棘球绦虫有明显的杀伤作用.%Objective To observe the impaired the effect of recombinant ferritin protein antiserum on the pro-toscolex (PSC) of Echinococcus granulosus in vitro. Methods Antiserum was made from New Zealand rabbits with purified Eg. ferritin immunization. Antiserum was divided into three different group with 10% ,15% , 20% concentration. PSC were cultured in the three antiserum groups. Living situation and mortality of PSC was investigated through microscope in different time points and sub structure of PSC was observed through transmission electronic microscope( TEM) . Results Impairment of PSC was positively related to concentration of anti - serum( P < 0. 01) , Lots of vacuole and myelinfigures were found inside of PSC in 20% anti - serum group. Conclusion The results confirmed that Eg. ferritin could impaire PSC in vitro.

  9. Prokaryotic expression and identification of porcine Mx1 gene and preparation of the mouse antiserum%猪源Mx1基因的原核表达及其抗血清制备

    Institute of Scientific and Technical Information of China (English)

    何丹妮; 周斌; 张小敏; 陈溥言; 庞然; 赵津


    Based on a pair of specific primers, the open reading frame of porcine Mxl (1 992 bp) gene (poMxl) was amplified from pT-poMxl by PCR. The poMxl gene was cloned into the prokaryotic expression vector of pGEX-6p-l and verified by enzyme digestion and DNA sequencing. Then this recombinant plasmid was transformed into Escherichia coli BL21 (DE3) for overexpression under opit imization conditions. The expression product had a molecular mass about 99×103 as expected. The poMxl protein was separated in gel slices and used to immunize Balb/c mice. Antiserum was prepared and specificity analyzed by Western blot. High titer of antiserum against porcine Mxl protein was obtained. The successful expression of porcine Mxl protein in E. Coli BL21 and the preparation of poMxl specific mouse antiserum laid foundation for the further study on the antiviral mechanism.%利用1对特异性引物,从pT-poMx1中扩增出Mx1基因开放式阅读框(1 992 bp),并将其编码区插入到携带GST标签的原核表达载体pGEX-6p-1中,经酶切和序列测定后,重组质粒pGEX-poMx1转化陶大肠杆菌BL21( DE3),利用IPTG诱导表达融合蛋白并分析表达效果.结果表明:经终浓度为0.8 mmol· L-1 IPTG诱导后4h,猪源Mx1基因(poMx1)在BL21(DE3)中获得高效表达,表达量占菌体蛋白的32.6%,SDS-PAGE显示融合蛋白相对分子质量为99×103.将目的蛋白切胶后免疫Balb/c小鼠,制备了特异性多抗血清,Western blot结果显示该抗血清与融合蛋白孵育反应后有明显的特异性条带.结论:poMx1基因表达成功.

  10. 绵羊肺腺瘤病毒CA基因的原核表达及抗原性检测%CA gene prokaryotic expression and the antiserum preparation of Jaagsiekte sheep retrovirus

    Institute of Scientific and Technical Information of China (English)

    付丽君; 李巍; 谢晓峰; 唐颖; 李晓云; 宋铭忻; 张子群


    绵羊肺腺瘤病(OPA)于1850年发现,但国内外至今尚未建立绵羊肺腺瘤病毒(JSRV)的体外培养方法及快速有效的实验室检测方法.为表达JSRV的衣壳蛋白(CA)基因,本研究根据JSRV CA基因编码序列设计特异性引物进行PCR扩增,克隆至pEASY载体中进行测序,并构建重组表达质粒pET-CA,在大肠杆菌中经IPTG诱导表达.重组蛋白经SDS-PAGE检测约为28 ku,主要以可溶形式表达.将纯化的重组蛋白免疫新西兰白兔,制备抗血清.以制备的抗血清对重组蛋白及病毒进行western blot鉴定.结果显示,该抗血清能够识别重组蛋白及天然的JSRV.本研究首次原核表达了完整的CA重组蛋白,并制备了能够与天然JSRV结合的抗CA蛋白的抗血清,为进一步建立JSRV ELISA方法奠定了基础.%To express the capsid antigen (CA) gene of Jaagsiekte sheep retrovirus (JSRV) and prepare the antiserum against the virus,the CA gene was amplified by PCR from JSRV proviral DNA with a pair of primers designed according to the CA gene of JSRV in GenBank.Subsequently,the CA gene was cloned into pET-28a(+) for expression in E.coli and the recombinant CA protein was about 28 ku,mainly in soluble form,detected by SDS-PAGE.In addition,the antiserum was prepared from rabbits immunized with the purified recombinant protein.Western blot showed that the antiserum was able to recognize both of the recombinant CA pretein and the native virus protein,which provided the basis for the establishment of the ELISA method to detect the JSRV infection in sheep.

  11. Preparation and Application of Antiserum Against Siraitia grosvenorii Isolate of Zucchini yellow mosaic virus%小西葫芦黄化花叶病毒罗汉果分离株抗血清的制备和应用

    Institute of Scientific and Technical Information of China (English)

    朱英芝; 韩英; 蒙姣荣; 廖咏梅; 邹承武; 陈保善


    将分离自罗汉果(Siraitia grosvenorii)的小西葫芦黄化花叶病毒(Zucchini yellow mosaic virus,ZYMV-LHG)在西葫芦上繁殖,利用差速离心法与PEG沉淀法相结合提纯病毒粒子,用于免疫新西兰大白兔,成功制备出效价为1:10 240的抗血清.分别用健康西葫芦叶片总蛋白及健康西葫芦叶片汁液吸附处理抗血清,比较2种处理后抗血清背景反应,以健康西葫芦汁液吸附法较佳.用间接ELISA对罗汉果果园及附近共46个科114种植物进行检测,其中9个科25种植物有阳性反应,以葫芦科、苋科、豆科、锦葵科等阳性率较高.田间病毒病发病率调查表明,宿根苗发病率比组培苗高,发病时间更早,是田间罗汉果病毒病的主要初侵染来源之一.%The Luohanguo (Siraiti-a grosvenorii) -infected by Zucchini yellow mosaic virus (ZYMV-LHG), was propagated on Cucurbita pepo and purified by differential centrifugation in combination with Polyethylene Glycol (PEG)precipitation. The purified ZYMV-LHG preparation was injected into New Zealand rabbits. The titer of the antiserum was 1:10 240 assayed by the indirect ELJSA. The antiserum was treated either by the leaf extract or total protein of zucchini squash to remove non specific reaction. The former was found better than the latter treatment. The treated antiserum was used to detect plants collected from the Luohanguo field and nearby lands with indirect enzyme-linked immunosorbent assay (ELISA). Of 114 plant species belonging to 46 families, 25 species from nine families were with positive reaction, among which plants of families Cucurbitaceae, Amaranthaceae, Amaranthaceae, Leguminosae and Malvaceae showed higher positive rates. A survey on the virus disease in the field revealed that the disease incidence of ratoon Luohanguo was higher than that of the tissue culture seeding planted annually, and the symptoms develops earlier in the ratoon. It suggested that the infected ratoon Luohanguo serve as the

  12. 香石竹斑驳病毒外壳蛋白基因的原核表达及抗血清制备%Prokaryotic Expression and Antiserum Preparation of the Coat Protein Gene of Carnation mottle virus

    Institute of Scientific and Technical Information of China (English)

    李晓鹏; 王连春; 彭杰军; 姬盼; 李准; 孔宝华; 陈海如


    香石竹斑驳病毒(Carnation mottle virus,CarMV)是侵染香石竹的主要病毒.本研究从已鉴定的CarMV毒源植物中提取总RNA,克隆外壳蛋白(cp)基因后构建pET30a-CP重组质粒,并转入BL21 (DE3) pLysS进行原核表达.通过对诱导温度、初菌浓度、IPTG的浓度等表达条件进行优化,目的蛋白在37℃,IPTG终浓度为1.0 mmol/L的条件下,诱导表达6h后获得最高表达量.采用割胶的方法纯化cp蛋白,纯化后的目的蛋白免疫小鼠获得了特异性抗血清.经Western-blot检测,结果表明所制备的抗血清与诱导表达的重组蛋白及接种CarMV的香石竹样品均发生特异性结合.利用所制备抗血清对昆明地区的48个香石竹样品进行抽检,结果表明香石竹样品的带毒率为79.2%.本研究为大规模抗血清生产、检测应用和深入研究该病毒cp基因与寄主的互作奠定基础.%Carnation mottle virus (CarMV) is one of the important viruses infecting carnation. In this study, RNA was extracted from CarMV infected carnation, and coat protein (cp) gene was cloned into pET30a ( + ) and transformed into BL21 (DE3) pLysS. The optimal conditionsor the highest expres-sion of coat protein was expressing at 37℃ for 6 h and inducing with 1. 0 mmol/L IPTG. The expressed protein was purified with gradient SDS-PAGE and the antiserum against the protein was raised in Kun-ming mice. The result of Western-blot analysis confirmed that the antiserum reacted strongly and spe-cifically recombinant CarMV cp and CarMV infected samples. Forty-eight carnation samples in Kun-ming were tested with the antibody made in this experiment, of which 79. 2% of the samples were in-fected by CarMV. This study provides a good basis for massive CarMV cp antiserum preparation CarMV detection and interaction studies between CarMV and host plants.

  13. Prokaryotic Expression and Antiserum Preparation of the Coat Protein of Cymbidium Mosaic Virus%建兰花叶病毒CP基因的原核表达及抗血清制备

    Institute of Scientific and Technical Information of China (English)



    通过间接酶联免疫检测和电镜观察对从福建省漳州市采集的卡特兰病样进行检测,证明样品感染了建兰花叶病毒.设计一对特异性引物,扩增并克隆病毒分离物的外壳蛋白基因,随后将目的基因插入pET-29a(+)中构建相应的原核表达载体.目的蛋白经诱导表达及纯化后免疫家兔并获得了特异性抗血清.Westem blot检测结果表明.抗血清与诱导表达的CyMV外壳蛋白发生特异性反应.间接酶联免疫法检测结果表明,抗血清可检测病汁液的最低稀释度达1:51 200,最佳工作浓度为1:1000,病汁液灵敏度为0.39 mg/mL,而与TMV等11种同源或异源病毒均无明显的血清学交叉反应.%Cymbidium mosaic virus (CyMV) is one of the most important and worldwide viruses attacking orchids. This virus causes the symptoms of mosaic,chlorosis,necrosis and malformation in the orchids,and has a high economic impact to the orchid industry. Cattleya plants contracted with a disease were collected as samples from Zhangzhou,Fujian,and were identified to be infected with Cymbidium mosaic virus by using ID -ELISA and electronic microscopy assay. One pair of specific primers was designed for amplification of the coat protein(CP) gene from the samples infected with CyMV. The open reading frame encoding CP of CyMV isolate obtained from Zhangzhou,Fujian is 672 bp,encoding a 23.6 ku protein with 223 aa. The expected CP gene was then inserted into the pET-29a(+)vector for prokaryotic expression. And the aimed protein was purified and used to immune the rabbit for antiserum preparation. According the result of ID-ELISA analysis,specific rabbit anti-CyMV serum was prepared with a high titre of 1:51 200,a working concentration of 1:1 000 and sap sensitivity of 0.39 mg/mL. Western blot analysis confirmed that the antiserum reacted strongly and specifically to the CP of CyMV. There were no cross reactions between the antiserums and 11 species of homologous or heterologous

  14. Produção de antissoro policlonal utilizando a proteína capsidial recombinante do Rupestris stem pitting-associated virus Production of polyclonal antiserum using recombinant coat protein of Rupestris stem pitting -associated virus

    Directory of Open Access Journals (Sweden)

    Marcos Fernando Basso


    Full Text Available O Rupestris stem pitting-associated virus (RSPaV é o agente causal das caneluras do lenho da videira. Este trabalho teve como objetivo produzir antissoro policlonal a partir da proteína capsidial (CP recombinante do RSPaV e avaliar a sua especificidade e sensibilidade. O gene da CP do RSPaV, com 780pb, foi previamente caracterizado. Esse gene foi subclonado no sítio de restrição EcoRI, no vetor de expressão pRSET-B e o plasmídeo recombinante foi utilizado para induzir a expressão da CP em Escherichia coli. A CP, ligada a uma cauda de seis histidinas, foi purificada por meio de cromatografia de afinidade em coluna de Ni-NTA a partir do extrato de proteínas totais extraídas de E. coli. A identidade da proteína purificada foi confirmada em SDS-PAGE e Western blot, utilizando-se anticorpos comerciais contra a cauda de seis histidinas. A CP recombinante expressada in vitro apresentou massa molecular de cerca de 31kDa. A proteína purificada foi quantificada e 2,55mg foram utilizados para a imunização de um coelho. O antissoro policlonal obtido reagiu com diferentes isolados deste vírus, extraídos de videiras em ELISA indireto.RSPaV is the causal agent of pitting in the grapevine woody cylinder. The aim of this research was to produce polyclonal antiserum against recombinant RSPaV coat protein (CP and evaluate its specificity and sensibility. The CP gene (780bp of RSPaV was previously characterized. This gene was subcloned into the EcoRI site of the pRSET-B expression vector and the recombinant plasmid was used to induce the expression of the CP in E. coli cells. The CP, fused to a 6-His-tag, was purified from E. coli total protein extract by affinity chromatography using Ni-NTA resin. Identity of the purified protein was confirmed by SDS-PAGE and Western blot, using antibodies against the histidine tail. The in vitro-expressed recombinant CP presented a MW of ca. 31kDa. The purified protein was quantified and 2.55mg used for the

  15. Cloning, Prokaryotic Expression of Echinococcus granulosus Heat Shock Protein 70 and Preparation of It's Antiserum%细粒棘球绦虫Hsp70基因的克隆、表达及抗体制备

    Institute of Scientific and Technical Information of China (English)

    赵莉; 薛晶; 石保新; 陈皓斐; 张文宝; 马正海; 张壮志; 张旭; 古努尔·吐尔逊; 米晓云; 金映红


    [Objective] The objective of the experiment is to express and purify E. granulosus (Eg) heat shock protein 70 (EgHsp70) in E. coli and prepare the antibody against E. granulosus. [ Methods] EgHsp70 gene was amplified by PCR and cloned into prokaryotic expression vector pMAL-p2x, and the recombinant plasmid was transformed into E. coil BL-21. The soluble expression conditions of fusion protein were optimized by induction with different concentrations, of IPTG different temperatures and cultivation times. The expressed fusion protein was purified by Mal-tag Magnetic Beads. To prepare the anti-serum, New Zealand white rabbits were immunized with purified EgHsp70 protein via hypodermic and volar. Western blot was used to determine the serum's specificity against EgHsp70 and native proteins. The serum titers were analyzed by ELISA. [Results] Full-length of EgHsp70 gene had an open reading frame of 765 bps encoding a protein mass of 68.6 kD. Restriction endonuclease analysis and DNA sequencing showed that EgHsp70 was cloned into the plasmid pMAL-p2x. Based on the optimization experiments, it was concluded that the best soluble expression conditions for the EgHsp70 protein are using 0.3 mmol· L-1 IPTG when bacterial cells growing to OD600 0.6 and induced for 4 h at 30℃. ELISA and Western blotting showed that the titers of the anti-serum were above 1 : 256 000, and the anti-serum could specifically bind with EgHsp70 protein and native proteins. [Conclusion] The EgHsp70 fusion protein was obtained by expressing in E.coli and purifying, and the antibody against EgHsp70 was prepared with the fusion protein immunized New Zealand white rabbits. This work will provide an antigen and detection antibody for further study on the EgHsp70 function. The protein is immunogenic and can be a vaccine candidate against Echinococcus infection.%[目的]克隆细粒棘球绦虫(Echinococcus granulosus,E.g)热休克蛋白家族基因Hsp70,在原核细胞中表达、纯化Hsp70蛋白并制

  16. 抗恩诺沙星抗血清的制备及其ELISA检测%Preparation of enrofloxacin antiserum and its determination by enzyme - linked immunosorbent assay (ELISA)

    Institute of Scientific and Technical Information of China (English)

    刘兵; 曾华金; 杨冉; 雷利芳; 屈凌波


    Objective;To develop a rapid and sensitive enzyme - linked immunosorbent assay(ELISA) for determination of enrofloxacin( ENR). Method; Immunogen enrofloxacin - bovine serum albumin( Enrofloxacin - BSA) and coating antigen enrofloxcin -ovum albumin (Enrofloxacin - OVA) were prepared by a succinic anhydride method. By subcutaneous injection, the polyclonal antiserum was produced from big ear rabbits immunized with conjugates ENR -BSA. Result; After the assay procedure was optimized,the standard curve of ENR was established and the practical measuring range of the ELISA extended from 5 ng ? mL-1 to 2. 5 |xg ? mL '' ( R2 =0. 9935 ) . In a comparison of the assay results obtained by ELISA and HPLC, there is a good correlation between these two methods (R2 = 0. 9892, n = 9 ) . The proposed method has been satisfactorily applied for the determination of ENR in rat plasma and Pharmaceuticals with recoveries in the range of 98. 4% to 105. 8% for milk sample,91. 7% to 101. 2% for rat plasma and 97.0% to 110.3% for rat urine. Conclusion; The experimental data indicated that in some extent the ELISA method was more suitable for high throughput and real - time ENR analysis in biological samples and animal food with lower detection limit,low background and no requirement of sample pre -treatment.%目的:建立快速、灵敏的恩诺沙星残留酶联免疫检测方法(ELISA).方法:采用混合酸酐法,将恩诺沙星与牛血清白蛋白和卵清蛋白分别合成免疫抗原(Enrofloxacin-BSA)和包被抗原(Enrofloxacin-OVA).通过背部多点免疫法免疫日本大白耳兔,制备抗恩诺沙星的特异性抗血清.结果:利用所获得的特异性抗血清,建立的恩诺沙星ELISA检测方法,其线性范围为5 ng·mL.~2.5μg·mL(R=0.9935),且与HPLC方法具有良好的相关性(R=0.9892,n=9).牛奶、大鼠血浆和尿液中的加样回收率分别为98.4%~105.8%,91.7%~101.2%,97.0%~110.3%.运用所建立的方法,对大鼠体内血浆及药品

  17. Effects of antiserums of cadherin, aminopeptidase N and alkaline phosphatase on the toxicities of Cry1Ac and Cry2Aa in Helicoverpa armigera (Lepidoptera: Noctuidae)%棉铃虫中肠钙粘蛋白、氨肽酶N及碱性磷酸酯酶的抗血清对Cry1Ac和Cry2Aa毒力的影响

    Institute of Scientific and Technical Information of China (English)

    王冰洁; 袁向东; 赵曼; 刘臣; 陈琳; 梁革梅


    [Aim] Cry1A and Cry2A toxins play insecticidal roles by specifically binding with receptor proteins on insect midgut,and are widely used in Bt transgenic crops now.This study aims to further understand the action mechanisms of Cry2A and the roles of the functional receptor proteins of Cry1 A in the toxicity of Cry2A.[Methods] We firstly extracted brush border membrane vesicles (BBMV) of midgut,prepared antibody and antiserums of cadherin (CAD),aminopeptidase N (APN),and alkaline phosphatase (ALP) in Helicoverpa armigera.Then,after detecting the antiserums of these three receptor proteins in BBMV by Western blot,we compared the effects of antiserums of these three receptor proteins on the toxicities of Cry1Ac and Cry2Aa in the susceptible and Cry1Ac-resistant H.armigera (BtR) by the antibody blocking technology.[Results] For the susceptible H.armigera,the antiserums of these three receptor proteins not only significantly reduced the toxicity of Cry1 Ac,but also remarkably reduced the toxicity of Cry2Aa.Among them,anti-APN antiserum had the biggest impact on the toxicity of Cry1Ac,causing the mortality of H.armigera larvae to be reduced by 84.44%,and anti-ALP antiserum had the greatest effect on the toxicity of Cry2Aa,causing the larval mortality to be reduced by 71.04% compared with the control (without treatment by antiserum).The toxicity of Cry1Ac to Cry1Ac-resistant H.armigera (BtR) was obviously reduced and the toxicity of Cry2Aa also became less to these resistant larvae.The influences of the antiserums of these three receptors on the toxicity of Cry1Ac to the Cry1Acresistant strain of H.armigera (BtR) were smaller than those to the susceptible strain.Especially,the inhibition percentage of anti-CAD and-APN antiserums to the toxicity of Cry1 Ac decreased significantly.The effects of anti-CAD and-ALP antiserums on the toxicity of Cry2Aa to the Cry1Ac-resistant strain (BtR) and the susceptible strain were not significantly different,but the anti-APN antiserum

  18. Production of second antibody for insulin and related peptides radioimmunoassay (RIA) (sheep anti-serum and guinea pig anti-IgG); Producao de segundo anticorpo para radioimunoensaio (RIE) de insulina e peptideos relacionados (antissoro de carneiro anti-IgG de cobaia)

    Energy Technology Data Exchange (ETDEWEB)

    Castanheira, Maria do Carmo; Silva, Sandra Rosa da; Borghi, Vania Caira [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil); Wajchenberg, Bernardo Leo [Sao Paulo Univ., SP (Brazil). Faculdade de Medicina


    A good RIA separation technique is essential to develop precise assays and the double antibody separation method is one of the most widely employed, satisfying the majority of the criteria required by RIA. However, its high cost is its main disadvantage, which leads to employ less expensive techniques, that are not so efficient. Therefore, our institution is producing a second antibody to be used in insulin assays, in which the first antibody is raised in guinea pig. Three sheep were immunized with 500 {mu}g of guinea pig IgG purified at our laboratory emulsified in Freund Complete Adjuvante and administered by multisite subcutaneous injection at 20 day intervals. Blood samples were taken from the jugular vein 10 days after boosts. After each four boosts a great bleeding was done by the same route. After these bleeding, the animals were subjected to a rest before being reimmunized. The antisera title were determined by the immuno diffusion method in comparison with a reference antiserum of know quality produced in goat by the Pel-Freez, USA. Approximately 3.5 L of antiserum were produced from the three sheep which presented title very similar to those exhibited by the commercial product, even presenting higher values. (author). 8 refs., 3 figs.

  19. Preparation High Titer Anti-serum of Porcine Circovirus Type Ⅱ Capsid Protein by Hydrodynamics Gentic Immunization%水流动力学基因免疫制备猪Ⅱ型圆环病毒核衣壳蛋白高效价抗血清

    Institute of Scientific and Technical Information of China (English)

    樊宝良; 张瑾; 代红星; 黄培华


    为了建立一个简捷有效的抗血清的制备方法,本研究选用猪Ⅱ型圆环病毒核衣壳蛋白基因,使用水流动力学基因免疫的方法制备高效价抗血清的可行性.应用无内提取试剂盒制备猪Ⅱ型圆环病毒核衣壳蛋白基因真核表达载体pcDNA-Cap的无内毒素质粒.将该质粒使用水流动力学尾静脉注射法对小鼠(Mus musculus)进行基因免疫,重复免疫5次后采血收集血清;以原核表达获得的N末端去除了核定位序列的猪圆环病毒核衣壳蛋白表达产物作为抗原蛋白,以制备的小鼠血清作为一抗进行酶联免疫吸附试验(ELISA)和Western blot检测.结果显示,应用水流动力学尾静脉注射法获得抗血清稀释5000倍通过Western blot至少能够检测到10 ng的抗原蛋白,ELISA分析表明,其效价可达到1∶1000000,说明获得的抗血清具有很好的效价水平.这一研究为猪Ⅱ型圆环病毒相关研究用抗体的制备提供了一个简洁有效的方法,也为猪Ⅱ型圆环病毒的防治方法的建立提供了一个值得尝试的策略.%In order to establish a simple and efficient anti-serum preparation method for molecular biology research, in this research, porcine circovirus type II capsid protein gene was selected to research on the possiblity of preparing high titer anti-serum by hydrodynamics gentic immunization. Endotoxin free plasmid of pcDNA-Cap, with would express porcine circovirus type II capsid protein in the exkaryotic cell, was prepared using endotoxin free plasmid preparing kit and was delivered by hydrodynamics tail vein injection method for genetic immunization of mice (Mus musculus). After 5 times continuous immunization, the blood serum was collected. Using porcine circovirus type II capsid protein which has been deleted its nuclear laction signal sequence at its N-terminal and expressed in Escherichia coli as antigen, the prepared anti-serum was tested by enzymeliked immuno sorbent assay(ELISA) and

  20. Expression and Purification of Heat Shock Protein 65(HSP65) of Mycobacterium leprae and Preparation of Anti-HSP65 Polyclonal Antiserum%麻风分枝杆菌热休克蛋白65的表达、纯化及其多克隆抗血清的制备

    Institute of Scientific and Technical Information of China (English)

    吴娟; 范小勇; 曲勍; 马辉; 许艳; 罗玉萍; Douglas B.Lowire


    目的 原核表达、纯化麻风分枝杆菌热休克蛋白65(Heat shock protein 65,HSP65),并制备其多克隆抗血清.方法 以含hsp65基因的真核表达质粒pCMV4.65为模板,PCR扩增hsp65基因,克隆入pET-28a(+)载体,构建重组表达质粒pET-28a-hsp65,转化大肠杆菌BL21(DE3),IPTG诱导表达.表达产物经镍离子亲和层析柱纯化后免疫BALB/c小鼠,制备多克隆抗血清,Western blot分析重组蛋白的反应原性.结果 重组表达质粒pET-28a-hsp65经双酶切及测序证实构建正确;表达的重组蛋白相对分子质量约65 000,表达量约占菌体总蛋白的30%,主要以包涵体形式表达;纯化的重组蛋白纯度可达95%,浓度约为1.0 mg/ml;制备的HSP65多克隆抗血清效价可达1:12 800;重组蛋白具有良好的反应原性.结论 已成功表达、纯化了麻风分枝杆菌重组HSP65蛋白,并制备了HSP65多克隆抗体,为进一步研究以HSP65为基础的亚单位疫苗和核酸疫苗奠定了基础.%Objective To express the heat shock protein 65 (HSP65) of Mycobacterium leprae in prokaryotic cells, purify the expressed product and prepare anti-HSP65 polyclonal antiserum.Methods The hsp65 gene was amplified by PCR using plasmid pCMV4.65 carrying the target gene as a template, and cloned into vector pET-28a(+).The constructed recombinant plasmid pET-28ahsp65 was transformed to E.coli BL21 (DE3) for expression under induction of IPTG.The expressed product was purified by nickel ion affinity chromatography and used for immunization of BALB / c mice to prepare antiserum.The reactogenicity of recombinant HSP65 was analyzed by Western blot.Results Both restriction analysis and sequencing proved that recombinant plasmid pET-28a-hsp65 was constructed correctly.The expressed recombinant HSP65, with a relative molecular mass of about 65 000, contained about 30% of total somatic protein and mainly existed in a form of inclusion body.The purity and concentration of purified recombinant HSP65 were 95

  1. H:z66抗体应激后伤寒沙门菌fliG基因缺陷株与野生株基因表达的差异%Difference of gene expression between the wild type and fliG of Salmonella enterica serovar Typhi in response to anti-z66 antiserum stress

    Institute of Scientific and Technical Information of China (English)

    张晓磊; 生秀梅; 张海方; 徐顺高; 黄新祥


    Objective: To explore the influence of flagella rotor protein gene fliG on gene expression regulation of Salmonella enterica serovar Typhi under anti-z66 antiserum stress. Methods: The fliG deleted mutant of S. Enterica serovar Typhi was prepared by homologous recombination mediated by suicide plas-mid; S. Enterica serovar Typhi genomic microarray was used to investigate the difference of gene expression between the wild type and fliG mutant; qRT-PCR was done to prove the results of microarray. Results: The fliG deleted mutant of S. Enterica serovar Typhi was constructed successfully. Gene expression profiles analysis revealed that expression of 21 genes were increased and expression of 7 genes were decreased in the fliG mutant under anti-z66 antiserum stress. Conclusion: The flagella rotor protein gene fliG probably played a role to regulate some gene expression in response to anti-z66 antiserum stress.%目的:观察伤寒沙门菌鞭毛转子蛋白基因 fliG 在H:z66抗体应激后对其他基因表达的影响.方法:利用自杀质粒介导的同源重组法制备伤寒沙门菌 fliG 基因缺陷变异株;利用伤寒沙门菌全基因组芯片分析技术,比较伤寒沙门菌野生株和 fliG 基因缺陷变异株在H:z66抗体应激后的基因表达谱差异,并选择部分表达有差异的基因进行实时定量PCR验证.结果:经PCR验证及序列比对,伤寒沙门菌 fliG 基因缺陷变异株制备成功;基因表达谱比较结果表明,与野生株相比,伤寒沙门菌 fliG 基因缺陷变异株在H:z66抗体应激后有21个基因表达上调,7个基因表达下调;实时定量PCR结果与芯片结果一致.结论:fliG 基因在伤寒沙门菌受到H:z66抗体刺激后的基因表达调控中发挥一定作用.

  2. 人14-3-3β蛋白的原核表达、抗血清制备及真核表达载体的构建%Prokaryotic expression, antiserum preparation and construction of eukaryotic expression vector of human 14-3-3β protein

    Institute of Scientific and Technical Information of China (English)

    杨学习; 孙敏英; 马瑞娟; 徐伟文; 李明


    目的 纯化原核表达的14-3-3β(YWHAB)重组蛋白并制备多抗血清,构建适用于哺乳动物细胞的真核表达载体.方法 将重组蛋白表达载体pET30a(+)/YWHAB转化大肠杆菌表达菌株BL21(DE3)感受态细胞,异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达,镍-四齿螯合剂(Ni-NTA)亲和层析柱纯化重组蛋白;以纯化的重组蛋白为抗原免疫BALB/c小鼠,应用ELISA和Western blot方法分别检测抗血清的效价和特异性;应用PCR扩增添加BamH Ⅰ和EcoR Ⅰ酶切位点把YWHAB的ORF亚克隆至真核表达载体pEGFP-N1,添加BamH Ⅰ和Hind Ⅲ酶切位点把YWHAB的开放阅读框(ORF)亚克隆至真核表达载体pCDNA3.1(+),对重组载体进行酶切和PCR鉴定.结果 YwHAB重组蛋白以可溶性形式表达,分子量为32 000,与预期分子量一致;纯化后的重组蛋白纯度达90%以上,ELISA结果显示其抗血清的效价为1:50 000,Western blot结果表明抗血清的特异性较好;酶切和PCR鉴定结果表明真核表达载体pEGFP-N1/YWHAB和pCDNA3.1(+)YWHAB构建成功.结论 通过亲和层析纯化获得人14-3-3β重组蛋白,进而免疫BALB/c小鼠制备多抗血清,为进一步研究人14-3-3β的功能成功构建了其真核表达载体.%Objective To purify human 14-3-3β (YWHAB) recombinant protein expressed in the E.coli, prepare its antiserum and construct the eukaryotic expression vector for transfecting mammalian cells. Methods The human 14-3-313 recombinant protein expression vector pET30a (+) /YWHAB constructed by the ORF of YWHAB gene and prokaryotic expression vector pET30a (+) was transformed into E.coli BL21 (DE3). The expression of the recombinant protein was induced by IPTG and the protein was purified by affinity chromatography on a Ni-NTA resin. BALB/c mice were immunized by the purified protein, and ELISA and Western blotting were employed to detect the titer and specificity of the antiserum. The open reading flame of YWHAB gene was obtained by PCR

  3. 类泛素修饰蛋白bISG15抗血清在体外修饰系统中的应用%Establishment of an in vitro Protein Modification System with Antiserum Against Ubiquitin-like Modifier bISG15

    Institute of Scientific and Technical Information of China (English)

    刘畅; 史颖姣; 宣成昊; 耿运琪; 乔文涛


    ISG15由干扰素刺激基因15编码,是最早被发现的类泛素修饰分子.病毒感染以及干扰素刺激可以强烈诱导其表达.与泛素类似,ISG15可以共价连接到其他蛋白分子上进行修饰,但ISG15及其连接修饰的功能作用还有很多尚未知.最近的研究表明,ISG15及其修饰作用在先天免疫中起着重要的作用.将牛类ISG15基因克隆进入pET28a(+)原核表达载体,并且表达了可溶的融合有His-tag标签的bISG15融合蛋白.使用Ni-NTA葡聚糖进行纯化浓缩.纯化蛋白免疫Balb/c小鼠并获得抗血清.Western印迹实验显示,抗血清可以特异地识别在真核细胞中表达的bISG15.浓缩的bISG15以及制备的抗血清用于建立bISG15的体外修饰系统.实验证明,使用该系统bISG15可以连接到细胞蛋白上进行修饰.%ISG15, the first ubiquitin-like molecule identified two decades ago, is encoded by interferon stimulated gene 15 ( ISG 15), where its robust expression can be induced by viral infections or interferon treatments. ISG 15 conjugate to other proteins as the ubiquitin and was found to be involved in innate immune response. However, the functions of ISG15 modification remained unclear. We cloned bovine ISG15(bisg15) into a prokaryotic expression vector pET28a( + ) with a His-tag to generate a soluble form of bISG15 fusion protein, and purified with Ni-NTA Sepharose chromatography. The purified protein was concentrated and used to immune Balb/c mice to raise the antiserum, which could specifically recognize bISG15 expressed in eukaryotic cells by Western blot analysis. The concentrated bISG15 protein and its antiserum were then used to establish an in vitro bISG15 modification system. Our studies have demonstrated that cellular proteins could be conjugated to bISG15 with this system.

  4. 小麦自噬标志分子ATG6的原核表达及其抗血清制备%Prokaryotic expression of recombinant ATG6 from wheat variety L10, a molecular marker of autophagy, and development of its antiserum

    Institute of Scientific and Technical Information of China (English)

    刘刚; 吴洪波; 王冬梅


    克隆小麦(Triticum aestivum L.)品种‘洛夫林10'(L10)中的ATG6(动物同源物为Beclin 1)基因,并构建该基因的原核表达载体,在大肠杆菌中诱导表达融合蛋白,经纯化后制备其兔抗血清.结果表明:从小麦品种L10中克隆获得1个ATG6的片段,长为1326 bp,利用所构建的大肠杆菌表达载体,经IPTG诱导,实现了对ATG6片段在原核系统的特异性表达.经蛋白纯化后,制备了其兔抗血清并通过Western blot鉴定了其特异性.ATG6抗体制备的成功,为小麦中ATG6基因和自噬功能的研究提供了研究基础.%Molecular cloning of ATG6 from wheat cultivar L10, whose prokaryotic expression vector was then constructed and expressed in Escherichia coil rosetta-gami B (DE3). The rabbit antiserum against ATG6 was prepared and characterized finally. The ATG6 was amplified from wheat variety L10 by PCR and then cloned into the pMD19-T vector, one of which was subcloned into the expression vector. The recombinant plasmid was identified by sequencing and digestion of restriction enzymes. The fusion protein was finally expressed by IPTG-induction in host bacteria-E. coli rosetta-gami B (DE3) and detected by SDS-PAGE. The rabbit anti-ATG6 antibody was prepared and detected by western blot analysis. The ATG6 was obtained partly and successfully expressed in the prokaryotic expression system. The rabbit anti-ATG6 antibody was prepared and detected by western blot. The experiment offered foundation to study autophagy and function of ATG6 gene in wheat.

  5. 猪带绦虫TSO45W-4B-TSOL18融合基因在大肠埃希菌ArcticExpress(DE3)中的表达、纯化和兔抗血清的制备%Expression and purification of a fusion gene TSO45W-4B-TSOL18 of Taenia solium in Escherichia coli ArcticExpress(DE3) and preparation of rabbit antiserum

    Institute of Scientific and Technical Information of China (English)

    周必英; 周泠; 刘美辰; 刘晖; 贺莉芳


    Objective To construct a recombinant plasmid pGEX-TSO45W-4B-TSOL18 of Taenia solium,to obtain purified recombinant protein TSO45W-4B-TSOL18 and to prepare rabbit antiserum against the recombinant fusion protein.Methods TSO45W-4B and TSOL18 encoding genes were connected with hydrophobic (Gly4Ser)3 linker.TSO45W-4B-TSOL18 fusion gene was synthesized and cloned into an expression vector pGEX-1λT to construct a recombinant plasmid pGEX-TSO45W-4B-TSOL18.The positive recombinants were transformed into Escherichia(E.) coli ArcticExpress (DE3),and the expression of recombinant protein was induced with isopropylβ-D-thiogalactopyranoside (IPTG) and analyzed by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE).The expressed recombinant fusion protein was purified through affinity chromatography.The purified recombinant protein was mixed with complete Freunds adjuvant at a dosage of 0.5 mg.Rabbit was intramuscularly and subcutaneously immunized in the hind leg and the back,respectively.After 2 weeks the rabbit was boosted with purified recombinant protein which was mixed with incomplete Freunds adjuvant at the same dosage.Rabbit can be boosted every 10 days until an adequate response was achieved.At the 6th day after the last immunization,blood was collected from the rabbit heart.Serum was separated to purify and prepare the antiserum.ELISA was applied to determine the titer of the antiserum and Western blot assay was used to determine the specificity of the antiserum.Results The size of the synthesized fusion gene TSO45W-4B-TSOL18 was 789 bp.The results of restriction enzyme digestion showed that the recombinant plasmid pGEX-TSO45W-4B-TSOL18 was successfully constructed.DNA sequencing showed that the size of the fusion gene TSO45W-4B-TSOL18 was 789 bp,which was consistent with expected result.As demonstrated by SDS-PAGE,relative molecular mass of the expressed recombinant fusion protein was approximately 55 × 103,and its purity was 85% after

  6. Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life. (United States)

    Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon


    The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion.

  7. A new ganglioside in human meconium detected by antiserum against the human milk sialyloligosaccharide, LS-tetrasaccharide b. (United States)

    Prieto, P A; Smith, D F


    Antibodies directed against human milk sialyloligosaccharides [D. F. Smith and V. Ginsburg (1980) J. Biol. Chem. 255, 55-59] are used to identify human meconium gangliosides by radioimmuneoverlay-thin-layer chromatography or by direct binding on nitrocellulose filters of sialyl[3H]oligosaccharide alditols obtained from gangliosides after ozonolysis and alkali-fragmentation. Thin-layer chromatograms of meconium monosialylgangliosides immunostained with rabbit antisera specific for LS-tetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) or LS-tetrasaccharide b (Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) reveal their corresponding gangliosides, 6'-LM1 and a previously undescribed ceramide derivative of LS-tetrasaccharide b, respectively. The sialyl[3H]oligosaccharides derived from the monosialylganglioside fraction of meconium are separated by paper chromatography and assayed for binding to specific anti-sialyloligosaccharide sera. Antisera specific for LS-tetrasaccharide c and 3'-sialyllactose (NeuAc alpha 2-3Gal beta 1-4Glc) identify their corresponding 3H-labeled haptens released from the major meconium gangliosides 6'-LM1 and GM3, respectively. Binding of a ganglioside-derived sialyl[3H]oligosaccharide by anti-LS-tetrasaccharide b serum is consistent with the presence in meconium of a monosialylganglioside with the following proposed structure: (formula; see text)

  8. Preliminary research on preparation of anti-serum against dimethoate%兔抗乐果抗血清制备的初步实验研究

    Institute of Scientific and Technical Information of China (English)

    张文元; 杨亚冬; 房国坚



  9. A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus (Coral Snake Venom.

    Directory of Open Access Journals (Sweden)

    Henrique Roman Ramos


    Full Text Available Envenoming by coral snakes (Elapidae: Micrurus, although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage.In this work we describe the mapping, by the SPOT-synthesis technique, of potential B-cell epitopes from five putative toxins from M. corallinus, which were used to design two multiepitope DNA strings for the genetic immunisation of female BALB/c mice. Results demonstrate that sera obtained from animals that were genetically immunised with these multiepitope constructs, followed by booster doses of recombinant proteins lead to a 60% survival in a lethal dose neutralisation assay.Here we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity.

  10. A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus (Coral Snake) Venom (United States)

    Ramos, Henrique Roman; Junqueira-de-Azevedo, Inácio de Loiola M.; Novo, Juliana Branco; Castro, Karen; Duarte, Clara Guerra; Machado-de-Ávila, Ricardo A.; Chavez-Olortegui, Carlos; Ho, Paulo Lee


    Background Envenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage. Methods and Findings In this work we describe the mapping, by the SPOT-synthesis technique, of potential B-cell epitopes from five putative toxins from M. corallinus, which were used to design two multiepitope DNA strings for the genetic immunisation of female BALB/c mice. Results demonstrate that sera obtained from animals that were genetically immunised with these multiepitope constructs, followed by booster doses of recombinant proteins lead to a 60% survival in a lethal dose neutralisation assay. Conclusion Here we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity. PMID:26938217


    Institute of Scientific and Technical Information of China (English)

    李向东; 范在丰; 李怀方; 裘维蕃


    本实验提纯了玉米矮花叶病毒(MDMV)的辅助成份-蛋白酶(HC-Pro),并制备了其抗血清.MDMV HC-Pro的分子量约为57 kD,提纯产量为0.3 mg/100g病叶.琼脂免疫双扩散测定证明MDMV HC-Pro抗血清的效价为1:16;微量免疫沉淀法测定时其效价为1:1024.该抗血清只与MDMV HC-Pro有明显的反应,与健康玉米汁液和MDMV无反应.MDMV HC-Pro抗血清可以专化性地抑制HC-Pro的蚜传活性.

  12. 猪带绦虫HSP70-4的真核表达及其抗血清的制备%Eukaryotic expression and anti-serum preparation of HSP70-4 from Taenia solium

    Institute of Scientific and Technical Information of China (English)

    殷静; 王帅; 刘光学; 何伟; 才学鹏


    为了研究猪带绦虫(Taenia solium)热休克蛋白70-4 (TsHSP70-4)基因序列的特征及其真核表达蛋白的抗原性,参考GeneDB中猪带绦虫基因组注释信息,设计特异性引物,RT-PCR扩增TsHSP70-4ORF序列.根据毕赤酵母密码子偏好性,对TsHSP70-4基因密码子进行优化,连接载体pPIC9K,在毕赤酵母中进行诱导表达,通过Western-blot及质谱测序进行蛋白鉴定.将纯化后的TsHSP70-4表达蛋白免疫新西兰大白兔,制备抗血清.结果显示,成功克隆出大小为1 953 bp的TsHSP70-4ORF序列.在毕赤酵母中进行了高效表达,获得大小约为95 ku的重组蛋白.Western-blot检测和质谱鉴定表明,获得的重组蛋白即为TsHSP70-4.免疫新西兰大白兔,制备出效价高达1∶409 600的抗血清.该研究为猪囊尾蚴病新疫苗的研制提供了依据.

  13. 鸡L-FABP抗血清制备及组织表达特性分析%Preparation of Antiserums against Chicken Liver-type Fatty Acid Binding Protein (L-FABP) and Tissue Expression Analyses of L-FABP

    Institute of Scientific and Technical Information of China (English)

    石慧; 王启贵; 王宇祥; 王宁; 李辉


    为制备鸡肝脏型脂肪酸结合蛋白(L-FABP)的多克隆抗血清,并分析L-FABP的组织表达特性,利用RT-PCR扩增L-FABP基因,构建鸡L-FABP基因的GST融合蛋白表达质粒pGEX-4T/L-FABP.将重组表达质粒转化大肠杆菌BL21,IPTG诱导产生GST/L-FABP融合蛋白,用亲和层析纯化目的蛋白,将纯化的GST/L-FABP融合蛋白免疫家兔制备抗血清,并利用此抗血清分析鸡L-FABP基因的组织表达特性.诱导得到了1个40 ku(14 ku L-FABP+ 26 ku GST)的融合蛋白,获得效价较高、特异性强的鸡L-FABP的抗血清.鸡L-FABP的组织表达特性研究结果表明,该基因在肝脏和小肠组织中有较高表达,但在心脏、脂肪、肌肉、肌胃、脾、肺和肾中没有检测到表达信号.

  14. NSSR-1蛋白抗血清的制备和研究应用%Preparation and Application of Antiserum against Neural-Salient Serine/Arginine Rich Protein 1

    Institute of Scientific and Technical Information of China (English)

    刘璇; 刘磊; 林万敏; 陈献华; 徐平


    神经细胞显著的丝氨酸/精氨酸富集蛋白-1(NSSR-1)是近期发现的可能的神经细胞特异的前体mRNA剪接蛋白.为了进一步对此基因进行研究,制备其特异的抗血清是必需环节.本文在通过电脑软件分析找出其特异片段序列的基础上进行多肽片段合成,和匙孔血蓝蛋白结合后作为抗原免疫家兔,使之产生特异性的抗血清.运用所获抗血清,对小鼠多种组织中NSSR-1的表达分布进行了Western blot分析.并且,对小鼠脑组织切片也进行了免疫组织化学分析,观察NSSR-1在神经系统细胞内的表达分布.Wester blot分析检测到的蛋白条带的分子量大小与推测的NSSR-1蛋白分子量相似,表明是NSSR-1的特异条带.NSSR-1蛋白的表达只存在于大脑和小脑,而不存在于肝、肺和肾中.免疫组织化学分析的结果表明NSSR-1蛋白的表达主要分布于神经细胞的细胞核,而在细胞质中表达水平较低.

  15. 香蕉苞片花叶病毒CP基因的原核表达及抗血清制备%Prokaryotic expression and antiserum preparation of Banana bract mosaic virus coat protein gene.

    Institute of Scientific and Technical Information of China (English)

    刘福秀; 陈秀; 韩玉春; 李伟东; 徐卫; 蔡波; 林明光


    Banana bract mosaic virus (BBrMV) coat protein (CP) gene was cloned, and prokaryotic expression recombinant plasmid pET- CP was constructed by inserting the cloned gene into pET -28b ( + ). Induced by IPTG,E. coli BL21 (DE3) containing pET- CP produced fusion proteins about 34 kDa in size. Soluble analysis of the fusion protein indicated that it was in the inclusion body. The highly purified interest protein was obtained by using the histidine labeling of N - terminus of the protein. The special antibody was generated to the protein through the purified protein immunizing healthy rabbit. Indirect enzyme immunoassay suggested antibody titre was higher than 1:51 200. The available antibody concentration for virus detection from plant material was 1:800 - 1 : 3 200.%本试验成功构建了香蕉苞片花叶病毒(Banana bract mosaicvirus,BBrMV)外壳蛋白(coat protein,CP)基因的原核表达载体,并诱导表达了34kDa的融合蛋白His.CP。对该原核表达蛋白的可溶性分析表明,该融合蛋白以包涵体形式存在。利用组氨酸标签纯化试剂盒对目的蛋白进行了纯化,获得了高纯度的融合蛋白。以纯化的蛋白为抗原免疫健康家兔,成功制备了抗BBrMV CP基因编码蛋白的兔抗血清。Western—blotting结果表明这种抗血清有很强的特异性。血清效价测定的效价在51200倍以上,对植物材料的合适检测浓度为1:800—1:3200。

  16. Expression of c-terminal of Parkin in E.coli and the preparation of antiserum%Parkin蛋白C端的表达及抗体的制备

    Institute of Scientific and Technical Information of China (English)

    欧阳珏; 夏昆; 郑多; 张灼华


    目的:构建Parkin基因3'端的表达质粒,在大肠杆菌中表达并制备多克隆抗体.方法:构建Parkin基因3'端(937~1959bp)的谷氨酰胺硫转移酶(glutathion-sulfate-transferase, GST)融合表达质粒,在JM 105中获得表达.用Triton-100(1%)和Tween-20(1%)处理,并用亲和层析纯化表达产物,将其免疫新西兰兔,用免疫印迹分析收获的兔抗血清.结果:构建的Parkin C表达质粒在JM 105中获得表达,以分子量为42 kD的包涵体存在;目的蛋白纯度为95%;得到效价为1∶64的抗血清;免疫印迹分析表明,制备的抗血清可与鼠脑细胞抽提物中51.6 kD的蛋白产生特异的免疫反应.结论:Parkin蛋白C端在JM 105中获得高效表达并得到特异性多克隆抗体.

  17. 斑马鱼Nfatc3基因多克隆抗体的制备及检测%Preparation and Detection of Polyclonal Antiserum of Nfatc3Gene in Zebrafish

    Institute of Scientific and Technical Information of China (English)

    冯德峰; 屈永贵; 袁佳佳; 陈发; 吴秀山; 李永青


    为了在斑马鱼模型中更深入地研究Nfatc3蛋白在心脏发育中的功能,采用PCR技术扩增斑马鱼Nfatc3基因的部分编码区并插入pET-28a表达载体中,之后将重组质粒转入大肠杆菌(E.coli)通过IPTG诱导表达His-Nfatc3融合蛋白,对该融合蛋白采用Ni-IDA凝胶柱层析纯化后,免疫新西兰兔制备了多克隆抗体,并用western blotting对抗体进行分析.结果表明获得了高效价的特异性兔抗Nfatc3多克隆抗体.这为Nfatc3功能的进一步研究奠定了基础.%To better understand the function of theNfatc3 protein in heart development by using zebrafish as a model, here, a fraction of the encoded region in the zebrafishNfatc3was obtained by PCR amplification, then the fragment was inserted into pET-28a vector to establish the expressing system. The recombinant plasmid was transformed into E.coli and fusion protein was induced by IPTG. The purified protein was obtained by treating New Zealand white rabbits to prepare antibody. The antibody was assayed by Western blotting. The result shows that the polyclonal antibody is of high sensitivity and specificity, which lays a solid foundation for the further studies ofNfatc3function.

  18. Metarhizium anisopliae var. Anisopliae Pr1A基因的克隆表达及抗血清研究%Cloning and Expressing Pr1A Gene of the Entomopathogene fungus Metarhizium anisopliae var. anisopliae and Researching Its Antiserum

    Institute of Scientific and Technical Information of China (English)

    宋妍; 张世清; 黄俊生


    采用RT-PCR方法从本实验室分离筛选到的金龟子绿僵小孢变种Metarhizium anisopliae var.anisopliae中,扩增得到Pr1A基因全长,此基因全长为1242bp,经Blastn分析此基因序列与M.anisopliae的Pr1A基因(M73795)同源率为98%.以pET-22b(+)为基础载体,构建pET-Pr1A重组表达载体,在大肠杆菌(Escherichia.coli)BL 21(DE3)中进行表达.经SDS-PAGE分析,获得了约42kDa大小的重组目的蛋白,目的蛋白占表达总蛋白含量的63.2%.将表达的Pr1A蛋白切胶回收后制备成抗原,免疫家兔4次后,采血收集抗血清,用ELISA测定效价为1/10 000.结果表明,获得的抗体可用于更进一步的研究,将有利于我们进一步了解M.anisopliaeis的侵染机理,弄清楚各Pr蛋白酶的作用方式和对寄主的选择优势,提高生防控制的有效性.

  19. Ação do soro de cabra anti-soro de coelho imunizado ou não com células linfóides do doador sobre o alotransplante cardíaco em ratos: immunosupression of goat antiserum against rabbit serum immunized or not with donor lymphoid cells Cardiac allograft in rats

    Directory of Open Access Journals (Sweden)

    Haylton Jorge Suaid


    Full Text Available INTRODUÇÃO: A rejeição imunológica é uma das principais causas da perda de órgãos transplantados. A tentativa do controle da reação imunológica é clinicamente feita através da imunossupressão inespecífica e experimentalmente também por bloqueio específico. O alotransplante cardíaco em ratos pela técnica de ONO,K é um bom método para avaliação clínica da rejeição e de estudos voltados para o controle da rejeição. Objetivo : estudar o efeito de um anti-antisoro linfocitário, anti-linfócitos do doador sobre a rejeição do alotransplante cardíaco de ratos Wistar para ratos Holtzman. MÉTODOS: o soro anti-linfocitário (SAL foi obtido através da imunização de coelhos com linfócitos obtidos de gânglios linfáticos da cadeia mesentérica de ratos Wistar, em solução de Tyrode, contendo 3x10(9 células/ ml. A inoculação de 3 coelhos foi feita com 1 ml da suspensão celular e 1 ml de adjuvante completo de Freund. Duas semanas após a primeira inoculação fez-se 4 doses semanais de reforço. Os coelhos foram sangrados na 5ª semana, quando então foram separados os soros. A titulação dos soros foi realizada pelo teste de citotoxicidade, sendo verificado que ambos apresentaram título de 1:1024. A dosagem de proteínas mostrou albumina com 3,1 e 2,7 g% e globulinas com 3,5 e 2,9 g%, sendo o normal 3,7 e 2,2 g% respectivamente. Os dois SAL foram misturados. Duas cabras foram inoculados, com 3 ml da mistura desses SAL, associados a 2 ml de adjuvante de Freund. As doses de reforço com 5 ml do SAL foram iniciadas 2 semanas após. A cabra A recebeu 8 doses (1,4 g de globulinas. A cabra B recebeu 4 doses de reforço (0,7 g de globulinas. Uma semana após a última inoculação retirou-se 125 ml de sangue de cada cabra, fazendo a separação dos anti-soro anti-SAL (ASAL. Uma terceira cabra C foi imunizada com soro normal de coelho. A determinação de precipitinas foi feita pelo método de OUCHTERLONY. O ASAL A teve título de 1:64 e B e C título de 1:128. Os ASAL A e B foram capazes de bloquear "in vitro" a atividade citotóxica do SAL até a diluição de 1:2 do SAL. O soro de cabra anti-soro normal de coelho (SCANC não foi capaz de bloquear a citotoxicidade do SAL. Os animais submetidos a transplante cardíaco foram divididos em 2 grupos controles um normal com 10 ratos (C1 e outro (C2 com 5 ratos que recebeu 1,0 ml endovenoso de SCANC. O grupo de ratos testes A foi composto por 19 ratos distribuídos em 3 subgrupos. Subgrupo A1 com 5 ratos recebeu 0,5 ml do ASAL A, via endovenosa, logo após a cirurgia,o subgrupo A2 com 7 ratos recebeu 1.0 ml do ASAL A nas mesmas condições e o subgrupo A3 também com 7 ratos recebeu 1,0 ml no dia da cirurgia e 1,0 ml nos outros 2 dias consecutivos. O grupo de ratos testes B que recebeu o ASAL B foi igual ao grupo A. A avaliação dos corações transplantados foi diária através da palpação abdominal. O tempo máximo de seguimento foi de 243 dias. Os corações considerados rejeitados foram retirados e feito estudos histológicos. RESULTADOS: o período de rejeição dos grupos foi : controles C1 e C2 foram 11,9 e 14,6 dias, respectivamente; no subgrupo A1 apenas um rato teve sobrevida cardíaca significante (153 dias, nos demais ela variou de 9 a 15 dias; no subgrupo A2 a sobrevida do coração foi significante e variou de 23 a 230 dias; no subgrupo A3 apenas 5 corações tiveram sobrevida significante que variou de 29 a 190 dias. A sobrevida dos corações transplantados do grupo B foi significante para um animal de cada subgrupo (120,132 e 129 dias. Os corações com sobrevida longa foram retirados batendo. Os demais corações foram rejeitados dentro do período de variação dos grupos controles. CONCLUSÕES: O soro de cabra anti-soro anti-linfócitos do doador, com maior período de imunização, foi capaz de bloquear a resposta imune de rejeição dos corações transplantados nas doses de 1,0 e 3,0 ml. Os ratos que não promoveram a rejeição aguda dos corações transplantados não apresentaram anticorpos citotóxicos circulantes. O fator causador do bloqueio parace n��o estar vinculado aos bloqueios de citotoxicidade "in vitro" e do teor de precepitinas do SAL.OBJECTIVE: To study the immunosupression efficacy an specific anti-antilymphocytic serum prepared in goats in a model of cardiac allografts in rats. METHODS: Three rabbits were immunized with lymphoid cells obtained from mesenteric lymphatic nodes of Wistar rats. Each one received subcutaneously 3x10(9 cells mixed with Freund's adjuvant. After 2 weeks, they were injected with the same amount of cells at weekly intervals for 4 additional times. In the 5th week they were bled and their serum were mixed. This serum, which had a cytotoxic titer of 1:1024, was used to immunize 2 goats that gave rise to the anti-antilymphocytic serum (AAS-1 and AAS-2. As control we immunized 1 additional goat with normal rabbit serum (ANS. The gel diffusion technique (AAS x rabbit serum showed precipitation bands against till the following dilution: AAS-1 - 1/64, AAS-2 - 1/128 and ANS 1/124. Both AAS were able to block the in vitro lymphocytotoxity of goat antilymphocytic serum till dilution of 1:2 while ANS did not. The hearts from Wistar rats (donors were transplanted in Holtzman rats. The transplanted rats were divide in groups: C1 - 11 animals (control that received no serum; C2 - 5 animals (control that received 1ml of goat normal serum; A- 19 animals - A1 with 5 rats injected intravenously in the day of surgery with 0.5ml of AAS-1, A2 with 7 rats injected with 1ml of AAS-1 only in the of surgery, and A3 with 7 rats that received 1ml of AAS-1 in days 0, 1 and 2 postoperatively; and group B with 19 rats (B1, B2 and B3 treated as group A except with the AAS-2 serum. RESULTS: Mean heart survival in groups C1 and C2 was respectively 11.9 and 14.6 days Survival range in the subgroups A1 and A2 were respectively 9 to 230 days and 23 to 230 days. In subgroup A3 heart survival was prolonged till 29 to 190 days in 5 animals. In group B only 3 animals had prolonged (120, 130 and 129 days heart survival in comparison with the control groups. CONCLUSION: Anti-antilymphocytic serum against donor antigen is able to suppress rejection of cardiac allograft in rats.

  20. 昆明麝香石竹斑驳病毒分离物的鉴定与提纯及高效价抗血清的制备%Identification,purification and high efficiency antiserum preparation of carnation mottle virus in Kunming

    Institute of Scientific and Technical Information of China (English)

    李婷婷; 方琦; 丁铭; 苏晓霞; 张丽珍; 张仲凯


    对引起云南鲜切花麝香石竹植株叶斑驳、花朵变小的病毒分离物应用酶联免疫吸附测定(ELISA)和电子显微镜技术(IEM)检测,直径大约28 nm.经TAS-ELISA测定仅与CsrMV标准抗体反应为阳性.鉴定为麝香石竹斑驳病毒(Carnation mottle vi-rus,CarMV)的分离株.对病株检测筛选后进行分离纯化,将提纯后的CarMV制备兔抗血清获得高效价的抗体,间接ELISA测定为1:10240.

  1. 丹参柯巴基焦磷酸合酶基因的优化表达、纯化及抗体制备%Optimizing expression and purification of recombinant Salvia miltiorrhiza copalyl diphosphate synthase protein in E. coli and preparation of rabbit antiserum against SmCPS

    Institute of Scientific and Technical Information of China (English)

    高伟; 崔光红; 孔建强; 程克棣; 王伟; 袁媛; 黄璐琦


    用含丹参柯巴基焦磷酸合酶(Salvia miltiorrhiza copalyl diphosphate synthase, SmCPS)基因的重组质粒pET32CPS转化大肠杆菌BL21trxB(DE3)进行诱导表达,SDS-PAGE结果表明SmCPS基因在大肠杆菌中获得了表达;对影响可溶性表达的4个因素,即诱导温度、IPTG诱导浓度、诱导时宿主菌的密度(A600)和诱导时间进行了优化.结果发现在宿主菌A600达到1.0时加入0.4mmol·L-1 IPTG,在20℃诱导培养8h表达的可溶性蛋白量最高,约占细胞总蛋白的35.6%.用Ni2+亲和色谱法纯化表达产物,纯化蛋白产率达到12.3mg·L-1.将纯化蛋白免疫制备兔源SmCPS抗血清,ELISA测定效价为1:24300,且经Western blotting鉴定该抗体可特异性识别SmCPS抗原,获得了效价高、免疫反应性好的SmCPS多克隆抗体,为进一步从蛋白水平研究SmCPS表达与丹参酮类有效成分生物合成相关性提供了物质基础.

  2. Serum sickness (United States)

    ... the problem should be stopped. Avoid using that medicine or antiserum in the future. ... Call your provider if you received medicine or antiserum in the last 4 weeks and have symptoms of serum sickness.

  3. Expression of human S100 protein and preparation of specific antiserum for S100 and establishment of a quantitative measurement for S100 protein in CSF specimens of patients with Creutzfeldt-Jakob disease%人S100蛋白的表达及其抗血清的制备和脑脊液中S100含量测定方法的建立

    Institute of Scientific and Technical Information of China (English)

    张福萍; 张瑾; 周伟; 董小平; 洪涛


    目的建立定量检测脑脊液(CSF)及血清中S100蛋白的方法,探讨S100蛋白的检测在辅助诊断克-雅病(CJD)中的应用.方法利用脑cDNA文库,经PCR获得了S100基因并克隆至原核表达载体pGEX-2T上,在大肠埃希菌中表达了谷胱甘肽-S-转移酶(GST)-S100融合蛋白;融合蛋白经亲和纯化后,免疫家兔,制备抗体;抗体经纯化后,用生物素(BNHS)标记,建立了可定量检测S100蛋白的生物素-亲和素系统ELISA方法,并初步用于临床脑脊液的检测中.结果所表达的GST-S100蛋白相对分子质量约为35 000,以其为抗原制备的S100特异性抗血清具有良好的免疫反应性.建立了定量检测脑脊液中S100蛋白的双抗体夹心ELISA方法,对3例"可能性的CJD"患者(14-3-3蛋白阳性)和15例无痴呆症状患者脑脊液进行检测,结果显示,3例CJD患者脑脊液S100含量均超过2.900 μg/L,而在无痴呆症状患者组中14例患者脑脊液S100含量都低于0.180 μg/L.对正常人和CJD患者血清进行检测,显示S100蛋白含量个体间差异很大.结论所建立的方法可用于脑脊液中S100蛋白的检测,进一步扩大标本量有助于明确脑脊液中S100蛋白的检测在辅助诊断CJD中的价值.

  4. Ultrastructural immunohistochemical localization of Clara cell secretory protein in pulmonary epithelium of rabbits.


    Patton, S E; Gupta, R. P.; Nishio, S.; Eddy, E M; Jetten, A. M.; Plopper, C. G.; Nettesheim, P; Hook, G E


    Highly purified Clara cells (93 +/- 3%) isolated from the lungs of rabbits were used to produce an antiserum against Clara cell secretory proteins. This antiserum was used to identify and study the biosynthesis and secretion of [35S]methionine-labeled proteins from isolated Clara cells. The antiserum recognized one major secretory protein with apparent molecular weight of 6 kDa and reacted weakly with a higher molecular weight protein of about 180 kDa. Biosynthesis and secretion of these prot...

  5. [Presence in the sub-esophageal ganglinnof the caterpillar (Thaumetopoea pityocampa Schiff) of cells containing anti-alpha-endorphins as revealed by immunofluorescence]. (United States)

    Rémy, C; Girardie, J; Dubois, M P


    Immunohistological investigations have been performed with Vertebrate neuropeptide antiserums in the subaesophageal ganglion of larval Thaumetopoea pityocampa (Lepidoptera). Positive immunoreaction was observed in two groups of cells only with an alpha-endorphin-antiserum. These cells are azocarmine ground positive earth and paraldehyde-fuchsin ground negative earth or Gomori ground negative earth. In the brain, immunohistological staining was completely negative.

  6. Subcellular location of the helper component-proteinase of Cowpea Aphid-Borne Mosaic Virus

    NARCIS (Netherlands)

    Mlotshwa, S.; Verver, J.; Sithole-Niang, I.; Gopinath, K.; Carette, J.; Kammen, van A.; Wellink, J.


    The helper component-proteinase (HC-Pro) of Cowpea aphid-borne mosaic virus (CABMV) was expressed in Escherichia coli and used to obtain HC-Pro antiserum that was used as an analytical tool for HC-Pro studies. The antiserum was used in immunofluorescence assays to study the subcellular location of H

  7. Prevention of edema disease in pigs by passive immunization

    DEFF Research Database (Denmark)

    Johansen, M.; Andresen, Lars Ole; Thomsen, L.K.


    The effect of treatment with verotoxin 2e (VT2e) specific antiserum was evaluated in 3 Danish pig herds with edema disease (ED). The antiserum was prepared by immunizing horses with a VT2e toroid. The study was performed as a randomized blind field trial with parallel treatment and control groups...

  8. Deoxyribonucleic-binding homeobox proteins are augmented in human cancer

    DEFF Research Database (Denmark)

    Wewer, U M; Mercurio, A M; Chung, S Y;


    the highly conserved 60 amino acid homeodomain. This peptide antiserum recognized a protein species of molecular weight 63,000 in immunoblots of nuclear extracts obtained from several tumor cell lines. The predominant molecular weight 63,000 nuclear protein recognized by the peptide antiserum...... the same patients exhibited little immunoreactivity. Both the peptide antiserum and the polyclonal antiserum against the native protein immunoblotted a molecular weight 63,000 protein in nuclear extracts of tumor tissue, but not significantly in extracts of normal tissue. At the molecular level......Homeobox genes encode sequence-specific DNA-binding proteins that are involved in the regulation of gene expression during embryonic development. In this study, we examined the expression of homeobox proteins in human cancer. Antiserum was obtained against a synthetic peptide derived from...

  9. Immunodiagnosis of episomal Banana streak MY virus using polyclonal antibodies to an expressed putative coat protein. (United States)

    Sharma, Susheel Kumar; Kumar, P Vignesh; Baranwal, Virendra Kumar


    A cryptic Badnavirus species complex, known as banana streak viruses (BSV) poses a serious threat to banana production and genetic improvement worldwide. Due to the presence of integrated BSV sequences in the banana genome, routine detection is largely based on serological and nucleo-serological diagnostic methods which require high titre specific polyclonal antiserum. Viral structural proteins like coat protein (CP) are the best target for in vitro expression, to be used as antigen for antiserum production. However, in badnaviruses precise CP sequences are not known. In this study, two putative CP coding regions (p48 and p37) of Banana streak MY virus (BSMYV) were identified in silico by comparison with caulimoviruses, retroviruses and Rice tungro bacilliform virus. The putative CP coding region (p37) was in vitro expressed in pMAL system and affinity purified. The purified fusion protein was used as antigen for raising polyclonal antiserum in rabbit. The specificity of antiserum was confirmed in Western blots, immunosorbent electron microscopy (ISEM) and antigen coated plate-enzyme linked immunosorbent assay (ACP-ELISA). The antiserum (1:2000) was successfully used in ACP-ELISA for specific detection of BSMYV infection in field and tissue culture raised banana plants. The antiserum was also utilized in immuno-capture PCR (IC-PCR) based indexing of episomal BSMYV infection. This is the first report of in silico identification of putative CP region of BSMYV, production of polyclonal antiserum against recombinant p37 and its successful use in immunodetection.

  10. Radioimmunoassay of buprenorphine

    Energy Technology Data Exchange (ETDEWEB)

    Bartlett, A.J.; Lloyd-Jones, J.G.; Rance, M.J.; Flockhart, I.R.; Dockray, G.J.; Bennett, M.R.D.


    Antisera to buprenorphine were obtained in rabbits immunised with 3-0-carboxymethylbuprenorphine and N-hemisuccinyl-norbuprenorphine conjugated to bovine serum albumin. Using the latter antiserum and tritium labelled buprenorphine a radioimmunoassay having good accuracy and precision was developed for concentrations as low as 50 picograms in 1 ml of plasma. The N-hemisuccinyl antiserum crossreacted with norbuprenorphine, and the 3-0-glucuronide conjugate with the 3-0-carboxy-methyl antiserum. Cross-reactivity of both antisera to other pharmacologically related compounds was negligible. The assay was employed to determine plasma buprenorphine concentration following its parenteral administration to dog and man.

  11. Immunochemical cross-reactivity between albumin and solid-phase adsorbed histamine

    DEFF Research Database (Denmark)

    Poulsen, L K; Nolte, H; Søndergaard, I


    For production of an antibody against histamine, this was coupled to human serum albumin (HSA) and used for immunization of rabbits. To test the antiserum, an immunoradiometric assay was developed comprising solid-phase bound histamine, antisera and radiolabelled protein A. Titration and inhibition...... experiments revealed that histamine adsorbed onto a solid-phase could bind the antiserum. However, neither free histamine nor histamine coupled to unrelated carriers could inhibit the binding of antiserum to the solid-phase histamine. Cross-reactivity was demonstrated between HSA and solid-phase bound...... histamine, as the immunoradiometric assay was inhibited by HSA. This unexpected cross-reactivity was established, as a commercially available antiserum with specificity to HSA without histamine also bound to the solid-phase bound histamine. It is suggested that preparations of antibodies against histamine...

  12. Distinct localization of FMRFamide- and bovine pancreatic polypeptide-like material in the brain, retrocerebral complex and suboesophageal ganglion of the cockroach Periplaneta americana L

    DEFF Research Database (Denmark)

    Verhaert, P; Grimmelikhuijzen, C J; De Loof, A


    One bovine pancreatic polypeptide (BPP) antiserum and two FMRFamide antisera were applied in the peroxidase-antiperoxidase (PAP) immunohistochemical technique on a complete series of sections of brains, suboesophageal ganglia (SOG), corpora cardiaca (CC) and corpora allata of Periplaneta american...

  13. A distinct tospovirus causing necrotic straek on alstroemeria sp. in Colombia

    NARCIS (Netherlands)

    Mehraban, A.; Botermans, M.; Verhoeven, J.Th.J.; Meekes, E.; Saaijer, J.; Peters, D.; Goldbach, R.W.; Kormelink, R.J.M.


    A tospovirus causing necrotic streaks on leaves was isolated from Alstroemeria sp. in Colombia. Infected samples reacted positively with tomato spotted wilt virus (TSWV) antiserum during preliminary serological tests. Further analysis revealed a close serological relationship to tomato chlorotic spo

  14. Passive immunization of pigs against experimental infection with Streptococcus suis serotype 2

    DEFF Research Database (Denmark)

    Andresen, Lars Ole; Tegtmeier, Conny


    The safety and protective efficacy of a horse antiserum raised against inactivated whole cell preparations of Streptococcus suis serotype 2 was investigated in pigs by experimental challenge. The antiserum was evaluated in two similar experiments each comprising 12 4-week-old pigs treated with 6 ...... indicate that passive immunization of pigs may be a way to reduce or control S. suis serotype 2 infections in pigs....

  15. Optimization of the radioimmunoassays for measuring fentanyl and alfentanil in human serum

    Energy Technology Data Exchange (ETDEWEB)

    Schuettler, J.; White, P.F.


    Measurement of serum fentanyl and alfentanil concentrations by radioimmunoassay (RIA) may result in significant errors and high variability when the technique described in the available fentanyl and alfentanil RIA kits is used. The authors found a 29-94% overestimation of measured fentanyl and alfentanil serum levels when 3H-fentanyl or 3H-alfentanil was added lastly to the mixture of antiserum and sample. This finding is related to a reduction in binding sites for the labeled compounds after preincubation of sample and antiserum. If this sequence is used, it becomes necessary to extend the incubation period up to 6 h for fentanyl and up to 10 h for alfentanil in order to achieve equilibration between unlabeled and labeled drug with respect to antiserum binding. However, when antiserum is added lastly to the mixture of sample and labeled drug, measurement accuracy and precision for fentanyl and alfentanil serum concentrations are enhanced markedly. In addition, it is important to perform the calibration curves and sample measurements using the same medium (i.e., serum alone or a serum/buffer dilution). In summary, to optimize the RIA for fentanyl and alfentanil, the authors recommend the following: 1) adding the antiserum lastly to the mixture of sample and labeled drug; 2) performing calibration curves using patient's blank serum when possible; 3) carefully examining and standardizing each step of the RIA procedure to reduce variability, and, finally; 4) comparing results with those of other established RIA laboratories.

  16. Identification and role of plasma membrane aquaporin in maize root

    Institute of Scientific and Technical Information of China (English)


    Using antiserum against expressed aquaporin fusion protein, GST-RD28, the distribution of aquaporin in the plasma membrane of maize root protoplasts has been examined under confocal laser scanning microscopy by indirect fluorescence staining. Results indicate that there are abundant aquaporins in maize roots, which are distributed in plasma membrane unevenly. Western blotting analysis of total protein solubilized from maize root plasma membrane shows that antiserum against GST-RD28 can cross-react with one protein around 55 ku. Another 28 ku protein can also be detected when the concentration of SDS and DTT in SDS-PAGE sample buffer is increased. The 55 and 28 ku proteins may be dimeric and monomeric of aquaporin respectively. Functional experiments show that aquaporin blocker HgCl2 and aquaporin antiserum can suppress the swelling of maize root protoplasts in hypotonic solution, indicating that aquaporin in plasma membrane of protoplast facilitates rapid transmembrane water flow.

  17. COTA (colon-ovarian tumor antigen). An immunohistochemical study. (United States)

    Pant, K D; Fenoglio-Preiser, C M; Berry, C O; Zamora, P O; Ram, M D; Fulks, R M; Rhodes, B A


    A goat anti-serum was prepared against mucinous ovarian cyst fluid and absorbed with normal colon and a variety of normal tissues until the only residual immunoreactivity was directed against colon cancer and ovarian tumor mucin. The set of antigenic determinants defined by this anti-serum has been called COTA, standing for colon-ovarian-tumor-antigen. This highly absorbed anti-serum (anti-COTA) was used for immunohistochemical staining of 42 different tissues in parallel with staining with a goat anti-CEA, which was also highly absorbed. The results suggest that COTA is a highly sensitive and specific antigen for colon carcinoma and may have potential for the early detection of malignant changes predictive of cancer of the colon.

  18. Immunoreactive neuronal pathways of growth hormone-releasing hormone (GRH) in the brain and pituitary of the teleost Gadus morhua. (United States)

    Pan, J X; Lechan, R M; Lin, H D; Jackson, I M


    Using an antiserum directed against the C-terminus of hGRH(1-44)NH2 and another recognizing the mid portion to C-terminal of hGRH(1-40)OH, we identify two immunocytochemically distinct GRH-immunoreactive systems in the brain of the codfish, Gadus morhua. The antiserum directed against GRF(1-44)NH2 stains cell bodies exclusively in the rostral pars distalis. The other antiserum immunoreactive with GRF(1-40)OH reacts with a population of parvocellular and magnocellular neuronal cell bodies in the hypothalamus and with two major axonal pathways which project toward the median eminence and terminate primarily in the pars nervosa. These results indicate the presence of at least two forms of hGRH-like peptides in the teleost which may have different roles in the regulation of pituitary function.

  19. Characterisation of non-maternal serum proteins in amniotic fluid at weeks 16 to 18 of gestation

    DEFF Research Database (Denmark)

    Drøhse, H; Christensen, H; Myrhøj, Vibeke;


    Proteins found in amniotic fluid are mainly serum proteins, probably of maternal origin. About 5% of the total protein concentration has the potential of being fetal or decidual in origin. Only a few of these proteins have been isolated and characterised. In order to describe the foetal...... and decidual components in amniotic fluid more extensively, a polyspecific antiserum to amniotic fluid at weeks 16-18 of gestation was raised. Specificities in the antiserum to serum proteins were removed by adsorption. Several proteins of non-serum protein origin reacted with the antiserum. Three...... of these proteins were chosen for isolation and further characterisation. With the use of immunological methods, SDS-PAGE and N-terminal sequencing we identified two of the proteins as C-terminal propeptides of procollagen Type I and Type III, which have not hitherto been described in amniotic fluid. The third...

  20. Isolation of a gene that is involved in Campylobacter jejuni 81116 cytotoxin activation. (United States)

    Liu, Kaiyan; Fry, Benjamin N; Coloe, Peter J


    Cytotoxin fractions were isolated from Campylobacter jejuni 81116 and semi-purified by size-exclusion liquid chromatography. The fraction showing the strongest toxicity was injected into mice to produce antiserum. The antiserum was used to screen a C. jejuni 81116 cosmid library. Nine genes were identified in overlapping cosmid inserts that induced reactivity with the antiserum. One of these genes showed high similarity to a periplasmic protein of unknown function and its isogenic mutant showed decreased toxicity compared to the C. jejuni 81116 wild type. This gene contains a Gram-negative bacterial RTX toxin-activating protein C signature, which suggests it may play a role in C. jejuni 81116 cytotoxin activation.

  1. Detection of feline coronavirus using microcantilever sensors (United States)

    Velanki, Sreepriya; Ji, Hai-Feng


    This work demonstrated the feasibility of detecting severe acute respiratory syndrome associated coronavirus (SARS-CoV) using microcantilever technology by showing that the feline coronavirus (FIP) type I virus can be detected by a microcantilever modified by feline coronavirus (FIP) type I anti-viral antiserum. A microcantilever modified by FIP type I anti-viral antiserum was developed for the detection of FIP type I virus. When the FIP type I virus positive sample is injected into the fluid cell where the microcantilever is held, the microcantilever bends upon the recognition of the FIP type I virus by the antiserum on the surface of the microcantilever. A negative control sample that does not contain FIP type I virus did not cause any bending of the microcantilever. The detection limit of the sensor was 0.1 µg ml-1 when the assay time was <1 h.

  2. Preparation and Application of Polyclonal Antibody against a Recombinant Laccase

    Institute of Scientific and Technical Information of China (English)

    Yinghai Xu; Yuzhi Hong; Yazhong Xiao; Wei Fang


    A laccase gene from Trametes sp. 420 was recombinantly expressed in Pichia pastoris, producing the enzyme rLacD.Six mutant enzymes were produced by site-directed mutation at six potential glycosylation sites in the enzyme rLacD respectively. To probe the mutants with lower activities sensitively and specifically, the antiserum containing specific polyclonal antibodies were prepared by immunizing healthy male rabbits, about 4-month-old and 2 kilogram weight, using pure rLacD as an immunogen. Antibodies were collected after the fifth immunization injection. The antiserum had titres of 1:32 in double immunodiffusion test and of 1:128,000 in enzyme-linked immunosorbent assay (ELISA). The results obtained by Western blot analysis showed that the antiserum could react with rLacD and its mutants with highly specific and sensitive affinities.

  3. Antigenic relatedness of equine herpes virus types 1 and 3. (United States)

    Gutekunst, D E; Malmquist, W A; Becvar, C S


    Antiserums prepared in specific pathogen free (SPF) ponies were used in direct and indirect immunofluorescence, immunodiffusion, complement fixation and serum neutralization procedures to study the interrelationships of the three types of equine herpes viruses (EHV-1, EHV-2, and EHV-3). Equine cell cultures infected with each type virus fluoresced when stained with homologous conjugated antiserum. In reciprocal tests EHV-1 and EHV-3 cross-fluoresced, but EHV-2 did not cross-fluoresce. Non-infected cell cultures did not fluoresce when stained with the 3 conjugates. EHV-1 and EHV-3 cross-fluoresced in reciprocal indirect fluorescent antibody tests, but no cross-fluorescence was shown with EHV-2. Antigens representing each type of equine herpes virus reacted with their homologous antiserum in the immunodiffusion test. In reciprocal tests, a common line(s) of identity formed with EHV-1 and EHV-3; however, the precipitin line(s) was not common with EHV-2. Antigen prepared from noninfected embryonic mule skin (EMS) cell cultures did not react with any of the antiserums. Specific complement-fixing antibodies were present in antiserums when tested against their homologous antigens. In reciprocal complement fixation tests EHV-1 and EHV-3 crossreacted, but no cross-reactivity was shown with EHV-2. Significant levels of neutralizing antibody were in an antiserum when tested against homologous virus, whereas cross-neutralization was not detectable in reciprocal tests. These studies indicate that each type of equine herpes virus contains specific antigenic components, and EHV-1 and EHV-3 share a common antigen(s) that is not shared with EHV-2.

  4. Localization of connexins in neurons and glia cells of the Helix aspersa suboesophageal brain ganglia by immunocytochemistry. (United States)

    Azanza, M J; Pes, N; Pérez-Bruzón, R N; Aisa, J; Raso, M; Junquera, C; Lahoz, J M; Maestú, C; Martínez-Ciriano, C; Pérez-Castejón, C; Vera-Gil, A; Del Moral, A


    The aim of the present study was to examine the distribution of cells expressing connexin 26 (Cx26) in the suboesophageal visceral, left and right parietal and left and right pleural ganglia of the snail Helix aspersa by immunocytochemistry. Altogether we have found approximately 452 immunoreactive neurons which represent the 4.7% of the total neurons counted. The stained large neurons (measured diameter 55-140 microm) occurred mostly on the peripheral surface of the ganglia while the small immunostained cells (5-25 microm diameter) were observed in groups near the neuropil. The number of large neurons giving positive Cx26-like immunostaining was small in comparison with that for medium (30-50 microm diameter) and small sized cells. The expression of Cx26 was also observed in the processes of glia cells localized among neurons somata and in the neuropil showing that the antiserum recognized epitopes in both protoplasmic and fibrous glia cells of Helix aspersa. The neuropils of all ganglia showed fibers densely immunostained. While we have observed a good specificity for Cx26-antiserum in neurons, a lack of reaction for Cx43 antiserum was observed in neurons and glia cells. The reaction for enolase antiserum in neurons was light and non-specific and a lack of reaction in glia cells and processes for GFAP antiserum was observed. Although the percentage of positive neurons for Cx26 antiserum was low is suggested that in normal physiological conditions or under stimulation the expression of connexin could be increased. The observed results can be considered of interest in the interpretation of Helix aspersa elemental two neuron networks synchronizing activity, observed under applied extremely low frequency magnetic fields.

  5. Do the ependymal cells contain vasoactive intestinal polypeptide? (United States)

    Kawano, H; Masuko, S


    Immunoreaction for vasoactive intestinal polypeptide (VIP) in the ependymal cells was investigated using two different commercially available polyclonal antisera for VIP. Immunostaining with an anti-VIP serum showed strong reaction products in the ependymal cells of the central canal and the third ventricle, in addition to immunoreactive neuronal elements in the spinal cord and in the suprachiasmatic nucleus. Staining of the ependymal cells was not reduced by preabsorption of the antiserum with synthetic VIP, while the immunoreactive neuronal elements disappeared. Such staining of the ependymal cells was not found using other antiserum.

  6. Pneumocystis pneumonia: importance of gallium scan for early diagnosis and description of a new immunoperoxidase technique to demonstrate Pneumocystis carinii

    Energy Technology Data Exchange (ETDEWEB)

    Levin, M.; McLeod, R.; Young, Q.; Abrahams, C.; Chambliss, M.; Walzer, P.; Kabins, S.A.


    Pneumocystis pneumonia presented in a homosexual with fever, a normal chest radiograph, and pulmonary gallium uptake. Bronchial washings yielded Mycobaterium tuberculosis, but despite antituberculosis therapy he remained febrile, and gallium uptake in the lung increased. Subsequently, silver stain of transbronchial lung biopsy obtained 2 months earlier at the time that tuberculosis was diagnosed showed many Pneumocystis cysts in alveolar spaces. In contrast to Pneumocystis cysts in infected lung tissue from other humans, our patient's Pneumocystis cysts reacted more avidly with antiserum to rat Pneumocystis than with antiserum to human pneumocystis, raising the possibility that organisms that infect humans may have varied surface antigenic properties.

  7. Immunohistochemical diagnosis of Neospora caninum in tissue sections. (United States)

    Lindsay, D S; Dubey, J P


    An avidin-biotin-peroxidase complex immunoperoxidase staining method was developed to detect Neospora caninum in formalin-fixed, paraffin-embedded tissue sections. Specific antiserum to N caninum was made in rabbits and used to probe tissues from dogs naturally and experimentally infected with N caninum. The test detected tachyzoites and bradyzoites of N caninum. A reaction was not observed to Toxoplasma gondii, Hammondia hammondi, Sarcocystis cruzi, S capricanis, S tenella, Besnoitia jellisoni, Caryospora bigenetica, Hepatazoon canis, Atoxoplasma sp, or the organism causing canine dermal coccidiosis. When antiserum made in rabbits to T gondii was used in the test, reaction to N caninum was not observed.

  8. Vasoactive intestinal peptide and nitric oxide promote survival of adult rat myenteric neurons in culture

    DEFF Research Database (Denmark)

    Sandgren, Katarina; Lin, Zhong; Svenningsen, Åsa Fex;


    of VIP, NO donor, VIP antiserum, or NOS inhibitor. A marked loss of neurons was noted during culturing. VIP and NO significantly promoted neuronal survival. Corroborating this was the finding of an enhanced neuronal cell loss when cultures were grown in the presence of VIP antiserum or NOS inhibitor....... adaptation. The aim of this study was to evaluate whether VIP and nitric oxide (NO) influence survival of cultured, dissociated myenteric neurons. Neuronal survival was evaluated after 0, 4, and 8 days in culture. Influence of VIP and NO on neuronal survival was examined after culturing in the presence...

  9. Comparison of the C-mediating killing activity and C-activating properties of mouse monoclonal and polyclonal antibodies against Trypanosoma cruzi

    Directory of Open Access Journals (Sweden)

    T. L. Kipnis


    Full Text Available A Mouse polyclonal antiserum against Trypanosoma cruzi or its IgG and IgM fractions and five monoclonal antibodies (two IgM, two IgG1 and one IgG2a recognize and combine with membrane components of trypomastigote forms of the parasite as revealed by immunofluorescence. Although all these antibodies sensitize trypomastigotes and prepare them to activate the complement (C system, as measured by consumption of total C, C4, B and C3, only the polyclonal antiserum or its IgG, IgM and Fabμ fragments were able to induce trypanosome lysis by the alternative C pathway.

  10. Preparation, Characterization, and Application of Antiharpinxoo Antibody

    Institute of Scientific and Technical Information of China (English)

    SHAO Min; LI Ming; PAN Xiao-mei; WANG Jin-sheng


    Polyclonal antiharpinxoo rabbit antibody has been prepared successfully using purified harpinxoo protein as an immunogen.The ELISA titer of the antiserum against harpinxoo was about 1:2 000. Western blot analysis showed that the antiserum could bind to the expression harpinxoo protein in particular. hrf1, encoding harpinxoo, is an expression in transgenic rice,detected by antiharpinxoo rabbit antibody. The rabbit antibody against harpinxoo can be used to study further about the biological function, harpinxoo localization, and hrf1 gene expression in other plants.

  11. B Lymphocyte Subpopulation Defined by a Rat Monoclonal Antibody, 14G8 (United States)


    Bethesda. MO). Proprtis an exresion f mmbrae atigns (, 2 In The goat anti-p antiserum from which the specifically purified antibodies and xpresio of embane...cell * Independent responses in B-cell defective CsA/N mice to Brucella abortus cycle by ProPiumn iodide staining. J. Cell. Bil. 66:188. and to TNP

  12. Radioimmunoassay for somatomedin C: comparison with radioreceptor assay in patients with growth-hormone disorders, hypothyroidism, and renal failure

    Energy Technology Data Exchange (ETDEWEB)

    Baxter, R.C.; Brown, A.S.; Turtle, J.R.


    An antiserum (Tr4) was raised in rabbits against a basic somatomedin C-like peptide preparation. Using high-immunoreactivity somatomedin C tracer, we compared the performance of radioimmunoassays in which we used the Tr4 antiserum distributed by the National Pituitary Agency (NPA) with that of the human placental-membrane somatomedin radioreceptor asay (RRA). In their cross reactivity towards various somatomedin-like and unrelated peptides, the two radioimmunoassay methods were almost identical, although NPA antiserum, with about fourfold higher titer than Tr4 antiserum, showed a slightly greater sensitivity for most peptides tested. Radioimmunoassay of acid-ethanol-extracted plasma samples from normal persons and acromegalic, hypopituitary, hypothyroid, and renal-failure patients revealed no analytical differences between the antisera (for 122 samples, r = 0.979 between methods). Somatomedin values for acromegalic and hypopituitary samples showed no overlap with normals. Values for hypothyroid and pre-dialysis renal-failure samples were significantly lower than normal. By comparison, the RRA showed greater cross reactivity towards some somatomedin-like peptides and gave significantly lower values than radioimmunoassay for acromegalic and hypothyroid plasma extracts, and significantly higher values for hypopituitary and renal-failure samples. We conclude that the radioimmunoassay methods clearly are of greater diagnostic value than RRA for clinical somatomedin measurement.

  13. Detection and localisation of picorna-like virus particles in tissues of Varroa destructor, an ectoparasite of the honey bee, Apis mellifera

    NARCIS (Netherlands)

    Zhang, Q.; Ongus, J.R.; Boot, W.J.; Calis, J.; Bonmatin, J.M.; Bengsch, E.; Peters, D.


    Virus-like particles, 27 nm in diameter, were observed in extracts of individual Varroa destructor mites and in sections of mite tissue. Application of a purification procedure resulted in virus preparations that were used to prepare an antiserum to detect the virus in individual mites. Immunohistol

  14. Isolation and characterization of human rhinovirus antigenic variants

    Energy Technology Data Exchange (ETDEWEB)

    Watson, D.G.


    Isolation of antigenic variants of human rhinovirus types 2, 14, and 17 was attempted by plaquing untreated virus (P-isolates), selecting variants in the presence of homologous antiserum (C-isolates), and by selecting variants in the presence of antibody following 5-fluorouracil mutagenesis (M-isolates). All viruses were triple-plaque purified and purity neutralization tested prior to isolate selection. Based on a fourfold reduction in neutralizing antibody titer to homologous antiserum, no antigenic variation was found in P-isolates from the three serotypes examined. Antigenic variants of all three serotypes could be isolated by the antiserum selection method (C-isolates). However, antigenic variants of RV17 were isolated at a much higher frequency and showed a larger degree of variation than those of RV2 and RV14. At least two of the variants selected, RV17 (C301) and RV2 (M803), failed to be neutralized by the known 89 rhinovirus antiserum. SDS-polyacrylamide gel electrophoresis of (/sup 35/S) methionine-labelled virion polypeptides revealed that each serotype had a characteristic pattern and that selected RV2 and RV17 isolates had patterns identical to those of the prototype strains. By isoelectric focusing an antigenic variant of RV2 was shown to contain altered virion polypeptides VP1 and VP2 whereas two RV17 antigenic variants demonstrated alterations only in the VP1 polypeptide.

  15. Amidated joining peptide in the human pituitary, gut, adrenal gland and bronchial carcinoids. Immunocytochemical and immunochemical evidence

    DEFF Research Database (Denmark)

    Bjartell, A; Fenger, M; Ekman, R;


    .g., ACTH, beta-endorphin, Pro-tau-MSH, in the pituitary gland and adrenal medulla. The JP-N immunoreactive cells in the adrenal medulla were identified as a subpopulation of adrenaline-producing cells by means of an antiserum against phenylethanolamine N-methyltransferase. In the gut immunoreactive JP...

  16. Ontogeny and localization of γ-crystallin antigen in the developing pigeon (Columba livia) lens

    NARCIS (Netherlands)

    Brahma, S.K.; Rabaey, M.; Doorenmaalen, W.J. van


    Ontogeny and localization of the lens γ-crystallin antigen were investigated in the embryonic and post-embryonic pigeon lenses by the indirect immunofluorescence with antiserum from rabbit immunized with isolated pigeon lens γ-crystallin. The results show that γ-crystallin appears for the first time

  17. Towards molecular detection methods for aphid-borne strawberry viruses

    NARCIS (Netherlands)

    Schoen, C.D.; Leone, G.


    Intact rhabdovirus particles of strawberry crinkle virus (SCV) mechanically transmitted from Fragaria vesca UC-S to Physalis pubescens plants were purified. When these particles were used as an immunogen, it was not possible to obtain either a virus-specific polyclonal antiserum after rabbit immuniz

  18. Tissue-type plasminogen activator in somatostatin cells of rat pancreas and hypothalamus

    DEFF Research Database (Denmark)

    Kristensen, P; Larsson, L I; Danø, K;


    -PA, and immunoblotting analysis demonstrated one band with a similar electrophoretic mobility. No urokinase-type PA immunoreactivity was found in the rat endocrine pancreas. A granular t-PA immunoreactivity resembling that found in adjacent sections with somatostatin antiserum was found in the median eminence...

  19. Locus-specific detection of HLA-DQ and -DR antigens by antibodies against synthetic N-terminal octapeptides of the beta chain

    DEFF Research Database (Denmark)

    Deufel, T; Grove, A; Kofod, Hans


    detected a 29 kDa component in immunoblots of Raji and AL-34 cell plasma membrane proteins separated by SDS gel electrophoresis. The binding of either N-terminal peptide antiserum was selectively inhibited only by the peptide used as antigen. Indirect immunofluorescence analysis by flow cytofluorometry...

  20. Specific, sensitive, precise, and rapid functional chromogenic assay of activated first complement component (C1) in plasma

    DEFF Research Database (Denmark)

    Munkvad, S; Jespersen, J; Sidelmann, Johannes Jakobsen;


    by using an amidolytic rate assay with a chromogenic substrate. We have optimized the assay conditions with respect to incubation time, concentration of antiserum to C1-inh, ionic strength, and pH. Our method determines specifically the concentration in plasma of free activated C1, not complexes...

  1. Chronic leptin infusion advances, and immunoneutralization of leptin postpones puberty onset in normally fed and feed restricted female rats

    NARCIS (Netherlands)

    Zeinoaldini, S.; Swarts, J.J.M.; Heijning, van de B.J.M.


    Does leptin play a vital role in initiating puberty in female rats and can it overrule a nutrionally imposed (i.e. a 30% feed restriction, FR) delay in puberty onset? Prepubertal female rats were chronically infused for 14 days with leptin (icv or sc) or leptin-antiserum (icv) while puberty onset wa

  2. Genetic Transformation of Citrus Paradisi with Antisense and untranslatable RNA-dependent RNA Polymerase Genes of Citrus Tristeza Closterovirus (United States)

    Expression of the RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) was studied in vivo and in vitro using a polyclonal antiserum raised against the recombinant CTV-RdRp protein. Although 56 kDa CTV-RdRp is thought to be expressed by a +1 translational frameshift at the carboxyl te...

  3. Expression of the recA gene of Pseudomonas aeruginosa PAO is inducible by DNA-damaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Miller, R.V.; Kokjohn, T.A.


    Western (immunoblot) analysis using Escherichia coli anti-RecA antiserum revealed that expression of the RecA protein of Pseudomonas aeruginosa PAO is induced upon exposure of the bacterium to UV irradiation or norfloxacin, a quinolone related to nalidixic acid.

  4. Effect of baculovirus infection on the mRNA and protein levels of the Spodoptera frugiperda eukaryotic initiation factor 4E

    NARCIS (Netherlands)

    Oers, van M.M.; Veken, van der L.T.J.N.; Vlak, J.M.; Thomas, A.A.M.


    The cDNA sequence of eukaryotic translation initiation factor eIF4E was derived from a Spodoptera frugiperda cDNA library. Eight tryptophan residues, typical for eIF4E, are strictly conserved in the encoded 210 amino acid protein. A polyclonal antiserum detected a 26 kDa protein in lepidopteran cell

  5. Fibronectin in cultured rat keratinocytes: distribution, synthesis, and relationship to cytoskeletal proteins

    DEFF Research Database (Denmark)

    Gibson, W T; Couchman, J R; Badley, R A;


    immunofluorescence staining of cultures grown in the absence of a feeder layer using an antiserum which had been cross-adsorbed with foetal calf serum proteins to remove antibodies which recognised serum fibronectin. The distribution of fibronectin in areas of cell-cell and cell-substratum contact...

  6. On the chemical characterization of colloid cyst contents

    NARCIS (Netherlands)

    Veerman, ECI; Go, KG; Molenaar, WM; Amerongen, AVN; Vissink, A


    Colloid cysts of the third ventricle have been investigated by chemical characterization of the cyst contents using ELISA with monoclonal antibodies for certain carbohydrate epitopes as well as a polyclonal antiserum against peptide domains, and immunohistochemistry on the cyst wall using the same a

  7. Subcellular localization of casein kinase I

    DEFF Research Database (Denmark)

    Grankowski, N; Issinger, O G


    An anti-yeast CKI antiserum was shown to cross-react with CKI isolated from Krebs II mouse ascites tumour cells. The mammalian CKI showed virtually the same molecular mass (app. 45 kDa) as the yeast enzyme. By immunofluorescence it could be shown that CKI is preferably located in the nucleolus....

  8. Discovery of Human IgGs against α-Cobratoxin for Development of Recombinant Antibody-based Antivenom

    DEFF Research Database (Denmark)

    Laustsen, Andreas Hougaard; Engmark, Mikael; Redsted Rasmussen, Arne

    , in which large mammals (typically horses) are immunized with snake venom and antiserum is derived from the animals blood. The incompatibility with the human immune system of these animal derived antivenoms leads to a range of side effects,such as serum sickness, anaphylaxis, and sometimes even death...

  9. Recombinant antivenoms based on mixtures of human antibodies against D. jamesoni toxins

    DEFF Research Database (Denmark)

    Pus, Urska; Harrison, Robert; Andersen, Mikael Rørdam

    Each year, more than 5 million people worldwide are affected by a snakebite, resulting in 150,000 deaths, and 400,000 amputations. The current medical treatment against envenoming is based on the administration of an animal-derived antiserum, containing antibodies against snake venom toxins. Due...

  10. Gemella bergeriae endocarditis in a boy. (United States)

    Logan, Latania K; Zheng, Xiaotian; Shulman, Stanford T


    We describe the first pediatric case of Gemella bergeriae endocarditis in a 15-year-old boy with tetralogy of Fallot and pulmonary atresia who presented with weight loss, chills, and cold intolerance. Blood cultures revealed Gram-positive cocci that failed to type with Lancefield group antiserum. The identification of the organism was confirmed by 16S rRNA gene sequencing.

  11. A New Hapten for Immunoassay of Aldicarb

    Institute of Scientific and Technical Information of China (English)

    Yan Feng ZHANG; Zhi Xian GAO; Qing Min ZHANG; Shu Gui DAI


    A new hapten, aldicarb oxime succinic ester (AOSE), was synthesized for immunoassay of aldicarb. It was conjugated to proteins by active ester method. Polyclonal antibody was raised against AOSE-BSA (bovine serum albumin) conjugate. Enzyme-linked immunosorbent assays (ELISAs) showed that this antiserum had high affinity to aldicarb and can be used for sensitive and selective immunoassay of aldicarb.

  12. Denmark: botulism in an infant or infant botulism?

    DEFF Research Database (Denmark)

    Paerregaard, A; Angen, O; Lisby, M;


    was noted. Botulism was suspected and confirmed by testing of patient serum in a bioassay. The condition of the patient improved following administration of botulism antiserum. The clinical picture was suggestive of intestinal (infant) botulism. However, botulism acquired from consumption of food...

  13. 9 CFR 130.14 - User fees for FADDL veterinary diagnostics. (United States)


    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false User fees for FADDL veterinary..., DEPARTMENT OF AGRICULTURE USER FEES USER FEES § 130.14 User fees for FADDL veterinary diagnostics. (a... 167.00 Rabbit antiserum, any agent 1 mL 179.00 185.00 190.00 196.00 (b) Veterinary diagnostics...

  14. Identification of Pro-Differentiation p53 Target Genes and Evaluation of Expression in Normal and Malignant Mammary Gland (United States)


    specific sequence in exon3’ of p63 gene downstream of △N promoter was used as antigen to immunize rabbit host. The antiserum was extracted from serum evaluate the prognostic significance of nestin. On the other hand, the reponse of nestin transcript to retinoic acid treatment is different from

  15. Avian Follicular and Interdigitating Dendritic Cells: Isolation and Morphologic, Phenotypic, and Functional Analyses (United States)

    An antiserum against Eimeria tenella sporozoites was used to localize and isolate Ag-binding cells in intestinal cecal tonsils of parasite-infected chickens. Based on their tissue localization, ultrastructural features, and expression of surface markers, two subpopulations of cells were isolated, C...

  16. Immunocytochemical localization of glutamic acid decarboxylase (GAD) and glutamine synthetase (GS) in the area postrema of the cat. Light and electron microscopy (United States)

    D'Amelio, Fernando E.; Mehler, William R.; Gibbs, Michael A.; Eng, Lawrence F.; Wu, Jang-Yen


    Morphological evidence is presented of the existence of the putative neurotransmitter gamma-aminobutyric acid (GABA) in axon terminals and of glutamine synthetase (GS) in ependymoglial cells and astroglial components of the area postrema (AP) of the cat. Purified antiserum directed against the GABA biosynthetic enzyme glutamic acid decarboxylase (GAD) and GS antiserum were used. The results showed that punctate structures of variable size corresponding to axon terminals exhibited GAD-immunoreactivity and were distributed in varying densities. The greatest accumulation occurred in the caudal and middle segment of the AP and particularly in the area subpostrema, where the aggregation of terminals was extremely dense. The presence of both GAD-immunoreactive profiles and GS-immunostained ependymoglial cells and astrocytes in the AP provide further evidence of the functional correlation between the two enzymes.

  17. Poliovirus RNA synthesis in vitro: structural elements and antibody inhibition

    Energy Technology Data Exchange (ETDEWEB)

    Semler, B.L.; Hanecak, R.; Dorner, L.F.; Anderson, C.W.; Wimmer, E.


    The poliovirus RNA polymerase complex has been analyzed by immunoautoradiography using antibody probes derived from purified replicase (P3) region viral polypeptides. Antibody preparations made against the polio RNA polymerase, P3-4b, detected a previously unreported cellular protein that copurifies with the RNA polymerase. An IgG fraction purified from rabbit antiserum to polypeptide P3-2, a precursor fo the RNA polymerase, specifically inhibits poliovirus RNA synthesis in vitro. The authors have also immunoprecipitated a 60,000-dalton protein (P3-4a) with antiserum to protein P3-4b and have determined the precise genomic map position of this protein by automated Edman degradation. Protein P3-4a originates by cleavage of the RNA polymerase precursor at a glutamine-glucine amino acid pair not previously reported to be a viral cleavage site.

  18. Methadone radioimmunoassay: two simple methods

    Energy Technology Data Exchange (ETDEWEB)

    Robinson, K.; Smith, R.N. (Metropolitan Police Forensic Science Laboratory, London (UK))


    Two simple and economical radioimmunoassays for methadone in blood or urine are described. Haemolysis, decomposition, common anticoagulants and sodium fluoride do not affect the results. One assay used commercially-available (1-/sup 3/H)(-)-methadone hydrobromide as the label, while the other uses a radioiodinated conjugate of 4-dimethylamino-2,2-diphenylpentanoic acid and L-tyrosine methyl ester. A commercially-available antiserum is used in both assays. Normethadone and ..cap alpha..-methadol cross-react to a small extent with the antiserum while methadone metabolites, dextropropoxyphene, dipipanone and phenadoxone have negligible cross-reactivities. The 'cut-offs' of the two assays as described are 30 and 33 ng ml/sup -1/ for blood, and 24 and 21 ng ml/sup -1/ for urine. The assay using the radioiodinated conjugate can be made more sensitive if required by increasing the specific activity of the label.

  19. [A new virus of rabbit. III. Study on morphological superstructure and antigenicity of rabbit hemorrhagic disease virus (RHDV)]. (United States)

    Zhao, L; Li, T; Song, B; Sun, F


    In the spring 1986, an acute infectious disease occurred in Wuhan Second Producing Medical Manufactory, and the rabbit almost died. We tested the mortal symptom and confirmed rabbit Hemorrhagic Disease (RHD) as same as Huang Yinyao report. Hubei Traditional Chinese Medicine Institute appear this RHD also. After we purified virus of above two source by low speed, high speed and sucrose density gradient centrifugation, they can react with antiserum of RHDV from Nanjing Agricultural University in agar gel immunodiffusion tests. These results proved that they belong to the same serotype. Data indicate RHDV have difference morphological superstructure, viral polypeptides and especially RHDV can't react with antiserum of standard Parvovirus of rabbit and so on, so we suggest RHDV is a new virus.

  20. Radioimmunoassay analysis of baculovirus granulins and polyhedrins

    Energy Technology Data Exchange (ETDEWEB)

    Summers, M.D.; Hoops, P.


    Granulin and polyhedrin proteins were purified by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis from the baculoviruses Autographa californica, Rachiplusia ou, Heliothis zea, Heliothis armigera. Trichoplusia ni, and Spodoptera frugiperda. Antisera were raised against Autographa californica (Ac) polyhedrin and Trichoplusia ni (Tn) granulin and analyzed for homologous and heterologous immunoreactivity by immunodiffusion and radioimmunoassay (RIA). Ac polyhedrin and Tn granulin antisera recognized antigenic determinants on several baculovirus polyhedrin and granulin proteins even though the heterologous proteins had different immunoreactivities when compared by competition radioimmunoassay. Antigenic differences among granulin and polyhedrin proteins were also detected by altered slopes of the competition reaction curves. Antiserum raised against Ac polyhedrin which was purified in the presence of SDS was tested by competition RIA for its ability to detect and react with native polyhedrin produced in the infected TN-368 cells. Ac polyhedrin antiserum had similar if not identical ability to bind to native polyhedrin and to polyhedrin purified in the presence of SDS.

  1. Genetic Analysis of the P1 Region of Human Enterovirus 71 Strains and Expression of the 55 F StrainVP1 Protein

    Institute of Scientific and Technical Information of China (English)

    Jian-qiang Li; Jun-jie Yang; Xiu-juan Fan; Zhen-peng Sun; Yan Sun; Huan Li; Zi-xin Meng; Wei Li


    Enterovirus 71 (EV71) is a member of the Entero-virus genus of the Picornaviridae family and is the major cause of Hand,foot,and mouth disease (HFMD) in children.Different strains from Gansu were cloned and the P1 protein was sequenced and analysed.Results indicate that there are three kinds of EV71 infections prevalent in Gansu.The VP1 protein from one of these strains,55F,was expressed.The recombinant protein was expressed with high level and reacted specifically with the EV71 patient antibody,the recombinant protein was also applied to raise antiserum in rabbits and after the fourth injection a high titer of antiserum was detected by ELISA assay.These data are useful for further clarification of prevalent EV71 strains in the north of China at the molecular level and provide a basis for EV71 diagnosis.

  2. Active and passive immunization with Pseudomonas aeruginosa ribosomal vaccines and antisera in the burned rat model. (United States)

    Lieberman, M M; Walker, H L; Ayala, E; Chapa, I


    Pseudomonas aeruginosa ribosomal vaccines were tested for their ability to protect rats subjected to a 20% total body surface burn against the lethal effects of infection with homologous organisms. When administered prior to burning, the vaccines provided 100% protection. When administered postburning, the vaccine from one strain also provided 100% protection when the time interval between vaccination and infection was 3 days. When this time interval was reduced to 1 or 2 days, approximately 50% protection was obtained with the same vaccine. The vaccine from a second strain tested provided about 50% protection with a 3-day time interval. In addition, passive immunization using antiserum to a ribosomal vaccine was also demonstrated to be effective in protecting burned and infected rats, especially when multiple doses of antiserum were used. In this case, 80% protection was obtained (with no protection observed using multiple doses of normal serum). Finally, a comparison of ribosomal and lipopolysaccharide vaccines and antisera was also performed.

  3. Amino acid residues 56 to 69 of HLA-A2 specify an antigenic determinant shared by HLA-A2 and HLA-B17. (United States)

    Ways, J P; Rothbard, J B; Parham, P


    The mouse monoclonal antibody MA2.1 was previously used to define an epitope shared by native HLA-A2 and HLA-B17 molecules and amino acid sequence comparison of nine HLA-A,B,C molecules identified residues 62 to 65 as the region most likely to form this epitope. An unabsorbed rabbit antiserum raised against a peptide corresponding to residues 56 to 69 of HLA-A2 gives highly specific reactions with HLA-A2 and HLA-B17 heavy chains in Western blots. No interactions with native HLA-A2 and B17 molecules were detected in a variety of assays. Although the topographic relationship between the epitopes recognized by the rabbit antiserum and the monoclonal antibody could not be determined, the results show that residues 56 to 69 of HLA-A2 can form epitopes with specificity for HLA-A2 and HLA-B17.

  4. Cloning,sequencing and prokaryotic expression of cDNAs for antifreeze protein family from Beetle Tenebrio molitor

    Institute of Scientific and Technical Information of China (English)

    Zhongyuan LIU; Yun WANG; Guodong LU; Xianlei WANG; Fuchun ZHANG; Ji MA


    Partial cDNA sequences coding for antifreeze proteins in Tenebrio molitor were obtained by RT-PCR.Sequence analysis revealed nine putative cDNAs with a high degree of homology to Tenebrio molitor antifreeze protein genes published in GenBank.The recombinant pGEX-4T-l-tmafp-XJ430 was introduced into E.coli BL21 to induce a GST fusion protein by IPTG.SDS-PAGE analysis for the fusion protein shows a band of 38 kDa.pCDNA3- tmafp-XJ430 was injected into mice to generate antiserum which was later detected by indirect ELISA.The titer of the antibody was 1:2000.Western blot-ting analysis shows that the antiserum was specifically against the antifreeze protein.Our results laid the founda-tion for further studies on the properties and functions of insect antifreeze proteins.

  5. The effects of nonidet P40 on the function of rat peritoneal mast cells in vitro. (United States)

    Batchelor, K W; Stanworth, D R


    1 Treatment of purified rat peritoneal mast cells at 37 degrees C with concentrations of the non-ionic detergent nonidet P40 (NP40) up to 0.005% (v/v) failed to reduce their viability. 2 There was a marked reduction in the histamine releasing capacity of NP40-treated mast cells upon challenge with a variety of selective (adrenocorticotrophic hormone 1-24 (Synacthen), rabbit anti-rat IgE antiserum, adenosine triphosphate (ATP) and the calcium ionophore, A 23187) and non-selective (rabbit anti-rat mast cell antiserum plus complement) histamine liberators. 3 Nonidet P40 (0.005%) was found to reduce the activity of a mast cell membrane 'ecto-enzyme', calcium-activated ATPase, by about 45% when presented at the time of its assay.

  6. Basement membrane changes in breast cancer detected by immunohistochemical staining for laminin

    DEFF Research Database (Denmark)

    Albrechtsen, R; Nielsen, M; Wewer, U


    The distribution of the basement membrane glycoprotein laminin was studied by the immunoperoxidase technique in benign and malignant human breast tissue and in axillary lymph nodes from patients with breast cancer. An antiserum prepared against rat laminin was used. The specificity of this antise......The distribution of the basement membrane glycoprotein laminin was studied by the immunoperoxidase technique in benign and malignant human breast tissue and in axillary lymph nodes from patients with breast cancer. An antiserum prepared against rat laminin was used. The specificity...... by laminin staining, but they were thinner and discontinuous. The poorly differentiated carcinomas lacked organized basement membranes detectable by laminin staining. Our studies suggest that staining for laminin may be a useful adjunct test for detection of micrometatases in lymph nodes. The correlation...

  7. Adsorption of antibody and globulin onto glass surfaces. (United States)

    Mizutani, T


    The amount of globulin adsorbed onto surfaces (97 m2) of porous glass (1 g) in phospate-buffered saline (pH 7.2) was estimated to be 83 mg by frontal analysis. In the adsorption chromatography of rabbit antiserum (the immunoglobulin G class) to horse serum albumin on a porous glass column, immunoglobulin G was not eluted with saline but was eluted with 0.2 M glycine (pH 9) with a recovery of 12%. The yield of immunoglobulin M antibody to sheep red blood cells recovered by elution with saline was 12.3%, and the total yield of immunoglobulin M was 15.8%. Thus, antibody and globulin were well adsorbed onto glass surfaces in physiological saline; immunoglobulin G had a stronger affinity to glass surfaces than did immunglobulin M. These facts should be considered when glass containers are used for purified antiserum.

  8. Improved method to raise polyclonal antibody using enhanced green fluorescent protein transgenic mice

    Institute of Scientific and Technical Information of China (English)

    Jianke Ren; Long Wang; Guoxiang Liu; Wen Zhang; Zhejin Sheng; Zhugang Wang; Jian Fei


    Recombinant fusion protein is widely used as an antigen to raise antibodies against the epitope of a target protein. However, the concomitant anticarrier antibody in resulting antiserum reduces the production of the desired antibody and brings about unwanted non-specific immune reactions. It is proposed that the carrier protein transgenic animal could be used to solve this problem. To validate this hypothesis, enhanced green fluorescent protein (EGFP) transgenic mice were produced. By immunizing the mice with fusion protein His6HAtag-EGFP, we showed that the antiserum from the transgenic mice had higher titer antibody against His6HA tag and lower titer antibody against EGFP compared with that from wild-type mice. Therefore, this report describes an improved method to raise high titer antipeptide polyclonal antibody using EGFP transgenic mice that could have application potential in antibodypreparation.

  9. Identification of a protein associated with circulative transmission of Barley yellow dwarf virus from cereal aphids, Schizaphis graminum and Sitobion avenae

    Institute of Scientific and Technical Information of China (English)

    WANG Xifeng; ZHOU Guanghe


    Using 2-D electrophoresis and virus overlay assay, a 50-kDa protein (P50) exhibiting specific binding to purified virus particles of BYDV-GAV was found in the protein extracts from Schizaphis graminum and Sitobion avenae,two aphid species transmitting BYDV-GAV. P50 in the extracts of S. graminum was isolated by preparation electrophoresis and electro-eluted proteins from the gel slices for antiserum preparation. After feeding the antiserum through membrane, the transmission efficiencies of S. graminun and S. avenae for BYDV-GAV decreased significantly. It was suggested that P50 should be related with transmission process. Location of P50 was found at the plasma membrane surrounding the accessory salivary gland (ASG) in the head tissues of S. graminum by immunogold-labelling experiment.The ascertainment of the protein associated with virus transmission has a significance influence on further understanding the transmission mechanism and genetic engineering for resistant to vector transmission.

  10. Thiobacillus ferrooxidans detection using immunoelectron microscopy. (United States)

    Coto, O; Fernández, A I; León, T; Rodríguez, D


    A specific, fast and very sensitive immunoelectron microscopy method was developed to morphologically and serologically distinguish different cultures of iron oxidizers. Bacteria isolated from the acidic waters of "Matahambre" and "Mina Delita" mines (Cuba) were characterized. An antiserum specific to Thiobacillus ferrooxidans did not react with other bacteria also present in the acidic waters of mine drainage. Our results suggest the occurrence of some strains of Thiobacillus ferrooxidans, Thiobacillus thiooxidans and Leptospirillum ferrooxidans in these waters.

  11. Determination of the genus-specific antigens in outer membrane proteins from the strains of Leptospira interrogans and Leptospira biflexa with different virulence

    Institute of Scientific and Technical Information of China (English)

    罗依惠; 严杰; 毛亚飞; 李淑萍


    Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.

  12. Evaluation of anti-pneumococcal capsular antibodies as adjunctive therapy in experimental pneumococcal meningitis

    DEFF Research Database (Denmark)

    Brandt, Christian; Frimodt-Moller, N; Lundgren, Jens Dilling;


    OBJECTIVE: Bacteraemia concomitant with meningitis has been shown to greatly affect outcome. Consequently, the efficacy of serotype-specific anti-pneumococcal antiserum (APAS) was investigated in a rat model of pneumococcal meningitis. METHODS: Rats were infected with Streptococcus pneumoniae...... at the time of infection whereas no effect was found when administered 26 h after infection. This work indicates that the clinical value of using APAS in pneumococcal meningitis may be limited...

  13. Purification in a functional form of the terminal protein of Bacillus subtilis phage phi 29.



    Phage phi 29 terminal protein, p3, essentially pure, was isolated in a denatured form from viral particles, and anti-p3 antiserum was obtained. A radioimmunoassay to detect and quantitate protein p3 was developed. By using this assay, native protein p3 was highly purified from Escherichia coli cells harboring a gene 3-containing recombinant plasmid. After three purification steps, the protein was more than 96% pure; its amino acid composition was very similar to that deduced from the nucleoti...

  14. Purification in a functional form of the terminal protein of Bacillus subtilis phage ø29



    Phage phi 29 terminal protein, p3, essentially pure, was isolated in a denatured form from viral particles, and anti-p3 antiserum was obtained. A radioimmunoassay to detect and quantitate protein p3 was developed. By using this assay, native protein p3 was highly purified from Escherichia coli cells harboring a gene 3-containing recombinant plasmid. After three purification steps, the protein was more than 96% pure; its amino acid composition was very similar to that deduced from the nucleoti...

  15. Antibodies against a synthetic peptide identify the Epstein-Barr virus-determined nuclear antigen.



    Five peptides corresponding to amino acid sequences predicted from all three reading frames of the nucleotide sequence of the third internal repeat array (IR3) of the Epstein-Barr virus (EBV) genome were synthesized chemically. All five peptides elicited antipeptide antibodies in rabbits. The antiserum raised against a 14-residue copolymer of glycine and alanine gave brilliant EBV-specific nuclear staining in the anticomplement immunofluorescence (ACIF) assay, in line with the original defini...

  16. Trivalent M-related protein as a component of next generation group A streptococcal vaccines (United States)


    Purpose There is a need to broaden protective coverage of M protein–based vaccines against group A streptococci (GAS) because coverage of the current 30-valent M protein vaccine does not extend to all emm types. An additional GAS antigen and virulence factor that could potentially extend vaccine coverage is M-related protein (Mrp). Previous work indicated that there are three structurally related families of Mrp (MrpI, MrpII, and MrpIII) and peptides of all three elicited bactericidal antibodies against multiple emm types. The purpose of this study was to determine if a recombinant form containing Mrp from the three families would evoke bactericidal antiserum and to determine if this antiserum could enhance the effectiveness of antisera to the 30-valent M protein vaccine. Materials and Methods A trivalent recombinant Mrp (trMrp) protein containing N-terminal fragments from the three families (trMrp) was constructed, purified and used to immunize rabbits. Anti-trMrp sera contained high titers of antibodies against the trMrp immunogen and recombinant forms representing MrpI, MrpII, and MrpIII. Results The antisera opsonized emm types of GAS representing each Mrp family and also opsonized emm types not covered by the 30-valent M protein–based vaccine. Importantly, a combination of trMrp and 30-valent M protein antiserum resulted in higher levels of opsonization of GAS than either antiserum alone. Conclusion These findings suggest that trMrp may be an effective addition to future constructs of GAS vaccines. PMID:28168173

  17. Muscle fibre types in the external eye muscles of the pigeon, Columba livia.


    McVean, A; Stelling, J; Rowlerson, A.


    Fibre typing with antisera raised against specific myosin types from muscles of known physiological properties were used to characterise the fibre types within the oculorotatory muscles of pigeons. Fibres reacting strongly to antiserum anti-ALD (specific for tonic fibre myosin) were found lying along the global margin of the muscle and also in a layer lying immediately beneath a discrete band of fibres running along the orbital margin. These fibres resembled those of the skeletal muscle ALD i...

  18. Expression of GPR177 (Wntless/Evi/Sprinter), a Highly Conserved Wnt-Transport Protein, in Rat Tissues, Zebrafish Embryos, and Cultured Human Cells


    Jin, Jay; Morse, Megan; Frey, Colleen; Petko, Jessica; Levenson, Robert


    GPR177 is an evolutionarily conserved transmembrane protein necessary for Wnt protein secretion. Little is currently known, however, regarding expression of GPR177, especially in vertebrate species. We have developed an antiserum against GPR177, and used it to examine expression of GPR177 in human tissue culture cells, adult mouse and rat tissues, as well as developing zebrafish embryos. In rodents, GPR177 is expressed in virtually all tissue types and brain regions examined. In zebrafish, GP...

  19. Loss of albumin and megalin binding to renal cubilin in rats results in albuminuria after total body irradiation. (United States)

    Yammani, Raghunatha R; Sharma, Mukut; Seetharam, Shakuntla; Moulder, John E; Dahms, Nancy M; Seetharam, Bellur


    The role of the renal apical brush-border membrane (BBM) endocytic receptors cubilin and megalin in the onset of albuminuria in rats exposed to a single dose of total body irradiation (TBI) has been investigated. Albuminuria was evident as immunoblot (IB) analysis of the urine samples from TBI rats revealed excretion of large amounts of albumin. IB analysis of the BBM proteins did not reveal any significant changes in cubilin or megalin levels, but (125)I-albumin binding to BBM from TBI rats declined by 80% with a fivefold decrease (from 0.5 to 2.5 microM) in the affinity for albumin. IB analysis of cubilin from the BBM demonstrated a 75% loss when purified using albumin, but not intrinsic factor (IF)-cobalamin (Cbl) ligand affinity chromatography. Immunoprecipitation (IP) of Triton X-100 extract of the BBM with antiserum to cubilin followed by IB of the immune complex with an antiserum to megalin revealed a 75% loss of association between megalin and cubilin. IP studies with antiserum to cubilin or megalin and IB with antiserum to the cation-independent mannose 6-phosphate/insulin-like growth factor II-receptor (CIMPR) revealed that CIMPR interacted with both cubilin and megalin. In addition, TBI did not disrupt the association of CIMPR with either cubilin or megalin in BBM. These results suggest that albuminuria noted in TBI rats is due to selective loss of albumin and megalin, but not CIMPR or IF-Cbl binding by cubilin. Furthermore, these results also suggest that albumin and IF-Cbl binding to cubilin occur at distinct sites and that in the rat renal BBM, CIMPR interacts with both cubilin and megalin.

  20. Prolactin-like activity of anti-prolactin receptor antibodies on casein and DNA synthesis in the mammary gland.


    Djiane, J; Houdebine, L M; Kelly, P A


    Prolactin receptors were partially purified from rabbit mammary gland membranes by using an affinity chromatography technique. Antibodies against this prolactin receptor preparation were obtained in guinea pig and sheep. Both antisera were able to inhibit the binding of 125I-labeled ovine prolactin to rabbit mammary gland membranes. When added to culture media of rabbit mammary explants, the anti-prolactin receptor antiserum inhibited the capacity of prolactin to initiate casein synthesis and...

  1. Determination of the genus-specific antigens in outer membrane proteins from the strains of Leptospira interrogans and Leptospira biflexa with different virulence

    Institute of Scientific and Technical Information of China (English)

    罗依惠; 严杰; 毛亚飞; 李淑萍


    Objective: To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups. The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence, and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972). OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results: All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa. A positive protein fragment with approximately 32 KDa confirmed by Western blot, was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira. Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.

  2. Expression of the glycoprotein gene from a fish rhabdovirus by using baculovirus vectors

    Energy Technology Data Exchange (ETDEWEB)

    Koener, J.F.; Leong, J.A.C. (Oregon State Univ., Corvallis (United States))


    A cDNA fragment containing the gene encoding the glycoprotein of infectious hematopoietic necrosis virus was inserted into Autographa californica baculovirus vectors under the control of the polyhedrin promoter. A 66-kilodalton protein, identical in size to the glycosylated glycoprotein of infectious hematopoietic necrosis virus, was expressed at high levels in Spodoptera frugiperda cells infected with the recombinant viruses. The expressed protein reacted with antiserum to the glycoprotein on Western blots.

  3. [Infant botulism]. (United States)

    Falk, Absalom; Afriat, Amichay; Hubary, Yechiel; Herzog, Lior; Eisenkraft, Arik


    Infant botulism is a paralytic syndrome which manifests as a result of ingesting spores of the toxin secreting bacterium Clostridium botulinum by infants. As opposed to botulism in adults, treating infant botulism with horse antiserum was not approved due to several safety issues. This restriction has led to the development of Human Botulism Immune Globulin Intravenous (BIG-IV; sells under BabyBIG). In this article we review infant botulism and the advantages of treating it with BIG-IV.

  4. Strain Identification of Burkholderia cepacia Palleroni and Holmes and Pseudomonas fluorescens Migula Associated to Maize Crop by Polyphasic Taxonomy


    Annia Hernández Rodríguez; María Esther González Vega; Alberto Caballero Núñez; Ariel Medina Concepción; Madelaine Quiñónez Pantoja; Mayra Heydrich Pérez; Ana Niurka Hernández Lauzardo; Monica Höfte


    A polyphasic taxonomic study, which included phenotypic characterization, the indirect immunofluorescence (IIF), and the polymerase chain reaction (PCR) was conducted for the identification of Burkholderia cepacia and Pseudomonas fluorescens strains previously isolated from maize rhizosphere. Conventional methods were used (API 20 NE, BioMeuriux), specific hyper-immune antiserum against the species under study, and specific primers were designed out of subunits 16S of the ribosomal RNA of B....

  5. 1株沙门样大肠埃希菌的分离与鉴定%Isolation and identification of one Salmonella-like Escherichia coli isolate

    Institute of Scientific and Technical Information of China (English)

    吴静怡; 刘栓奎; 刘然; 董路宁; 李明; 党荣理


    Objective To identify and characterize one Enterobacteriaceae isolate which could cross-agglutinate with Salmonella diagnostic anti-serum. Methods Phenotype and genotype of the isolate were determined by culture, metabolic assay, agglutination reaction, and 16S rRNA sequence phylogenetic analysis, respectively. Results The isolate was consistent with Escherichia coli on culture and biochemical features, but could cross-agglutinate with multiple types or group-specific Salmonella anti-serum rather than Escherichia coli anti-serum. 16S rRNA sequence analysis revealed that the isolate was identical with Escherichia coli, but was obviously different from Salmonella spp. Conclusion One Escherichia coli isolate which can cross-agglutinate with Salmonella diagnostic anti-serum has been obtained in this study.%目的 对1株与沙门菌抗血清发生交叉凝集的肠道菌分离株进行鉴定.方法 分别通过生化培养、抗血清凝集试验与16S rRNA序列比对分析,对该菌株进行鉴定.结果 该菌株的分离培养、生化代谢特征均与大肠埃希菌一致,但能与沙门菌属多型、群特异性抗血清发生凝集,与大肠埃希菌抗血清无凝集反应.序列分析结果表明,该菌株16S rRNA与大肠埃希菌一致,与沙门菌属存在明显差异.结论 本实验分离获得1株具有沙门菌血清凝集特征的大肠埃希菌.

  6. [Comparative immunochemical study of the serum proteins of Lacertilia (Reptilia)]. (United States)

    Blanc, F


    The immunochemical relationships of the serum proteins of Uromastix acanthinurus, Agama mutabilis, Oplurus cuvieri, O. quadrimaculatus, Chamaeleo chamaeleon, Chalcides ocellatus and Lacerta lepida were studied by means of immunoelectrophoresis with an antiserum anti-Uromastix successively absorbed. Homogeneity of the Iguania species was pointed out and they were found more closely related to the Scincid Chalcides ocellatus than to the Lacertid Lacerta lepida. Within the Iguania, the two Agamids are immunologically more closely related to the Chamaeleon than to the two Iguanids.

  7. A specific immunoassay for the determination of morphine and its glucuronides in human blood. (United States)

    Beike, J; Blaschke, G; Mertz, A; Köhler, H; Brinkmann, B


    The development of specific antisera for immunochemical determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide is described. Morphine was N-demethylated to normorphine and N-alkylated to give N-aminopropyl-normorphine as hapten for antisera against morphine. As haptens for antisera against morphine-3-glucuronide and morphine-6-glucuronide, N-aminopropyl-nor-morphine was glucuronidated in position 3 or 6 respectively. Each of these three haptens were coupled to BSA employing the glutaraldehyde method to obtain three different immunogens. Immunisation of rabbits with these conjugates gave anti-morphine, anti-morphine-3-glucuronide and anti-morphine-6-glucuronide antisera, which were tested in a competitive, heterogeneous radioimmunoassay. Tracers for this radioimmunoassay procedure were synthesised by substitution of morphine and morphine-6-glucuronide in position 2 with 125I and indirect iodination of the morphine-3-glucuronide hapten according to the method of Bolton and Hunter. The resulting antisera show very specific reactions with morphine, morphine-3-glucuronide and morphine-6-glucuronide. Cross reactivities of each antiserum with structurally related opiates and opioides are very low. The cross reactivities of the anti-morphine antiserum against morphine-3-glucuronide, morphine-6-glucuronide, codeine, codeine-6-glucuronide or dihydrocodeine were less than 0.3%, the anti-morphine-3-glucuronide antiserum against morphine, morphine-6-glucuronide, codeine, codeine-6-glucuronide or dihydrocodeine less than 0.1% and the anti-morphine-6-glucuronide antiserum against morphine, morphine-3-glucuronide, codeine or dihydrocodeine less than 0.1%, against codeine-6-glucuronide less than 2.3%. The determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in blood samples (limit of detection= 3, 1, 0.5 ng/g) of nine cases of fatal heroin overdose with this radioimmunoassay method and the comparison with a GC/MS method is described.

  8. Production of recombinant capsid protein of Macrobrachium rosenbergii nodavirus (r-MCP43) of giant freshwater prawn, M. rosenbergii (de Man) for immunological diagnostic methods. (United States)

    Farook, M A; Madan, N; Taju, G; Majeed, S Abdul; Nambi, K S N; Raj, N Sundar; Vimal, S; Hameed, A S Sahul


    White tail disease (WTD) caused by Macrobrachium rosenbergii nodavirus (MrNV) and extra small virus (XSV) is a serious problem in prawn hatcheries. The gene for capsid protein of MrNV (MCP43) was cloned into pRSET B expression vector. The MCP43 protein was expressed as a protein with a 6-histidine tag in Escherichia coli GJ1158 with NaCl induction. This recombinant protein, which was used to raise the antiserum in rabbits, recognized capsid protein in different WTD-infected post-larvae and adult prawn. Various immunological methods such as Western blot, dot blot and ELISA techniques were employed to detect MrNV in infected samples using the antiserum raised against recombinant MCP43 of MrNV. The dot blot assay using anti-rMCP43 was found to be capable of detecting MrNV in WTD-infected post-larvae as early as at 24 h post-infection. The antiserum raised against r-MCP43 could detect the MrNV in the infected samples at the level of 100 pg of total protein. The capsid protein of MrNV estimated by ELISA using anti-rMCP43 and pure r-MCP43 as a standard was found to increase gradually during the course of infection from 24 h p.i. to moribund stage. The results of immunological diagnostic methods employed in this study were compared with that of RT-PCR to test the efficiency of antiserum raised against r-MCP43 for the detection of MrNV. The Western blot, dot blot and ELISA detected all MrNV-positive coded samples as detected by RT-PCR.

  9. Single-reagent one-step procedures for the purification of ovine IgG, F(ab')2 and Fab antivenoms by caprylic acid. (United States)

    Al-Abdulla, Ibrahim; Casewell, Nicholas R; Landon, John


    Antivenoms are typically produced in horses or sheep and often purified using salt precipitation of immunoglobulins or F(ab')2 fragments. Caprylic (octanoic) acid fractionation of antiserum has the advantage of not precipitating the desired antibodies, thereby avoiding potential degradation that can lead to the formation of aggregates, which may be the cause of some adverse reactions to antivenoms. Here we report that when optimising the purification of immunoglobulins from ovine antiserum raised against snake venom, caprylic acid was found to have no effect on the activity of the enzymes pepsin and papain, which are employed in antivenom manufacturing to digest immunoglobulins to obtain F(ab')2 and Fab fragments, respectively. A "single-reagent" method was developed for the production of F(ab')2 antivenom whereby whole ovine antiserum was mixed with both caprylic acid and pepsin and incubated for 4h at 37°C. For ovine Fab antivenom production from whole antiserum, the "single reagent" comprised of caprylic acid, papain and l-cysteine; after incubation at 37°C for 18-20h, iodoacetamide was added to stop the reaction. Caprylic acid facilitated the precipitation of albumin, resulting in a reduced protein load presented to the digestion enzymes, culminating in substantial reductions in processing time. The ovine IgG, F(ab')2 and Fab products obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step conventional salt fractionation with sodium sulphate.

  10. Health Education Authority's first mass media AIDS campaign. (United States)


    The DNA sequence of the early E3 transcription unit of adenovirus 2 (Ad2) (J. Hérissé et al., Nucleic Acids Res. 8:2173-2192, 1980), indicates that an open reading frame exists between nucleotides 1860 and 2163 that could encode a protein of Mr 11,600 (11.6K). We have determined the DNA sequence of the corresponding region in Ad5 (closely related to Ad2) and have established that this putative gene is conserved in Ad5 (a 10.5K protein). To determine whether this protein is expressed, we prepared an antiserum in rabbits against a synthetic peptide corresponding to amino acids 66 to 74 in the 11.6K protein of Ad2. The peptide antiserum immunoprecipitated a ca. 13K-14K protein doublet, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from [35S]methionine-labeled Ad2- or Ad5-early-infected KB cells. The antiserum also immunoprecipitated a 13K-14K protein doublet translated in vitro from Ad2 or Ad5 early E3-specific mRNA purified by hybridization to Ad2 EcoRI-D (nucleotides -236 to 2437). The synthetic peptide successfully competed with the 13K-14K protein doublet in immunoprecipitation experiments, thereby confirming the specificity of the antiserum. As deduced from the DNA sequence, the 11.6K protein (and the corresponding 10.5K Ad5 protein) has a conserved 22-amino-acid hydrophobic domain, suggesting that the protein may be associated with membranes. We conclude that a gene located at nucleotides 1860 to 2143 in the Ad2 E3 transcription unit (nucleotides 1924 to 2203) in the Ad5 E3 transcription unit) encodes an 11.6K protein (10.5K in Ad5).

  11. A 52 Kilodalton Protein Vaccine Candidate for Francisella tularensis (United States)


    du vaccin vivant F. tularensis (LVS). Soixante pourcent (60%) des souris vaccindes ont survdcu la dose ltale multiple alors que toutes les souris non...le lysat des cellules de cultures vivantes du vaccin vivant F. tularensis. Plusieurs composants de Francisella tularensis ont dt6 identifids par cet...antiserum. Le s6rum de souris provenant de souris vaccin6es avec F. tularensis non- vivant n’a pas identifid ces composants. A partir de ces prot6ines

  12. Presence of Arp Specifically Contributes to Joint Tissue Edema Associated with Early-Onset Lyme Arthritis


    Hove, Petronella R.; Haldorson, Gary J; Magunda, Forgivemore; Bankhead, Troy


    Antiserum to the Borrelia burgdorferi arthritis-related protein, Arp, has been shown to prevent or reduce arthritis in immunodeficient mice. To directly investigate the requirement for this lipoprotein in the generation of Lyme arthritis, we utilized targeted deletion to generate a B. burgdorferi clone that lacked only the arp gene locus. Infection of Lyme disease-susceptible C3H/HeN mice with the arp deletion mutant demonstrated significantly reduced tibiotarsal joint swelling during the fir...

  13. The sessile drop method for immunohistochemical processing of unmounted sections of nervous tissue. (United States)

    Nadelhaft, I


    A novel method for the immunohistochemical processing of free-floating tissue sections is described. Sections are immersed within drops of solution arranged on a hydrophobic surface. The procedure consists of sequentially suctioning away one fluid drop and replacing it by another, while the section remains in place. The technique permits easy testing of different antiserum dilutions, comparisons among different immunohistochemical protocols, and comparison of different antisera on serial tissue sections. Comparison is made to processing mounted sections.

  14. Hyaluronidase, phospholipase A2 and protease inhibitory activity of plants used in traditional treatment of snakebite-induced tissue necrosis in Mali, DR Congo and South Africa

    DEFF Research Database (Denmark)

    Schmidt, Marianne Molander; Nielsen, Line Hagner; Søgaard, Søren Vinter


    ETHNOPHARMACOLOGICAL RELEVANCE: Snakebite envenomation, every year, causes estimated 5-10,000 mortalities and results in more than 5-15,000 amputations in sub-Saharan Africa alone. Antiserum is not easily accessible in these regions or doctors are simply not available, thus more than 80% of all p...... patients seek traditional practitioners as first-choice. Therefore it is important to investigate whether the plants used in traditional medicine systems contain compounds against the necrosis-inducing enzymes of snake venom....

  15. Detection of Bifidobacteriurn longum in faecal samples after orogastric intubation using immunological test systems


    Dreher, R M; Gerhard, J.; Schönborn, W.; Lauer, E.


    A simple method based on a sandwich-ELISA was developed to study the passage and the viability of a bifidobacterial species after oral application of lyophilized preparation. Rabbits were immunized with a formaldehyde-inactivated strain of the species Bifidobacterium longum resulting in a highly specific antiserum. The cross-reaction of most of 46 selected, representative strains of 14 bifidobacterial species from animal and human sources was negligibly low. Only two strains of B. angulatum a...

  16. Three-Dimensional Computer Graphics Brain-Mapping Project. (United States)


    carboxymethylcellulose (CMC). A mound of CMC was built up around the specimen to complete the embed- ding. In order to reduce the reflectance off the frozen CMC...University of Texas Health Center, Dallas, Texas). Details of the produc- tion and characterization of this antiserum have been recently described...cell density. This reduces the prospect for characterizing the cell types on the basis of their dendritic morphology within the IPL. The nAChR staining

  17. Immunotherapy of Congenital SIV Infection. (United States)


    incubating the membrane with goat anti-RLV antiserum followed by horseradish peroxidase-conjugated rabbit anti goat IgG. 4. Stable transfection amd generation...after oral macaques (unpublished data). Consequently, we believe virus challenge. Epidemiological data indicate that that host factors are responsible...heterosexual partners of seropositive drug abusers in 96. Roques PA, Gras G, Parnet-Mathieu F, et al.: Clearance of HIV Spain . Lancet 1990, 335:860

  18. Light-microscopic immunocytochemistry for Gentamicin and its use for studying uptake of the drug in kidney

    DEFF Research Database (Denmark)

    Fujiwara, Kunio; Shin, Masashi; Matsunaga, Hayato


    Gentamicin (GM) is a widely used antibiotic but shows renal toxicity. We produced a serum against GM (anti-GM) conjugated to bovine serum albumin with N-(gamma-maleimidobutyryloxy)succinimide. The antiserum was monospecific for GM and did not cross-react with the analog streptomycin, tobramycin...... compartments. This approach should be useful for accurately detecting the uptake and toxicity of the antibiotic in different tissues....

  19. Characterization and evaluation of a Sarcoptes scabiei allergen as a candidate vaccine


    Zhang Runhui; Jise Quwu; Zheng Wanpeng; Ren Yongjun; Nong Xiang; Wu Xuhang; Gu Xiaobin; Wang Shuxian; Peng Xuerong; Lai Songjia; Yang Guangyou


    Abstract Background Sarcoptic mange caused by the mite Sarcoptes scabiei is a worldwide disease affecting both humans and animals. Here we report the molecular characterization and evaluation of a recombinant S. scabiei tropomyosin (SsTm) protein in a vaccination trial in rabbits. Methods The full-length cDNA was cloned in a bacterial pET vector, and the recombinant protein was expressed in BL21 (DE3) cells and purified. Using specific rabbit antiserum, tropomyosin was localized immunohistoch...

  20. Updates on tetanus toxin: a fundamental approach

    Directory of Open Access Journals (Sweden)

    Md. Ahaduzzaman


    Full Text Available Clostridium tetani is an anaerobic bacterium that produces second most poisonous protein toxins than any other bacteria. Tetanus in animals is sporadic in nature but difficult to combat even by using antibiotics and antiserum. It is crucial to understand the fundamental mechanisms and signals that control toxin production for advance research and medicinal uses. This review was intended for better understanding the basic patho-physiology of tetanus and neurotoxins (TeNT among the audience of related field.

  1. Characterizing mouse male germ cell-specific actin capping protein α3 (CPα3): dynamic patterns of expression in testicular and epididymal sperm

    Institute of Scientific and Technical Information of China (English)

    Keizo Tokuhiro; Yasushi Miyagawa; Hiromitsu Tanaka


    Aim: To characterize mouse capping protein α3 (CPα3) during spermatogenesis and sperm maturation. Methods: We produced rat anti-CPα3 antiserum and examined the expression of CPα3 in various mouse tissues using Western blot analysis and the localization of CPα3 in testicular and epididymal sperm using immunohistochemical analyses. We also examined how the localization of CPα3 and β-actin (ACTB) in sperm changed after the acrosomal reaction by performing immunohistochemical analyses using anti-CPα3 antiserum and anti-actin antibody. Results: Western blot analysis using specific antiserum revealed that CPα3 was expressed specifically in testes. Interestingly, the molecular weight of CPα3 changed during sperm maturation in the epididymis. Furthermore, the subcellular localization of CPα3 in sperm changed dynamically from the flagellum to the post-acrosomal region of the head during epididymal maturation. The distribution of ACTB was in the post-acrosomal region of the head and the flagellum. After inducing the acrosomal reaction, the CPα3 and ACTB localization was virtually identical to the localization before the acrosomal reaction.Conclusion: CPα3 might play an important role in sperm morphogenesis and/or sperm function.

  2. Distribution and biological activity of obestatin in the rat. (United States)

    Dun, Siok L; Brailoiu, G Cristina; Brailoiu, Eugen; Yang, Jun; Chang, Jaw Kang; Dun, Nae J


    Obestatin, a 23 amino acid peptide recently isolated from the rat stomach, is encoded by the same gene that encodes ghrelin. With the use of an antiserum directed against the mouse/rat obestatin, obestatin immunoreactivity (irOBS) was detected in cells of the gastric mucosa, myenteric plexus, and in Leydig cells of the testis in Sprague-Dawley rats. Double labeling the myenteric plexus with obestatin antiserum and choline acetyltransferase (ChAT) antiserum revealed that nearly all irOBS neurons were ChAT positive and vice versa. For comparative purposes, myenteric ganglion cells, cells in the gastric mucosa, and Leydig cells of the testis were shown to be immunoreactive to preproghrelin. The biological activity of obestatin on rat central neurons was assessed by the calcium microfluorimetric Fura-2 method. Obestatin (100 nM) administered to dissociated and cultured rat cerebral cortical neurons elevated cytosolic calcium concentrations [Ca2+]i in a population of cortical neurons. The result provides the first immunohistochemical evidence that obestatin is expressed in cells of the gastric mucosa and myenteric ganglion cells, and also in Leydig cells of the testis; the peptide is biologically active on central neurons.

  3. Muscle fibre types in the external eye muscles of the pigeon, Columba livia. (United States)

    McVean, A; Stelling, J; Rowlerson, A


    Fibre typing with antisera raised against specific myosin types from muscles of known physiological properties were used to characterise the fibre types within the oculorotatory muscles of pigeons. Fibres reacting strongly to antiserum anti-ALD (specific for tonic fibre myosin) were found lying along the global margin of the muscle and also in a layer lying immediately beneath a discrete band of fibres running along the orbital margin. These fibres resembled those of the skeletal muscle ALD in their type properties. Using another antiserum, anti-I, specific for slow twitch and to a lesser extent, slow tonic myosins, it was possible to identify another slow fibre type which formed the orbital layer and also lay scattered randomly through the body of the muscle. No equivalent to this type was found in the skeletal muscles ALD or iliofibularis. The remaining fibres which did not react with either anti-ALD or anti-I formed 58% of the fibre population and reacted with an antiserum specific for fast myosin. However, their response to alkali preincubation suggests that the fast fibres of eye muscles also contain a myosin which is different from those in skeletal muscle.

  4. Development and precise characterization of phospho-site-specific antibody of Ser(357) of IRS-1: elimination of cross reactivity with adjacent Ser(358). (United States)

    Waraich, Rizwana Sanaullah; Zaidi, Nousheen; Moeschel, Klaus; Beck, Alexander; Weigert, Cora; Voelter, Wolfgang; Kalbacher, Hubert; Lehmann, Rainer


    Antibodies that recognize specifically phosphorylated sites on proteins are widely utilized for studying the regulation and biological function of phosphoproteins. The proposed strategy is a powerful, analytical tool allowing the generation of phospho-site specific antibodies albeit adjacent phosphorylation sites are present. Here, we demonstrate the assessment and elimination of cross reactivity of phospho-site-specific-Ser(357) IRS-1 antibody. While determining the specificity of p-Ser(357) antiserum we came across the cross reactivity of the antiserum with adjacent Ser(358) which was successfully abolished by an improved immuno-purification method. The specificity of the purified antiserum was then verified by indirect ELISA, results of ELISA were also mirrored in the experiments carried out in BHK-IR cells using different mutants of IRS-1 carrying mutations at either Ser(357)/Ser(358)/Ser(357/358). Immuno-purified-p-Ser(357) did not react with IRS-1 Ala(357) and IRS-1 Ala(357/358). In conclusion, the present study describes generation and characterization of p-Ser(357) IRS-1 antibody, which reacts with IRS-1 in site specific and phosphorylation state-dependent manner without showing cross reactivity to adjacent Ser(358). This antibody can be effectively used to further clarify the inhibitory role of Ser(357) in insulin signal transduction.

  5. [An easy way to purify the inclusion body protein with high purity from prokaryotic expression cells]. (United States)

    Liu, Rong; Zhong, Qin-Ping; Jiang, Ming-Sen; Dong, Hui-Fen


    To clone partial ORF of SjBMP and to construct the recombinant SjBMP-pET-28a(+) plasmids, and then to transform them into the competent cells E. coli BL21 (DE3), finally a positive clone was used to be induced by IPTG. The bacterial aggregates with target protein expressed as inclusion bodies were purified by the methods of Ni(2+)-NTA affinity purification under denaturation condition and SDS-PAGE gel extraction. The purified protein was used to immune rabbits and make antiserum against the SjBMP, and the antiserum were then used to identify the rSjBMP by Western blotting. The target protein obtained by Ni(2+)-NTA Agarose affinity purification was not pure with unspecific proteins, but the protein further purified by SDS-PAGE gel extraction and the dialysis bag horizontal electrophoresis was quite pure, and the recovery rate was more than 11.0%. Meanwhile, Western blotting was used to identify the recombinant SjBMP protein by antiserum, only a specific single strip appeared, which suggested the protein purified by this method kept its antigenicity, and could be used for common immunological studies. Therefore, the SDS-PAGE gel extraction combining with electroosmosis and dialysis recycling are good and easy to purify the inclusion body proteins.

  6. Characterization of polyclonal antibodies against nonstructural protein 9 from the porcine reproductive and respiratory syndrome virus

    Directory of Open Access Journals (Sweden)

    Mengmeng ZHAO,Juanjuan QIAN,Jiexiong XIE,Tiantian CUI,Songling FENG,Guoqiang WANG,Ruining WANG,Guihong ZHANG


    Full Text Available Porcine reproductive and respiratory syndrome (PRRS is considered to be one of the most important infectious diseases impacting the swine industry and is characterized by reproductive failure in late term gestation in sows and respiratory disease in pigs of all ages. The nonstructural protein 9 gene, Nsp9, encoding the RNA-dependent RNA polymerase, is generally regarded as fairly conserved when compared to other viral proteins. Antibodies against Nsp9 will be of great importance for the diagnosis and treatment of the causal agent, PRRS virus. A study was undertaken to generate polyclonal antibodies against the immunodominant Nsp9. For this purpose, the Nsp9 was expressed in Escherichia coli and subsequently used as an antigen to immunize New Zealand rabbits. Antiserum was identified via an indirect ELISA, and then verified based on the ability to react with both naturally and artificially expressed Nsp9. Results of virus neutralization test showed that this antiserum could not neutralize the PRRSV. Nevertheless, this antiserum as a diagnostic core reagent should prove invaluable for further investigations into the mechanism of PRRS pathogenesis.

  7. Effect of gamma irradiation on toxicity and immunogenicity of Androctonus australis hector venom

    Energy Technology Data Exchange (ETDEWEB)

    Abib, L. [Univ. des Sciences et de la Technologie, Lab. de Biologie Cellulaire et Moleculaire, Faculte des Sciences Biologiques, Houari Boumedienne, Bab Ezzouar (Algeria); Laraba-Djebari, F. [Univ. des Sciences et de la Technologie, Lab. de Biologie Cellulaire et Moleculaire, Faculte des Sciences Biologiques, Houari Boumedienne, Bab Ezzouar, Alger (Algeria); Institut Pasteur d' Algerie, Lab. de Recherche et de Developpement sur les Venins, Alger (Algeria); E-mail:


    An investigation was made of the radiosensitivity of the toxic and immunological properties of Androctonus australis hector venom. This venom was irradiated with two doses of gamma rays (1 and 2 kGy) from a {sup 60}Co source. The results showed that venom toxicity was abolished for the two radiation doses (1 and 2 kGy) with, respectively, 10 and 25 times its initial LD50 value. However, irradiated venoms were immunogenic, and the antibodies elicited by them were able to recognize the native venom by enzyme-linked immunosorbent assay. Antisera raised against these toxoids (1 and 2 kGy) had a higher neutralizing capacity and immunoreactivity against all components of native venom than did the antiserum produced against the native venom. The antiserum of rabbits immunized with 2-kGy-irradiated venom was more efficient than 1-kGy-irradiated toxoid antiserum. Indeed, in vivo protection assays showed that the mice immunized with 2-kGy-irradiated venom resisted lethal doses (i.p.) of A. australis hector venom. (author)

  8. Evolution of quasispecies diversity for porcine reproductive and respiratory syndrome virus under antibody selective pressure. (United States)

    Zhao, Peng; Ma, ChengTai; Dong, Xuan; Cui, ZhiZhong


    To study the quasispecies diversity of porcine reproductive and respiratory syndrome virus (PRRSV), open reading frame 5 (ORF5) of strain SD0612 was amplified and cloned. Sixty clones of ORF5 were sequenced and analyzed with DNAStar software. Nucleic acid sequence homology was 97.7%-100%, with 78 mutations observed. Among these 60 clones, the sequences of 17 clones were identical and recognized as the dominant quasispecies of strain SD0612. Evolution of SD0612 quasispecies diversity under antibody selective pressure was also studied. SD0612 was passed continuously in the Marc-145 cell line over 40 passages in 6 independent lineages. SD0612 antiserum was not added to lineage A, B, and C cultures; however, antiserum was added to culture medium for lineages D, E, and F. PRRSV ORF5 was then amplified, cloned, and sequenced from each of the 6 lineages, designated as A40-F40. F40 was further passed in Marc-145 cells using 6 independent lineages with or without F40 antiserum for another 40 passages. ORF5 from the 6 newly-derived virus lineages, which we designated as a40-f40, were amplified, cloned and sequenced. The proportion of dominant quasispecies increased with passage number in cell cultures supplemented with antibodies, but decreased when antibodies were lacking. Our work has demonstrated a diversity of quasispecies for ORF5 in PRRSV SD0612. Antibody selective pressure was able to significantly influence quasispecies diversity and promote a dominant quasispecies that was able to evade immune reactions.

  9. Screening of papaya accessions resistant to Papaya lethal yellowing virus and capacity of Tetranychus urticae to transmit the virus

    Directory of Open Access Journals (Sweden)

    Marcos Fernando Basso


    Full Text Available The objective of this work was to produce a polyclonal antiserum against the coat protein (CP of Papaya lethal yellowing virus (PLYV and to determine its specificity and sensibility in the diagnosis of the virus, as well as to evaluate the genetic resistance to PLYV in papaya (Carica papaya accessions and to investigate the capacity of the two-spotted spider mite Tetranychus urticae to acquire and transmit PLYV to the plants. Sixty-five papaya accessions were evaluated. For each accession, ten plants were mechanically inoculated using PLYV-infected plant extracts, and three plants were mock inoculated with phosphate buffer alone and used as negative controls. Ninety days after inoculation, newly-emerging systemic leaves were collected from the inoculated plants, and viral infection was diagnosed by indirect Elisa, using polyclonal antiserum sensible to the in vitro-expressed PLYV CP. Viral transmission by T. urticae was evaluated in greenhouse. The experiments were repeated twice. Polyclonal antiserum recognized the recombinant PLYV CP specifically and discriminated PLYV infection from infections caused by other plant viruses. Out of the 65 papaya accessions evaluated, 15 were considered resistant, 18 moderately resistant, and 32 susceptible. The two-spotted spider mite T. urticae was capable of acquiring PLYV, but not of transmitting it to papaya.


    Institute of Scientific and Technical Information of China (English)

    Ming-shan Ren; Chuan-zhen Lu; Jian Qiao; Hui-min Ren; Ren Xu; Ren-bao Gan


    Objective To express and purify the protein coded by the TRAF-type zinc finger domain ofmyasthenia gravis (MG)-related gene P9 ( P9-ZFD ) and to prepare P9-ZFD antiserum for detecting expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of patient with MG.Methods The cDNA encoding P9-ZFD was amplified by RT-PCR. The cloned P9-ZFD cDNA was ligated into pET-24a, and the P9-ZFD recombinant protein was induced via E. coli. BL21 (DE3) and purified by histidine affinity chromatography. P9-ZFD antiserum was prepared and its fiter and specificity were determined by ELISA and Western blot. Expression and subcellular distribution of P9-ZFD protein in the skeletal muscles of MG and control were studied.Results The molecular weight of purified P9-ZFD protein was about 30 kD. Its purity was more than 95%. Antiserum specific for P9-ZFD was excellent. P9-ZFD protein is fully confined to the cytoplasm membrane of skeletal muscle cell of MG, obvious immunostaining was absent in the A, I, and Z bands of cytoplasm and no immunoreactivity was observed in the skeletal muscle cell of control.Conclusion P9-ZFD protein is expressed as a cytoplasm membrane-bound protein and has obvious distribution difference in the skeletal muscle cells of patient with MG and normal control.

  11. Spectra analysis of coating antigen: A possible explanation for difference in anti-AFB1 polyclonal antibody sensitivity (United States)

    Ye, Yang; Liu, Aiping; Wang, Xiaohong; Chen, Fusheng


    For the detection of small hapten molecules, indirect competitive enzyme-linked immunosorbent assay (icELISA) is a preferred method. However, diverse coating antigen might bring different antiserum titer and sensitivity for the identical antiserum. In the present study, four AFB1-protein (aflatoxin B1-carrier protein) conjugates were prepared by activated ester method (AFB1O-BSA/AFB1O-OVA) and mannich method (AFB1-cBSA/AFB1-cOVA), and then applied as coating antigen for titer and sensitivity detection of the identical antiserum obtained from rabbit immunized by AFB1-KLH. Afterwards, the ultraviolet-visible, fluorescence and far-ultraviolet circular dichroism (far-UV CD) spectra were recorded for understanding the difference in titer and sensitivity obtained. Results revealed that AFB1O-BSA/AFB1O-OVA showed a strong intrinsic fluorescence band centered at 450 nm that originated from the emission of AFB1, which differed from AFB1-cBSA/AFB1-cOVA, while the decrease of α-helical and increase of β-sheet in AFB1-cBSA was the most remarkable. This indicated that the better sensitivity obtained by using AFB1O-BSA as coating antigen might be caused by its extended structure, because such structure affect the binding between AFB1 and antibody. The study might offer structural information for understanding the titer and sensitivity difference caused by coating antigen.

  12. Plasma membrane fatty acid-binding protein and mitochondrial glutamic-oxaloacetic transaminase of rat liver are related

    Energy Technology Data Exchange (ETDEWEB)

    Berk, P.D.; Potter, B.J.; Sorrentino, D.; Zhou, S.L.; Isola, L.M.; Stump, D.; Kiang, C.L.; Thung, S. (Mount Sinai School of Medicine, New York, NY (USA)); Wada, H.; Horio, Y. (Univ. of Osaka (Japan))


    The hepatic plasma membrane fatty acid-binding protein (h-FABP{sub PM}) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate. Sinusoidally enriched liver plasma membranes and purified h-FABP{sub PM} have GOT enzymatic activity. Monospecific rabbit antiserum against h-FABP{sub PM} reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of ({sup 3}H)oleate but not that of ({sup 35}S)sulfobromophthalein or ({sup 14}C)taurocholate. The inhibition of oleate uptake produced by anti-h-FABP{sub PM} can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABP{sub PM} and mGOT are closely related.

  13. Purification of the major outer membrane protein of Azospirillum brasilense, its affinity to plant roots, and its involvement in cell aggregation. (United States)

    Burdman, S; Dulguerova, G; Okon, Y; Jurkevitch, E


    The major outer membrane protein (MOMP) of the nitrogen-fixing rhizobacterium Azospirillum brasilense strain Cd was purified and isolated by gel filtration, and antiserum against this protein was obtained. A screening of the binding of outer membrane proteins (OMPs) of A. brasilense to membrane-immobilized root extracts of various plant species revealed different affinities for the MOMP, with a stronger adhesion to extracts of cereals in comparison with legumes and tomatoes. Moreover, this protein was shown to bind to roots of different cereal seedlings in an in vitro adhesion assay. Incubation of A. brasilense cells with MOMP-antiserum led to fast agglutination, indicating that the MOMP is a surface-exposed protein. Cells incubated with Fab fragments obtained from purified MOMP-antiserum immunoglobulin G exhibited significant inhibition of bacterial aggregation as compared with controls. Bacteria preincubated with Fab fragments showed weaker adhesion to corn roots in comparison to controls without Fab fragments. These findings suggest that the A. brasilense MOMP acts as an adhesin involved in root adsorption and cell aggregation of this bacterium.

  14. Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vassinated mice

    Energy Technology Data Exchange (ETDEWEB)

    Dalton, J.P.; Tom, T.D.; Strand, M.


    Spleen cells of mice vaccinated with radiation-attenuated Schistosoma mansoni cercariae were used to produce monoclonal antibodies directed against newly transformed schistosomular surface antigens. One of these monoclonal antibodies recognized a polypeptide of 18 kDa. Binding was measured by radioimmunoassay. This glycoprotein was purified by monoclonal antibody immunoaffinity chromatography and a polyclonal antiserum was prepared against it. Immunofluorescence assays showed that the polyclonal antiserum bound to the surface of newly transformed schistosomula and lung-stage organisms but not to the surface of liver-stage and adult worms. Using this polyclonal antiserum we isolated recombinant clones from an adult worm cDNA expression library constructed in lambdagt11. Clone 654.2 contained an insert of 0.52 kilobase and hybridized to a 1.2-kilobase mRNA species from adult worms. Most importantly, clone 654.2 produced a fusion protein of 125 kDa that was reactive with sera of vaccinated mice that are capable of transferring resistance. This result encourages future vaccination trials with the fusion protein.

  15. cDNA cloning and recombinant expression of the general odorant binding protein Ⅱ from Spodoptera litura

    Institute of Scientific and Technical Information of China (English)


    A cDNA encoding the general odorant binding protein Ⅱ(GOBP Ⅱ) was isolated from the antennae of Spodoptera litura(SlGOBP Ⅱ,GenBank Accession No.EU086371) by homologous cloning and rapid amplification of cDNA ends(RACE).Sequencing and structural analyses revealed that the open reading frame(ORF) of SlGOBP Ⅱ was 489 bp,encoding 162 amino acids with a predicted MW of 18.2 kD and pI of 5.72.SlGOPB Ⅱ shared typical structural features of odorant binding proteins with other insects,including the six conservative cysteine residues.The deduced amino acid sequence of SlGOPB Ⅱ shared significant identity with the GOBP Ⅱ from S.frugiperda and S.exigua.RT-PCR and Northern blot analyses showed that SlGOBP Ⅱ was specifically expressed in the antennae.cDNA encoding SlGOBP Ⅱ was constructed into the pET-32a vector and the recombinant protein was highly expressed in Escherichia coli BL21(DE3) after induction with IPTG.SDS electrophoresis and Western blot analysis confirmed the molecular weight of the recombinant SIGOBPⅡ i.e,32 kD,which has a 6×His tag at the N-terminus.The recombinant SlGOBP Ⅱ was purified by single-step Ni-NTA affinity chromatography and used to raise antiserum in rabbits.ELISA showed that the titer of antiserum was 1:12800,while Western blot analysis showed that the recombinant SlGOBP Ⅱ was recognized as anti-SlGOBP Ⅱ antiserum.

  16. Utilização da serologia na identificação de Ascia monuste orseis (Latreille, 1819 (Lepidoptera: Pieridae - DOI: 10.4025/actascibiolsci.v31i2.515 Using the serological technique to identify Ascia monuste orseis (Latreille, 1819 (Lepidoptera: Pieridae - DOI: 10.4025/actascibiolsci.v31i2.515

    Directory of Open Access Journals (Sweden)

    Carlos Roberto Sousa-Silva


    Full Text Available O presente trabalho teve como objetivo obter antissoro específico paraAscia monuste orseis (Latreille, 1819 com a finalidade de identificar seus predadores. Para a obtenção do antissoro, um coelho foi imunizado, via linfonódulo, com uma injeção de antígeno obtido pela maceração de lagartas do 4o e 5o instares de A. monuste orseis. Reações serológicas homólogas, realizadas por meio do método de dupla difusão em ágar, foram positivas após 21 dias da 1a inoculação do antígeno. Ovos, lagartas do 1o, 2o e 3o instares, pupas e adultos de A. monuste orseis também reagiram positivamente ao antissoro obtido.This study aimed to obtain specific antiserum for Ascia monuste orseis (Latreille, 1819, with the aim of identifying the predators associated with this insect. The preparation of specific antiserum was performed by immunizing a rabbit via a lymph nodule injection containing antigen obtained from the maceration of 4th and 5th instars of A. monuste orseis caterpillars. Serological homologous tests performed using double diffusion in agar gel were positive 21 days after the first antigen inoculation. Eggs, 1th, 2th and 3th instar caterpillars, pupae and adults of A. monuste orseis also reacted positively to the obtained antiserum.

  17. Utilização da serologia na identificação de Ascia monuste orseis (Latreille, 1819 (Lepidoptera: Pieridae = Using the serological technique to identify Ascia monuste orseis (Latreille, 1819 (Lepidoptera: Pieridae

    Directory of Open Access Journals (Sweden)

    Fabiano de Mello Costa


    Full Text Available O presente trabalho teve como objetivo obter antissoro específico para Ascia monuste orseis (Latreille, 1819 com a finalidade de identificar seus predadores. Para a obtenção do antissoro, um coelho foi imunizado, via linfonódulo, com uma injeção de antígeno obtido pela maceração de lagartas do 4o e 5o instares de A. monuste orseis. Reações serológicas homólogas, realizadas por meio do método de dupla difusão em ágar, foram positivas após 21 dias da 1a inoculação do antígeno. Ovos, lagartas do 1o, 2o e 3o instares, pupas e adultos de A. monuste orseis também reagiram positivamente ao antissoro obtido.This study aimed to obtain specific antiserum for Ascia monuste orseis (Latreille, 1819, with the aim of identifying the predators associated with this insect. The preparation of specific antiserum was performed by immunizing a rabbit via a lymph nodule injection containing antigen obtained from the maceration of 4th and 5th instars of A. monuste orseis caterpillars. Serological homologous tests performed using double diffusion in agar gel were positive 21 days after the first antigen inoculation. Eggs, 1th, 2th and 3th instar caterpillars, pupae and adults of A. monuste orseis also reacted positively to the obtained antiserum.

  18. Specific humoral immune responses in rhesus monkeys vaccinated with the Alzheimer's disease-associated β-amyloid 1-15 peptide vaccine

    Institute of Scientific and Technical Information of China (English)

    LI Shao-bing; WANG Hua-qiao; LIN Xian; XU Jie; XIE Yao; YUAN Qun-fang; YAO Zhi-bin


    Background Alzheimer's disease(AD) is a neurodegenerative disorder characterized by overproduction of β-amyloid (Aβ), with the subsequent pathologic deposition of Aβ which is important for memory and cognition. Recent studies showed murine models of AD and AD patients inoculated with Aβ1-42 peptide vaccine had a halted or delayed pathological progression of AD. Unfortunately, the clinical phase Ⅱa trial of Aβ1-42 peptide vaccine (AN1792) was halted prematurely because of episodes of menigoencephalitis in 18 of the vaccinated patients. The vaccination of BALB/c or Tg2576 transgenic mouse with Aβ1-15 peptide vaccine is safe and the immune effects are satisfactory. This study further characterizes the specific humoral immune responses in adult rhesus monkeys induced by Aβ1-15 peptide vaccine.Methods Five male adult rhesus monkeys were injected intramuscularly with Aβ1-15 peptide vaccine at baseline and at weeks 2, 6, 10, 14, 18 and 22. The titers and IgG isotypes of the antibody against Aβ1-42 in serum was measured by Enzyme-linked Immunosorbent Assay (ELISA). The specificity of the antibody against Aβ1-42 was determined by Western blot. The Aβ plaques in Tg2576 transgenic mouse brain were stained with the antiserum using immunohistochemistry method.Results At the eighth week after the vaccination, antibody against Aβ1-42 began to develop significantly in serum. The titers of the antibody increased following vaccine boosted and reached 1∶3840 at the twenty-fourth week, then decreased after the termination of inoculation. The IgG1 was accounted for the highest level in the antiserum pool. The antibody against Aβ1-42 showed high specificity. The Aβ plaques in Tg2576 transgenic mouse brain were labeled with the antiserum.Conclusion Aβ1-15 vaccine can induce vigorously specific humoral immune responses in adult rhesus monkey.

  19. Ureaplasma urealyticum binds mannose-binding lectin. (United States)

    Benstein, Barbara D; Ourth, Donald D; Crouse, Dennis T; Shanklin, D Radford


    Mannose-binding C-type lectin (MBL) is an important component of innate immunity in mammals. Mannose-binding lectin (MBL), an acute phase protein, acts as an opsonin for phagocytosis and also activates the mannan-binding lectin complement pathway. It may play a particularly significant role during infancy before adequate specific protection can be provided by the adaptive immune system. Ureaplasma urealyticum has been linked to several diseases including pneumonia and chronic lung disease (CLD) in premature infants. We therefore investigated the ability of U. urealyticum to bind MBL. A guinea pig IgG anti-rabbit-MBL antiserum was produced. An immunoblot (dot-blot) assay done on nitrocellulose membrane determined that the anti-MBL antibody had specificity against both rabbit and human MBL. Pure cultures of U. urealyticum, serotype 3, were used to make slide preparations. The slides containing the organisms were then incubated with nonimmune rabbit serum containing MBL. Ureaplasma was shown to bind rabbit MBL with an immunocytochemical assay using the guinea pig IgG anti-rabbit MBL antiserum. Horseradish peroxidase (HRP)-labeled anti-guinea pig IgG was used to localize the reaction. The anti-MBL antiserum was also used in an immunocytochemical assay to localize U. urealyticum in histological sections of lungs from mice specifically infected with this organism. The same method also indicated binding of MBL by ureaplasma in human lung tissue obtained at autopsy from culture positive infants. Our results demonstrate that ureaplasma has the capacity to bind MBL. The absence of MBL may play a role in the predisposition of diseases related to this organism.

  20. Identification and characterization of two distinct ligand binding regions of cubilin. (United States)

    Yammani, R R; Seetharam, S; Seetharam, B


    Using polymerase chain reaction-amplified fragments of cubilin, an endocytic receptor of molecular mass 460 kDa, we have identified two distinct ligand binding regions. Region 1 of molecular mass 71 kDa, which included the 113-residue N terminus along with the eight epidermal growth factor (EGF)-like repeats and CUB domains 1 and 2, and region 2 of molecular mass 37 kDa consisting of CUB domains 6-8 bound both intrinsic factor-cobalamin (vitamin B(12); Cbl) (IF-Cbl) and albumin. Within these two regions, the binding of both ligands was confined to a 110-115-residue stretch that encompassed either the 113-residue N terminus or CUB domain 7 and 8. Ca(2+) dependence of ligand binding or the ability of cubilin antiserum to inhibit ligand binding to the 113-residue N terminus was 60-65%. However, a combination of CUB domains 7 and 8 or 6-8 was needed to demonstrate significant Ca(2+) dependence or inhibition of ligand binding by cubilin antiserum. Antiserum to EGF inhibited albumin but not IF-Cbl binding to the N-terminal cubilin fragment that included the eight EGF-like repeats. While the presence of excess albumin had no effect on binding to IF-Cbl, IF-Cbl in excess was able to inhibit albumin binding to both regions of cubilin. Reductive alkylation of the 113-residue N terminus or CUB 6-8, CUB 7, or CUB 8 domain resulted in the abolishment of ligand binding. These results indicate that (a) cubilin contains two distinct regions that bind both IF-Cbl and albumin and that (b) binding of both IF-Cbl and albumin to each of these regions can be distinguished and is regulated by the nonassisted formation of local disulfide bonds.

  1. Development of fluoroimmunoassay methods for delta-9-tetrahydrocannabinol

    Energy Technology Data Exchange (ETDEWEB)

    Mason, A.P.


    Heterogeneous, competitive, labelled-ligand solid-phase primary antibody fluoroimmunoassay methods for the detection of THC in blood and plasma were proposed, and the required assay components were produced and characterized. These components included polyclonal rabbit antisera and monoclonal antibodies reactive with tetrahydrocannabinols, solid-phase immunoglobin reagents, a fluoroligand, and protein conjugates of THC for immunization and immunoassay response amplification. The stereoselective rabbit anti-THC antiserum F-444-12 was found to have a high binding titer, a high affinity (K/sub D/ = 3.4 x 10/sup -/exclamation/sup 1/ M for 5'-iodo/sup -125/ 2/-THC), and high specificity versus a large number of cannabinoid compounds. Immobilization of the immunoglobulin fraction of the antiserum on hydrophilic polyacrylamide microspheres resulted in only a four fold increase in K/sub D/, and a two fold increase in the concentration of binding sites required for the production of equivalent binding titers. Specificity for small ligands was not affected, but the binding of THC-protein conjugates was reduced in potency. Two monoclonal hybridoma cell lines were produced that secrete monoclonal antibodies which bind the radioligand. The fluoroligand was synthesized from 5' 2/-THC and FITC using a diamimoethane linkage structure. While the compound had the fluorescence properties of FTIC, it was bound to the antiserum F-144-12 with a cross-reactive potency 1.4x greater than the radioligand, and 10x greater than THC.

  2. cDNA cloning and recombinant expression of the gen-eral odorant binding protein Ⅱ from Spodoptera litura

    Institute of Scientific and Technical Information of China (English)

    JIN FengLiang; DONG XiaoLin; XU XiaoXia; REN ShunXiang


    A cDNA encoding the general odorant binding protein Ⅱ (GOBP Ⅱ) was isolated from the antennae of Spodoptera fitura (SiGOBP Ⅱ, GenBank Accession No. EU086371) by homologous cloning and rapid amplification of cDNA ends (RACE). Sequencing and structural analyses revealed that the open reading frame (ORF) of SIGOBP Ⅱ was 489 bp, encoding 162 amino acids with a predicted MW of 18.2 kD and pl of 5.72. SIGOPB Ⅱ shared typical structural features of odorant binding proteins with other insects, including the six conservative cysteine residues. The deduced amino acid sequence of SIGOPB Ⅱ shared significant identity with the GOBP Ⅱ from S. frugiperda and S. exigua. RT-PCR and Northern blot analyses showed that SIGOBP Ⅱ was specifically expressed in the antennae, cDNA encoding SIGOBP Ⅱ was constructed into the pET-32a vector and the recombinant protein was highly expressed in Es-cherichia coil BL21 (DE3) after induction with IPTG. SDS electrophoresis and Western blot analysis confirmed the molecular weight of the recombinant SIGOBPⅡ i.e, 32 kD, which has a 6xHis tag at the N-terminus. The recombinant SIGOBP U was purified by single-step Ni-NTA affinity chromatography and used to raise antiserum in rabbits. ELISA showed that the titer of antiserum was 1 : 12800, while Western blot analysis showed that the recombinant SIGOBP Ⅱ was recognized as anti-SiGOBP Ⅱ an-tiserum.

  3. Hemoglobin switching in man and chicken is mediated by a heteromeric complex between the ubiquitous transcription factor CP2 and a developmentally specific protein. (United States)

    Jane, S M; Nienhuis, A W; Cunningham, J M


    The human stage selector protein (SSP) has been implicated in the developmental regulation of the globin genes. Binding of SSP to the stage selector element (SSE) in the proximal gamma-globin promoter is integral to the competitive silencing of a linked beta-promoter in embryonic/fetal stage erythroleukemia (K562) cells. We now report the biochemical purification of SSP from K562 cell nuclear extract and demonstrate that the ubiquitously expressed transcription factor CP2 is pivotal to, but not sufficient for, SSP binding activity. Although addition of anti-CP2 antiserum disrupts the formation of the SSP-SSE complex in the electrophoretic mobility shift assay (EMSA), recombinant CP2 fails to bind to the SSE. Binding of CP2 to the SSE requires a heterodimeric partner present in K562 cells. We have defined the molecular weight of the partner protein as 40-45 kDa in UV and protein cross-linking experiments. An element analogous to the human SSE has previously been demonstrated in the chicken beta A-gene-promoter. The effects of this element are dependent on the binding of the chicken stage selector protein, NF-E4. Comparative studies between human CP2 and chicken NF-E4 demonstrate homology between the protein complexes. SSP binds to the chicken SSE and formation of this complex is ablated by the addition of anti-CP2 antiserum or a monoclonal antibody to NF-E4. Western analysis of partially purified NF-E4 using anti-CP2 antiserum or the NF-E4 monoclonal antibody both demonstrate a dominant band at 66 kDa. Similarly, the NF-E4 antibody recognizes the 66 kDa human CP2 protein in Western analysis of the SSP-SSE complex.(ABSTRACT TRUNCATED AT 250 WORDS)

  4. Antigens of Streptococcus mutans: isolation of a serotype-specific and a cross-reactive antigen from walls of strain V-100 (serotype e). (United States)

    Wetherell, J F; Bleiweis, A S


    Two cell wall-associated polysaccharide antigens were extracted from purified cell walls of Streptococcus mutans serotype e strain V-100. One of these purified antigens (I) is specific for serotype e, whereas the other (II) has antigenic determinants reactive with both heterologous anti-serotype c serum (GS-5) and the homologous (e) serum. When crude formamide extracts of V-100 cell walls were loaded onto a Cellex-D column and eluted with a linear gradient of ammonium carbonate (0.02 to 0.40 M), the two products mentioned above could be recovered. The purified, antigenically reactive products (I and II) were each composed only of rhamnose and glucose in approximately a 2:1 molar ratio. Immunoelectrophoresis of the crude formamide extract, peak I, and peak II showed the purified fractions to have opposite mobilities and the crude extract to have a mobility that encompassed both purified peaks when reacted with homologous antiserum (V-100). When these three fractions were immunoelectrophoresed and reacted with heterologous anti-serotype c serum (GS-5), only the anodic portion of the crude V-100 formamide extract and purified peak II formed precipitates. Ouchterlony analysis with homologous antiserum produced precipitin patterns between the crude formamide extract and both purified peaks, indicating complete identity. However, only crude extracts of V-100 and the purified peak II material reacted with heterologous (c) antiserum; peak I did not cross-react in these Ouchterlony assays. Hapten inhibition studies revealed that a beta-glucosyl moiety is the immunodeterminant for serotype e and is present on each purified fraction. The basis of the cross-reaction between anti-c sera and the purified antigen II of e is discussed.

  5. Molecular cloning, functional characterization and localization of an annexin from a fish gill fluke Microcotyle sebastis (Platyhelminthes: Monogenea). (United States)

    Choi, Seung Hyuk; Kwon, Se Ryun; Lee, Eun Hye; Kim, Ki Hong


    The full cDNA of an annexin gene from Microcotyle sebastis (MsANX) was cloned for the first time in monogeneans. The cDNA of MsANX comprises 1199bp with a 29bp 5' untranslated region, an open reading frame of 1062bp, and a 108bp 3' untranslated region. The recombinantly produced MsANX bound phosphatidylserine vesicles in the presence of Ca2+, whereas no MsANX was precipitated in the absence of free Ca2+. Phylogenetically, MsANX formed a cluster with human annexin A13, known as the earliest annexin in vertebrates and expressed mainly in the intestine. The localization of MsANX in M. sebastis was analyzed by Western blotting and immunohistochemistry using the antiserum raised against the recombinant MsANX. In Western blot analysis, rat antiserum bound to a protein corresponding to the MsANX in size when worm crude extracts were used as antigens, but no bands were detected by the antiserum when the excretory/secretory proteins of worms were used as antigens. In immunohistochemistry analysis, significant antibody binding annexin was found in the ovarian region, the pharynx and the intestinal caecum of the worm. Interestingly, the alimentary canal location of MsANX was similar to the location of human annexin A13, and further research is needed to trace evolutionary relationship among helminthic annexins and human annexin A13. Also it remains to be investigated whether immunization of naïve fish with the recombinant MsANX can induce protective immune responses against M. sebastis infection.

  6. Immunohistochemical localization of two types of choline acetyltransferase in neurons and sensory cells of the octopus arm. (United States)

    Sakaue, Yuko; Bellier, Jean-Pierre; Kimura, Shin; D'Este, Loredana; Takeuchi, Yoshihiro; Kimura, Hiroshi


    Cholinergic structures in the arm of the cephalopod Octopus vulgaris were studied by immunohistochemistry using specific antisera for two types (common and peripheral) of acetylcholine synthetic enzyme choline acetyltransferase (ChAT): antiserum raised against the rat common type ChAT (cChAT), which is cross-reactive with molluscan cChAT, and antiserum raised against the rat peripheral type ChAT (pChAT), which has been used to delineate peripheral cholinergic structures in vertebrates, but not previously in invertebrates. Western blot analysis of octopus extracts revealed a single pChAT-positive band, suggesting that pChAT antiserum is cross-reactive with an octopus counterpart of rat pChAT. In immunohistochemistry, only neuronal structures of the octopus arm were stained by cChAT and pChAT antisera, although the pattern of distribution clearly differed between the two antisera. cChAT-positive varicose nerve fibers were observed in both the cerebrobrachial tract and neuropil of the axial nerve cord, while pChAT-positive varicose fibers were detected only in the neuropil of the axial nerve cord. After epitope retrieval, pChAT-positive neuronal cells and their processes became visible in all ganglia of the arm, including the axial and intramuscular nerve cords, and in ganglia of suckers. Moreover, pChAT-positive structures also became detectable in nerve fibers connecting the different ganglia, in smooth nerve fibers among muscle layers and dermal connective tissues, and in sensory cells of the suckers. These results suggest that the octopus arm has two types of cholinergic nerves: cChAT-positive nerves from brain ganglia and pChAT-positive nerves that are intrinsic to the arm.

  7. Effects of Static Magnetic Field on Growth of Leptospire, Leptospira interrogans serovar canicola: Immunoreactivity and Cell Division

    CERN Document Server

    Triampo, W; Triampo, D; Wong-Ekkabut, J; Tang, I M; Triampo, Wannapong; Doungchawee, Galayanee; Triampo, Darapond; Wong-Ekkabut, Jirasak


    The effects of the exposure of the bacterium, Leptospira interrogans serovar canicola to a constant magnetic field with magnetic flux density from a permanent ferrite magnet = 140 mT were studied. Changes in Leptospira cells after their exposure to the field were determined on the basis of changes in their growth behavior and agglutination immunoreactivity with a homologous antiserum using darkfield microscopy together with visual imaging. The data showed that the exposed Leptospira cells have lower densities and lower agglutination immunoreactivity than the unexposed control group. Interestingly, some of the exposed Leptospira cells showed abnormal morphologies such as large lengths. We discussed some of the possible reasons for these observations.

  8. Dermatophytosis caused by Trichophyton spp. in a Tenerife Lizard (Gallotia galloti): an immunohistochemical study. (United States)

    Orós, J; Hernández, J D; Gallardo, J; Lupiola, P; Jensen, H E


    Reports of dermatophytosis in reptiles are rare. This report describes the microscopical and immunohistochemical findings in a case of dermatophytosis caused by Trichophyton spp. in a 2-year-old Tenerife lizard (Gallotia galloti) with ulcerative and pustular skin lesions. Microscopically, the lesions were characterized by superficial epidermal pustules containing heterophils with numerous fungal hyphae that stained by periodic acid-Schiff and Grocott's stain. Fungal culture was not performed, but a panel of polyclonal antibodies specific for different fungal genera was applied to tissue sections. These immunohistochemical studies demonstrated reactivity of the hyphae only with antiserum specific for Trichophyton spp.

  9. The effects of nonidet P40 on the function of rat peritoneal mast cells in vitro.


    Batchelor, K. W.; Stanworth, D R


    1 Treatment of purified rat peritoneal mast cells at 37 degrees C with concentrations of the non-ionic detergent nonidet P40 (NP40) up to 0.005% (v/v) failed to reduce their viability. 2 There was a marked reduction in the histamine releasing capacity of NP40-treated mast cells upon challenge with a variety of selective (adrenocorticotrophic hormone 1-24 (Synacthen), rabbit anti-rat IgE antiserum, adenosine triphosphate (ATP) and the calcium ionophore, A 23187) and non-selective (rabbit anti-...

  10. La relación de Helicobacter pylori con la displasia y el cáncer gástrico en Costa Rica


    Miranda, M; Cháves, M; Orozco, L; San Román, María A; Durán, S; Vargas, G.; Jiménez, G.; Peña, E.; L. Rodríguez; Barrantes, E


    Occurrence of the bacterium Helicobacter pylori was compared for two Costa Rican sites with contrasting levels of gastric cancer incidence, Poás (incidence 15. 13%) and Puriscal (83. 53%). A sample of 1 85 adults of similar age and sex proportions was studied in each site, using both H. pylori antiserum tests and gastroscopy to collect two biopsies per case. No c1ear association between H. pylori and gastric cancer was found. Se comparó la incidencia de labaéteria Hdicobacter pylori en un ...

  11. Molecular cloning, expression and in situ hybridization of rat brain glutamic acid decarboxylase messenger RNA. (United States)

    Julien, J F; Legay, F; Dumas, S; Tappaz, M; Mallet, J


    A cDNA library was generated in the expression vector lambda GT11 from rat brain poly(A)+ RNAs and screened with a GAD antiserum. Two clones reacted positively. One of them was shown to express a GAD activity which was specifically trapped on anti-GAD immunogel and was inhibited by gamma-acetylenic-GABA. Blot hybridization analysis of RNAs from rat brain revealed a single 4 kilobases band. Preliminary in situ hybridizations showed numerous cells labelled by the GAD probe such as the Purkinje and stellate cells in the cerebellar cortex and the cells of the reticular thalamic nucleus.


    Directory of Open Access Journals (Sweden)

    Murad Lesmana


    Full Text Available Suatu modifikasi uji CAMP digunakan bersama dengan reaksi biokimiawi untuk identifikasi Vibrio cholerae pada sampel klinis. Dari 579 usap dubur penderita diare, 92 (16% memberikan hasil isolasi V. cholerae 01 biotipe El Tor dan 34 (6% V. cholerae non-01. Semua isolat V. cholerae 01 El Tor menunjukkan reaksi CAMP positif kuat dengan gambaran hemolisis sinergistik lengkap berbentuk sosis; sedangkan V. cholerae non-01 memberikan reaksi CAMP yang sempit dengan pola hemolisis menyerupai bulan sabit. Hasil uji CAMP yang dilakukan bersama dengan reaksi biokimiawi sesuai dengan metode biakan konvensional yang menyertakan tes aglutinasi dengan antiserum V. cholerae 01 untuk mengidentifikasi V. cholerae.

  13. Misidentification of Vibrio cholerae O155 isolated from imported shrimp as O serogroup O139 due to cross-agglutination with commercial O139 antisera

    DEFF Research Database (Denmark)

    Dalsgaard, A.; Mazur, J.; Dalsgaard, Inger


    Fish and shellfish products imported into Denmark are routinely analyzed for pathogenic Vibrio spp., particularly Vibrio cholerae, if products originate from subtropical or tropical areas. A V. cholerae strain that agglutinated commercial O139 antiserum but not the O1, Inaba, or Ogawa antisera...... was isolated from imported raw frozen shrimp. The toxigenicity of the strain was analyzed, and the results of a polymerase chain reaction showed that the V. cholerae strain did not contain the virulence genes ctx, tcp9, and zot, which are normally found in V. cholerae O1 and O139. The strain was resistant...

  14. Human Immune Responses to Dengue Viruses. (United States)


    FA titer of these antisera. We found using these hyper- immunized murine ascitis fluids that the homologous antiserum was most active in augmenting...statistically significant (pɘ.05). CHyperimmune mouse ascitis fluid was used as a source of anti-dengue 2 anti- body at a 1:20 dilution. PBL without anti-dengue 2 antibody. *statistically significant (pɘ.05), l1not significant. bHyperimmune mouse ascitis fluid was used as a source

  15. Chemical composition of phase I Coxiella burnetii soluble antigen prepared by trichloroacetic acid extraction. (United States)

    Lukácová, M; Brezina, R; Schramek, S; Pastorek, J


    Optimal conditions of extraction (time and temperature) by trichloroacetic acid of soluble antigen from phase I Coxiella burnetii (TCAE), possessing protective properties and used as a chemovaccine against Q fever in men, were studied. Extracts prepared under various conditions were analysed for their polysaccharide, protein and phosphorus contents. Forty-five min of extraction at 0 degrees C were sufficient to obtain a soluble antigen reacting in immunodiffusion with hyperimmune rabbit antiserum. The polysaccharide contents decreased with prolonged extraction at 0 degrees C. At higher extraction temperatures (37 and 100 degrees C), the polysaccharide contents increased while that of proteins decreased. TCAE prepared at 100 degrees C gave no positive immunodiffusion reaction.

  16. Bovine coronavirus hemagglutinin protein. (United States)

    King, B; Potts, B J; Brian, D A


    Treatment of purified bovine coronavirus (Mebus strain) with pronase destroyed the integrity of virion surface glycoproteins gp140, gp120, gp100, reduced the amount of gp26 and destroyed the hemagglutinating activity of the virus. Bromelain, on the other hand, destroyed the integrity of gp120, gp100 and gp26 but failed to remove gp140 and failed to destroy viral hemagglutinating activity. These experiments suggest that gp140 is the virion hemagglutinin. Immunoblotting studies using monospecific antiserum demonstrate that gp140 is a disulfide-linked dimeric structure reducible to monomers of 65 kDa.

  17. Laboratoire de Chimie Bactérienne C.N.R.S., Marsielle, France. (United States)

    Chippaux, M; Giudici, D; Abou-Jaoudé, A; Casse, F; Pascal, M C


    Mutants of E. coli, completely devoid of nitrite reductase activity with glucose or formate as donor were studied. Biochemical analysis indicates that they are simultaneously affected in nitrate reductase, nitrite reductase, fumarate reductase and hydrogenase activities as well as in cytochrome C552 biosynthesis. The use of an antiserum specific for nitrate reductase shows that the nitrate reductase protein is probably missing. A single mutation is responsible for this phenotype: the gene affected, nir R, is located close to tyr R i.e. at 29 min on the chromosomal map.

  18. Reticuloendotheliosis virus: Detection of immunological relationship to mammalian type C retroviruses. [/sup 125/I tracer technique

    Energy Technology Data Exchange (ETDEWEB)

    Charman, H.P.; Gilden, R.V.; Oroszlan, S.


    Reticuloendotheliosis virus (REV) p30 shares cross-reactive determinants and a common NH/sub 2/-terminal tripeptide with mammalian type C viral p30's. An interspecies competition radioimmunoassay was developed, using iodinated REV p30 and a broadly reactive antiserum to mammalian virus p30's. The avian leukosis-sarcoma viruses and mammalian non-type C retroviruses did not compete in this assay. Previous data indicating that the REV group is not represented completely in normal avian cell DNA lead us to speculate that this may be the first example of interclass transmission, albeit in the remote past, among the Retroviridae.

  19. The light organ symbiont Vibrio fischeri possesses a homolog of the Vibrio cholerae transmembrane transcriptional activator ToxR.


    Reich, K A; Schoolnik, G K


    A cross-hybridizing DNA fragment to Vibrio cholerae toxR was cloned from the nonpathogenic light organ symbiont Vibrio fischeri, and three proteins homologous to V. cholerae ToxR, ToxS, and HtpG were deduced from its DNA sequence. V. fischeri ToxR was found to activate a V. cholerae ToxR-regulated promoter, and an antiserum raised against the amino-terminal domain of V. cholerae ToxR cross-reacts V. fischeri ToxR.

  20. Novel /sup 125/I radioimmunoassay for the analysis of. delta. /sup 9/-tetrahydrocannabinol and its metabolites in human body fluids

    Energy Technology Data Exchange (ETDEWEB)

    Law, B.; Mason, P.A.; Moffat, A.C.; King, L.J.

    A cannabinoid radioimmunoassay (RIA) that detects some of the major 9/-THC metabolites is developed and evaluated for use in forensic science. It incorporates a novel /sup 125/I radiotracer, is sensitive, reliable, relatively quick, and simple to use. The RIA uses a commercially available antiserum and detects a number of cannabinoid metabolites, including 9/-THC-11-oic acid and its glucuronide conjugate in biological fluids. The method was successfully applied to the analysis of blood and urine samples submitted for forensic analysis.

  1. Loss of high-molecular-weight cytokeratin antigenicity in prostate tissue obtained by transurethral resections

    DEFF Research Database (Denmark)

    Multhaupt, H A; Fessler, J N; Warhol, M J


    OBJECTIVE: Staining of prostatic basal cells for the expression of high-molecular-weight cytokeratin has been suggested as a way of distinguishing benign from malignant prostate glands. We evaluated the utility of high-molecular-weight cytokeratin in the diagnosis of malignancy in prostate...... specimens obtained in various ways. DESIGN: Prostate tissues obtained from needle biopsies, transurethral resections, and total prostatectomies were immunostained with monoclonal antibody 34betaE12, an antibody directed against high-molecular-weight cytokeratins. RESULTS: Antiserum to high...

  2. Radioimmunoassay of sodium cromoglycate in human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Brown, K.; Gardner, J.J.; Lockley, W.J.S.; Preston, J.R.; Wilkinson, D.J. (Fisons plc, Loughborough (UK). Pharmaceutical Div.)


    A sensitive radioimmunoassay method for sodium cromoglycate in human plasma is described. The lowest quantifiable concentration of sodium cromoglycate is 0.93 nmol/l when 0.1 ml plasma samples are analysed. The range of the method is limited; both 0.01 and 0.1 ml volumes of plasma must be analysed to encompass the concentration range 0.93-139 nmol/l which may be encountered in plasma samples from patients and human volunteers. The method is specific for sodium cromoglycate as indicated by a low cross-reactivity of the anti-cromoglycate antiserum with a number of drugs.

  3. An immunohistochemical study of Flexibacter psychrophilus infection in experimentally and naturally infected rainbow trout (Oncorhynchus mykiss) fry

    DEFF Research Database (Denmark)

    Evensen, O.; Lorenzen, Ellen


    was compared in naturally and experimentally (intraperitoneal injections) infected fry by use of immunohistochemistry. This study showed that F. psychrophilus could be detected in paraffin-wax-embedded tissue of rainbow trout fry by immunohistochemistry. The principal immunohistochemical findings in naturally......An immunohistochemical method is described for the detection of Flexibacter psychrophilus in formalin-fixed, parafiin-wax-embedded fry of rainbow trout. Rabbit antiserum as well as rainbow trout hyperimmune serum were used in the study. The distribution and tissue localization of the bacterium...

  4. Investigating the Functional Role of Prostate-Specific Membrane Antigen and Its Enzymatic Activity in Prostate Cancer Metastasis (United States)


    DOD founding, previously, we successfully generated several photo -stable fluorescent probes (Cy3, Cy5, Alexa488, Alexa546 and Alexa633)-conjugated...M.P., V.M., S.P., U.A., and S.X.L. performed research; C.S.G. and Z.Y. contributed new reagents/analytic tools ; M.P., K.J.W., and E.A.F. analyzed data...Sigma. [35S]methionine/cysteine ([35S]met/cys) was from Perkin–Elmer. Collage - nase was from Worthington Biochemical. Rabbit polyclonal antiserum against

  5. GnRH-II receptor-like antigenicity in human placenta and in cancers of the human reproductive organs. (United States)

    Eicke, Nicola; Günthert, Andreas R; Viereck, Volker; Siebold, Doreen; Béhé, Martin; Becker, Tamara; Emons, Günter; Gründker, Carsten


    We have recently demonstrated that the antiproliferative activity of GnRH-II on human endometrial and ovarian cancer cell lines is not mediated through the GnRH-I receptor. A functional receptor for human GnRH-II has not yet been identified. In this study, we have generated a polyclonal antiserum to the putative human GnRH-II receptor using a peptide (YSPTMLTEVPPC) corresponding to the third extracellular domain coupled to keyhole limpet haemocyanin via the Cys residue. A database search showed no identical peptide sequences in any other human gene. To avoid cross-reactions against two similar amino acid sequences the antiserum was pre-absorbed using these peptides. Immune histological sections of human placenta and human endometrial, ovarian and prostate cancers using rabbit anti-human GnRH-II receptor antiserum showed GnRH-II receptor-like staining. Western blot analysis of cell membrane preparations of human endometrial and ovarian cancer cell lines yielded a band at approximately 43 kDa whereas Western blot analysis of cell membrane preparations of ovaries obtained from the marmoset monkey (Callithrix jacchus) yielded a band at approximately 54 kDa. To identify the GnRH-II receptor-like antigen we used the photo-affinity labelling technique. Photochemical reaction of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II (10(-9) M) with cell membrane preparations of human endometrial and ovarian cancer cells yielded a band at approximately 43 kDa. In competition experiments, the GnRH-I agonist Triptorelin (10(-7) M) showed a weak decrease of (125)I-labelled (4-azidobenzoyl)-N-hydroxysuccinimide-[d-Lys(6)]-GnRH-II binding to its binding site. The GnRH-I antagonist Cetrorelix (10(-7) M) showed a clearly stronger decrease, whereas GnRH-II agonist [d-Lys(6)]-GnRH-II (10(-7) M) was the most potent competitor. Western blot analysis of the same gel using rabbit anti-human GnRH-II receptor antiserum identified this band as GnRH-II receptor

  6. Produção e purificação de anticorpos policlonais para Salmonella Enteritidis (Enterobacteriaceae Production and purification of polyclonal antibodies for Salmonella Enteritidis (Enterobacteriaceae

    Directory of Open Access Journals (Sweden)

    Mario Augusto Ono


    Full Text Available O objetivo deste trabalho foi produzir e purificar anticorpos policlonais específicos para Salmonella Enteritidis (Enterobacteriaceae. O anti-soro foi produzido em coelhos, empregando-se flagelina purificada. O título e a especificidade foram determinados através do ensaio imunoenzimático - ELISA e a purificação por cromatografia de afinidade com sepharose Proteína A. As suspensões bacterianas foram cultivadas em cinco diferentes meios de cultura (infusão de cérebro coração - BHI, caldo tripticase soja, caldo lactosado, caldo nutriente - CN e água peptonada. Observou-se que dependendo do meio o título do anti-soro pode variar e os melhores resultados foram obtidos com BHI e CN. O anti-soro foi específico para Salmonella Enteritidis, apresentando porcentagens de reações cruzadas com Salmonella Typhimurium, Salmonella Infantis e Salmonella Newport de 16,0, 11,9 e 6,4%, respectivamente. Menores porcentagens foram obtidas com outras enterobactérias testadas. Esses resultados indicam a possibilidade da utilização desses anticorpos na padronização de ensaios imunológicos para a detecção de Salmonella EnteritidisThe purpose of this study was to produce and to purify specific polyclonal antibodies for Salmonella Enteritidis (Enterobacteriaceae. The anti-serum was raised in rabbits using purified flagelin. Anti-serum titer and specificity were determined by an immunoassay - ELISA and its purification was performed by sepharose protein A affinity chromatography. The bacteria suspensions were cultivated in five different media (brain heart infusion - BHI, tripticase soy broth, nutrient broth - NB, peptone water. Results have showed that anti-serum titers varied depending on which media type was used and BHI and NB media yielded the most significant results. The anti-serum produced was specific for Salmonella Enteritidis. Its cross-reactivity with Salmonella Thyphimurium, Salmonella Infantis and Salmonella Newport were 16.0, 11

  7. Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening


    DellaPenna, Dean; Alexander, Danny C.; Bennett, Alan B


    The expression of a gene encoding the cell wall-degrading enzyme polygalacturonase [poly(1,4-α-D-galacturonide) glucanohydrolase, EC] was characterized during tomato fruit ripening. Polygalacturonase was purified from ripe tomato fruit and used to produce highly specific antiserum. Immunoblot analyses detected a 45- and a 46-kDa protein in ripe fruit but immunoprecipitation of in vitro translation products of mRNA from ripe tomato fruit yielded a single 54-kDa polypeptide, suggesting...

  8. Sarcocystis canis n. sp. (Apicomplexa: Sarcocystidae), the etiologic agent of generalized coccidiosis in dogs. (United States)

    Dubey, J P; Speer, C A


    Sarcocystis canis n. sp. is proposed for the protozoon associated with encephalitis, hepatitis, and generalized coccidiosis in dogs. Only asexual stages are known in macrophages, neurons, dermal, and other cells of the body. The parasite is located free in the host cell cytoplasm without a parasitophorous vacuole; schizonts divide by endopolygeny. Schizonts are 5-25 x 4-20 microns and contain 6-40 merozoites. Merozoites are approximately 5-7 microns x 1 micron and do not contain rhoptries. The parasite is PAS-negative and reacts with Sarcocystis cruzi antiserum but not with Toxoplasma gondii, Neospora caninum, or Caryospora bigenetica antisera in an immunohistochemical test.


    Flexner, S; Amoss, H L


    Claims of efficiency have been made at two widely separated periods for the chemical treatment of epidemic meningitis, in the first instance for lysol and in the second for protargol. The use of lysol was long since abandoned; the recommendation for protargol is based on a single series of cases, small in number. Because of the variable severity of epidemics of meningitis, small reliance can be placed on results of treatment limited in extent to small numbers of cases and to one locality. A more uniform and accurate measure of the value of a method of treatment is provided by animals infected experimentally with pathogenic cultures of meningococci. Young guinea pigs respond in a definite manner to intraperitoneal inoculation of virulent meningococci. Neither protargol nor lysol proved to have any curative action on the experimental infection thus produced in these animals. Monkeys respond in a characteristic manner to the inoculation of virulent cultures into the subarachnoid space. Protargol displayed no curative action on the experimental infection thus produced in these animals. On the contrary, both lysol and protargol exert antileukotactic and antiphagocytic effects, and are also potent protoplasmic poisons, and the leukocytes with which they come in contact are injured and made to degenerate. According to the extent to which these harmful properties are exerted, the chemicals promote the advance rather than restrain the progress of meningococcic infection. Recovery from meningococcic infection in man and animals is accomplished chiefly through the process of phagocytosis. The specific antiserum acts curatively by increasing the emigration of leukocytes, by promoting phagocytosis directly, and by agglutinating the meningococci, and also by neutralizing endotoxin. Any means which interfere with and reduce these essential processes retard or prevent recovery. Both lysol and protargol interfere with and diminish the emigration of leukocytes and the phagocytosis

  10. Immunochemical and biological quantification of peanut extract

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Pedersen, Mona H; Platzer, Michael;


    Biological standardization of allergen extracts is one of the steps in the characterization of an extract. The gold standard for determination of biological potency is the skin prick test, but histamine release (HR) has been used as a convenient ex vivo method for analyzing a large number...... of samples. We describe the use of rabbit basophils as a tool in biological standardization. Using peanut as a model allergen, it is described how rabbits immunized for production of antiserum may become sensitized and their basophils used for histamine release experiments. It is also possible to use rabbit...

  11. Studies on the surface coat of Paramecium aurelia. II. Relationship to the immobilization antigen. (United States)

    Wyroba, E


    Correlations between the presence of surface coat and immobilization antigen of Paramecium tetraurelia were studied. Supravital, partial removal of the surface coat resulted in accelerated response of monobacterially and axenically grown cells to the homologous antiserum. Ciliates pretreated with trypsin or pronase (0.5 mg/ml for 45 min at 0-4 degrees C) were immobilized approximately twice as fast as untreated control cells. The probable localization of at least part, of the immobilization antigen within the surface coat of P. tetraurelia is discussed.

  12. Development and characterization of new polyclonal antibodies specific for three polychlorinated biphenyls

    Institute of Scientific and Technical Information of China (English)

    Han Yu Chen; Hui Sheng Zhuang; Chun Zhou


    Three polychlorinated biphenyls(PCBs)congeners and their corresponding haptens bearing four carbon length carboxylic groups that can be linked to a protein for raising antibodies were synthesized.The three resultant immunogens were fabricated and used to stimulate immune responses in rabbits to survey the characteristics of the haptens.Three of the resultant polyclonal antibodies(Pabs)were obtained.The antiserum exhibited relatively high antibody titres(1:32-64)in double agar diffusion.◎2008 Hui Sheng Zhuang.Published by Elsevier B.V.on behalf of Chinese Chemical Society.All rights reserved.

  13. Isolation of L-3-phenyllactyl-Leu-Arg-Asn-NH2 (Antho-RNamide), a sea anemone neuropeptide containing an unusual amino-terminal blocking group

    DEFF Research Database (Denmark)

    Grimmelikhuijzen, C J; Rinehart, K L; Jacob, E


    -phenyllactyl-Leu-Arg-Asn-NH2. By using reversed-phase HPLC and a chiral mobile phase, it was shown that the 3-phenyllactyl group had the L configuration. Immunocytochemical staining with antiserum against Arg-Asn-NH2 showed that L-3-phenyllactyl-Leu-Arg-Asn-NH2 (Antho-RNamide) was localized in neurons of sea...... anemones. The L-3-phenyllactyl group has not been found earlier in neuropeptides of vertebrates or higher invertebrates. We propose that this residue renders Antho-RNamide resistant to nonspecific aminopeptidases, thereby increasing the stability of the peptide after neuronal release....


    Institute of Scientific and Technical Information of China (English)

    HE Qian; ZHANG Shu-qun; CHU Yong-lie; JIA Xiao-li; JIANG Jian-tao


    Objective To investigate the marker protein of human breast carcinoma from nuclear matrix proteins (NMPs).Methods NMPs were injected subcutaneously into rabbit to get antiserum, which was used to detect the NMPs specificity for breast carcinoma.Results There was an apparent positive band (100kD) in the NMPs of breast carcinoma, which did not exist in normal breast and other tumors that were detected.Conclusion One or one group of 100kD NMPs were found to be related to human breast carcinoma, which may be involved in the carcinogenesis and development of human breast carcinoma and valuable for breast carcinoma diagnosis.

  15. Triatomíneos rupestres infectados por Trypanosomatidae, coletados em Quaraí, Rio Grande do Sul, 2003 Rupestrian triatomines infected by Trypanosomatidae, collected in Quaraí, Rio Grande do Sul, 2003

    Directory of Open Access Journals (Sweden)

    Luciamáre Perinetti Alves Martins


    Full Text Available Foram capturados triatomíneos rupestres em seis localidades do município de Quaraí-RS, com o intuito de verificar o índice de infecção por Trypanosomatidae, bem como o animal reservatório. A captura ocorreu no ambiente silvestre, sendo coletados 453 exemplares, os quais foram identificados e separados por estádio ninfal. Coletaram-se 421 (92,9% exemplares de Triatoma rubrovaria, 26 (5,7% de Triatoma circummaculata e 6 (1,3% de Panstrongylus tupynambai. Dentre as três espécies coletadas, somente Triatoma rubrovaria mostrou-se positivo para Trypanosomatidae, num total de 13 (4,2% exemplares. Após a inoculação em camundongos e meio de cultura LIT, isolaram-se cinco cepas de Trypanosoma cruzi. Dos triatomíneos infectados com Trypanosomatidae, 4 (30,8% tiveram a reação de precipitina não reagente para os anti-soros testados, 4 (30,8% positivos para anti-soro de roedor, 4 (30,8% para anti-soro de cabra e 1 (7,7% para anti-soro de porco e humano.Rupestrian triatomines were captured in six Quaraí city localities, RS, to verify the level of Trypanosomatidae infection, as well as the animal reservoir. The capture occurred in a wild environment and 453 samples were collected, which were identified and separated by nymphal instar. 421 (92.9% samples of Triatoma rubrovaria, 26 (5.7% of Triatoma circummaculata and 6 (1.3% of Panstrongylus tupynambai were collected. Only 13 samples (4.2% of Triatoma rubrovaria presented Trypanosomatidae infection. After mice and LIT culture inoculation, five strains of Trypanosoma cruzi were isolated. Of these triatomines, 4 (30.8% displayed no reagent precipitin for the antiserum tested, 4 (30.8% were positive for rodent antiserum, 4 (30.8% were positive for goat antiserum and 1 (7.7% were positive for human and pig antiserum.

  16. Counter-immunoelectro-osmophoresis for the detection of infantile gastroenteritis virus (orbi-group) antigen and antibody. (United States)

    Middleton, P J; Petric, M; Hewitt, C M; Szymanski, M T; Tam, J S


    A moderatley sensitive, rapid, and economical test scheme for the detection of infantile gastroenteritis virus (IGV) in stool or antibody in serum has been developed and evaluated. The test scheme with minor modifications was an adaptation of a counter-immunoelectro-osmophoresis system we once used for the detection of hepatitis B antigen. Large numbers of stool samples may be screened during half a working day for the presence of IGV using reference antiserum to IGV prepared in guinea-pigs. Serological studies of a diagnostic but not epidemiological nature may also be performed with equal facility by this same test scheme using highly purified IGV antigen derived from stool.

  17. Cloning, expression and immunogenicity of the avian pneumovirus (Colorado isolate) F protein. (United States)

    Tarpey, I; Huggins, M B; Davis, P J; Shilleto, R; Orbell, S J; Cook, J K


    The F protein of the Colorado isolate of avian pneumovirus (APV), expressed from a DNA plasmid, was recognized by antiserum to both A and B subgroup APVs. After two intramuscular injections of turkeys with this plasmid, a homologous antibody response was detected by enzyme-linked immunosorbent assay. This antibody also recognized subgroup A APV. However, there was no neutralization of the Colorado isolate or of subgroup A or B viruses. Although no significant clinical protection was detected following homologous challenge of poults, an anamnestic serological response was seen, suggesting that a systemic antibody response but no local mucosal immunity was induced.

  18. Preparation and purification of 7-Iodoclonazepam for use in radioimmunoassay

    Energy Technology Data Exchange (ETDEWEB)

    Goddard, C.P.; Law, B.; Mason, P.A.; Stead, A.H.


    A method is described for the preparation and purification of 7-(/sup 125/I)-Iodoclonazepam (5-(o-Chlorophenyl)-2,3-Dihydro-7-(/sup 125/I)-Iodo-1H-1,4-Benzodiazepin-2-one). The structure was confirmed by mass spectrometry using 7-(/sup 127/I)iodoclonazepam prepared by the same method. 7-(/sup 125/I)-Iodoclonazepam binds well to a benzodiazepine antiserum. Although readily displaced by all the benzodiazepines commercially available in the UK, it is not displaced by structurally related nonbenzodiazepines except at very high concentrations. 7-(/sup 125/I) Iodoclonazepam should therefore be useful for the development of a screening radioimmunoassay (RIA) for benzodiazepines.

  19. Partial biological and molecular characterization of a Cucumber mosaic virus isolate naturally infecting Cucumis melo in Iran. (United States)

    Rasoulpour, Rasoul; Afsharifar, Alireza; Izadpanah, Keramat


    Melon seedlings showing systemic chlorotic spots and mosaic symptoms were collected in central part of Iran, and a virus was isolated from diseased plants by mechanical inoculation. The virus systemically infected the most inoculated test plants by inducing mosaic symptoms, while, in the members of Fabaceae family and Chenopodium quinoa induced local lesions. Agar gel diffusion test using a polyclonal antiserum against a squash Cucumber mosaic virus (CMV) isolate showed the presence of CMV in the mechanically inoculated plants (designated CMV-Me). The virus was purified by polyethylene glycol precipitation and differential centrifugation. A polyclonal antiserum was produced against the virus that reacted specifically with virus antigen in ELISA and agar gel diffusion tests. The virus was molecularly characterized by PCR amplification of the full length of the coat protein gene using cucumovirus genus specific primer pair CPTALL-3/CPTALL-5 and sequence analysis of the resulting product. No RNA satellite was detected using the primer pair CMVsat3H/sat5T7P. Phylogenetic analysis based on the coat protein amino acid sequences showed that CMV-Me belongs to Subgroup IB. These results may be helpful in melon breeding programs, focusing on plant resistance to plant viruses including CMV.

  20. Immunoradiometric assay for the determination of E. coli proteins in recombinant dna derived human growth hormone produced at IPEN-CNEN/SP; Ensaio imunorradiometrico para a determinacao de proteinas bacterianas contaminantes em lotes de hormonio de crescimento humano recombinante produzido no IPEN-CNEN/SP

    Energy Technology Data Exchange (ETDEWEB)

    Soares, Carlos R.J.


    An immunoradiometric assay (IRMA) for the determination of multiple antigens was set up in order to quantify E. coli (ECP) in lots of purified recombinant human growth hormone (rec-hGH). SDS-PAGE and Western Blotting techniques were carried out, in parallel, to confirm the results obtained by IRMA and to provide more information about the contaminants. Anti-ECP antibodies were obtained by rabbit immunization with ECP, which were submitted to the same purification process utilized for rec-hGH with the exception of the last step. A strain-process-specific assay was thus set up. The antiserum obtained was purified through an affinity column prepared with the same ECP used for immunization, this provided an highly sensitive assay (0,03 ng ECP/mL). This IRMA was shown to be specific, not presenting any cross reaction with hGH and studies carried out on precision, accuracy and linearity of response with dilution confirmed its validity as one of the fundamental purity tests for rec-hGH produced at IPEN-CNEN/SP, whose principles can be easily extended to the analysis of other similar products. These studies have also shown that the utilization of an affinity column, prepared with the described anti-ECP antiserum was very effective, providing rec-hGH lots with less then 10 parts per million (0,001%) of contaminating proteins. (author). 45 refs., 15 figs., 11 tabs.

  1. Localization of motilin-immunopositive cells in the rat intestine by light microscopic immunocytochemistry. (United States)

    Sakai, T; Satoh, M; Koyama, H; Iesaki, K; Umahara, M; Fujikura, K; Itoh, Z


    Motilin-immunopositive cells (Mo cells) are known to exist in the upper small intestine of many species including man. However, the possible presence of Mo cells in the rat gastrointestine has remained obscure because antiserum against it raised in rabbit was found not to cross-react with motilin in the rat gastrointestine. The present study was designed to investigate the distribution of Mo cells in the rat gastrointestine by the peroxidase-conjugated second antibody method using newly raised chicken anti-motilin serum (CPV3). This antiserum was suggested to recognize the N-terminal region of the motilin molecule by enzyme-linked immunosorbent assays and immunocytochemical absorption test. Mo cells detected in the rat gastrointestine by immunocytochemistry were found to be distributed in the duodenum (1.5 cells/mm2), jejunum (2.2 cells/mm2), and ileum (0.028 cells/mm2), and no positive cells were found in the gastric body, gastric antrum, cecum, colon, or pancreas. The immunopositive cells in the rat intestine were spindle shaped or polygonal, scattered throughout the epithelium of the villi and crypts, and similar to those commonly observed in the upper small intestine of other species. These results indicate for the first time that motilin-immunopositive cells do exist in the rat intestine.

  2. cDNAs encoding for storage proteins in the tubers of taro (Colocasia esculenta Schott). (United States)

    Hirai, M; Nakamura, K; Imai, T; Sato, T


    Two major protein groups of taro (Colocasia esculenta) tuber were purified, and their antisera were used for the screening of the cDNA library constructed from poly(A)+ RNA of taro tuber. A cDNA clone obtained by screening with an anti-12 kD protein antiserum had an insert 1058 bp-long, and an open reading frame for a peptide of 268 amino acids. The analyses of the N-terminal amino acid sequence and in vitro translation product suggested that the protein was synthesized as a peptide with a molecular weight of 27 kD, and then processed into two mature peptides with a molecular weight of 12.5 and 13.9 kD and an extra peptide with a molecular weight of 0.6 kD. The cDNA clones obtained using the anti-25 kD protein antiserum were highly homologous with each other. One of them had an insert 958 bp-long and an open reading frame for a peptide with 209 amino acids. The amino acid sequence deduced from the nucleotide sequence of this clone indicated that the 25 kD proteins were homologous to the trypsin inhibitors of soybean and winged bean as well as sporamins, the storage proteins of sweet potato.

  3. Production of adenosine from extracellular ATP at the striatal cholinergic synapse. (United States)

    James, S; Richardson, P J


    The components of the ectonucleotidase pathway at the immunoaffinity-purified striatal cholinergic synapse have been studied. The ecto-ATPase (EC had a Km of 131 microM, whereas the ecto-ADPase (EC had a Km of 58 microM, was Ca(2+)-dependent, and was inhibited by the ATP analogue 5'-adenylylimidodiphosphate (AMPPNP). The ecto-5'-nucleotidase (EC had a Km of 21 microM, was inhibited by AMPPNP and alpha,beta-methylene ADP, and by a specific antiserum. The Vmax values of the ATPase, ADPase, and 5'-nucleotidase enzymes present at this synapse were in a ratio of 30:14:1. Very little ecto-adenylate kinase activity was detected on these purified synapses. The intraterminal 5'-nucleotidase enzyme, which amounted to 40% of the total 5'-nucleotidase activity, was inhibited by AMPPNP, alpha,beta-methylene ADP, and the antiserum, and also had the same kinetic properties as the ectoenzyme. The time course of ATP degradation to adenosine outside the nerve terminals showed a delay, followed by a period of sustained adenosine production. The delay in adenosine production was proportional to the initial ATP concentration, was a consequence of feedforward inhibition of the ADPase and 5'-nucleotidase, and was inversely proportional to the ecto-5'-nucleotidase activity. The function and characteristics of this pathway and the central role of 5'-nucleotidase in the regulation of extraterminal adenosine concentrations are discussed.

  4. Purification of tracer for somatomedin C radioimmunoassay by hydrophobic interaction chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Baxter, R.C.; Brown, A.S.


    A tracer for use in the somatomedin C radiommunoassay by hydrophobic interaction chromatography was purified. Material showing greatest immunoreactivity binds to Octyl Sepharose CL-4B (Pharmacia) in a buffer mixture consisting of 130 mL of acetonitrile and 870 mL of 0.1 mol/L NH/sub 4/HCO/sub 3/, pH 7.8, but is eluted by increasing the acetonitrile content to 180 mL/L. As compared with tracer purified by binding to specific antiserum in liquid phase, precipitating the complex with second antibody, and then dissociating by gel chromatography at acid pH, this tracer shows equal immunoreactivity against specific somatomedin C antiserum. Either preparation allows excellent discrimination between extracts of normal, acromegalic, and hypopituitary plasma samples; thus either is suitable for use in the somatomedin C radioimmunoassay. Tracer purification by hydrophobic interaction chromatography is rapid and inexpensive. It may be useful in preparing highly immunoreactive tracers for other peptide radioimmunoassays.

  5. DTDP-rhamnosyl transferase RfbF, is a newfound receptor-related regulatory protein for phage phiYe-F10 specific for Yersinia enterocolitica serotype O:3. (United States)

    Liang, Junrong; Li, Xu; Zha, Tao; Chen, Yuhuang; Hao, Huijing; Liu, Chang; Duan, Ran; Xiao, Yuchun; Su, Mingming; Wang, Xin; Jing, Huaiqi


    Bacteriophages and their hosts are continuously engaged in evolutionary competition. Here we isolated a lytic phage phiYe-F10 specific for Yersinia enterocolitica serotype O:3. We firstly described the phage receptor was regulated by DTDP-rhamnosyl transferase RfbF, encoded within the rfb cluster that was responsible for the biosynthesis of the O antigens. The deletion of DTDP-rhamnosyl transferase RfbF of wild type O:3 strain caused failure in phiYe-F10 adsorption; however, the mutation strain retained agglutination with O:3 antiserum; and complementation of its mutant converted its sensitivity to phiYe-F10. Therefore, DTDP-rhamnosyl transferase RfbF was responsible for the phage infection but did not affect recognition of Y. enterocolitica O:3 antiserum. Further, the deletions in the putative O-antigen biosynthesis protein precursor and outer membrane protein had no effect on sensitivity to phiYe-F10 infection. However, adsorption of phages onto mutant HNF10-ΔO-antigen took longer time than onto the WT, suggesting that deletion of the putative O-antigen biosynthesis protein precursor reduced the infection efficiency.

  6. Antigenic homogeneity of male Müllerian gland (MG) secretory proteins of a caecilian amphibian with secretory proteins of the mammalian prostate gland and seminal vesicles: evidence for role of the caecilian MG as a male accessory reproductive gland. (United States)

    Radha, Arumugam; Sree, Sreesha; Faisal, Kunnathodi; Kumar, G Pradeep; Oommen, Oommen V; Akbarsha, Mohammad A


    Whereas in all other vertebrates the Müllerian ducts of genetic males are aborted during development, under the influence of Müllerian-inhibiting substance, in the caecilian amphibians they are retained as a pair of functional glands. It has long been speculated that the Müllerian gland might be the male accessory reproductive gland but there has been no direct evidence to this effect. The present study was undertaken to determine whether the caecilian Müllerian gland secretory proteins would bear antigenic similarity to secretory proteins of the prostate gland and/or the seminal vesicles of a mammal. The secretory proteins of the Müllerian gland of Ichthyophis tricolor were evaluated for cross-reactivity with antisera raised against rat ventral prostate and seminal vesicle secretory proteins, adopting SDS-PAGE, two-dimensional electrophoresis and immunoblot techniques. Indeed there was a cross-reaction of five Müllerian gland secretory protein fractions with prostatic protein antiserum and of three with seminal vesicle protein antiserum. A potential homology exists because in mammals the middle group of the prostate primordia is derived from a diverticulum of the Müllerian duct. Thus this study, by providing evidence for expression of prostatic and seminal vesicle proteins in the Müllerian gland, substantiates the point that in caecilians the Müllerian glands are the male accessory reproductive glands.

  7. Structural studies of the vacuolar membrane ATPase from Neurospora crassa and comparison with the tonoplast membrane ATPase and Zea mays

    Energy Technology Data Exchange (ETDEWEB)

    Bowman, E.J.; Mandala, S.; Taiz, L.; Bowman, B.J.


    The H translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of M/sub r/ approx. = 70,000 and approx. = 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-( UC)ethylmaleimide and 7-chloro-4-nitro( UC)benzo-2-oxa-1,3-diazole, labeled the M/sub r/ approx. = 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-( UC)dicyclohexylcarbodiimide labeled a polypeptide of M/sub r/ approx. = 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of M/sub r/ 5.2 x 10V, 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochrondrial ATPase but stongly crossreacted with antiserum against a polypeptide of M/sub r/ approx. = 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F0F1 ATPases.

  8. A radioimmunoassay for rat ghrelin: evaluation of method and effects of nonylphenol on ghrelin secretion in force-fed young rats. (United States)

    Chang, Yen-Jui; Huang, Wei-Ju; Lin, Han-Wei; Chang, Ling-Ling; Chang, Full-Young; Wang, Paulus S


    Antiserum YJC 13-31 against the rat ghrelin conjugated to bovine serum albumin (BSA) was produced in the rabbit and a double antibody radioimmunoassay (RIA) for ghrelin has been developed. Characterization results of this antiserum revealed no cross-reaction with human growth hormone and somatostatin. Weak cross-reactions with insulin (0.1%), rat growth hormone (0.1%) and glucagon (0.3%) were observed, which scarcely interfered the assay system. The sensitivity of this RIA was 5 pg per assay tube. With the rat serum samples, the within-assay precision was 7.1% and the between-assay precision was 12.3%. The RIA was also available to detect the ghrelin in rat tissue extracts with good parallelism to the rat ghrelin standard. In application, the serum ghrelin and corticosterone levels in weaned rats were measured by RIA. Gavage of saline was sufficient to raise serum ghrelin from 2.6 +/- 0.18 to 6.7 +/- 0.7 ng/ml (P ghrelin levels in a dose-dependent manner. Besides, gavages of saline elevated the serum levels of corticosterone from 108.8 +/- 13.5 to 188.7 +/- 23.5 ng/ml (P < 0.01) but the elevation effects of corticosterone from gavages were overcome by NP in the low dose of 50 mg/kg. It can be speculated that ingestion of NP is harmful to young animals during growth and environmental adaptation.

  9. Radioimmunoassay of fragment E-related neoantigen: validation studies and clinical application. [Fibrinogen-fibrin degradation products

    Energy Technology Data Exchange (ETDEWEB)

    Chen, J.P.; Hanna, W.T.; Williams, T.K.; Krauss, S. (Tennessee Univ., Knoxville (USA). Memorial Research Center and Hospital)


    An E-neoantigen radioimmunoassay (Eneo RIA) is described which can determine normal and pathological plasma levels of E-related fibrinogen-fibrin degradation products (FDP). The assay employs rabbit antiserum produced against fragment E derived from a plasmin digest of fibrinogen and subsequently absorbed with fibrinogen. The absorbed antiserum contains antibodies which are equally reactive with fibrinogen derived E (Fg-E) and fibrin derived E(Fb-E) but not with fibrinogen at 1 mg/ml. The Eneo RIA was validated by assay parallelism and by recovery experiments. Plasma Eneo immunoreactivities in 14 normals were 4-22 ng/ml (mean 12.7 ng/ml). Plasma Eneo levels in 23 of 24 patients with neoplastic and haematological diseases were elevated above normal (range 27-2027 ng/ml). Unusually high Eneo values were observed with three patients whose diseases were complicated by either disseminated intravascular coagulation (DIC) or deep vein thrombosis. After heparin therapy, the Eneo level of a patient with chronic DIC declined. A pathological plasma was eluted from a Sephadex G-200 column and Eneo immunoreactivity was determined on the eluates. The gel filtration pattern of Eneo indicates that E-related FDP is a family of plasmic fragments derived from crosslinked fibrin.

  10. Hereditary cerebral hemorrhage with amyloidosis in patients of Dutch origin is related to Alzheimer disease

    Energy Technology Data Exchange (ETDEWEB)

    van Duinen, S.G.; Castano, E.M.; Prelli, F.; Bots, G.T.A.B.; Luyendijk, W.; Frangione, B.


    Hereditary cerebral hemorrhage with amyloidosis in Dutch patients is an autosomal dominant form of vascular amyloidosis restricted to the leptomeninges and cerebral cortex. Clinically the disease is characterized by cerebral hemorrhages leading to an early death. Immunohistochemical studies of five patients revealed that the vascular amyloid deposits reacted intensely with an antiserum raised against a synthetic peptide homologous to the Alzheimer disease-related ..beta..-protein. Silver stain-positive, senile plaque-like structures were also labeled by the antiserum, yet these lesions lacked the dense amyloid cores present in typical plaques of Alzheimer disease. No neurofibrillary tangles were present. Amyloid fibrils were purified from the leptomeningeal vessels of one patient who clinically had no signs of dementia. The protein had a molecular weight of approx. 4000 and its partial amino acid sequence to position 21 showed homology to the ..beta..-protein of Alzheimer disease and Down syndrome. These results suggest that hereditary cerebral hemorrhage with amyloidosis of Dutch origin is pathogenetically related to Alzheimer disease and support the concept that the initial amyloid deposition in this disorder occurs in the vessel walls before damaging the brain parenchyma. Thus, deposition of ..beta..-protein in brain tissue seems to be related to a spectrum of diseases involving vascular syndromes, progressive dementia, or both.


    Institute of Scientific and Technical Information of China (English)

    刘新峰; 金泳清; 陈光辉


    Mongolian gerbils were used as delayed neuronal damage (DND) animal models.At the end of 15 minute cerebral ischermia and at various reperfusion time ranging from 1 to 96 hours,the content of water and arginine vasopressin (AVP) in the CA1 sector of hippocampus were measured by the specific gravity method and radioimmunoassy.Furthermore,we also examined the effect of intracerebroventricular (ICV) injection of AVP,AVP antiserum on calcium,Na+,K+-ATP ase activity in the CA1 sector after ischemia and 96 hour reperfusion.The results showed that AVP Contents of CA1 sector of hippocampus during 6 to 96 hour recirculation,and the water content of CA1 sector during 24 to 96 hour were significantly and continuously increased.After ICV injection of AVP,the water content and calcium in CA1 sector of hippocampus at cerebral ischemia and 96 hour recirculation further increased,and the Na+,K+-AT-tion of AVP antiserum,the water contenr and calcium in CA1 sector were significantly decreased as compared with that of control.These suggested that AVP was involved in the pathopysiologic process of DND in hippocampus following cerbral ischemia and reprfusion.Its mechanism might be through the change of intracellular action mediated by specific AVP receptor to lead to Ca inos over-load of neuron and inhibit the Na+,K+-ATPase activity,thereby to exacerbate the DND in hippocampus.

  12. Cloning and characterization of nanos gene in silkworm Bombyx mori

    Institute of Scientific and Technical Information of China (English)

    Guoli Zhao; Keping Chen; Qin Yao; Weihua Wang


    Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Droso phila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5'-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mori and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5. 38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee (Apis mellifera). This gene has been registered in the GenBank under the accession number EF647589. One encoding se quence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.

  13. Development of a specific radioimmunoassay to measure physiological changes of circulating leptin in cattle and sheep. (United States)

    Ehrhardt, R A; Slepetis, R M; Siegal-Willott, J; Van Amburgh, M E; Bell, A W; Boisclair, Y R


    Studies of leptin in large domestic ruminants have been limited to measurements of gene expression because methods to measure circulating levels are not available. To develop a bovine leptin radioimmunoassay, we produced recombinant bovine leptin and used it to immunize rabbits, and to prepare bovine leptin tracer and standards. A single antiserum with sufficient affinity and titer was identified. Using this antiserum, logit-transformed binding of (125)I-labeled bovine leptin was linearly related (R(2)= 0.99) to the log of added bovine or ovine leptin between 0.1 to 2.0 ng. Serial dilution of bovine and ovine plasma, chicken serum and bovine milk gave displacement curves that were parallel to those of bovine or ovine leptin. Recoveries of external addition of bovine leptin in ewe and cow plasma ranged between 94 and 104%. Plasma leptin concentration measured by this assay was directly related to the plane of! nutrition in growing calves and lambs. At 11-14 weeks of age, ewe lambs had a higher circulating leptin concentration than ram lambs. Finally, plasma leptin concentration was linearly related to the fat content of the empty carcass in growing cattle and to body condition score in lactating dairy cows. We conclude that circulating leptin in sheep and cattle is increased by fatness and plane of nutrition, consistent with results in humans and rodents. This assay provides an important tool to investigate mechanisms that regulate plasma leptin in cattle and sheep.


    Directory of Open Access Journals (Sweden)

    S. Ullah, M. Ashfaque, S.U. Rahman, M. Akhtar and A. Rehman


    Full Text Available Two hundred intestines pieces (100 each of broilers and layers of about 8 cm length were collected from the poultry sale shops in Faisalabad city. These pieces were opened, scratched and vigorously shaken into sterilized normal saline, the suspension was centrifuged and supernatants were subjected to spot haemagglutination with 2% chicken RBC’s. Out of 200 samples, 95% samples of layers and 75% of the broilers showed positive spot haemagglutination. Micro haemagglutination inhibition with Newcastle disease (ND antiserum revealed, 85 and 66 samples positive in layers and broilers respectively. A total of 10% samples of the layers and 9% of the broilers were not inhibited by ND antiserum suggesting other HA viruses. A total of 20 samples were used to isolate the virus in embryonated eggs (allantoic route. These isolates were confirmed as NDV by haemagglutination inhibition test. Five isolates were tested for intracerebral pathogenicity index (ICPI in day old chicks. The ICPI values obtained were 0.28, 0.31, 0.37, 0.38 and 0.46. The isolates were found to be lentogenic.

  15. Identification of "tumor-associated" nucleolar antigens in human urothelial cancer. (United States)

    Yu, D; Pietro, T; Jurco, S; Scardino, P T


    Nucleoli isolated from HeLa S3 cells were used to produce rabbit antisera capable of binding nucleoli of transitional cell carcinomas (TCCa) of the bladder. Cross-reactivity of the rabbit antiserum with normal nucleoli was reduced by absorption with fetal calf serum, normal human serum, and human placental nucleoli. This antinucleolar antiserum exhibited strong reactivity in immunoperoxidase assays performed on specimens of human bladder cancer. In frozen tissue sections of 24 patients with TCCa and eight individuals without tumor, nucleolar staining was observed in all malignant specimens, but was not observed in seven of the normal specimens. Cytologic examination of bladder washing specimens from 47 normal individuals showed absence of nucleolar staining in 43 (91%) of 47 normal specimens while 12 (86%) of 14 specimens from patients with TCCa were positive. These results suggest that there are antigens associated with the nucleoli of HeLa cells and transitional cell carcinomas which are generally absent (or in low concentration) in normal human urothelial cells, and that antisera to these antigens may be useful in the cytologic diagnosis of human transitional cell carcinoma.

  16. Biochemical and immunological characterization of the microfibrillar ecto-endoplasmic boundary in the ciliate Isotricha prostoma. (United States)

    Vigues, B; Metenier, G; Groliere, C A


    The cytoplasm of the ciliated protozoan Isotricha prostoma is compartmented by a continuous fibrillar system made up of a double layer of 4 nm-diameter filaments: the microfibrillar ecto-endoplasmic boundary (EEB). Isolation of this structure after treatment of the cells in a buffer of low ionic strength in the presence of the detergent Triton X-100 evidenced connections linking the two filamentous layers. One dimensional electrophoresis on SDS-polyacrylamide gel of EEB fractions revealed several major proteins with apparent molecular weights between 11 and 23 K. Of these, two neighboring bands of MW22 and 23 K were removed from gels and used as antigens to obtain rabbit antibodies. The antiserum obtained reacted specifically with injected proteins as shown by the technique of immunological detection on nitrocellulose sheets using the peroxidase reaction product. Electron microscopy localization of the antigens with anti-IgG coupled with colloidal gold showed significant labeling of the EEB within the cortex of Isotricha permeabilized with Triton X-100. We hope that the 22-23 K antiserum will prove to be a useful tool for the comparative study of other non-actin filament systems in Protozoa.

  17. Large-scale purification of synaptophysin and quantification with a newly established enzyme-linked immunosorbent assay. (United States)

    Schlaf, G; Göddecke, M; Wolff, J R; Felgenhauer, K; Mäder, M


    Synaptophysin (SYP I), an integral membrane protein, was purified on a large scale (0.55 - 2.7 mg) from isolated small synaptic vesicles (SSV) of porcine cortex. In order to achieve this, a conventional purification procedure which consists of size exlusion chromatography, hydrophobic interaction chromatography and chromatofocusing has been developed. This procedure was compared with purification of SYP I by immunoaffinity chromatography. The elution patterns of both procedures were monitored using sodium dodecylsulfate gel electrophoresis (SDS-PAGE) with subsequent Coomassie blue staining of proteins and simultaneous immunoblotting with SYP I-specific antibody. Contaminating proteins with relative molecular masses (M(r)) very similar to SYP I could be removed during the process of purification, demonstrating that the 38 kDa protein found after Triton X-100 lysis of enriched SSV does not exclusively represent SYP I. A specific antiserum was raised in rabbits using a highly purified preparation of SYP I. This antiserum was used in combination with a monoclonal antibody to establish a specific and sensitive enzyme-linked immunosorbent assay (ELISA) which allowed rapid and reliable quantification of this hydrophobic membrane protein in all purification steps, starting with Triton X-100-lysed brain homogenates. Using this ELISA, the concentration of SYP I in highly purified SSV was determined to be 5.8% of solubilized protein.

  18. Development of an ELISA kit using monoclonal antibody to Clostridium difficile toxin A

    Institute of Scientific and Technical Information of China (English)

    Si-Wu Fu; Ya-Li Zhang; Dian-Yuan Zhou


    AIM: To establish an ELISA kit using monoclonal antibodiesagainst Clostridium difficile ( C. difficile) toxin A.METHODS: An indirect sandwich ElISA was described using the purified rabbit monospecific antiserum as capturing antibody. After the polystyrene microtitre plates with 96 fiat-bottomed wells were coated with rabbit antiserum, the wells were blocked with 100 g/L BSA in PBS-T. C. difficiletoxin A or culture filtrates were added to each well and then monoclonal antibodies IgG-horseradish peroxidase conjugate was added as detecting antibody, tetramethylbenzidine was used as substrate and A450 of the stopped reacting product was recorded in an automated plate reader. RESULTS: The tested specimens included culture filtrates of 2 strains of toxigenic C. difficile, 2 strains of non-toxigenic C. difficile, 26 strains of E. coli, 2 strains of S. dysenteriae, 1 strain of Bif infantis, 5 strains of V. cholera, 2 strains ofS. typhi, 7 strains of C. botulinum, 1 strain of toxigenic C. sordllii, and 1 strain of C. butyricum. A total of 47 strains of culture filtrates were all negative except for 2 strains of toxigenic C. difficile. The detective limitation of toxin A was 0.1 ng/mL.CONCLUSION: An ELISA kit with high specificity and excellent sensitivity for the rapid detection of C. difficile toxin A was established. It will be a useful tool for diagnostic test of C. difficile toxin A.

  19. A radioimmunoassay for lignin in plant cell walls

    Energy Technology Data Exchange (ETDEWEB)

    Dawley, R.M.


    Lignin detection and determination in herbaceous tissue requires selective, specific assays which are not currently available. A radioimmunoassay (RIA) was developed to study lignin metabolism in these tissues. A {beta}-aryl ether lignin model compound was synthesized, linked to keyhole limpet hemocyanin using a water-soluble carbodiimide, and injected into rabbits. The highest titer of the antiserum obtained was 34 {eta}g/mL of model derivatized BSA. An in vitro system was developed to characterize the RIA. The model compound was linked to amino activated polyacrylamide beads to mimic lignin in the cell walls. {sup 125}I Radiolabelled protein A was used to detect IgG antibody binding. The RIA was shown in the in vitro system to exhibit saturable binding. The amount of antibody bound decreased when the serum was diluted. Immunoelectrophoresis and competitive binding experiments confirmed that both aromatic rings of the lignin model compound had been antigenic. Chlorogenic acid, a phenolic known to be present in plant cells, did not compete for antibody binding. The RIA was used to measure lignin in milled plant samples and barley seedlings. Antiserum binding to wheat cell walls and stressed barley segments was higher than preimmune serum binding. Antibody binding to stressed barley tissue decreased following NaClO{sub 2} delignification. The RIA was found to be less sensitive than expected, so several avenues for improving the method are discussed.

  20. Rhesus monkeys kidney cells persistently infected with Simian Virus 40: production of defective interfering virus and acquisition of the transformed phenotype. (United States)

    Norkin, L C


    Monolayer cultures of LLC-MK2 rhesus monkey kidney cells became persistently infected with simian virus 40 (SV40) when infected at a multiplicity of infection of 100 plaque-forming units/cell. A stable carrier state developed characterized by extensive viral proliferation without obvious cytopathic effect other than the slow growth of these cultures. By 11 weeks all cells produced the SV40 T antigen. In contrast, less than 5% of the cells produced V antigen. Virus-free clonal isolates were obtained by cloning in SV40 antiserum. Continuous cultivation in antiserum resulted in a temporary cure of unclone cultures. When virus did eventually reappear in the "cured" cultures the titers remained low. The virus produced by the carrier culture was defective at both 31 and 37% c, and it interfered with the growth of standard s40 during mixed infection of CV-1 green monkey kidney cells. All of the interfering activity in carrier culture homogenates could be sedimented by centrifugation at 109,000 x g for 3 h. These cultures were completely susceptible to vesicular stomatitis virus. Extensive viral deoxyribonucleic acid synthesis occurred in CV-1 cells infected with carrier culture virus. Carrier culture homogenates are only slightly less cytopathic to CV-1 cells than standard SV40. The carrier culture express several properties of SV40 transformation.

  1. Protection against Salmonella typhi infection in mice after immunization with outer membrane proteins isolated from Salmonella typhi 9,12,d, Vi. (United States)

    Isibasi, A; Ortiz, V; Vargas, M; Paniagua, J; González, C; Moreno, J; Kumate, J


    The current studies were undertaken to assess the ability of the outer membrane proteins (OMPs) of Salmonella typhi to induce protection against challenge with the bacteria in mucin. OMPs were isolated as described by Schnaitman (J. Bacteriol. 108:553-556, 1971) and were found to be contaminated with approximately 4% lipopolysaccharide (LPS). Immunization with as little as 30 micrograms of OMPs conferred 100% protection to mice challenged with up to 1,000 50% lethal doses (LD50) of two strains of S. typhi (9,12,d, Vi and Ty2). In addition, 30% protection against challenge with up to 500 LD50 of Salmonella typhimurium was achieved. Immunization with LPS at doses equivalent to those found in the OMPs was considerably inferior to the OMPs in the induction of an immune status. Moreover, LPS was effective only when the challenge was performed with S. typhi 9,12,d, Vi (40% protection to 100 LD50). An antiserum raised in rabbits reacted mainly against the bands of the molecular weights corresponding to the so-called porins contained in the OMP preparation as shown by Western blotting (immunoblotting). This rabbit antiserum protected 100% of mice against challenge with 100 LD50 of either strain of S. typhi and 80% of mice against challenge with the same LD50 of S. typhimurium. These results indicate the usefulness of OMPs in the induction of active immunity against S. typhi in mice. Images PMID:2844676

  2. Maturation of the inhibitory response of growth hormone secretion to ether stress in postnatal rat. (United States)

    Strbák, V; Jurcovicová, J; Vigas, M


    To study the maturation of inhibitory influences on growth hormone (GH) secretion the effect of ether stress on plasma GH levels was studied during postnatal ontogenesis in female rats. Ether stress did not affect plasma GH levels in 1-day-old pups. A distinct decrease of plasma GH was found in 3- and 9-day-old pups, and the response was prevented by treatment of 3-day-old animals with somatostatin antiserum. No effect of ether stress on plasma GH was noted in 12-, 15-, 18- and 21-day-old rats. Treatment of intact 12-day-old pups with the somatostatin antiserum increased plasma GH level under basal conditions. The inhibitory effect of ether stress on plasma GH was noted again at the age 30 days and in adult animals. It is concluded that the hypothalamus of 3-day-old rats is able to release enough somatostatin to inhibit GH secretion after stress. At the period 12-18 days a phase of pituitary refractoriness was noted: ether stress as well as TRH injection (our previous observation) fail to affect plasma GH in female pups, probably due to high somatostatin secretion under basal conditions and (or) low capacity of pituitary to release GH. It is suggested that regulation of GH secretion is not mature until after the 21st day of life.

  3. Molecular cloning and nucleotide sequence of a full-length cDNA for human alpha enolase. (United States)

    Giallongo, A; Feo, S; Moore, R; Croce, C M; Showe, L C


    We previously purified a 48-kDa protein (p48) that specifically reacts with an antiserum directed against the 12 carboxyl-terminal amino acids of the c-myc gene product. Using an antiserum directed against the purified p48, we have cloned a cDNA from a human expression library. This cDNA hybrid-selects an mRNA that translates to a 48-kDa protein that specifically reacts with anti-p48 serum. We have isolated a full-length cDNA that encodes p48 and spans 1755 bases. The coding region is 1299 bases long; 94 bases are 5' noncoding and 359 bases are 3' noncoding. The cDNA encodes a 433 amino acid protein that is 67% homologous to yeast enolase and 94% homologous to the rat non-neuronal enolase. The purified protein has been shown to have enolase activity and has been identified to be of the alpha type by isoenzyme analysis. The transcriptional regulation of enolase expression in response to mitogenic stimulation of peripheral blood lymphocytes and in response to heat shock is also discussed. Images PMID:3529090

  4. Application of different /sup 125/I tracers in radioimmunoassays of estradiol-17. beta

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    Bienert, R.; Flentje, H.; Herzmann, H. (Akademie der Wissenschaften der DDR, Berlin-Buch. Zentralinstitut fuer Isotopen- und Strahlenforschung; Akademie der Wissenschaften der DDR, Leipzig. Zentralinstitut fuer Isotopen- und Strahlenforschung)


    Some different /sup 125/I-labelled estradiol tracers were produced by direct radioiodizing of estradiol and also of the histamine and tyramine conjugates of estradiol-3-carboxymethylether (E/sub 2/-3-CM) by means of the chloramine-T method. The linkage properties of these tracers were investigated in relation to the /sup 3/H-labelled estradiol opposite to the antisera, which were produced against the cow serum albumin (RSA) conjugates of E/sub 2/-3-CM and estradiol-6-carboxymethyloxime (E/sub 2/-6-CMO). As suitable system for the radioimmunological estradiol determination could be revealed 4-/sup 125/I-iodine estradiol in connection with one antiserum in each case of the radioligand antiserum combinations against E/sub 2/-3-CM-RSA- and E/sub 2/-6-CMO-RSA-conjugate. The double antibody method is used for separation in optimized RIA systems. The first and the second antibody reaction take place simultaneously.

  5. A serological study of the HLA-B17 cross-reactive group. (United States)

    Darke, C


    The HLA-B17 cross-reactive group and the participation of the subdivisions of B17 ( Bw57 and Bw58 ) in cross-reactivity were investigated by the serological analysis of 81 cytotoxic HLA antisera (produced by pregnancy alone), the HLA typing of the antiserum donors and the identification of their immunizing antigens. The sera, all of which contained B17 activity, were produced in response to one of 10 HLA antigens (A2, Bw44, Bw49, Bw51, Bw55 , Bw56 , Bw57 , Bw58 , Bw62 and Bw63 ). Antisera stimulated by Bw57 and Bw58 cross-reacted with Bw49 and both subdivisions of B5 and B15, with bidirectional cross-reactivity occurring in many instances. Bidirectional cross-reactivity was also observed between Bw57 and A2 (an A2 stimulated antiserum also reacted with Bw58 ), and Bw57 and Bw55 . Immunization by Bw62 produced some antisera which showed strong cross-reactivity with Bw57 but no reactivity with Bw58 . Significant HLA-B antigen frequency disturbances were found in the responders to the B17 cross-reactive group antigens. Twenty-five HLA antigens were found to comprise the B17 cross-reactive group and its related cross-reactions. The multideterminant nature of the HLA antigens is again emphasized by these findings.

  6. Detection and characterization of the S. typhimurium HilA protein

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    Schechter Lisa M


    Full Text Available Abstract Background Virulence genes on Salmonella pathogenicity island 1 (SPI1 are coordinately regulated by HilA, a member of the OmpR/ToxR family of transcription factors. Although a great deal is known about the complex regulation of hilA gene expression, very little is known about the HilA protein. Results In order to detect and localize the HilA protein in S. typhimurium, we raised polyclonal antiserum against purified His-tagged HilA. This allowed us to study the effect of environmental conditions on the production of HilA. We also used the antiserum to examine the fractionation properties and SDS-PAGE mobility of native HilA. Our results indicate that S. typhimurium initiates translation of HilA from the first AUG codon in the hilA open-reading frame (ORF, producing a soluble 553 amino acid (63 kDa protein product. Conclusion Materials and methods are now available to study the environmental regulation of the HilA protein in S. typhimurium. Our results also indicate that future in vitro studies of the interaction between HilA and DNA should utilize soluble preparations of HilA. Previous analyses used preparations of HilA in which the protein fractionated with the membrane, greatly limiting the types of experiments that could be conducted.

  7. Magnetoreception: activated cryptochrome 1a concurs with magnetic orientation in birds. (United States)

    Nießner, Christine; Denzau, Susanne; Stapput, Katrin; Ahmad, Margaret; Peichl, Leo; Wiltschko, Wolfgang; Wiltschko, Roswitha


    The radical pair model proposes that the avian magnetic compass is based on radical pair processes in the eye, with cryptochrome, a flavoprotein, suggested as receptor molecule. Cryptochrome 1a (Cry1a) is localized at the discs of the outer segments of the UV/violet cones of European robins and chickens. Here, we show the activation characteristics of a bird cryptochrome in vivo under natural conditions. We exposed chickens for 30 min to different light regimes and analysed the amount of Cry1a labelled with an antiserum against an epitope at the C-terminus of this protein. The staining after exposure to sunlight and to darkness indicated that the antiserum labels only an illuminated, activated form of Cry1a. Exposure to narrow-bandwidth lights of various wavelengths revealed activated Cry1a at UV, blue and turquoise light. With green and yellow, the amount of activated Cry1a was reduced, and with red, as in the dark, no activated Cry1a was labelled. Activated Cry1a is thus found at all those wavelengths at which birds can orient using their magnetic inclination compass, supporting the role of Cry1a as receptor molecule. The observation that activated Cry1a and well-oriented behaviour occur at 565 nm green light, a wavelength not absorbed by the fully oxidized form of cryptochrome, suggests that a state other than the previously suggested Trp/FAD radical pair formed during photoreduction is crucial for detecting magnetic directions.

  8. An important developmental role for oligosaccharides during early embryogenesis of cyprinid fish. (United States)

    Bakkers, J; Semino, C E; Stroband, H; Kijne, J W; Robbins, P W; Spaink, H P


    Derivatives of chitin oligosaccharides have been shown to play a role in plant organogenesis at nanomolar concentrations. Here we present data which indicate that chitin oligosaccharides are important for embryogenesis in vertebrates. We characterize chitin oligosaccharides synthesized in vitro by zebrafish and carp embryos in the late gastrulation stage by incorporation of radiolabeled N-acetyl-D-[U14C]glucosamine and by HPLC in combination with enzymatic conversion using the Bradyrhizobium NodZ alpha-1, 6-fucosyltransferase and chitinases. A rapid and sensitive bioassay for chitin oligosaccharides was also used employing suspension-cultured plant cells of Catharanthus roseus. We show that chitin oligosaccharide synthase activity is apparent only during late gastrulation and can be inhibited by antiserum raised against the Xenopus DG42 protein. The DG42 protein, a glycosyltransferase, is transiently expressed between midblastula and neurulation in Xenopus and zebrafish embryogenesis. Microinjection of the DG42 antiserum or the Bradyrhizobium NodZ enzyme in fertilized eggs of zebrafish led to severe defects in trunk and tail development.

  9. Purification and characterization of cytotoxin produced by a clinical isolate of Vibrio cholerae O54 TV113. (United States)

    Isac, Sree Renjini; Nair, Gopinath Balakrish; Singh, Durg Vijai


    Vibrio cholerae O54 TV113 isolated from a diarrheal patient produces an extracellular cytotoxin that caused alteration in the morphology of Chinese hamster ovary cells manifested as cell shrinkage with intact cell boundaries and finally causing cell death. Syncase medium supplemented with lincomycin (50 μg/ml), pH 7.2, and 18 h incubation with shaking at 37 °C supported optimal cytotoxin production. We isolated and purified this cytotoxin to homogeneity by ultrafiltration, 40-80 % ammonium sulfate precipitation, gradient-anion exchange chromatography, stepwise-anion exchange chromatography, and size exclusion chromatography increasing the specific activity by 866-fold. The cytotoxin is heat-labile, sensitive to protease and papain, and has a molecular weight of 64 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and enterotoxic activity in rabbit ileal loop assay. Both cytotoxic and enterotoxic activity could be inhibited or neutralized by antiserum raised against purified cytotoxin but not by preimmune serum. Immunodiffusion test between purified cytotoxin and its antiserum gave a single well-defined precipitin band showing reaction of complete identity and a well-defined single band in an immunoblot assay. This study thus indicate that the cytotoxin expressed by strain TV113 has both cytotoxic and enterotoxic activity and appears to contribute in pathogenesis of non-O1, non-O139 strains.

  10. Effect of radiation on normal hematopoiesis and on viral induced cancers of the hematopoietic system. Technical progress report, August 1, 1974--May 1, 1975. [Mice, x radiation

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    Okunewick, J.P.


    Studies carried out during the above period on viral leukemia have conclusively shown that the pluripotent hematopoietic colony forming stem cell (CFU-S) is a target cell for the leukemia virus. Treatment of this cell population with antiserum prepared in syngeneic mice against the disease resulted in inactivation of up to 50 percent of the CFU-S obtained from the spleens of viral leukemic mice. At the same time, normal serum had no effect on these cells, nor did the antiserum have any effect on normal CFU-S. Data indicated that a considerable time delay, on the order of a week, preceded the expression of the viral antigen in the leukemic CFU-S, but that it could be seen at all times after that up to the terminal point of the disease. We examined the effect of the virus on DNA synthesis (S-phase cells) in the CFU-S immediately after virus injection. The results showed that a doubling of the number of cells in S could be seen as early as four hours after introduction of the virus into the animal. Studies with ethidium bromide, an inhibitor of viral reverse transcriptase, were found to be in agreement with this observation. When given to viral leukemic animals in combination with fractionated exposure to x-ray, the data suggested that ethidium bromide did act to extend survival somewhat, but not much over that seen through the use of x-ray alone.

  11. Epitope selection to male specific antigens for sex selection in swine. (United States)

    Mohammadi, Azarm Akhavien; Tetro, Jason A; Filion, Lionel G


    Immunological approaches to gender selection have been contemplated since the discovery of the family of male-specific H-Y antigens found only on the surface of male cells. H-Y antigens are able to elicit an immune reaction when cells or tissues from a male donor are grafted to a female recipient. We describe here the development and testing of an inexpensive approach using polyclonal antibodies against four specific H-Y outer membrane proteins male enhanced antigen 1 (MEA 1), male enhanced antigen 2 (MEA 2), sex determining region Y (SRY) and testis determining factor (TDF). Epitopes based on hydrophilic primary sequences of the proteins were synthesized, N-terminal biotin-labeled, linked to streptavidin and mixed with a Ribi adjuvant prior to immunization in rabbits. The antiserum was tested to determine affinity to swine spermatozoa using anti-motility, flow cytometry and motility and sedimentation chambers. Fluorescent microscopy and fluorescent in situ hybridization (FISH) was used to identify the percentage of motile spermatozoa that contained the Y chromosome. We found that the polyclonal antibodies had high affinity to the spermatozoa leading to a cessation of motility. Furthermore, the majority of these non-motile spermatozoa contained the Y chromosome. We conclude that the use of polyclonal antiserum against synthetic H-Y peptide antigens may be an inexpensive and simple means to inhibit the motility of swine spermatozoa bearing the Y chromosome.

  12. Potential threat of a new pathotype of Papaya leaf distortion mosaic virus infecting transgenic papaya resistant to Papaya ringspot virus. (United States)

    Bau, H-J; Kung, Y-J; Raja, J A J; Chan, S-J; Chen, K-C; Chen, Y-K; Wu, H-W; Yeh, S-D


    A virus identified as a new pathotype of Papaya leaf distortion mosaic virus (PLDMV, P-TW-WF) was isolated from diseased papaya in an isolated test-field in central Taiwan, where transgenic papaya lines resistant to Papaya ringspot virus (PRSV) were evaluated. The infected plants displayed severe mosaic, distortion and shoe-stringing on leaves; stunting in apex; and water-soaking on petioles and stems. This virus, which did not react in enzyme-linked immunosorbent assay with the antiserum to the PRSV coat protein, infected only papaya, but not the other 18 plant species tested. Virions studied under electron microscope exhibited morphology and dimensions of potyvirus particles. Reverse transcription-polymerase chain reaction conducted using potyvirus-specific primers generated a 1,927-nucleotide product corresponding to the 3' region of a potyvirus, showing high sequence identity to the CP gene and 3' noncoding region of PLDMV. Search for similar isolates with the antiserum against CP of P-TW-WF revealed scattered occurrence of PLDMV in Taiwan. Phylogenetic analysis of PLDMV isolates of Taiwan and Japan indicated that the Taiwan isolates belong to a separate genetic cluster. Since all the Taiwan isolates infected only papaya, unlike the cucurbit-infecting Japanese P type isolates, the Taiwan isolates are considered a new pathotype of PLDMV. Susceptibility of all our PRSV-resistant transgenic papaya lines to PLDMV indicates that the virus is an emerging threat for the application of PRSV-resistant transgenic papaya in Taiwan and elsewhere.

  13. Characterisation and quantitation of pilus antigens of Moraxella bovis by ELISA. (United States)

    Lepper, A W; Hermans, L R


    Hyperimmune serums raised in rabbits to purified pili from 9 Australian and 2 American strains of Moraxella bovis from infectious bovine keratoconjunctivitis (IBK) affected herds were used to study the degree of binding between combinations of antigen and antiserum in a conventional enzyme linked immunosorbent assay (ELISA). With the aid of appropriate absorption tests major antigenic differences among pili were found permitting 6 distinct serogroups to be recognised. Further, production of specific antiserums to representative strains of each serogroup in goats facilitated the development of a double antibody sandwich ELISA which could be used to quantitate pilus expression of a given strain of M. bovis, or to differentiate pilus serogroups of 22 strains of M. bovis obtained from a total of 12 Australian herds. Most isolates were found to belong to serogroups designated IV and V. One strain from the United States of America showed total homology with Australian serogroup IV while the other showed some cross-reactivity with serogroups V and VI.

  14. Role of aVb3 integrin in embryo implantation in the mouse

    Institute of Scientific and Technical Information of China (English)


    Integrin, a heterodimeric adhesive molecule composed of a and b subunits, can regulate cell adhesion and trafficking. Recent data have documented that, at the "implantation window" stage, aVb3 integrin participates in the maternal-fetal interaction and becomes a potential marker of uterine receptivity. Furthermore, it can affect invasiveness of embryo. This work made a further study about its action mechanism. Results of indirect immunofluorescence and laser scanning confocal microscopy showed that aVb3 integrin was clearly expressed in the mouse blastocyst. Injection of aVb3 integrin antiserum into a uterine horn of a pregnant mouse on day 3 markedly decreased the number of embryos implanted (P < 0.001). In a co-culture model, aVb3 integrin antisera at 1:100 and 1:200 dilutions significantly depressed the attachment and outgrowth reactions of blastocysts on monolayer of uterine epithelial cells. Analysis of correlation manifested that the inhibitory effect of aVb3 integrin antiserum was dosage/dilution-dependent. Thus, aVb3 integrin is an essential factor in the uterine endometrium for embryo implantation in the mouse. This integrin distinctly expressed in the mouse blastocyst at "implantation" stage affected the process of embryo implantation by route of mediating both the attachment and the outgrowth processes of blastocyst on uterine epithelial cells.

  15. A radioimmunoassay for alpha- and beta-gliadins. (United States)

    Ciclitira, P J; Lennox, E S


    1. A rapid, sensitive specific radioimmunoassay for alpha- and beta-gliadin has been developed using an antiserum raised in rabbits to A-gliadin, a component of alpha-gliadin. 2. The antigen used in the assay was alpha-gliadin labelled with 125I; antigen-antibody complexes were collected after adsorption to Staphylococcus aureus in suspension. 3. The sensitivity of the assay, as judged by competitive binding with unlabelled antigen, was 1 ng of alpha- or beta-gliadin, which show complete cross-reaction with this antiserum. 4. Cross-reactivity to other wheat proteins was less than 1% and no cross-reactivity to extracts of rye, barley or oats was observed. 5. This radioimmunoassay for alpha- and beta-gliadin has been used to measure their amount in different varieties of wheat flour, several foods prepared from flour, e.g. bread, biscuits and products prepared as 'gluten free'. The possibility of assaying for alpha-gliadin in prepared foods is of special value since alpha-, beta-, gamma- and omega-gliadin have been shown to exacerbate coeliac disease.

  16. Variations in Loxosceles spider venom composition and toxicity contribute to the severity of envenomation. (United States)

    de Oliveira, Kátia C; Gonçalves de Andrade, Rute M; Piazza, Roxane M F; Ferreira, Jorge M C; van den Berg, C W; Tambourgi, Denise V


    Envenomation by Loxosceles spiders causes two main clinical manifestations: cutaneous and systemic loxoscelism. The factors contributing to the severity of loxoscelism are not fully understood. We have analysed biochemical and toxicity variations in venom of L. laeta and L. intermedia, with the aim to find a correlation with the seriousness of loxoscelism. Differences in expression of proteins, glycoproteins and sphingomyelinase activity were observed between venom from male and female spiders and between venom from the two species. These differences were reflected in the toxicity of the venoms including the capacity to induce complement-dependent haemolysis, dermonecrosis and lethality. Comparative analysis of gender and species, showed that these biological activities were more prominent in venom from female spiders, especially from L. laeta. Antiserum raised against venom from females L. laeta spiders had the highest efficacy in neutralizing venoms of males and females of both species. These results indicate that the severity of loxoscelism depends, at least partially, on the species and sex of the spider and suggest that for accidents involving L. laeta an specific serum therapy is necessary. Furthermore, it emphasizes the efficacy of the antiserum produced against L. laeta female venom in neutralizing Loxosceles venoms from different species and gender.

  17. Molecular cloning of tomato fruit polygalacturonase: Analysis of polygalacturonase mRNA levels during ripening. (United States)

    Dellapenna, D; Alexander, D C; Bennett, A B


    The expression of a gene encoding the cell wall-degrading enzyme polygalacturonase [poly(1,4-alpha-D-galacturonide) glucanohydrolase, EC] was characterized during tomato fruit ripening. Polygalacturonase was purified from ripe tomato fruit and used to produce highly specific antiserum. Immunoblot analyses detected a 45- and a 46-kDa protein in ripe fruit but immunoprecipitation of in vitro translation products of mRNA from ripe tomato fruit yielded a single 54-kDa polypeptide, suggesting post-translational processing. A plasmid cDNA library was prepared from poly(A)(+) RNA isolated from ripe tomato fruit. The cDNA library was inserted into a lambda-based expression vector, and polygalacturonase cDNA clones were identified by immunological screening. Hybrid-select translation experiments indicated that the cDNAs encode a 54-kDa in vitro translation product that is specifically immunoprecipitated with polygalacturonase antiserum. RNA-blot analysis indicated that the 1.9-kilobase polygalacturonase mRNA was virtually absent from immature-green fruit, accumulated steadily during the ripening process, and was at its highest level in red-ripe fruit. There was at least a 2000-fold increase in the level of polygalacturonase mRNA between immature-green and red-ripe tomato fruit. These studies show that the levels of polygalacturonase mRNA are developmentally regulated during tomato fruit ripening.

  18. Development of enzyme-linked immunosorbent assay for estimation of urinary albumin. (United States)

    Shrivastav, Tulsidas G; Kariya, Kiran P; Prasad, Pramod K V; Chaube, Shail K; Kumar, Dinesh


    Yearly estimation of urinary albumin is a prerequisite for predicting renal status in Diabetes Type II patients with negative dipstick results for overt proteinuria. A simple, sensitive, and cost-effective enzyme linked immunosorbent assay (ELISA) for urinary albumin has been developed using human serum albumin antiserum (HSA-antiserum), HSA-biotin, and streptavidin-horseradish peroxidase (SA-HRP) conjugates. To the antibody-coated wells, 100 μL of HSA standards followed by 1:100 diluted urine samples in duplicate were added and then 50 μL of HSA-biotin conjugates was added in all the wells. 100 μL of SA-HRP was added after washing. Bound enzyme activity was measured by adding 100 μL TMB/H2O2. The analytical sensitivity and ED50 of the developed method was found to be 0.01 μg/mL and 0.35 μg/mL, respectively. The percent recovery of the HSA from exogenously spiked urine pools were in the range of 98.13-100.29%. The intra- and inter-assay coefficient of variation (CVs) ranged from 3.38-10.32 % and 4.22-11.01%, respectively. The antibody showed 4.4% and 3.2% cross reactivity with monkey and horse serum albumin, respectively. There was no cross reaction with human β2-microglobulin, γ-globulin, and haemoglobulin.

  19. Antibodies to lipooligosaccharide of a Brazilian purpuric fever isolate of Haemophilus influenzae biogroup aegyptius lack bactericidal and protective activity. (United States)

    Peters, V B; Rubin, L G


    The immunological basis for protection against Brazilian purpuric fever (BPF), a fulminant infection of young children associated with bacteremia with Haemophilus influenzae biogroup aegyptius, is unknown. Candidate antigens to which protective antibodies may be directed include cell surface proteins and lipooligosaccharide (LOS). We studied the activity of antisera to LOS purified from a BPF H. influenzae biogroup aegyptius isolate. Anti-LOS antisera contained anti-LOS antibody by enzyme immunoassay and immunoblot and no detectable anti-outer membrane protein antibodies by immunoblot. Anti-LOS antisera had minimal bactericidal activity and were not protective against the homologous strain in an infant rat model of bacteremia. Antiserum to whole bacterial cells had a titer of anti-LOS antibody similar to that of anti-LOS antisera and was bactericidal and protective. Removal of anti-LOS antibodies from anti-whole cell antiserum by affinity chromatography did not result in a loss of bactericidal activity. Serum from a normal adult contained anti-LOS antibodies and had bactericidal activity. However, anti-LOS antibodies purified from this serum did not have detectable bactericidal activity. These studies suggest that anti-LOS antibodies produced in rats are not bactericidal and do not contribute to protection against experimental bacteremia with BPF strains of H. influenzae biogroup aegyptius.

  20. Antibody to a 145-kilodalton outer membrane protein has bactericidal activity and protective activity against experimental bacteremia caused by a Brazilian purpuric fever isolate of Haemophilus influenzae biogroup aegyptius. The Brazilian Purpuric Fever Study Group. (United States)

    Rubin, L G; Rizvi, A


    The immunologic basis for protection against Brazilian purpuric fever, a septicemic infection associated with Haemophilus influenzae biogroup aegyptius bacteremia, is unknown. Passive immunization of infant rats with antiserum to whole bacterial cells of the homologous strain protects them from experimental bacteremia following bacterial challenge. In immunoblotting, antibody to a 145-kDa protein (P145) was present in protective antisera but not in nonprotective antisera. As judged by analysis of the antibodies eluted from whole bacterial cells and the agglutination of bacteria by antisera to P145, this protein is surface exposed. We prepared monospecific rat antisera to this protein by three methods: (i) immunization with whole bacterial cells and absorption with a Brazilian purpuric fever strain not expressing P145, (ii) immunization with gel-purified P145, and (iii) immunization with a P145-expressing transformant of a laboratory H. influenzae strain expressing this protein and absorption of the antiserum with the laboratory H. influenzae strain. These antisera had low antilipooligosaccharide antibody titers, were reactive only with P145, and had bactericidal activity in vitro. Following passive immunization, these antisera partially protected infant rats from bacteremia resulting from intraperitoneal challenge with bacteria. As assessed by immunoblotting, pooled adult human sera contained antibodies reactive with P145. Antibody to P145 may contribute to protection against Brazilian purpuric fever.

  1. Synaptic connectivity in the midget-parvocellular pathway of primate central retina. (United States)

    Jusuf, Patricia R; Martin, Paul R; Grünert, Ulrike


    The synaptic connectivity of OFF midget bipolar cells was investigated in the central retina of two primate species, the New World common marmoset monkey, Callithrix jacchus, and the Old World macaque monkey, Macaca fascicularis. In marmosets, dichromatic and trichromatic animals were compared. Bipolar output synapses were identified with antibodies against ribbon proteins (kinesin, C-terminal binding protein 2) or with an antiserum that recognizes postsynaptic glutamate receptor clusters (GluR4). The midget bipolar cells were identified immunocytochemically with antibodies to CD15 (marmoset) or an antiserum to recoverin (macaque). In marmosets, midget ganglion cells were retrogradely labeled from the parvocellular layers of the dorsal lateral geniculate nucleus. Consistent with previous studies of Old World primates, in marmoset, midget bipolar cells contacted midget ganglion cells at a ratio of 1:1. The number of output synapses made by OFF midget bipolar cells was quantified for 104 cells in two dichromatic marmosets, 108 cells in one trichromatic marmoset, and 118 cells in one macaque. The number of output synapses was comparable for all animals, ranging from 10-71 in the dichromatic marmoset (average 29.7 +/- 12.4 SD), 12-86 in the trichromatic marmoset (average 28.6 +/- 11.7 SD) and 9-48 in the macaque (average 26.5 +/- 9.3 SD) per axon terminal. In all animals the number of output synapses per axon terminal showed a unimodal distribution. Our results suggest that the midget circuitry is comparable in dichromatic and trichromatic animals.

  2. Streptococcus pneumoniae PstS production is phosphate responsive and enhanced during growth in the murine peritoneal cavity (United States)

    Orihuela, C. J.; Mills, J.; Robb, C. W.; Wilson, C. J.; Watson, D. A.; Niesel, D. W.


    Differential display-PCR (DDPCR) was used to identify a Streptococcus pneumoniae gene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment as pstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the complete pstS gene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identify pstS expression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation of pstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.

  3. Production of specific antisera for radioimmunoassay of human luteinizing hormone (LH) in the presence of human chorionic gonadotropin (hCG). [/sup 125/I

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    Thorell, J.I.; Jeppsson, S.; Holmstrom, B.


    A specific radioimmunoassay for LH, which measures plasma LH in the presence of human chorionic gonadotropin (hCG) is described. Rabbits were immunized with highly purified native LH. One of the antisera with a difference in its reactivity against LH and hCG was further purified by affinity chromatography on a column with hCG coupled to Sepharose 4B. The adsorbed antiserum and /sup 125/I-LH was used in a double antibody assay. The LH standard (MRC/68/40) efficiently inhibited the binding of /sup 125/I-LH, and the standard curve showed a sensitivity of 0.5 ng/ml in the sample. hCG up to 10,000 ng/ml did not inhibit the binding of /sup 125/I-LH. The plasma level of LH in pregnant women in the first trimester was low (1.3 +- 0.1 ng/ml). When LH was measured in fertile or menopausal women with or without stimulation with LH/FSH releasing hormone (LH-RH)/sup x/ the results agreed to those found with our conventional LH-assay based on antiserum against hCG.

  4. Sequence motifs and prokaryotic expression of the reptilian paramyxovirus fusion protein (United States)

    Franke, J.; Batts, W.N.; Ahne, W.; Kurath, G.; Winton, J.R.


    Fourteen reptilian paramyxovirus isolates were chosen to represent the known extent of genetic diversity among this novel group of viruses. Selected regions of the fusion (F) gene were sequenced, analyzed and compared. The F gene of all isolates contained conserved motifs homologous to those described for other members of the family Paramyxoviridae including: signal peptide, transmembrane domain, furin cleavage site, fusion peptide, N-linked glycosylation sites, and two heptad repeats, the second of which (HRB-LZ) had the characteristics of a leucine zipper. Selected regions of the fusion gene of isolate Gono-GER85 were inserted into a prokaryotic expression system to generate three recombinant protein fragments of various sizes. The longest recombinant protein was cleaved by furin into two fragments of predicted length. Western blot analysis with virus-neutralizing rabbit-antiserum against this isolate demonstrated that only the longest construct reacted with the antiserum. This construct was unique in containing 30 additional C-terminal amino acids that included most of the HRB-LZ. These results indicate that the F genes of reptilian paramyxoviruses contain highly conserved motifs typical of other members of the family and suggest that the HRB-LZ domain of the reptilian paramyxovirus F protein contains a linear antigenic epitope. ?? Springer-Verlag 2005.

  5. Transforming growth factor-beta modulates plasminogen activator activity and plasminogen activator inhibitor type-1 expression in human keratinocytes in vitro. (United States)

    Wikner, N E; Elder, J T; Persichitte, K A; Mink, P; Clark, R A


    Transforming growth factor beta (TGF-beta) is a multifunctional mediator with effects on cellular growth, differentiation, and extracellular matrix (ECM) metabolism. Because TGF-beta stimulates fibronectin expression in cultured human keratinocytes, we wished to determine whether it might also affect ECM degradation through the plasminogen activator (PA)-plasminogen activator inhibitor (PAI) system. Immunofluorescence of human keratinocytes using a monospecific antiserum to type 1 PAI (PAI-1) showed enhanced cellular and ECM staining when they were cultured in the presence of TGF-beta. The antiserum also identified an Mr 50,000 protein in conditioned media that was markedly enhanced by TGF-beta. A corresponding stimulation of PAI-1 mRNA was demonstrated by quantitative RNA blot analysis. Total plasminogen activating activity of conditioned medium was markedly decreased by TGF-beta. Zymography showed this to be at least partially due to decreased secreted urokinase activity. TGF-beta may play an important role in stabilizing the provisional matrix synthesized by keratinocytes in healing wounds.

  6. Cryptochrome 1 in Retinal Cone Photoreceptors Suggests a Novel Functional Role in Mammals. (United States)

    Nießner, Christine; Denzau, Susanne; Malkemper, Erich Pascal; Gross, Julia Christina; Burda, Hynek; Winklhofer, Michael; Peichl, Leo


    Cryptochromes are a ubiquitous group of blue-light absorbing flavoproteins that in the mammalian retina have an important role in the circadian clock. In birds, cryptochrome 1a (Cry1a), localized in the UV/violet-sensitive S1 cone photoreceptors, is proposed to be the retinal receptor molecule of the light-dependent magnetic compass. The retinal localization of mammalian Cry1, homologue to avian Cry1a, is unknown, and it is open whether mammalian Cry1 is also involved in magnetic field sensing. To constrain the possible role of retinal Cry1, we immunohistochemically analysed 90 mammalian species across 48 families in 16 orders, using an antiserum against the Cry1 C-terminus that in birds labels only the photo-activated conformation. In the Carnivora families Canidae, Mustelidae and Ursidae, and in some Primates, Cry1 was consistently labeled in the outer segment of the shortwave-sensitive S1 cones. This finding would be compatible with a magnetoreceptive function of Cry1 in these taxa. In all other taxa, Cry1 was not detected by the antiserum that likely also in mammals labels the photo-activated conformation, although Western blots showed Cry1 in mouse retinal cell nuclei. We speculate that in the mouse and the other negative-tested mammals Cry1 is involved in circadian functions as a non-light-responsive protein.

  7. Isolation of a unique membrane protein from Naegleria fowleri. (United States)

    Réveiller, F L; Suh, S J; Sullivan, K; Cabanes, P A; Marciano-Cabral, F


    Naegleria fowleri, an amoeboflagellate, is the causative agent of Primary Amoebic Meningoencephalitis, a fulminating disease of the central nervous system. In order to elucidate the mechanisms of pathogenicity of this amoeba, a cDNA expression library was prepared from N. fowleri RNA. A specific protein was found to be expressed from a cDNA clone designated Mp2CL5. Northern blot analysis showed that the Mp2CL5 mRNA was expressed in pathogenic N. fowleri but was not expressed in non-pathogenic Naegleria species nor in Acanthamoeba. Western blot analysis using anti-N. fowleri antiserum demonstrated that IPTG-induced Escherichia coli Mp2CL5 expressed a 23-kDa recombinant protein. The Mp2CL5 recombinant protein was histidine-tagged and purified to homogeneity from E. coli. A polyclonal rabbit antiserum was prepared against the purified Mp2CL5 recombinant protein. This antibody was used to further characterize the Mp2CL5 native protein expressed by N. fowleri. Western blot analysis in conjunction with immunofluorescence microscopy demonstrated the presence of a native protein of 17 kDa on the plasma membrane of N. fowleri trophozoites. The native N. fowleri protein was expressed in the logarithmic phase of trophozoite growth and the production of this protein increased through the stationary phase of growth. Studies are in progress to examine further its role as a virulence factor.

  8. 抗莱克多巴胺多克隆抗体的制备%Preparation for Ractopamine Polyclonal Antibodies

    Institute of Scientific and Technical Information of China (English)

    张红琳; 陈昌云; 周红霞; 于小莲; 杨剑波; 石国庆


    为了制备抗莱克多巴胺多克隆抗体,试验通过连接剂butane-1,4-diol diglyciydyl ether把莱克多巴胺和载体蛋白BSA和0VA偶联成免疫抗原和包被抗原,免疫新西兰大白兔,饱和硫铵沉淀方法纯化抗体,间接ELISA法检测抗血清效价.结果通过免疫兔获得了抗莱克多巴胺的多克隆抗体.经ELISA测定,其效价为1:12800.%The RCT (Ractopamine) was coupled with BSA and OVA to produce immunogen and envelope antigen by using coupling agent butane-l, 4-diol diglyciydyl ether, respectively. The polyclonal antibodies was obtained by immuning New Zealand rabbits with BSA-RCT and purified by saturated ammonium sulfate precipitation. The indirect ELISA method was used to detect its antiserum titer. The results showed that the polyclonal antibodies against RCT had been obtained from immunized rabbits and the titer of the polyclonal antiserum was 1:12 800.

  9. Liver fatty acid binding protein is the mitosis-associated polypeptide target of a carcinogen in rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bassuk, J.A.; Tsichlis, P.N.; Sorof, S.


    Hepatocytes in normal rat liver were found previously to contain a cytoplasmic 14,000-dalton polypeptide (p14) that is associated with mitosis and is the principal early covalent target of activated metabolites of the carcinogen N-2-fluorenylacetamide (2-acetylaminofluorene). The level of immunohistochemically detected p14 was low when growth activity of hepatocytes was low, was markedly elevated during mitosis in normal and regenerating livers, but was very high throughout interphase during proliferation of hyperplastic and malignant hepatocytes induced in rat liver by a carcinogen (N-2-fluorenylacetamide or 3'-methyl-4-dimethylaminoazobenzene). The authors report here that p14 is the liver fatty acid binding protein. The nucleotide sequence of p14 cDNA clones, isolated by screening a rat liver cDNA library in bacteriophage lambdagt11 using p14 antiserum, was completely identical to part of the sequence reported for liver fatty acid binding protein. Furthermore, the two proteins shared the following properties: size of mRNA, amino acid composition, molecular size according to NaDodSO/sub 4/ gel electrophoresis, and electrophoretic mobilities in a Triton X-100/acetic acid/urea gel. The two polypeptides bound oleic acid similarly. Finally, identical elevations of cytoplasmic immunostain were detected specifically in mitotic hepatocytes with either antiserum. The collected findings are suggestive that liver fatty acid binding protein may carry ligands that promote hepatocyte division and may transport certain activated chemical carcinogens.

  10. Establishment of Indirect ELISA for Special Serum of Dairy-derived Staphylococcal enterotoxin A%乳源性金黄色葡萄球菌肠毒素A的间接ELISA检测方法建立

    Institute of Scientific and Technical Information of China (English)

    孙茂忠; 薛秀恒; 王菊花; 梅林; 吕高婧; 徐洪军


    Staphylococcal enterotoxin was collected and purified from Staphylococcus aureus,which was isolated from milk sample.In this way,a specific anti-serum of Staphylococcal enterotoxin A was prepared and the indirect ELISA method for detecting Staphylococcal enterotoxin was developed.The result showed that the optimal concentration of coating antigen is 10 μg/mL,the optimal dilution of anti-serum examined is 1︰2 000,ELISA titer is 1︰3 200.The foundation was provided for dairy-derived Staphylococcus aureus detection through this method.%从乳品中筛选出金黄色葡萄球菌,并对其所产的肠毒素进行分离纯化。制备出金黄色葡萄球菌肠毒素的特异性抗血清,建立金黄色葡萄球菌肠毒素A特异性血清间接ELISA方法。结果表明,最佳抗原包被浓度为10μg/mL,抗体最佳稀释倍数为1︰1 600,抗血清效价达到1︰3 200。通过建立此检测方法可以为乳源金黄色葡萄球菌肠毒素的检验提供依据。

  11. Metarhizium anisopliae host-pathogen interaction: differential immunoproteomics reveals proteins involved in the infection process of arthropods. (United States)

    Santi, Lucélia; Silva, Walter O B; Pinto, Antônio F M; Schrank, Augusto; Vainstein, Marilene H


    Metarhizium anisopliae is an entomopathogenic fungus well characterized for the biocontrol of a wide range of plagues. Its pathogenicity depends on the secretion of hydrolytic enzymes that degrade the host cuticle. To identify proteins involved in the infection process and in host specify, immunoproteomic analysis was performed using antiserum produced against crude extract of M. anisopliae cultured in the presence of Rhipicephalus (Boophilus) microplus and Dysdercus peruvianus cuticles. Spots detected using antisera produced against M. anisopliae cultured in cuticles and spore surface proteins, but not with antiserum against M. anisopliae cultured in glucose, were identified so as to give insights about the infection process. An MS/MS allowed the identification of proteases, like elastase, trypsin, chymotrypsin, carboxypeptidase and subtilisin (Pr1A, Pr1I and PR1J), chitinases, DNase I and proline-rich protein. Chymotrypsin and Pr1I were inferred as host specific, being recognized in D. peruvianus infection only. This research represents an important contribution to the understanding the adaptation mechanisms of M. anisopliae to different hosts.

  12. Schistosoma mansoni polypeptides immunogenic in mice vaccinated with radiation-attenuated cercariae

    Energy Technology Data Exchange (ETDEWEB)

    Dalton, J.P.; Strand, M.


    We compared the humoral immune response of mice protected against Schistosoma mansoni by vaccination with radiation-attenuated cercariae to that of patently infected mice, and we identified antigens that elicit a greater, or unique, immune response in the vaccinated mice. These comparisons were based upon radioimmunoprecipitations and immunodepletion of (/sup 35/S)methionine-labeled schistosomular and adult worm polypeptides, followed by one- and two-dimensional polyacrylamide gel analyses. The humoral responses of patently infected mice and of mice vaccinated once were remarkably similar and were directed against schistosome glycoproteins ranging in molecular size from greater than 300 to less than 10 kDa. Exposing mice to a second vaccination resulted in a marked change in the immune response, to one predominantly directed toward high molecular size glycoproteins. Sequential immunodepletion techniques identified five schistosomular and seven adult worm antigens that showed a greater or unique immunogenicity in vaccinated mice as compared with patently infected mice. These adult worm antigens were purified by preparative sequential immunoaffinity chromatography and used to prepare a polyclonal antiserum, anti-irradiated vaccine. This antiserum bound to the surface of live newly transformed and lung-stage schistosomula, as assessed by immunofluorescence assays, and was reactive with a number of /sup 125/I-labeled schistosomular surface polypeptides, including a doublet of 150 kDa that was also recognized by sera of vaccinated mice but not by sera of patently infected mice.

  13. [Construction of genomic library of L. interrogans serovar lai using lambda gt11 as the vector and a study of recombiant plasmid pDL121]. (United States)

    Liu, H; Dai, B; Jing, B; Wu, W; Li, S; Fang, Z; Zhao, H; Ye, D; Yan, R; Liu, J; Song, S; Yang, Y; Zhang, Y; Liu, F; Tu, Y; Yang, H; Huang, Z; Liang, L; Hu, L; Zhao, M


    A genomic library of L. interrogans serovar lai strain 017 has been constructed using lambda gt11 as the vector. DNA was partially digested by two blunt-end restriction enzymes, then methylated with EcoR I methylase; after EcoR I linker was added to the DNA, the linker-ended DNA was ligated to the dephosphorylated EcoR I digested lambda gt11 arms. The recombined DNA was packaged in vitro, and used to transduct E. coli Y1090 for amplification. There were 2.1 x 10(6) recombinant bacteriophages as recognized by their ability to form white plaques plated on Lac host in the presence of both IPTG and X-Ga1. A positive clone, designated lambda DL12, was screened with a rabbit anti-serum against L. interrogans serovar lai from the genomic library. The DNA from lambda DL12 was subcloned into plasmid pUC18. A recombinant (designated as pDL121) was obtained. SDS-PAGE analysis indicated that a 23 kd was expressed in E. coli JM 103 harboring pDL121. Western blotting analysis showed that a specific protein band molecular weight of 23 kd could be recognized by the rabbit antiserum against L. interrogans serovar lai strain 017.

  14. White spot syndrome virus envelope protein VP124 involved in the virus infection

    Institute of Scientific and Technical Information of China (English)

    ZHU Yanbing; WU Chenglin; YANG Feng


    White spot syndrome virus(WSSV)is one of the major shrimp pathogens causing large economic losses to shrimp farming.In an attempt to identify the envelope proteins involved in the virus infection,purified WSSV virions were mixed with three antisera against WSSV envelope proteins(VP39,VP124 and VP187),individually.And then they were injected intramuscularly into crayfish (Procambarus clarkii) to conduct in vivo neutralization assays.The results showed that for groups injected with virions only and groups injected with the mixture of virions and antiserum against VP124,the crayfish mortalities were 100% and 60% on the 8th day postinfection,individually.The virus infection could be delayed or neutralized by antibody against the envelope pro-tein VP124.Quantitative PCR was used to further investigate the influence of three antisera described above on the virus infec-tion.The results showed that the antiserum against VP124 could restrain the propagation of WSSV in crayfish.All of the results suggested that the viral envelope protein VP124 played a role in WSSV infection.

  15. Analysis of Leptospira interrogans ompA gene and immunological identification of its recombinant expression product%问号钩端螺旋体ompA基因分析及其重组表达产物的免疫学鉴定

    Institute of Scientific and Technical Information of China (English)

    丁威; 董海艳; 薛峰; 严杰; 楼永良


    Objective To investigate the distribution of ompA gnne in 15 Chinese reference standard strains belonging to 15 serogroups of Leptospira interrogate, and to express recombinant OmpA ( rOmpA ) and to identify immunogenicity and immunoprotection of rOmpA. Methods Genomic DNAs from different leptospiral strains were extracted by phenol-chloroform method. Entire ompA gene fragments from the strains were amplified by PCR and then sequenced after T-A cloning. A prokaryotic expression system of ompA gene from L. interrogans strain 56601 was constructed, and the expression and yield of rOmpA were determined by SOS-PAGE plus Bio-Rad Agarose Image Analyser. Rabbits were immunized with rOmpA for obtaining antiserum, and immunodiffusion test was used to measure the antiserum's titer. Western blot assay was performed to determine the immunoreaetivity of rOmpA with the antiserum against rOmpA and antiserum against whole cell of L. interrogans strain 56601, while mi-croscopic agglutination test (MAT) was applied to detect the cross agglutination to the 15 L. interrogans strains. A leptospire adhering cell model and a leptospire infecting guinea pigs model were used to determine the adhesion-bloc-king effect of rOmpA antiserum and immunoprotection of rOmpA. Results All the 15 L. interrogans strains, but not L. biflexa strain Patoe Ⅰ , had sequence conserved ompA genes. The yield of rOmpA was approximate 20% of the total bacterial proteins, rOmpA could induce rabbits to produce antibody and immunodiffusion titer of the anti-serum was 1:4. Both antisera against rOmpA and against whole cell of L. interrogans strain 56601 were able to pro-duce positive Western blot signs to rOmpA, and the former offered 1 : 20-1 : 320 MAT titers to the 15 L. interrogans strains. 1: 10-1:160 dilutions of rOmpA antiserum could efficiently block L. interrogans strain 56601 adhering to J774A. 1 cells, and 100 μg and 200 μg rOmpA displayed 50.0% and 75.0% immunoprotective rates in the infee-ted guinea

  16. 南方鲇血型的初步鉴定%Preliminary Studies on the Blood Group of Southern Catfish Silurus meridionalis

    Institute of Scientific and Technical Information of China (English)

    黄林; 金丽; 张耀光


    To identify the blood group of the southern catfish Silurus meridionalis, cross-reactions were conducted between the fish's serum and red blood cells. The results showed that there was no agglutination in all the cross-reactions between the southern catfish's serum and individual red blood cells. This indicated that southern catfish may not have blood groups or that they may have blood groups but lack enough lectin in the serum. Using southern catfish red blood cells as the antigen to immune Japanese white rabbit to prepare antiserum, the prepared antiserum was used to cross-react with southern catfish red blood cells. The results showed that there were various degrees of agglutination which indicated the blood group existed in southern catfish. We could infer that southern catfish may have four blood groups which were named as N~A, N~B, N~(AB), N~O, and the method of preparing antiserum to cross-react with red blood cells is more reliable to identify the blood group of southern catfish.%本研究旨在通过观察南方鲇血清与其红细胞的交叉反应以鉴定南方鲇的血型.实验结果表明:南方鲇的血清与同种其他个体的红细胞进行交叉反应时均未出现凝集现象,这表明南方鲇可能不存在血型或南方鲇具备血型但血清中相应的凝集素含量不足.以南方鲇的红细胞为抗原免疫日本种大耳白兔制备的抗血清与南方鲇的红细胞进行交叉反应,出现了不同程度的凝集反应,这表明南方鲇存在血型.据上述两个实验结果可以推断,南方鲇可能存在4种血型,分别命名为N~A、N~B、N~(AB)和N~O型;同时也证实,在鉴定南方鲇血型的研究中,通过制备抗血清与红细胞进行交叉反应的方法更为可靠.

  17. Development of a high-yield reassortant influenza vaccine virus derived from the A/Anhui/1/2013 (H7N9) strain. (United States)

    Nakamura, Kazuya; Shirakura, Masayuki; Suzuki, Yasushi; Naito, Tadasuke; Fujisaki, Seiichiro; Tashiro, Masato; Nobusawa, Eri


    In April 2013, the first three fatal cases of human infection with an avian influenza A virus (H7N9) were reported in China. Because of a pandemic threat by this virus, we have commenced to develop candidate vaccine viruses (CVVs). Three 6:2 genetic reassortant viruses with different hemagglutinin (HA) sequences, NIIDRG-10, -10.1 and -10.2, were generated by a reverse genetics technique between the high egg-growth master virus, A/Puerto Rico/8/34 (H1N1) and A/Anhui/1/2013 (H7N9), kindly provided by the Chinese Center for Disease Control and Prevention. The different HA gene sequences of the three CVVs were derived from the original virus stock. NIIDRG-10 possesses HA, whose sequence is identical to that of the original A/Anhui/1/2013 (H7N9) in the Global Initiative on Sharing Avian Influenza Data (EPI439507), while NIIDRG-10.1 and -10.2 possess amino acid differences, A125T and N123D/N149D, respectively, compared with NIIDRG-10. NIIDRG-10 replicated in embryonated chicken eggs with low hemagglutination titer 128, whereas NIIDRG-10.1 and -10.2 grew well with hemagglutination titer 1024. These viruses reacted well with a ferret antiserum raised against the original A/Anhui/1/2013 virus. Ferret antiserum against NIIDRG-10.1 reacted well with A/Anhui/1/2013 similar to the homologous virus NIIDRG-10.1. These results indicated that NIIDRG-10.1 passed the two-way test of antigenic identity. In contrast, the ferret antiserum against NIIDRG-10.2 reacted with A/Anhui/1/2013 at an 8-fold lower hemagglutination inhibition titer than with the homologous virus NIIDRG-10.2, indicating an antigenic change. The total and HA protein yields of NIIDRG-10.1 were 14.7 and 6.9 μg/ml, respectively, similar to those levels of high-yield seed viruses of seasonal influenza vaccines. NIIDRG-10.1 was approved as one of the CVVs for H7N9 viruses by the WHO in 2013. The candidate vaccine derived from NIIDRG-10.1 is currently being evaluated in a phase II clinical study in Japan.

  18. Sequence analysis and characterization of a 40-kilodalton Borrelia hermsii glycerophosphodiester phosphodiesterase homolog. (United States)

    Shang, E S; Skare, J T; Erdjument-Bromage, H; Blanco, D R; Tempst, P; Miller, J N; Lovett, M A


    We report the purification, molecular cloning, and characterization of a 40-kDa glycerophosphodiester phosphodiesterase homolog from Borrelia hermsii. The 40-kDa protein was solubilized from whole organisms with 0.1% Triton X-100, phase partitioned into the Triton X-114 detergent phase, and purified by fast-performance liquid chromatography (FPLC). The gene encoding the 40-kDa protein was cloned from a B. hermsii chromosomal DNA lambda EXlox expression library and identified by using affinity antibodies generated against the purified native protein. The deduced amino acid sequence included a 20-amino-acid signal peptide encoding a putative leader peptidase II cleavage site, indicating that the 40-kDa protein was a lipoprotein. Based on significant homology (31 to 52% identity) of the 40-kDa protein to glycerophosphodiester phosphodiesterases of Escherichia coli (GlpQ), Bacillus subtilis (GlpQ), and Haemophilus influenzae (Hpd; protein D), we have designated this B. hermsii 40-kDa lipoprotein a glycerophosphodiester phosphodiesterase (Gpd) homolog, the first B. hermsii lipoprotein to have a putative functional assignment. A nonlipidated form of the Gpd homolog was overproduced as a fusion protein in E. coli BL21(DE3)(pLysE) and was used to immunize rabbits to generate specific antiserum. Immunoblot analysis with anti-Gpd serum recognized recombinant H. influenzae protein D, and conversely, antiserum to H. influenzae protein D recognized recombinant B. hermsii Gpd (rGpd), indicating antigenic conservation between these proteins. Antiserum to rGpd also identified native Gpd as a constituent of purified outer membrane vesicles prepared from B. hermsii. Screening of other pathogenic spirochetes with anti-rGpd serum revealed the presence of antigenically related proteins in Borrelia burgdorferi, Treponema pallidum, and Leptospira kirschneri. Further sequence analysis both upstream and downstream of the Gpd homolog showed additional homologs of glycerol metabolism

  19. Robert Feulgen Prize Lecture. Distribution and role of gap junctions in normal myocardium and human ischaemic heart disease. (United States)

    Green, C R; Severs, N J


    In the heart, individual cardiac muscle cells are linked by gap junctions. These junctions form low resistance pathways along which the electrical impulse flows rapidly and repeatedly between all the cells of the myocardium, ensuring their synchronous contraction. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera were raised to three synthetic peptides, each matching different cytoplasmically exposed portions of the sequence of connexin43, the major gap-junctional protein reported in the heart. The specificity of each antiserum for the peptide to which it was raised was established by dot blotting. New methods were developed for isolating enriched fractions of gap junctions from whole heart and from dissociated adult myocytes, in which detergent-treatment and raising the temperature (potentially damaging steps in previously described techniques) are avoided. Analysis of these fractions by SDS-polyacrylamide gel electrophoresis revealed major bands at 43 kDa (matching the molecular mass of connexin43) and at 70 kDa. Western blot experiments using our antisera indicated that both the 43-kDa and the 70-kDa bands represent cardiac gap-junctional proteins. Pre-embedding immunogold labelling of isolated gap junctions and post-embedding immunogold labelling of Lowicryl-embedded whole tissue demonstrated the specific binding of the antibodies to ultrastructurally defined gap junctions. One antiserum (raised to residues 131-142) was found to be particularly effective for cytochemical labelling. Using this antiserum for immunofluorescence labelling in combination with confocal scanning laser microscopy enabled highly sensitive detection and three-dimensional mapping of gap junctions through thick slices of cardiac tissue. By means of the serial optical sectioning ability of the confocal microscope, images of the entire gap junction population of complete en face-viewed disks were reconstructed. These reconstructions reveal

  20. Prevention of edema disease in pigs by passive immunization. (United States)

    Johansen, M; Andresen, L O; Thomsen, L K; Busch, M E; Wachmann, H; Jorsal, S E; Gyles, C L


    The effect of treatment with verotoxin 2e (VT2e) specific antiserum was evaluated in 3 Danish pig herds with edema disease (ED). The antiserum was prepared by immunizing horses with a VT2e toxoid. The study was performed as a randomized blind field trial with parallel treatment and control groups. There were approximately 50 piglets in each group in each of the 3 herds and 741 piglets were included in the study (244 from herd A, 249 from herd B, and 247 from herd C). Treatment groups received 2, 4, or 6 mL anti-VT2e serum intramuscularly the day before weaning. Control groups were treated with 6 mL normal horse serum or 6 mL RPMI 1640 medium as placebo. All pigs that died in the trial period (1 d before weaning to 44 d after weaning) were examined pathologically and microbiologically. Mortality due to ED, mortality due to other causes, and adverse effects due to treatment were recorded. As there was no mortality due to ED, herd B was excluded from statistical calculations on mortality. The content of horse antibodies specific to VT2e in serum from pigs was analyzed in an indirect ELISA. A higher dose of anti-VT2e serum was reflected in higher optical density values in the indirect ELISA. Transient adverse reactions, seen as vomiting, ataxia, and cyanosis, occurred shortly after the injection of horse serum in 1.5% of the pigs, and one pig died. There were no statistically significant differences in mortality due to other causes among the 3 treatment groups in herds A and C. Only pigs from which F18+, VT2e+, ST-, LT- hemolytic E. coli (0139 or O-rough) was isolated were diagnosed as dead due to ED. Deaths due to ED in the control groups were 8.1% and 12.0% in herds A and C, respectively, compared with 0% and 0.7% in the corresponding serum groups. The difference between treatment and control groups was statistically significant (P<0.0001). It was not possible to establish an effect of dose (2, 4, or 6 mL) of anti-VT2e serum, because only one pig died of ED in the

  1. Comparison of gel filtration and ammonium sulphate precipitation in the purification of diphtheria toxin and toxoid. (United States)

    Møyner, K; Christiansen, G


    Crude diphtheria toxin and toxoid were subjected to purification by gel filtration and stepwise ammonium sulphate precipitation. The various fractions obtained by the purification procedures were studied by immunological methods. A high molecular weight fraction of glycoprotein nature was present in all of the crude preparations studied. The fraction was antigenically non-identical with the real toxin or toxoid and did not have its origin in the culture medium. It showed a long flocculation time when tested against equine diphtheria toxoid antiserum. The fraction could be removed from the crude preparations by gel filtration or by precipitation with 21% (w/v) ammonium sulphate. When comparing toxoids purified by each of these methods, the method of gel filtration resulted in a somewhat higher degree of purity, suggesting that this method would be more suitable than the AS precipitation method for the purification of diphtheria toxoid.

  2. Evaluation of anti-pneumococcal capsular antibodies as adjunctive therapy in experimental pneumococcal meningitis

    DEFF Research Database (Denmark)

    Brandt, Christian; Frimodt-Moller, N; Lundgren, Jens Dilling


    OBJECTIVE: Bacteraemia concomitant with meningitis has been shown to greatly affect outcome. Consequently, the efficacy of serotype-specific anti-pneumococcal antiserum (APAS) was investigated in a rat model of pneumococcal meningitis. METHODS: Rats were infected with Streptococcus pneumoniae...... serotype 3. All rats received ceftriaxone starting 26 h post-infection. APAS was administered either at the time of infection or 26 h post-infection and effects were compared with rats treated with antibiotics only. RESULTS AND CONCLUSION: A significant clinical benefit was found when APAS was given...... at the time of infection whereas no effect was found when administered 26 h after infection. This work indicates that the clinical value of using APAS in pneumococcal meningitis may be limited...

  3. ScII类似蛋白是洋葱根端细胞核、染色体和染色体骨架的组成成分%ScⅡ-like Protein is Localized in the Nuclei, Chromosomes and Chromosome Scaffolds of Allium cepa

    Institute of Scientific and Technical Information of China (English)

    王岩; 邢苗; 阎石


    The nuclei were isolated from the root meristematic cells of Allium cepa and the nuclear matrices were prepared. A 135kD polypeptide, which is equivalent to Sc Ⅱ in molecular weight, was revealed in the nuclei by SDS-PAGE and was then demonstrated to be an Sc Ⅱ-like protein by Western blot with an anti-chicken Sc antiserum. Neither the 135kD polypeptide nor the positive labelling of the anti-Sc antiserum was found in the nuclear matrices. The immuno-fluorescence tests showed that the nuclei labelled with the anti-Sc Ⅱ antiserum emanated strong, specific fluorescence, while the fluorescence of the nuclear matrices was too weak to be detected. The results of immunoelectron microscopy indicated that a large number of the gold particles were concentrated in the condensed chromatin of the nuclei, but very few gold particles were distributed in cytoplasm and nucleoplasm. These results strongly suggested that an Sc Ⅱ like protein is a component of the nuclei of A. cepa and is mainly located in the condensed chromatin regions, but the nuclear matrices contain no or very little amount of that protein. By means of immunofluorescence and immunoelectron microscopy, the chromosomes and chromosomal scaffolds of A. cepa labelled with the anti-chicken Sc Ⅱ antiserum were observed to send off specific fluorescence and have many gold particles representing the presence of the Sc Ⅱ-like protein distributed among them. The significance of the Sc Ⅱ-like protein as a novel component in the nuclei, chromosomes and chromosome scaffolds of higher plants is discussed.%采用机械破碎和蔗糖梯度离心方法从洋葱根端分生组织中分离出细胞核并制备出核骨架。细胞核SDS-PAGE谱带中135kD处有一多肽,免疫印迹实验结果表明,该多肽可被抗鸡Sc Ⅱ抗体标记,核骨架中没有此多肽。经抗Sc Ⅱ抗体和FITC偶联的二抗标记后,细胞核发出代表Sc Ⅱ的特异性荧光,而核骨架中无荧光发出。经抗Sc

  4. Daboia russellii and Naja kaouthia venom neutralization by lupeol acetate isolated from the root extract of Indian sarsaparilla Hemidesmus indicus R.Br. (United States)

    Chatterjee, Ipshita; Chakravarty, A K; Gomes, A


    The present study reports the isolation and purification of lupeol acetate from the methanolic root extract of Indian medicinal plant Hemidesmus indicus (L.) R.Br. (family: Asclepiadaceae) which could neutralize venom induced action of Daboia russellii and Naja kaouthia on experimental animals. Lupeol acetate could significantly neutralize lethality, haemorrhage, defibrinogenation, edema, PLA(2) activity induced by Daboia russellii venom. It also neutralized Naja kaouthia venom induced lethality, cardiotoxicity, neurotoxicity and respiratory changes in experimental animals. Lupeol acetate potentiated the protection by snake venom antiserum action against Daboia russellii venom induced lethality in male albino mice. Venom induced changes in lipid peroxidation and super oxide dismutase activity was antagonized by lupeol acetate. Snake venom neutralization by lupeol acetate and its possible mechanism of action has been discussed.

  5. Study and development of a radioimmunoassay of antidiuretic hormone sensitive at 10/sup -12/M

    Energy Technology Data Exchange (ETDEWEB)

    Caillens, H.; Rousselet, F. (Faculte des Sciences Pharmaceutiques, (France)); Paillard, F. (Hopital Tenon, Paris (France))


    A radioimmunoassay of antidiuretic hormone is described. The antiserum was obtained by immunization of rabbits with lysine vasopressin conjugated to hemocyanine. The specificity of the antibody was selective and directed against the pentapeptide ring of the vasopressin molecule: oxytocin showed no cross-reactivity at 10/sup -9/M. The labelled hormone (/sup 125/I-AVP) prepared using the chloramine-T method had a high specific activity (1860 Ci/mmol). Incubation was performed in an equilibrium system. Comparative studies of different separation methods of bounds and free /sup 125/I-AVP showed that the sensitivity and the precision of the standard curve were better using charcoal dextran. The limit of detection of the assay was 1,6 pg per ml.

  6. Specificity of a cytochemical bioassay for arginine-vasopressin and its validation for plasma measurement. (United States)

    Smith, A; McIntosh, N


    The total Na+/K+ ATP-ase activity of the thick ascending limb of the loop of Henle may be stimulated by arginine-vasopressin (AVP). Lysine-vasopressin (LVP), oxytocin (OT), and arginine-vasotocin (AVT) produce less than 5% of the enzyme activity induced by the same concentration of AVP. Physiological concentrations of a mixture of other hormones with known activity on the kidney (T3, T4, aldosterone, angiotensin II, and OT) did not significantly increase total Na+/K+ ATP-ase activity. Specific AVP antiserum consistently removed greater than 90% of the stimulatory effect of plasma. The concentration of AVP in plasmas from dehydrated subjects was greater than 10 times that of the same subjects hydrated. Intra-assay coefficient of variation was 35% and 52% from 200 microliters and 20 microliters of plasma respectively. The interassay coefficient of variation was 53% and 55% from plasma pools with high and low AVP content.

  7. Toxigenic characteristics of Clostridium perfringens type C in enterotoxemia of domestic animals. (United States)

    Niilo, L


    Eleven Clostridium perfringens type C strains isolated from fatal cases of hemorrhagic enterotoxemia of Canadian calves, a piglet, and a foal were studied for the production of soluble antigens. All the isolates from calves and a foal failed to produce delta toxin, but were capable of producing large amounts of lethal beta toxin. A strain isolated from a piglet produced delta, but very little beta toxin. Other differences were relatively minor. The results indicated that young domestic animals may be susceptible to all subtypes of C. perfringens type C. A simple method of using blood agar plates coated with type A antiserum for demonstration of hemolytic patterns was found advantageous in differentiation of C. perfringens strains.

  8. Direct visualization of retinoic acid in the rat hypothalamus: an immunohistochemical study. (United States)

    Mangas, A; Bodet, D; Duleu, S; Yajeya, J; Geffard, M; Coveñas, R


    In order to increase our knowledge about the distribution of vitamins in the mammalian brain, we have developed a highly specific antiserum directed against retinoic acid with good affinity (10(-8) M), as evaluated by ELISA tests. In the rat brain, no immunoreactive fibers containing retinoic acid were detected. Cell bodies containing retinoic acid were only found in the hypothalamus. This work reports the first visualization and the morphological characteristics of cell bodies containing retinoic acid in the mammalian paraventricular hypothalamic nucleus and in the dorsal perifornical region, using an indirect immunoperoxidase technique. The restricted distribution of retinoic acid in the rat brain suggests that this vitamin could be involved in very specific physiological mechanisms.

  9. Purification and separation of multiple forms of lactophorin from bovine milk whey and their immunological and electrophoretic properties. (United States)

    Kanno, C


    Lactophorin is designated as a glycoprotein, which is present in bovine milk whey and reacts to the antiserum of the soluble glycoprotein of bovine milk fat globule membrane. The lactophorin was purified by DEAE-cellulose (pH 7.7), Sephadex G-100, and then Bio Gel A-15m from the component-3 fraction of the proteose-peptone fraction of bovine milk whey. The purified lactophorin was separated into seven components by DEAE-cellulose chromatography at pH 8.6. The seven components (LP-1 to -7) of lactophorin were almost homogeneous, but the respective bands were somewhat broad and varied in mobilities on disc electrophoresis. The seven lactophorin components fused completely to the antisoluble glycoprotein of milk fat globule membrane on double immunodiffusion but showed different mobilities of precipitation lines on immunoelectrophoresis. The results indicated that lactophorin consisted of multiple forms but had a common set of antigenic determinant groups against anti-soluble glycoprotein.

  10. Comparative studies of canine colipase and lipases from bovine, porcine, canine, human and rat pancreases. (United States)

    Lee, P C


    1. Colipase was purified from canine pancreatic juice and found to have certain specificity in its reaction with various pancreatic lipases. 2. This colipase will stimulate the lipolytic activities of lipases isolated from canine, bovine and porcine pancreas but not lipases from a fungus, or from human and rat pancreases. 3. Characterization of these lipases showed (a) the molecular dimension of rat lipase is very different from the other lipases; (b) the pIs of canine, porcine and bovine lipases are almost identical but different from the pIs of rat, human and Candida (a fungus) lipases; and (c) the antiserum prepared against canine lipase will also react with lipases from human, hog and cow pancreases but not with rat and Candida lipases. 4. These physical differences can explain partly the difference in reaction between the various lipases and the canine colipase.

  11. Two cases of viper bite: still an important health problem

    Directory of Open Access Journals (Sweden)

    Adrija Hajra


    Full Text Available Viper venoms act mainly as hemotoxic. Manifestations of snakebites depend on specific toxins that constitute the venom. The local and systemic snake bite related symptoms are directly linked to the toxicity of the venom. Edema, ecchymoses, hematoma, and gangrenous lesions are reported to occur as local symptoms. Systemic symptoms may include fever, nausea, vomiting, delirium, jaundice, circulatory collapse, convulsions, and coma. Death from secondary infections, neurotoxicity, disseminated intravascular coagulation (DIC, intracranial hemorrhage, and acute renal failure are the well-known facts. For reduction of morbidity and mortality, it is important that antiserum is administered at the appropriate dose as early as possible after snake bite. There are several case reports about various complications of viperid bite. Here we are discussing two cases of viper bite. These cases are unique because of the extensive tissue necrosis. One of them succumbed to septicemia after acute pancreatitis. [Int J Res Med Sci 2016; 4(4.000: 1274-1277

  12. Role of laminin receptor in tumor cell migration

    DEFF Research Database (Denmark)

    Wewer, U M; Taraboletti, G; Sobel, M E;


    Polyclonal antisera were made against biochemically purified laminin receptor protein as well as against synthetic peptides deduced from a complementary DNA clone corresponding to the COOH-terminal end of the laminin receptor (U.M. Wewer et al., Proc. Natl. Acad. Sci. USA, 83: 7137-7141, 1986......). These antisera were used to study the potential role of laminin receptor in laminin-mediated attachment and haptotactic migration of human A2058 melanoma cells. The anti-laminin receptor antisera reacted with the surface of suspended, nonpermeabilized melanoma and carcinoma cells. The anti-laminin receptor...... antisera blocked the surface interaction of A2058 cells with endogenous laminin, resulting in the inhibition of laminin-mediated cell attachment. The A2058 melanoma cells migrated toward a gradient of solid phase laminin or fibronectin (haptotaxis). Anti-laminin antiserum abolished haptotaxis on laminin...

  13. Isolation of Aureimonas altamirensis, a Brucella canis-like bacterium, from an edematous canine testicle. (United States)

    Reilly, Thomas J; Calcutt, Michael J; Wennerdahl, Laura A; Williams, Fred; Evans, Tim J; Ganjam, Irene K; Bowman, Jesse W; Fales, William H


    Microbiological and histological analysis of a sample from a swollen testicle of a 2-year-old Border Collie dog revealed a mixed infection of the fungus Blastomyces dermatitidis and the Gram-negative bacterium Aureimonas altamirensis. When subjected to an automated microbial identification system, the latter isolate was provisionally identified as Psychrobacter phenylpyruvicus, but the organism shared several biochemical features with Brucella canis and exhibited agglutination, albeit weakly, with anti-B. canis antiserum. Unequivocal identification of the organism was only achieved by 16S ribosomal RNA gene sequencing, ultimately establishing the identity as A. altamirensis. Since its first description in 2006, this organism has been isolated infrequently from human clinical samples, but, to the authors' knowledge, has not been reported from a veterinary clinical sample. While of unknown clinical significance with respect to the pathology observed for the polymicrobial infection described herein, it highlights the critical importance to unambiguously identify the microbe for diagnostic, epidemiological, infection control, and public health purposes.

  14. Serological studies on chloridazon-degrading bacteria. (United States)

    Layh, G; Böhm, R; Eberspächer, J; Lingens, F


    Agglutination tests and immunofluorescence tests with antisera against four strains of chloridazon-degrading bacteria revealed the serological uniformity of a group of 22 chloridazon-degrading bacterial strains. No serological relationship could be found between chloridazon-degrading bacteria and representatives of other Gram-negative bacteria. This was demonstrated by agglutination tests, including testing of the antiserum against Acinetobacter calcoaceticus, and by immunofluorescence tests, including testing of the sera against Pseudomonas and Acinetobacter strains. The tests were performed with 31 representatives of different Gram-negative bacteria, and with 22 strains of chloridazon-degrading bacteria as antigens. Differences in the extent of agglutination reactions and antibody titres among chloridazon-degrading bacterial strains together with cross-adsorption xperiments, suggest a rough classification of chloridazon-degrading bacteria into two subgroups. On the basis of immunofluorescence data, a linkage-map was worked out to represent serological relationships in the group of chloridazon-degrading strains.

  15. Purification in a functional form of the terminal protein of Bacillus subtilis phage phi 29. (United States)

    Prieto, I; Lázaro, J M; García, J A; Hermoso, J M; Salas, M


    Phage phi 29 terminal protein, p3, essentially pure, was isolated in a denatured form from viral particles, and anti-p3 antiserum was obtained. A radioimmunoassay to detect and quantitate protein p3 was developed. By using this assay, native protein p3 was highly purified from Escherichia coli cells harboring a gene 3-containing recombinant plasmid. After three purification steps, the protein was more than 96% pure; its amino acid composition was very similar to that deduced from the nucleotide sequence of gene 3. The purified protein was active in the formation of the covalent p3-dAMP initiation complex when supplemented with extracts of B. subtilis infected with a sus mutant of phi 29 in gene 3. No DNA polymerase or ATPase activities were present in the final preparation of protein p3.

  16. A distinct tospovirus causing necrotic streak on Alstroemeria sp. in Colombia. (United States)

    Hassani-Mehraban, Afshin; Botermans, Marleen; Verhoeven, J Th J; Meekes, Ellis; Saaijer, Janneke; Peters, Dick; Goldbach, Rob; Kormelink, Richard


    A tospovirus causing necrotic streaks on leaves was isolated from Alstroemeria sp. in Colombia. Infected samples reacted positively with tomato spotted wilt virus (TSWV) antiserum during preliminary serological tests. Further analysis revealed a close serological relationship to tomato chlorotic spot virus (TCSV) and groundnut ringspot virus (GRSV). A major part of the S-RNA segment, encompassing the nucleocapsid (N) protein gene, the 5' untranslated region and a part of the intergenic region 3' of the N gene, was cloned and sequenced. The deduced N protein sequence showed highest amino acid identity (82%) to that of TCSV, indicating that the virus represents a new tospovirus species, for which the name Alstroemeria necrotic streak virus (ANSV) is coined. Phylogenetic analysis based on the N protein sequence revealed that this Alstroemeria-infecting tospovirus clustered with tospoviruses from the American continent. Frankliniella occidentalis was identified as potential vector species for ANSV.

  17. [A rare cause of compartment syndrome of the forearm and hand following snake bite injury]. (United States)

    Schnecker, K


    With the intention to commit suicide a 25 year old patient was bitten by his own rattle snake. At the time of the admission the skin of the right forearm was dark, a hemorrhagic necrotizing colour, and the patient was in shock. He was immediately taken to the intensive care unit and the shock symptoms were treated there. Parasthesias in the area of the nervus medianus were also noticed. The treatment included an antiserum and the release of the tourniquet which caused a further increase of the swelling of the forearm. The lesion led to a hemorrhagic necrotizing inflammation. The surgical incision of the loge of Guyon, the carpal channel, the forearm and proximal of the lacertus fibrosus was persuaded. The circulation improved immediately and after three weeks the nerval function had recovered. The skin defect was covered 14 days after the first operation with meshgraft.

  18. Characterization of Elongation Factor Tu of Mycoplasma ovipneumoniae

    Directory of Open Access Journals (Sweden)

    Xuan Zhang, Yue-feng Chu, Ping Zhao, Peng-cheng Gao, Ying He, Nu Wang and Zhong-xin Lu*


    Full Text Available Mycoplasma ovipneumoniae is considered as an important pathogen of small ruminants, but its antigenic proteins are not well known so far. In this study, we cloned the EF-Tu gene of M. ovipneumoniae and analyzed the molecular features of the gene and its coding protein for the first time. The gene was then expressed in E.coli and the antigenicity of the coding protein was evaluated as well. The EF-Tu gene of M. ovipneumoniae is 1209 bp in length, encodes 402 amino acids, and shares the highest DNA sequence identity of 87.5% and deduced amino acid sequence identity of 97.8% with those of M. hyopneumoniae, respectively. The recombinant EF-Tu protein can react with the polyclonal antiserum of M. ovipneumoniae and can induce humoral immune responses in mice, which indicated that the EF-Tu may be used as a candidate protein in developing the technologies to control the disease.

  19. Nuclease enhancement of specific cell agglutination in a serodiagnostic test for Neisseria gonorrhoeae. (United States)

    Arko, R J; Wong, K H; Peacock, W L


    Antiserum to a purified type R lipopolysaccharide antigen isolated from Neisseria gonorrhoeae was used in a slide agglutination test and compared with conventional carbohydrate utilization and fluorescent antibody tests to confirm the identity of laboratory cultures classified as typical or "atypical" N. gonorrhoeae. Cultures of Corynebacterium vaginalis, N. meningitidis, N. catarrhalis, N. sicca, and N. lactamicus were also tested in the slide agglutination procedure. The addition of both deoxyribonuclease and ribonuclease (1 mg/ml) to the cell suspension medium of phosphate-buffered saline improved the sensitivity and specificity of the agglutination reaction for N. gonorrhoeae. Problems relating to the agglutination test as an aid in identification of N. gonorrhoeae are discussed. PMID:110830

  20. Studies on the Feeding Habits of Lutzomyia (Lutzomyia longipalpis (Lutz & Neiva, 1912 (Diptera: Psychodidae: Phlebotominae Populations from Endemic Areas of American Visceral Leishmaniasis in Northeastern Brazil

    Directory of Open Access Journals (Sweden)

    Margarete Martins dos Santos Afonso


    Full Text Available The aim of this study was to identify potential blood feeding sources of L. (L. longipalpis specimens from populations in Northeastern Brazil, endemic areas of American Visceral Leishmaniasis (AVL and its correlation with the transmission of L. (L. i. chagasi. The ELISA technique was applied using bird, dog, goat, opossum, equine, feline, human, sheep, and rodent antisera to analyze 609 females, resulting in an overall positivity of 60%. In all municipalities, females showed higher positivity for bird followed by dog antiserum and sand fly specimens were also positive for equine, feline, human, sheep, goat, opossum, and rodent antisera. The finding for 17 combinations of two or three types of blood in some females corroborates the opportunistic habit of this sand fly species. The results demonstrating the association between L. (L. longipalpis and opossum suggest the need for further evaluation of the real role of this synanthropic mammal in the eco-epidemiology of AVL.

  1. Preliminarily functional analysis of a cloned novel human gene ADAM29

    Institute of Scientific and Technical Information of China (English)


    ADAM is a family of type I integral membrane proteins which are characterized by sharing a disintegrin and metalloprotease domain and involved in many important physiological processes such as fertilization, neurogenesis and inflammatory response. A novel human ADAM gene--ADAM29, which was cloned in our laboratory, is exclusively expressed in human testis and contains a potential fusion domain. A full-length cDNA of ADAM29 was obtained by using multiple-step PCR. Phylogenetic tree of known mammalian ADAMs specifically expressed in testis was reconstructed. Polyclonal antiserum was raised by immunizing the rabbits with sub-peptide of ADAM29 (Leu268-Asp374) as immunogen. The result of immunohistochemical test on human testis showed that ADAM29 is expressed in different stages of spermatogenesis and in interstitial cells. ADAM29 may play a certain role in the signal transduction during the maturation of testis-associated cells.

  2. Anti-enrofloxacin Antibody Production by Using Enrofloxacin-screened HSA as an Immunogen

    Institute of Scientific and Technical Information of China (English)

    LIU Chune; LIN Hong; CAO Limin; JIANG Jie


    A two-step zero-length cross-linking procedure using active esters was successfully adopted for conjugating enrofloxacin (EF) to human serum albumin (HSA). The derived conjugate was characterized by UV spectrum and then used for immunization of BALB/C mice. In enzyme-linked immunosorbent assay (ELISA) and competitive inhibition ELISA experiments, the derived antiserum exhibited high antibody titer (greater than 1: 250 000) as well as varied cross-reactivity (from 97.8% to 161.7%) to three analogs of EF belonging to fluoroquinolones family. But over the concentration range studied, no significant cross-reactivity was observed to other group of antibiotics (chloramphenicol, oxytetracycline, sulphamethoxazole and nysfungin). It was confirmed that the synthesized immunogen was highly antigenic and elicited specific antibody responses in BALB/C mice against EF.

  3. Distribution of two basement membrane proteoglycans through hair follicle development and the hair growth cycle in the rat

    DEFF Research Database (Denmark)

    Couchman, J R; King, J L; McCarthy, K J


    The distribution of two distinct populations of basement membrane proteoglycans has been monitored through hair growth development in the rat embryo and subsequent hair growth cycle. An antiserum against a small heparan sulfate proteoglycan uniformly stained the dermal-epidermal junction...... of embryonic rats throughout the period of hair follicle formation. On the other hand, monoclonal antibodies recognizing a basement membrane-specific chondroitin sulfate proteoglycan only weakly stained 16-d embryo dermal-epidermal junction, but strong staining was associated with hair follicle buds...... as they developed. Through the hair growth cycle, it was found that the heparan sulfate proteoglycan persisted around the follicles, while the chondroitin sulfate proteoglycan decreased in amount through catagen until it was undetectable at the base and dermal papilla of the telogen follicle. As anagen commenced...

  4. Isolation of porcine reproductive and respiratory syndrome (PRRS) virus in a Danish swine herd and experimental infection of pregnant gilts with the virus

    DEFF Research Database (Denmark)

    Bøtner, Anette; Nielsen, Jens; Bille-Hansen, Vivi


    The first case of porcine reproductive and respiratory syndrome (PRRS) in Denmark was diagnosed in March 1992 by the detection of specific antibodies against PRRS virus in serum samples originating from sows in a herd located on the island of Als. Subsequently, PRRS virus was isolated from a 200......-sow farrow-to-finish herd with clinical signs consistent with PRRS. The virus was isolated by inoculation of pleural fluid from a stillborn piglet onto porcine pulmonary alveolar macrophages. The isolate was identified as PRRS virus by staining with a specific antiserum. By electron microscopy...... infection was demonstrated by the re-isolation of PRRS virus from approximately 45% of the piglets from the experimentally infected gilts. However, the experimental infection produced no significant reproductive disorders or other clinical signs. At autopsy, histopathological examination revealed slight...

  5. [AS-1L - a newly discovered strain of Cyanophage AS-1 in GDR]. (United States)

    Gorjusin, V A; Stenz, E; Averkiev, A A


    In samples, taken from waters in the surroundings of Leipzig (GDR) in 1978, we found cyanophages in Central Europe for the first time. Among other cyanophages we isolated the new strain AS-1L. Out of 20 tested cultures of unicellular cyanobacteria seven strains belonging to the genus Synechococcus proved to be susceptible for this cyanophage. In morphology AS-1L corresponds to the cyanophage AS-1 found in the U.S.A., to which it is related serologically, too. AS-1L differs from the other strains of AS-1 by a shorter growth cycle, especially a shorter latent period, by the kinetics of inactivation by antiserum, and by a somewhat narrower pH scope of stability. Consequently the isolated cyanophage is to be looked at as a new strain of the cyanophage AS-1.

  6. Selective extraction of isolated mitotic apparatus. Evidence that typical microtubule protein is extracted by organic mercurial. (United States)

    Bibring, T; Baxandall, J


    Mitotic apparatus isolated from sea urchin eggs has been treated with meralluride sodium under conditions otherwise resembling those of its isolation. The treatment causes a selective morphological disappearance of microtubules while extracting a major protein fraction, probably consisting of two closely related proteins, which constitutes about 10% of mitotic apparatus protein. Extraction of other cell particulates under similar conditions yields much less of this protein. The extracted protein closely resembles outer doublet microtubule protein from sea urchin sperm tail in properties considered typical of microtubule proteins: precipitation by calcium ion and vinblastine, electrophoretic mobility in both acid and basic polyacrylamide gels, sedimentation coefficient, molecular weight, and, according to a preliminary determination, amino acid composition. An antiserum against a preparation of sperm tail outer doublet microtubules cross-reacts with the extract from mitotic apparatus. On the basis of these findings it appears that microtubule protein is selectively extracted from isolated mitotic apparatus by treatment with meralluride, and is a typical microtubule protein.

  7. Evaluation of a multiplex PCR for detection of serotypes K1, K2 and K5 in Klebsiella sp. and comparison of isolates within these serotypes. (United States)

    Turton, Jane F; Baklan, Hatice; Siu, L K; Kaufmann, Mary E; Pitt, Tyrone L


    A multiplex PCR using targets within the serotype-specific region of the capsular polysaccharide synthesis gene cluster of serotypes K1, K2 and K5 was evaluated using the 77 reference serotype strains of Klebsiella, and a panel of clinical isolates subjected previously to conventional serotyping. The PCR was highly specific for these serotypes, which are those most associated with virulence in humans and horses. PCR confirmed that isolates of the K5 serotype had cross-reacted with antiserum for other serotypes, particularly for K7. K5 isolates received by our laboratory were almost exclusively from thoroughbred horses, and were submitted for screening prior to breeding programmes. Most, including a reference strain isolated in 1955, belonged to a cluster of genetically similar isolates of sequence type (ST) 60. K1 isolates, all from humans, belonged to a previously identified cluster of ST 23.

  8. Sperm-Egg Interaction: Evidence for Boar Sperm Plasma Membrane Receptors for Porcine Zona Pellucida (United States)

    Peterson, Rudolph N.; Russell, Lonnie; Bundman, Donna; Freund, Matthew


    Freshly ejaculated, noncapacitated boar sperm bind rapidly and in large numbers to pig egg zona pellucida in vitro. In the present study, the number of sperm bound decreased sharply when sperm motility was lowered by energy poisons or by reducing the temperature. Highly motile sperm from humans, guinea pigs, and rats, added at concentrations ten times higher than control sperm, did not bind to the porcine zona. At the same high concentration, a small number of hamster and bull sperm bound to the zona. Binding of boar sperm to the zona pellucida was blocked almost completely by diluted whole antiserum to sperm plasma membranes and by univalent (Fab) antibody to these membranes. When antibody to sperm plasma membrane was first absorbed with plasma membrane vesicles, sperm binding was not inhibited. These results provide direct evidence for the existence of sperm plasma membrane receptors for the zona pellucida of the pig.

  9. Hormone-sensitive lipase (HSL) expression and regulation in skeletal muscle

    DEFF Research Database (Denmark)

    Langfort, J; Ploug, T; Ihlemann, J;


    Because the enzymatic regulation of muscle triglyceride metabolism is poorly understood we explored the character and activation of neutral lipase in muscle. Western blotting of isolated rat muscle fibers demonstrated expression of hormone-sensitive lipase (HSL). In incubated soleus muscle...... epinephrine increased neutral lipase activity by beta-adrenergic mechanisms involving cyclic AMP-dependent protein kinase (PKA). The increase was paralleled by an increase in glycogen phosphorylase activity and could be abolished by antiserum against HSL. Electrical stimulation caused a transient increase...... in activity of both neutral lipase and glycogen phosphorylase. The increase in lipase activity during contractions was not influenced by sympathectomy or propranolol. Training diminished the epinephrine induced lipase activation in muscle but enhanced the activation as well as the overall concentration...

  10. Serologic test-systems development: immunoassays for antibiotics. Progress report, May 16, 1980-September 30, 1981

    Energy Technology Data Exchange (ETDEWEB)

    Brake, R.; Hollstein, U.; Hindman, K.


    Progress on development of immunoassays for the antibiotics gentamicin, tetracycline, and tylosin is discussed. The development of the gentamicin assay was completed and the assay was transferred to the Beltsville Laboratories of the US Department of Agriculture (USDA) Food Safety and Quality Service (FSQS). The sensitivity (1 ppB) is 50-fold greater than we have previously reported, and is satisfactory for the current needs. At year's end, work was still in progress on both the tetracycline and tylosin assays. Tetracycline presents a more difficult problem of chemistry than we had anticipated. Immunoassay reagents synthesized by diazonium coupling were only weakly immunoreactive; analysis suggests that this coupling procedure damages the tetracycline structure. Several tetracycline derivatives that offer promising alternative coupling procedures were synthesized. Tylosin was successfully coupled to peroxidase. With this conjugate and antiserum, obtained from R. Mageau of the USDA, some immune-specific binding was demonstrated. Several problems with the tylosin assay remain to be resolved.

  11. Sex differences in the photoperiodic regulation of RF-Amide related peptide (RFRP) and its receptor GPR147 in the syrian hamster

    DEFF Research Database (Denmark)

    Henningsen, Jo B; Poirel, Vincent-Joseph; Mikkelsen, Jens D;


    RF-(Arg-Phe) related peptides (RFRP-1 and -3) are considered to play a role in the seasonal regulation of reproduction; however, the effect of the peptides depends on species and gender. This study aimed at comparing the RFRP system in male and female Syrian hamsters over long and short photoperi......RF-(Arg-Phe) related peptides (RFRP-1 and -3) are considered to play a role in the seasonal regulation of reproduction; however, the effect of the peptides depends on species and gender. This study aimed at comparing the RFRP system in male and female Syrian hamsters over long and short...... photoperiods to investigate the neuroanatomical basis of these differential effects. The neuroanatomical distribution of RFRP neurons and fibers, revealed using an antiserum recognizing RFRP-1 and -3, as well as GPR147 mRNA, are similar in male and female Syrian hamsters. RFRP neurons are mainly found...

  12. Increased muscle glucose uptake during contractions

    DEFF Research Database (Denmark)

    Ploug, Thorkil; Galbo, H; Richter, E A


    We reinvestigated the prevailing concept that muscle contractions only elicit increased muscle glucose uptake in the presence of a so-called "permissive" concentration of insulin (Berger et al., Biochem. J. 146: 231-238, 1975; Vranic and Berger, Diabetes 28: 147-163, 1979). Hindquarters from rats...... in severe ketoacidosis were perfused with a perfusate containing insulin antiserum. After 60 min perfusion, electrical stimulation increased glucose uptake of the contracting muscles fivefold. Also, subsequent contractions increased glucose uptake in hindquarters from nondiabetic rats perfused for 1.5 h......-methylglucose uptake increased during contractions and glucose uptake was negative at rest and zero during contractions. An increase in muscle transport and uptake of glucose during contractions does not require the presence of insulin. Furthermore, glucose transport in contracting muscle may only increase if glycogen...

  13. Construction of genomic libraries of Cryptosporidium parvum and identification of antigen-encoding genes. (United States)

    Dykstra, C C; Blagburn, B L; Tidwell, R R


    Genomic libraries have been constructed from bovine C. parvum DNA in the lambda ZAP and lambda DASH vectors. Based on an estimated genome size of 2 x 10(4) kilobases (kb), each recombinant library contains greater than 10 genomic equivalents. The average recombinant size for the lambda ZAP library is 2.1 kb and for the lambda DASH library is 14 kb. We have identified genes to major antigens recognized by hyperimmune bovine antiserum. These recombinants are currently being purified and characterized. Limited DNA sequence analysis of random C. parvum clones confirms suggestions that the genome is quite AT-rich. The DNA sequence of random lambda ZAP fusion proteins has identified a potential ATPase, a structural protein and a DNA-binding protein.

  14. Neuron specific enolase demonstration in the diagnosis of a solid-cystic (papillary cystic) tumour of the pancreas. (United States)

    Chott, A; Klöppel, G; Buxbaum, P; Heitz, P U


    Immunoreactivity to neuron specific enolase (NSE) was demonstrated in a solid-cystic (papillary cystic) tumour of the human pancreas, employing immunohistochemical methods. Positive staining for NSE was found with two different antisera. In addition, sodium-dodecyl-sulphate-polyacrylamide-gel-electro-phoresis (SDS-PAGE) of tumour homogenate revealed a distinct band reacting with a NSE antiserum. However, we failed to detect any hormonal products or neuroendocrine granules in the tumour. Therefore the authors advise caution in using the enzyme as a differential diagnostic tool, especially in surgical pathology of epithelial pancreatic neoplasms occurring in young females. In individual cases electron microscopy will be necessary since solid-cystic tumours of the pancreas consistently show large intracytoplasmic zymogen-like granules.

  15. Comparação da eficiência dos caldos de enriquecimento seletivo no isolamento de Salmonella Dublin Comparison of the efficiency of selective enrichment broths for Salmonella Dublin isolation

    Directory of Open Access Journals (Sweden)

    D.G. Silva


    Full Text Available The aim of this study was to compare three different selective enrichment broths: Rappaport-Vassiliadis (RV, selenite cystine (SC and Muller-Kauffmann tetrathionate (MKT for Salmonella Dublin isolation from faecal samples of calf experimentally infected. The bacteriological procedure involved pre-enrichment stages in Hajna-GN broth (only for the samples inoculated in RV broth, selective enrichment, culture in modified brilliant green agar (BGA, presumptive biochemistry tests (using triple-sugar-iron agar and lysine-agar and slide agglutination test with poli-O and poli-H Salmonella antiserum. The effects of enrichment temperatures using RV broth were also evaluated (37ºC and 42ºC. SC broth was significantly more efficient in the isolation of Salmonella Dublin (P<0,05, whereas RV broth incubated at 42ºC had a lower efficiency in the microbiological isolation.

  16. Cell-Free, De Nova Synthesis of Poliovirus (United States)

    Molla, Akhteruzzaman; Paul, Aniko V.; Wimmer, Eckard


    Cell-free translation of poliovirus RNA in an extract of uninfected human (HeLa) cells yielded viral proteins through proteolysis of the polyprotein. In the extract, newly synthesized proteins catalyzed poliovirus-specific RNA synthesis, and formed infectious poliovirus de novo. Newly formed virions were neutralized by type-specific antiserum, and infection of human cells with them was prevented by poliovirus receptor-specific antibodies. Poliovirus synthesis was increased nearly 70-fold when nucleoside triphosphates were added, but it was abolished in the presence of inhibitors of translation or viral genome replication. The ability to conduct cell-free synthesis of poliovirus will aid in the study of picornavirus proliferation and in the search for the control of picornaviral disease.

  17. [An avian strain of Escherichia coli with antigens common to the genus Salmonella]. (United States)

    Terzolo, H R; Zoratti de Verona, A; d'Empaire, M; Furowicz, A J


    On a commercial poultry farm, a large percentage (9%) of clinically healthy fowls had positive reaction to the plate test, with commercial polyvalent pullorum antigens. We could not isolate Salmonella from the positive birds. An strain, of Escherichia coli Balcarce (E. coli B) was isolated from the feces of one of the birds. The isolate was identified biochemically and the antigenic study showed correlation with E. coli 044 and the somatic fraction 1, 2, 8, 14 and 23 of the Salmonella genus. The common antigens were studied by agglutination, absorption and crossed immunodiffusion tests, comparing the isolated strain and the different Salmonella serotypes. Four pullorum polyvalent commercial antigens reacted with sera containing somatic agglutinins 1, and with the E. coli B antiserum. These observations confirm the high antigenic correlation between the genus of the Enterobacteriaceae family. It is indicated that for the diagnosis of avian salmonelosis rather than using a single serological tests, the isolation and identification of the etiological agent is required.

  18. Transduction of plasmid DNA in Streptomyces spp. and related genera by bacteriophage FP43. (United States)

    McHenney, M A; Baltz, R H


    A segment (hft) of bacteriophage FP43 DNA cloned into plasmid pIJ702 mediated high-frequency transduction of the resulting plasmid (pRHB101) by FP43 in Streptomyces griseofuscus. The transducing particles contained linear concatemers of plasmid DNA. Lysates of FP43 prepared on S. griseofuscus containing pRHB101 also transduced many other Streptomyces species, including several that restrict plaque formation by FP43 and at least two that produce restriction endonucleases that cut pRHB101 DNA. Transduction efficiencies in different species were influenced by the addition of anti-FP43 antiserum to the transduction plates, the temperature for cell growth before transduction, the multiplicity of infection, and the host on which the transducing lysate was prepared. FP43 lysates prepared on S. griseofuscus(pRHB101) also transduced species of Streptoverticillium, Chainia, and Saccharopolyspora.

  19. Development and validation of a direct sandwich chemiluminescence immunoassay for measuring DNA adducts of benzo[a]pyrene and other polycyclic aromatic hydrocarbons

    DEFF Research Database (Denmark)

    Georgiadis, Panagiotis; Kovács, Katalin; Kaila, Stella;


    We have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)-DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated...... with rabbit antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and subsequently trapped by goat anti-rabbit IgG bound to a solid surface. Anti-single-stranded (ss) DNA antibodies binds in a quantity proportional to the adduct levels...... and is detected by chemiluminescence. The BPDE-DNA SCIA has a limit of detection of 3 adducts per 10(9) nucleotides with 5 µg DNA per well. We have validated the BPDE-DNA SCIA using DNA modified in vitro, DNA from benzo[a]pyrene (BP)-exposed cultured cells and mice. The levels of adduct measured by SCIA were...

  20. Luminal proteinases from Plodia interpunctella and the hydrolysis of Bacillus thuringiensis CryIA(c) protoxin. (United States)

    Oppert, B; Kramer, K J; Johnson, D; Upton, S J; Mcgaughey, W H


    The ability of proteinases in gut extracts of the Indianmeal moth, Plodia interpunctella, to hydrolyze Bacillus thuringiensis (Bt) protoxin, casein, and rho-nitroanilide substrates was investigated. A polyclonal antiserum to protoxin CryIA(c) was used in Western blots to demonstrate slower protoxin processing by gut enzymes from Bt subspecies entomocidus-resistant larvae than enzymes from susceptible or kurstaki-resistant strains. Enzymes from all three strains hydrolyzed N-alpha-benzoyl-L-arginine rho-nitroanilide, N-succinyl-ala-ala-pro-phenylalanine rho-nitroanilide, and N-succinyl-ala-ala-pro-leucine rho-nitroanilide. Zymograms and activity blots were used to estimate the apparent molecular masses, number of enzymes, and relative activities in each strain. Several serine proteinase inhibitors reduced gut enzyme activities, with two soybean trypsin inhibitors, two potato inhibitors, and chymostatin the most effective in preventing protoxin hydrolysis.

  1. Identification and characterization of the murine cell surface receptor for the urokinase-type plasminogen activator

    DEFF Research Database (Denmark)

    Solberg, H; Løber, D; Eriksen, J;


    -hybridization and pronounced sequence similarity with human u-PAR cDNA [Kristensen, P., Eriksen, J., Blasi, F. & Danø, K. (1991) J. Cell Biol. 115, 1763-1771]. A rabbit antiserum raised against this peptide specifically recognized two polypeptide bands with electrophoretic mobilities identical to those identified by ligand...... of this variant yielded a polypeptide with an apparent M(r) of about 30,000, which corresponds to the Mr calculated from the cDNA derived protein sequence of mouse u-PAR. Receptor-bound mouse u-PA could be released by phosphatidylinositol-specific phospholipase C treatment, indicating that mouse u-PAR is attached...... by ligand-blotting analysis.(ABSTRACT TRUNCATED AT 400 WORDS)...

  2. Radioimmunoassay of sodium cromoglycate in human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Brown, K.; Gardner, J.J.; Lockley, W.J.S.; Preston, J.R.; Wilkinson, D.J. (Fisons Ltd., Loughborough (UK). Pharmaceutical Div.)


    A sensitive radioimmunoassay method for sodium cromoglycate in human plasma is described. The lowest quantifiable concentration of sodium cromoglycate is 0.93 nmol/1 when 0.1 ml plasma samples are analysed. Direct analysis of sodium cromoglycate concentrations in plasma samples collected up to several hours after the administration of single therapeutic doses of the compound is possible. The range of the method is limited; both 0.01 and 0.1 ml volumes of plasma must be analysed to encompass the concentration range 0.93-139 nmol/1 which may be encountered in plasma samples from patients and human volunteers. The method is specific for sodium cromoglycate as indicated by a low cross-reactivity of the anti-cromoglycate antiserum with a number of drugs.

  3. Light-independent and light-dependent protochlorophyllide-reducing activities and two distinct NADPH-protochlorophyllide oxidoreductase polypeptides in mountain pine (Pinus mugo). (United States)

    Forreiter, C; Apel, K


    Lower plants and gymnosperms synthesize chlorophyll and develop photosynthetically competent chloroplasts even when grown in the dark. In cell-free extracts of pine (Pinus mugo, Turra, ssp. mugo) seedlings, light-independent and light-dependent protochlorophyllide-reducing activities are present. Two distinct NADPH-protochlorophyllide-oxidoreductase (POR) polypeptides can be detected immunologically with an antiserum raised against the POR of barley. The subcellular localization and amounts of the two POR polypeptides are differentially affected by light: one of them is predominantly present in prolamellar bodies of etiochloroplasts and its abundance rapidly declines once the pine seedlings are exposed to light; the other is found in thylakoid membranes and its amount does not change during illumination of dark-grown seedlings. Two types of cDNA sequences are identified that encode two distinct POR polypeptides in pine. The relevance of these POR polypeptides for the two chlorophyll biosynthetic pathways active in gymnosperms is discussed.

  4. VAMP-1: a synaptic vesicle-associated integral membrane protein. (United States)

    Trimble, W S; Cowan, D M; Scheller, R H


    Several proteins are associated with, or are integral components of, the lipid bilayer that forms the delineating membrane of neuronal synaptic vesicles. To characterize these molecules, we used a polyclonal antiserum raised against purified cholinergic synaptic vesicles from Torpedo to screen a cDNA expression library constructed from mRNA of the electromotor nucleus. One clone encodes VAMP-1 (vesicle-associated membrane protein 1), a nervous-system-specific protein of 120 amino acids whose primary sequence can be divided into three domains: a proline-rich amino terminus, a highly charged internal region, and a hydrophobic carboxyl-terminal domain that is predicted to comprise a membrane anchor. Tryptic digestion of intact and lysed vesicles suggests that the protein faces the cytoplasm, where it may play a role in packaging, transport, or release of neurotransmitters. Images PMID:3380805

  5. Radioimmunoassay of nicergoline in biological material

    Energy Technology Data Exchange (ETDEWEB)

    Bizollon, C.A.; Rocher, J.P.; Chevalier, P.


    A method for radioimmunosassay of nicergoline (Sermion) in plasma and urine is described. The antiserum was produced in rabbits by administration of an immunogen obtained by coupling bovine serum albumin to the nicergoline molecule at the indole nitrogen. The resulting antibodies reacted well with nicergoline and the 1-demethyl derivative and did not cross-react with the principal metabolites of the substrances nor with rye ergot derivatives, in particular dihydroergotamine, methysergide and bromocriptine. The tracer was nicergoline labelled with iodine-125. The assay was sensitive because concentrations as low as as 125x10/sup -12/ mol/l nicergoline could be directly determined in plasma and urine without prior extraction. The marked specificity and high sensitivity allowed easy determination of plasma and urine levels of this drug following administration in man.

  6. Mechanisms of Cytotoxicity of the Aids Virus (United States)


    G1 X G2 X GI X G1 X G2 X G1 X Antiserum M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 M Lane -69 kDa - P55 p40 4 -3OkDa 14 A~ FIG. 4. Specificity of...supplemeinted with (0.5", Nonidet P-40t iiid NIHI AIDS Research and Reference Reagent Pogram incubated for- 301 miii onl ice. The nule~li w crc pelee...eili-dianilter (󈧉-7 plates twice, and thle suIpernatants were reoilc. 1 liiid, thre Nonidet with 1(0 Ig (if recombinant proviral clonie NLHXAD.-\\V(iG

  7. Comparative studies of HSV-1 antigens solubilised from infected cells by using non-ionic or zwitterionic detergents. (United States)

    Jennings, R; Erturk, M


    HSV-1 antigen preparations solubilised from Vero cells by using either the non-ionic detergent Nonidet P40 or the zwitterionic detergent Empigen BB, and purified on sucrose density gradients or over a sucrose cushion, were tested by ELISA with anti-HSV-1 glycoprotein monoclonal antibodies and by radioimmunoprecipitation (RIP) with polyclonal HSV-1 antiserum. Amongst several proteins detected in these preparations, the four major HSV-1 glycoproteins, gB, gC, gD, and gE, were found to be present. Differences between NP40 or Empigen-solubilised HSV-1 antigen preparations with respect to two of these glycoproteins, gB and gE, were detected by using a small panel of monoclonal antibodies. Comparative studies in mice showed the Empigen-solubilised HSV-1 antigen preparations elicited greater antibody responses and greater protection against lethal HSV-1 challenge infection than the NP40-solubilised preparation.

  8. Plasmodium falciparum: characterization of toxin-associated proteins and identification of a hemoglobin containing parasite cytokine stimulator

    DEFF Research Database (Denmark)

    Kristensen, G; Jakobsen, P H


    ]-methionine and immunoprecipitated the labeled antigens with an antiserum against IMP which blocks malaria parasite-induced TNF production. We detected four proteins associated with IMP when the immunoprecipitates were separated by SDS-PAGE and analyzed by autoradiography. To evaluate the capacity of different P. falciparum...... antigens to induce cytokine production we separated a mixture of exoantigens by SDS-PAGE gels. Antigen fractions of 43-71 kDa and of a low molecular mass of TNF alpha interleukin 1 alpha, and interleukin 6 production from human mononuclear cells. The low......Previous studies have indicated the inositol monophosphate (IMP) is a component of the malaria parasite toxin that induces cytokines such as tumour necrosis factor (TNF). To further characterize the toxin we have labeled Plasmodium falciparum in vitro cultures with [14C]inositol or [35S...


    Directory of Open Access Journals (Sweden)

    N. A. Isutina


    Full Text Available The method of gas-liquid chromatography investigates composition of polyunsaturated fatty acids of membrane of erythrocytes, discharged of navel bloods neonatal from mothers who have transferred in the season gestation exacerbation herpes of an infection contamination and his influence on microviscosity of membrane. Essential infringements of data exchange of bonds in navel bloods neonatal with an exacerbation  herpes  infection  contaminations  (antiserum  capacity  IgG  to  virus  of  simple  herpes  of  1  type 1 : 12 800 which show deficiency essential ω-3 acids at simultaneous augmentation of the precursor proinflammatory eicosanoid ω-6 arachidonic acids, promoting augmentation of relative microviscosity of membrane of erythrocytes that will be one of probable causes of development of hypoxia are found.

  10. Adherence of Moraxella bovis to cell cultures of bovine origin. (United States)

    Annuar, B O; Wilcox, G E


    The adherence of five strains of Moraxella bovis to cell cultures was investigated. M bovis adhered to cultures of bovine corneal epithelial and Madin-Darby bovine kidney cells but not to cell types of non-bovine origin. Both piliated and unpiliated strains adhered but piliated strains adhered to a greater extent than unpiliated strains. Antiserum against pili of one strain inhibited adherence of piliated strains but caused only slight inhibition of adherence to the unpiliated strains. Treatment of bacteria with magnesium chloride caused detachment of pili from the bacterial cell and markedly inhibited adherence of piliated strains but caused only slight inhibition of adherence by the unpiliated strains. The results suggested that adhesion of piliated strains to cell cultures was mediated via pili but that adhesins other than pili may be involved in the attachment of unpiliated strains of M bovis to cells.

  11. Biochemical identification of the bovine blood group M' antigen as a major histocompatibility complex class I-like molecule

    DEFF Research Database (Denmark)

    Hønberg, L S; Larsen, B; Koch, C;


    . To elucidate the structural relationship between BoLA-A16 and blood group antigen M', immunoprecipitation experiments on red and white cell lysates isolated from M'-A16 positive and negative cattle were carried out. These results showed that M(r) 44,000 and M(r) 12000 polypeptides can be precipitated from both......Absorption and elution experiments showed that it was impossible to separate antibodies against blood group factor M' from antibodies against bovine lymphocyte antigen (BoLA) A16 in an antiserum showing haemolytic activity against M' as well as lymphocytotoxic activity against BoLA-A16...... red and white cells isolated from M'-A16 positive animals, whereas no bands were seen in M'-A16 negative animals in precipitations with the same antibody. Precipitation with a crossreacting human beta 2-microglobulin (beta 2-m) specific antibody confirmed a class-I-like structure associated with beta...

  12. Fate of (D-Ala2-deltorphin-I-like immunoreactive neurons in 6-hydroxydopamine lesioned rat brain

    Directory of Open Access Journals (Sweden)

    A Casini


    Full Text Available The use of a polyclonal antiserum specific to C-terminal tetrapeptide amide of (D-Ala2deltorphin-I, a naturally occurring amphibian skin opioid peptide, has already demonstrated the presence of immunoreactive neurons in rat midbrain. Double immunostaining identified these neurons as a subpopulation of the mesencephalic dopaminergic neurons that were also tyrosine hydroxylase-immunopositive and calbindin- D28kD- negative, namely, the neurons predominantly affected in Parkinson disease. We followed the fate of these neurons after a monolateral injection of 6-hydroxy-dopamine into rat brain. Almost all the immunopositive neurons and their nigrostriatal, mesolimbic and mesocortical projections on the side ipsilateral to the lesion disappeared. Only a few scattered immunopositive neurons within the substantia nigra, pars compacta, and those of supramammillary nucleus remained unaffected. The consistent overlap of dopamine and this new molecule provides a further key to identifying the mammalian counterpart of these amphibian skin opioid peptides.

  13. Preparation of a polyvalent antivenom against various Mexican scorpion Centruroides species. (United States)

    Garcia y Perez, G; Martin, M F; Rochat, H


    Antisera were obtained from rabbits injected with four different immunogens from the Mexican scorpion Centruroides suffusus suffusus i.e. the crude venom, a telson extract, a toxic fraction obtained from this telson extract by gel filtration and the same toxic fraction subjected to acetylation. The neutralizing capacity of these antisera are compared: it appears that a telson extract can be used instead of the crude venom to produce an efficient antiserum. The immunological properties of ground telsons obtained from three other species of the Mexican scorpion Centruroides (Centruroides noxius, Centruroides limpidus limpidus, Centruroides limpidus tecomanus) are studied with the antisera raised against Centruroides suffusus suffusus immunogens: an almost total cross-neutralization is observed.


    Directory of Open Access Journals (Sweden)

    L. D. Varbanets


    Full Text Available The lipopolysaccharide from Escherichia coli L-19 was isolated, chemically identified and its nontoxicity but pyrogenicity were shown. The investigations of lipopolysaccharide influence on T- and B-lymphocytes indicated that it might be used as a mitogen in blasttransformation reactions since it was only in insignificant degree less active in comparison with the commercial one. It was shown that in reactions of Ouchterlony double immunodiffusion in agar LPS from E. coli L-19 exerted an activity of antigen in homological system. There were not established serological cross reactions between antiserum to heated E.coli L-19 cells and LPS from another of E.coli strain М-17 аnd also the representatives of Enterobacteriaceae species such as Budvicia aquatica, Rahnella aquatilis and Pragia fontium. These results indicate the absence of common antigenic determinants between the tested strains.

  15. Evaluation of Lama5 as a candidate for the mouse ragged (Ra) mutation

    DEFF Research Database (Denmark)

    Durkin, M E; Albrechtsen, R; Chambers, D M;


    The laminin alpha5 chain is a component of the basement membranes of many developing and adult tissues. The mouse laminin alpha5 chain gene (Lama5) has been mapped close to the locus of the semidominant ragged (Ra) mutation on distal chromosome 2. The cause of the Ra mutation, which is usually...... lethal in the homozygous state, has not been determined. We have investigated whether a defect in Lama5 is responsible for the ragged mutation, using the RaJ strain. No differences in the level of the laminin alpha5 chain transcript were found in placental RNA from homozygous RaJ mutant embryos compared...... to normal littermates. Antiserum raised against a recombinant laminin alpha5 chain polypeptide stained the basement membranes of both normal and homozygous mutant embryos to a similar extent. More precise mapping of Lama5 on an interspecific Ra backcross indicated that Lama5 is proximal to the Ra locus...

  16. Investigation on inhibition of biological effects of endothelin

    Institute of Scientific and Technical Information of China (English)

    田青; 赵东; 张继峰; 高连如; 刘胜昔; 杨军; 苏静怡; 张肇康; 汤健; 唐朝枢


    The effects of a series of substances on the biological function of endothelin (ET) are reported. The substances used are: synthetic inhibitors of endothelium derived relaxing factors (EDRFs), inhibitor of big-endothelin converting enzyme phosphoramidon, antiserum of endothelin, antagonists of endothelin A receptor BQ123 and JKC301, and two Chinese anti-snake venom herb medicines Lobelia radians Thumb and Taris polyphylla Smith var. chinensis (Franch) Hara. The results showed that inhibiting the production of nitric oxide (NO) could stimulate ET release from vascular endothelium, elevate plasma ET and increase blood pressure. These changes could be reversed by L-arginine (L-Arg), the substrate of nitric oxide synthase (NOS). The amount of ET released by arterial endothelium could be increased or inhibited by inhibiting or stimulating the synthesis of prostacyclin (PGI2). The plasma ET level and blood pressure in both SHR and WKY rats could be decreased by giving phosphoramidon (PhR). The above results i

  17. Isolation of the etiological agent of primary amoebic meningoencephalitis from artificially heated waters

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, A.R.; Tyndall, R.L.; Coutant, C.C.; Willaert, E.


    To determine whether artificial heating of water by power plant discharges facilitates proliferation of the pathogenic free-living amoebae that cause primary amoebic meningoencephalitis, water samples (250 ml) were taken from discharges within 3,000 feet (ca. 914.4 m) of power plants and were processed for amoeba culture. Pathogenic Naegleria fowleri grew out of water samples from two of five lakes and rivers in Florida and from one of eight man-made lakes in Texas. Pathogenic N. fowleri did not grow from water samples taken from cooling towers and control lakes, the latter of which had no associated power plants. The identification of N. fowleri was confirmed by pathogenicity in mice and by indirect immunofluorescence analyses, by using a specific antiserum.

  18. Early cytoplasmic vacuolization of African green monkey kidney cells by SV40. (United States)

    Miyamura, T; Kitahara, T


    As early as 3--4 hours after infection with SV40 at a high input multiplicity, African green monkey (Cercopithecus aethiops) kidney (AGMK) cells developed cytoplasmic vacuolization. At 10--20 hours after infection, the vacuolization reached its maximal level, then disappeared and SV40 specific cytopathic change followed. This vacuolization developed before the synthesis of the specific T and V antigens. This early cytoplasmic vacuolization (ECV) was prevented by preincubating the virus with specific antiserum, or by heating the virus with MgCl2. The ECV could be induced by UV-irradiated SV40. Addition of metabolic inhibitors had no effect on the induction of the ECV. These results suggest that the capacity to induce the ECV resides in a structural component(s) of SV40 virion and the vacuolization is not associated with the replication of SV40.

  19. Early cytoplasmic vacuolization of African green monkey kidney cells by SV40. [uv radiation

    Energy Technology Data Exchange (ETDEWEB)

    Miyamura, T.; Kitahara, T.


    As early as 3 to 4 hours after infection with SV 40 at a high input multiplicity, African green monkey (Cercopithecus aethiops) kidney (AGMK) cells developed cytoplasmic vacuolization. At 10 to 20 hours after infection, the vacuolization reached its maximal level, then disappeared and SV 40 specific cytopathic change followed. This vacuolization developed before the synthesis of the specific T and V antigens. This early cytoplasmic vacuolization (ECV) was prevented by pre-incubating the virus with specific antiserum, or by heating the virus with MgCl/sub 2/. The ECV could be induced by uv-irradiated SV 40. Addition of metabolic inhibitors had no effect on the induction of the ECV. These results suggest that the capacity to induce the ECV resides in a structural component(s) of SV 40 virion and the vacuolization is not associated with the replication of SV 40.

  20. Chlamydia trachomatis antigen in female genital tract infection

    Directory of Open Access Journals (Sweden)

    Badrinath S


    Full Text Available Thirty cases of female genital tract infection were investigated for the presence of Chlamydia trachomatis antigen. Endocervical swabs obtained were subjected to antigen detection by enzyme immunoassay. Rabbit antiserum to chlamydial lipopolysaccharide was used in a card test. Anti rabbit immunoglobulin G conjugated to alkaline phosphatase with a chromogenic substrate 5 bromo-4 chloro-3-indolyl phosphate and nitro blue tetrazolium were used for the enzymatic reaction. Chlamydial antigen could be detected in four out of thirty samples (13.3%. In contrast direct immunofluorescence detected 5 cases (16.6%. Although less sensitive, enzyme immunoassay can be used as a rapid diagnostic tool in detecting Chlamydia trachomatis antigen in genital infections.

  1. [Brazilian purpuric fever. Fast characterization of invasive strains of Haemophilus aegyptius]. (United States)

    Brandileone, M C; Vieira, V S; Tondella, M L; Sacchi, C T; Landgraf, I M; Zanella, R C; Bibb, W F; Irino, K


    Strains of H. aegyptius isolated during outbreak of Brazilian Purpuric Fever (BPF) in Brazil were characterized antigenically by slide agglutination test utilizing antiserum produced with a H. aegyptius strain isolated from blood culture from a patient with BPF. By means of this method, it were identified H. aegyptius strains responsible for outbreaks of conjunctivitis with identical antigenic characteristics to strains isolated from BPF. The sensitivity and specificity of slide seroagglutination test was 97.7% and 89.6% respectively; therefore this assay was efficient to be used as a screening method in the studies of purulent conjunctivitis for detecting high risk populations for BPF, and to implement measures that will increase the efficiency of epidemiologic surveillance.

  2. Obtenção e avaliação de antígenos de Aspergillus fumigatus Obtention and evaluation of Aspergillus fumigatus antigens extraction

    Directory of Open Access Journals (Sweden)

    Vanda de Sá Lirio


    Full Text Available Antígenos de três amostras de A. fumigatus (354, 356, JIG e antissoro contra a mistura destes antígenos foram produzidos e avaliados imunoquimicamente. Os antígenos de filtrado de cultura foram obtidos após concentração com acetona conforme adaptação da técnica descrita por Coleman & Kaufman. Em prova de ID obteve-se 100% de positividade com os soros de pacientes com aspergilose estudados. Com relação aos soros heterólogos encontramos reatividade com soro de um paciente com candidíase e com soro de um paciente com histoplasmose; foi encontrado padrão idêntico de resposta quando se utilizou o antígeno de referência. O antissoro foi titulado por ID, CIE e RFC MI contra o antígeno específico apresentando títulos respectivos de 1:32, 1:32 e 1:128, e utilizado para reagir contra o mesmo antígeno por IEF, demonstrando 8 linhas de precipitação, sendo 5 na região anódica e 3 na região catódica. O perfil de bandeamento do antígeno em eletroforese utilizando gel de poliacrilamida (SDS-PAGE a 12,5% apresentou-se complexo com 26 sub-unidades protéicas, cujos pesos moleculares variaram de 18 a > 100kDa. Quando estes componentes foram eletrotransferidos e reagidos com o antissoro específico ("immunoblotting", verificou-se imunogenicidade em todas as frações bandeadas.Antigens from three strains of Aspergillus fumigatus (354, 356, and JIG and an antiserum against the mixing of these antigens have been produced, and evaluated immunochemically. The antigens were obtained through a modified Coleman & Kaufman technique (culture filtrate concentrated by acetone. Analysis by the immunodiffusion test (ID against homologous serum has yielded 100% sensitivity (with the studied sera. Concerning heterologous sera we found reactivity with a serum of a patient of candidiasis and another with histoplasmosis. The same result was obtained with a reference antigen in immunodiffusion, showing similar standards of response. Titration of the

  3. Use of an indirect haemagglutination test, for the detection of Clostridium perfringens type A enterotoxin

    Directory of Open Access Journals (Sweden)

    Marlene B. Serafim


    Full Text Available An indirect haemagglutination (IH test is described for the detection of Clostridium perfringens type A enterotoxin, produced by strains isolated from human cases of food poisoning and from contaminated food. Though no strict relationship could be observed between titers in the IH test and the time it took mice to die from the intravenous inoculation of mice (IIM, results of the supernatants examined by both methods demonstrated that the IH test was more sensitive than the ILM one. No unspecific reaction was obtanined int he IH wirh a negative control and the inhibitions of the IH and IIM tests by specific antiserum against C. perfringens enterotoxin showed that the IH test is very spcific. The IH assay is recommended for its sensitivity and easy performance by less-equipped laboratories, by these and other data.

  4. Recombinant Gluconacetobacter diazotrophicus containing Cry1Ac gene codes for 130-kDa toxin protein. (United States)

    Subashini, M; Moushumi Priya, A; Sundarakrishnan, B; Jayachandran, S


    Recombinant Gluconacetobacter diazotrophicus containing Cry1Ac gene from Bacillus thuringiensis var. kurstaki borne on pKT230, shuttle vector, was generated. PCR amplification of Cry1Ac gene present in recombinant G. diazotrophicus yielded a 278-bp DNA product. The nitrogenase assay has revealed that the recombinant G. diazotrophicus in sugarcane stem produced similar levels of nitrogenase compared to wild-type G. diazotrophicus. The presence of 130-kDa protein in apoplastic fluid from sugarcane stem harvested from pots inoculated with recombinant G. diazotrophicus shows that the translocated G. diazotrophicus produces 130-kDa protein which is recognized by the hyperimmune antiserum raised against 130-kDa protein. The first instar Eldana saccharina neonate larvae that fed on artificial medium containing recombinant G. diazotrophicus died within 72 h after incubation.

  5. Development and evaluation of one-step rRT-PCR and immunohistochemical methods for detection of Rift Valley fever virus in biosafety level 2 diagnostic laboratories. (United States)

    Drolet, Barbara S; Weingartl, Hana M; Jiang, Jieyuan; Neufeld, James; Marszal, Peter; Lindsay, Robbin; Miller, Myrna M; Czub, Markus; Wilson, William C


    Rift Valley fever virus (RVFV) is a zoonotic insect transmitted virus endemic to Africa and the Arabian Peninsula. Infection causes abortions and high mortality in newborn ruminants. The overall human infection rate is BSL-4 conditions and require inactivation and safety testing for use outside of containment. In this study, antiserum against recombinant RVFV-nucleocapsid (N) was produced to develop an immunohistochemical (IHC) assay which was subsequently evaluated on formalin fixed lamb and calf tissues at BSL-2 laboratory conditions. Antigen was detected by IHC in 79% of rRT-PCR-positive sheep and 70% of rRT-PCR-positive calf tissues tested. Once validated and approved by national regulatory agencies, these assays can be safely produced and distributed to regional diagnostic laboratories, providing capacity for early detection of RVFV in suspected ruminant samples.

  6. Production and Characterization of Polyclonal Antibody for the N-Methylcarbamate Insecticide Metolcarb

    Institute of Scientific and Technical Information of China (English)

    ZHANG Qi; LI Tie-jun; ZHU Xiao-xia; XU Li-na; LIU Feng-quan; HU Bai-shi; JIANG Ying-hua; CAO Bin


    The hapten, 3-{[1-(3-(methyl)phenyloxy)-carbonyl]amino}propanoic acid (HOM), mimicking the analyte metolcarb, was synthesized and verified by mass spectrometry (MS) and 1H-nuclear magnetic resonance spectrometry (1H-NMR). Then,HOM was conjugated with the carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA) with stoichiometric amounts of N-hydroxysuccinimide/dicyclohexylcarbodimide (NHS/DCC) using the activated ester method. Polyclonal antibodies were raised against the conjugate of HOM-BSA in rabbits. Antiserum titres were determined by noncompetitive indirect ELISA procedures and the titer of pAb01 reached 1.28 × 106. The cross-reactivities of the structurally related Nmethylcarbamate insecticides were 0.0% except for dimethacarb. These results indicate that the antibody pAb01 with strong affinity and high specificity can be used to develop a sensitive and rapid detection protocol for metolcarb residue.

  7. Production of the Polyclonal Anti-human Metallothionein 2A Antibody with Recombinant Protein Technology

    Institute of Scientific and Technical Information of China (English)

    Faiz M.M.T.MARIKAR; Qi-Ming SUN; Zi-Chun HUA


    Metallothionein 2A (MT2A) is a small stress response protein that can be induced by exposure to toxic metals. It is highly expressed in breast cancer cells. In this study, the eDNA encoding the human MT2A protein was expressed as glutathione S-transferase (GST) fusion protein in Escherichia coli.Recombinant MT2A proteins were loaded onto 12% sodium dodecylsulfate-polyacrylamide gel and separated by electrophoresis, the recombinant protein was visualized by Coomassie blue staining and the 33 kDa recombinant GST-MT2A fusion protein band was cut out from the gel. The gel slice was minced and used to generate polyclonal antisera. Immunization of rabbit against MT2A protein allowed the production of high titer polyclonal antiserum. This new polyclonal antibody recognized recombinant MT2A protein in Western blot analysis. This low-cost antibody will be useful for detection in various immuno-assays.

  8. Renalase-specific polyclonal antibody and its application in the detection of renalase's expression. (United States)

    Wang, Feng; Zhao, Qing; Xing, Tao; Li, Junhui; Wang, Niansong


    Renalase is generated mainly by the kidneys, and renalase's expression in chronic kidney disease patients is reduced due to renal dysfunction. In this study, human renalase recombinant protein with prokaryotic expression was used for immunization of male New Zealand rabbits, and polyclonal antibodies against human renalase were obtained. To prepare and verify renalase polyclonal antibody, renalase recombinant protein was used as antigen and male New Zealand rabbits were immunized to obtain anti-serum for identification. On the basis of renalase antibody, the expression of renalase in renal tubular epithelial cells and renal tissue was detected. Two anti-renalase polyclonal antibodies were obtained. Renalase was constitutively expressed in human renal tubular epithelial cells, with the maximum expression in proximal convoluted tubules in renal tissue, and a small amount of expression in the glomeruli. Anti-renalase polyclonal antibodies were successfully prepared, which lays a foundation for further studies.

  9. In vivo expression and variation of Escherichia coli type 1 and P pili in the urine of adults with acute urinary tract infections. (United States)

    Kisielius, P. V.; Schwan, W. R.; Amundsen, S. K.; Duncan, J. L.; Schaeffer, A. J.


    In vivo expression of pili by Escherichia coli in the urine of 41 adults with lower urinary tract infections was analyzed by immunostaining with polyclonal antiserum to type 1 and P pili. Type 1 pili were detected in 31 of 41 urine specimens, while P pili were detected in 6 of 18 specimens. The piliation status of bacterial populations in urine was heterogeneous, varying from predominantly piliated to a mixture of piliated and nonpiliated cells. Bacteria frequently adhered to exfoliated uroepithelial cells and leukocytes in urine. Expression of pili in vivo did not always correlate with the hemagglutination phenotype after growth in vitro. Strains isolated from different sites in the urogenital tract of two individuals showed phenotypic variation in the state of piliation. The results demonstrate that E. coli type 1 and P pili are expressed and are subject to variation in vivo during acute urinary tract infections in adults. Images PMID:2566580

  10. Nucleotide sequence of maize dwarf mosaic virus capsid protein gene and its expression in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    赛吉庆; 康良仪; 黄忠; 史春霖; 田波; 谢友菊


    The 3’-terminal 1 279 nucleotide sequence of maize dwarf mosaic virus (MDMV) genome has been determined. This sequence contains an open reading frame of 1023 nudeotides and a 3’ -non-coding region of 256 nucleotides. The open reading frame includes all of the coding regions for the viral capsid protein (CP) and part of the viral nuclear inclusion protein (Nib). The predicted viral CP consists of 313 amino acid residues with a calculated molecular weight of 35400. The amino acid sequence of the viral CP derived from MDMV cDNA shows about 47%-54% homology to that of 4 other potyviruses. The viral CP gene was constructed in frame with the lacZ gene in pUC19 plasmid and expressed in E. coli cells. The fusion polypeptide positively reacted in Western blot with an antiserum prepared against the native viral CP.

  11. Giardia lamblia: intracellular localization of alpha8-giardin. (United States)

    Wei, Chao Jun; Tian, Xi Feng; Adam, Rodney D; Lu, Si Qi


    Alpha8-giardin (α8-giardin) is a member of the multi-gene α-giardin family in the intestinal parasitic protozoan, Giardia lamblia. This gene family shares an ancestry with the annexin super family, whose common characteristic is calcium dependent binding to membranes that contain acidic phospholipids. In the present study, the antigenicity, hydrophilicity, flexibility, surface probability, and secondary structure of α8-giardin amino acids were predicted by bioinformatics applications. A specific anti-peptide antiserum, anti-P3, was used to determine the intracellular location of α8-giardin with confocal immunofluorescence microscopy and immunoelectron microscopy. The results indicated that α8-giardin was located on the plasma membrane and flagella, but not on the ventral disk. Reduction of α8-giardin transcript levels by ribozyme-mediated cleavage decreased trophozoite motility and growth rate, indicating the functional importance of α8-giardin to Giardia trophozoite biology.

  12. Changes in RFamide related peptide-1 (RFRP-1)-immunoreactivity during postnatal development and the estrous cycle

    DEFF Research Database (Denmark)

    Jørgensen, Sara Rubek; Andersen, Mille Dahl; Overgaard, Agnete;


    in hypothalamic neurons that innervate and inhibit GnRH neurons. The RFRP precursor is processed into two mature peptides RFRP-1 and RFRP-3. These are characterized by a conserved C-terminal motif Arg-Phe-NH2 but display highly different N-terminals. Even though the two peptides are equally potent in vitro......, little is known about their relative distribution and their distinct roles in vivo. In this study, we raised an antiserum selective for RFRP-1 and defined the distribution of RFRP-1-immunoreactive (ir) neurons in the rat brain. Next, we analyzed the level of RFRP-1-immunoreactivity during postnatal...... in between, the dorsomedial hypothalamic, ventromedial hypothalamic, and arcuate nuclei. The number of RFRP-1-ir neurons and the density of cellular immunoreactivity were unchanged from juvenile to adulthood in male rats during the postnatal development. However, both parameters were significantly increased...

  13. Biological characterisation of Sarcocystis neurona isolated from a Southern sea otter (Enhydra lutris nereis) (United States)

    Lindsay, D.S.; Thomas, N.J.; Dubey, J.P.


    Sarcocystis neurona was isolated from the brain of a juvenile, male southern sea otter (Enhydra lutris nereis) suffering from CNS disease. Schizonts and merozoites in tissue sections of the otter's brain reacted with anti-S. neurona antiserum immunohistochemically. Development in cell culture was by endopolyogeny and mature schizonts were first observed at 3 days postinoculation. PCR of merozoite DNA using primer pairs JNB33/JNB54 and restriction enzyme digestion of the 1100 bp product with Dra I indicated the organism was S. neurona. Four of four interferon-γ gene knockout mice inoculated with merozoites developed S. neurona-associated encephalitis. Antibodies to S. neurona but not Sarcocystis falcatula, Toxoplasma gondii, or Neospora caninum were present in the serum of inoculated mice. This is the first isolation of S. neurona from the brain of a non-equine host.

  14. Rabbit antibodies against Toxoplasma Hsp20 are able to reduce parasite invasion and gliding motility in Toxoplasma gondii and parasite invasion in Neospora caninum. (United States)

    Coceres, Veronica M; Alonso, Andrés M; Alomar, M Lis; Corvi, María M


    Toxoplasma gondii Hsp20 is a pellicle-associated functional chaperone whose biological role is still unknown. Hsp20 is present in different apicomplexan parasites, showing a high degree of conservation across the phylum, with Neospora caninum Hsp20 presenting an 82% identity to that of T. gondii. Hence rabbit anti-T. gondii Hsp20 serum was able to recognize the N. caninum counterpart. Interestingly, both N. caninum and T. gondii Hsp20 localized to the inner membrane complex and to the plasma membrane. Incubation of T. gondii and N. caninum tachyzoites with an anti-TgHsp20 serum reduced parasite invasion at rates of 57.23% and 54.7%, respectively. This anti-serum also reduced T. gondii gliding 48.7%. Together, all this data support a role for Hsp20 in parasite invasion and gliding motility.

  15. Effects of calmodulin on DNA-binding activity of heat shock transcription factor in vitro

    Institute of Scientific and Technical Information of China (English)


    The DNA-binding activity of heat shock transcription factor (HSF) was induced by heat shock (HS) of a whole cell extract. Addition of antiserum, specific to CaM, to a whole cell extract reduced bind of the HSF to the heat shock element (HSE) with maize, and the re-addition of CaM to the sample restored the activity of the HSF for binding to HSE. In addition, DNA-binding activity of the HSF was also induced by directly adding CaM to a whole cell extract at non-HS temperature with maize. Similar results were obtained with wheat and tomato. Our observations provide the first example of the involvement of CaM in regulation of the DNA-binding activity of the HSF.

  16. Production and characterisation of polyclonal antisera against feline IgE. (United States)

    Gilbert, S; Halliwell, R E


    Cats, naturally or experimentally infected with Toxocara cati were immunised with dinitrophenylated ascaris antigen (DNP-Asc). All cats developed immediate skin reactivity to DNP coupled to bovine serum albumin (DNP-BSA) and the sera of the nine cats had a heat labile homocytotropic antibody detectable by homologous Prausnitz-Küstner (PK) tests. Reagin-rich fractions were prepared from these sera and used for the preparation of polyclonal antisera in rabbits. Resultant antisera were passed through a immunoabsorbent column of Sepharose 4B coupled to heated normal cat serum. An immunoabsorbent column prepared with the resultant antisera removed the PK reactivity from the cat sera, and the activity was recovered following acid elution. The antiserum failed to detect any recognised immunoglobulin in cat sera, but precipitated with a heat labile protein with gamma-1 electrophoretic mobility in the sera of parasited cats. These findings support the contention that the antisera are specific for feline IgE.

  17. The Role of Mosquitoes in the Diet of Adult Dragon and Damselflies (Odonata). (United States)

    Pfitzner, Wolf Peter; Beck, Matthias; Weitzel, Thomas; Becker, Norbert


    The flood plains of the Upper Rhine Valley provide excellent conditions for the proliferation of mosquitoes as well as for the development of dragon and damselflies. It could be assumed that mosquitoes belong to the diet of the Odonata and that the latter could be harmed by the reduction of the mosquito population with the purpose of diminishing the massive nuisance for the people living there. A total of 41 adult dragonflies and damselflies were examined by immunoblot for remnants of mosquitoes in their guts. A rabbit antiserum against Aedes vexans proteins was used for the immunoblot. Only 3 Aeshna cyanea and 1 Platycnemis pennipes could be shown to have fed on mosquitoes. In specimens of the genus Sympetrum no mosquitoes were detected. It seems very doubtful that mosquitoes are an essential part of the Odonata diet.

  18. Antibody-based biological toxin detection

    Energy Technology Data Exchange (ETDEWEB)

    Menking, D.E.; Goode, M.T. [Army Edgewood Research, Development and Engineering Center, Aberdeen Proving Ground, MD (United States)


    Fiber optic evanescent fluorosensors are under investigation in our laboratory for the study of drug-receptor interactions for detection of threat agents and antibody-antigen interactions for detection of biological toxins. In a direct competition assay, antibodies against Cholera toxin, Staphylococcus Enterotoxin B or ricin were noncovalently immobilized on quartz fibers and probed with fluorescein isothiocyanate (FITC) - labeled toxins. In the indirect competition assay, Cholera toxin or Botulinum toxoid A was immobilized onto the fiber, followed by incubation in an antiserum or partially purified anti-toxin IgG. These were then probed with FITC-anti-IgG antibodies. Unlabeled toxins competed with labeled toxins or anti-toxin IgG in a dose dependent manner and the detection of the toxins was in the nanomolar range.

  19. Fibronectin distribution in epithelial and associated tissues of the rat

    DEFF Research Database (Denmark)

    Couchman, J R; Gibson, W T; Thom, D


    Specific antiserum was used to investigate the distribution of the extracellular glycoprotein, fibronectin, in rat skin and tongue tissue by light and electron microscopy with immunofluorescence and immunoperoxidase techniques. We conclude that fibronectin is absent from stable, differentiated...... parts of tissues, such as the sebaceous glands or the matrix, medulla, cortex, and cuticles of the hair and the inner and outer root sheaths, or even in tissues in which there is some cell movement, such as the epidermis. It is, however, characteristic of sites at which cell division is occurring...... in contact with an extracellular scaffolding, such as basement membrane or loose connective tissue. Conspicuous examples were in the glassy membrane and connective tissue sheath associated with the follicular epithelium, the basement membrane underlying vascular endothelial cells, the connective tissues...

  20. Immune reactivity of cells from long-term rat renal allograft survivors

    Energy Technology Data Exchange (ETDEWEB)

    Weiss, A.; Stuart, F.P.; Fitch, F.W.


    Lewis rats receiving an LBN kidney allograft demonstrate no signs of rejection if they are pretreated with donor spleen cells and antiserum reactive with the donor alloantigen. We examined the cellular reactivity of long-term kidney allograft survivors. Normal proliferative and cytolytic responses were obtained with spleen cells from long-term survivors, in marked contrast to the diminished responses of cells from neonatally tolerant rats or the heightened cytolytic response of cells from rats that had rejected a renal allograft. Serum from long-term renal allograft survivors as well as serum obtained from rats at the time of transplantation did not suppress proliferative or cytolytic responses of normal cells. The results of this study suggest that long-term renal allograft survivors possess the precursors of those cells which are responsible for proliferative and cytolytic responses in mixed leukocyte cultures, but that they have not been sensitized to their renal allograft.

  1. Characterization of a 7-kilodalton subunit of vaccinia virus DNA-dependent RNA polymerase with structural similarities to the smallest subunit of eukaryotic RNA polymerase II. (United States)

    Amegadzie, B Y; Ahn, B Y; Moss, B


    A previously unrecognized 7-kDa polypeptide copurified with the DNA-dependent RNA polymerase of vaccinia virus virions. Internal amino acid sequences of the small protein matched a viral genomic open reading frame of 63 codons. Antipeptide antiserum was used to confirm the specific and complete association of the 7-kDa protein with RNA polymerase. The amino acid sequence predicted from the viral gene, named rpo7, was 23% identical to that of the smallest subunit of Saccharomyces cerevisiae RNA polymerase II, and a metal-binding motif, Cys-X-X-Cys-Gly, was located at precisely the same location near the N terminus in the two proteins. RNA analyses demonstrated early transcriptional initiation and termination signals in the rpo7 gene sequence. The viral RNA polymerase subunit was synthesized during the early phase of infection and continued to accumulate during the late phase.

  2. Competitive enzyme immunoassay for urinary vanillylmandelic acid. (United States)

    Taran, F; Bernard, H; Valleix, A; Créminon, C; Grassi, J; Olichon, D; Deverre, J R; Pradelles, P


    An enzyme immunoassay for urinary vanillylmandelic acid (VMA) using polyclonal antiserum and VMA-acetylcholinesterase conjugate as enzymatic tracer is described. Two different strategies for immunogen preparation were developed and enantioselectivity was demonstrated. Selected EIA allowed direct measurement of urinary VMA using D(-)-VMA as standard with good sensitivity (MDC = 0.1 mumol/l) and precision (CV less than 7% in 0.2-2.25 mumol/l range). Cross-reactivity with homovanillic acid (HVA) was 0.8% and less than 0.4% with other structurally related catecholamine metabolites. Intra- and inter-assay repeatability were less than 10% and recovery was 97.3% +/- 3%. Good correlation was obtained for EIA and HPLC analysis with normal and pathologic human urine samples (EIA = 0.895 HPLC-7.085, r2 = 0.98, n = 47).

  3. Immunoreactive transforming growth factor alpha and epidermal growth factor in oral squamous cell carcinomas

    DEFF Research Database (Denmark)

    Therkildsen, M H; Poulsen, Steen Seier; Bretlau, P


    , the cells above the basal cell layer were positive for both TGF-alpha and EGF. The same staining pattern was observed in oral mucosa obtained from healthy persons. In moderately to well differentiated carcinomas, the immunoreactivity was mainly confined to the cytologically more differentiated cells, thus......Forty oral squamous cell carcinomas have been investigated immunohistochemically for the presence of transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF). The same cases were recently characterized for the expression of EGF-receptors. TGF-alpha was detected...... with a monoclonal mouse antibody and EGF with polyclonal rabbit antiserum. Thirty-five of the tumours were positive for TGF-alpha and 26 of the tumours for EGF. None of the poorly differentiated tumours was positive for EGF, but they all were for TGF-alpha. In sections including normal differentiated oral mucosa...

  4. The Mycoplasma hominis P120 membrane protein contains a 216 amino acid hypervariable domain that is recognized by the human humoral immune response

    DEFF Research Database (Denmark)

    Nyvold, Charlotte Guldborg; Birkelund, Svend; Christiansen, Gunna


    domain. Based on restriction endonuclease cleavage patterns of the hypervariable domain the 18 isolates could be divided into four cases. Reactivity with both mAb 26.7D and pAb 121 confirmed these classes. The hypervariable, but not the constant, part of P120 was recognized by the human humoral immune...... and found to have a sequence identity of 91% with the gene of strain 7488. One hypervariable and two semivariable regions were detected. The epitope for mAb 26.7D was mapped to the hypervariable domain by expression of various parts of this domain in Escherichia coli using expression vector systems....... A polyclonal antiserum (pAb 121) generated against the hypervariable region of P120 from PG21 identified the P120 homologue in M. hominis PG21. Fusion proteins of the hypervariable and constant parts of the proteins were constructed and tested for reactivity with 21 human sera. Twelve sera reacted...

  5. [In relation to Cleopatra and snake bites]. (United States)

    Espinoza, R


    Cleopatra VII, one of the last Egyptian sovereigns of the ptolomeic dynasty, is envisioned as a mythic figure, surrounded by intrigues and mystery. her mysterious death was caused, according to history, by a snake bite. This article shows some instances of great Cleopatra's life and the state of the art on snake venoms. Even at the present time, snake bites are a public health problem in Asia, Africa, Central and South America, causing more than 25,000 deaths every year. Most snake venoms have a protein structure and cause neurotoxic and hemolytic effects, altering coagulation and fibrinolysis. The mortality due to snake bites fluctuates between 1 and 22%. Specific treatment includes the use of specific antiserums with highly purified components.

  6. Zea mI, the maize homolog of the allergen-encoding Lol pI gene of rye grass. (United States)

    Broadwater, A H; Rubinstein, A L; Chay, C H; Klapper, D G; Bedinger, P A


    Sequence analysis of a pollen-specific cDNA from maize has identified a homolog (Zea mI) of the gene (Lol pI) encoding the major allergen of rye-grass pollen. The protein encoded by the partial cDNA sequence is 59.3% identical and 72.7% similar to the comparable region of the reported amino acid sequence of Lol pIA. Southern analysis indicates that this cDNA represents a member of a small multigene family in maize. Northern analysis shows expression only in pollen, not in vegetative or female floral tissues. The timing of expression is developmentally regulated, occurring at a low level prior to the first pollen mitosis and at a high level after this postmeiotic division. Western analysis detects a protein in maize pollen lysates using polyclonal antiserum and monoclonal antibodies directed against purified Lolium perenne allergen.

  7. Iodine-125 radioimmunoassay for the direct detection of benzodiazepines in blood and urine

    Energy Technology Data Exchange (ETDEWEB)

    Goddard, C.P.; Stead, A.H.; Mason, P.A.; Law, B.; Moffat, A.C.; McBrien, M.; Cosby, S.


    A radioimmunoassay (RIA) for the direct detection of benzodiazepines in blood and urine is described. It is based on a commercially available antiserum and an easily synthesised radio-iodinated derivative of clonazepam that allows the use of relatively simple gamma-counting procedures. The assay can detect low therapeutic levels of all of the benzodiazepines currently available in the UK in samples of blood and urine (1-50 ng ml/sup -1/, depending on the drug); no prior sample preparation is required. It is inexpensive, rapid, simple to perform and is broadly specific for the benzodiazepine class of drugs. The assay offers a most suitable means of screening large numbers of samples of forensic interest for the presence of the benzodiazepines.

  8. Radioimmunoassay for pantothenic acid in blood and other tissues. [/sup 3/H and /sup 14/C tracer techniques

    Energy Technology Data Exchange (ETDEWEB)

    Wyse, B.W.; Wittwer, C.; Hansen, R.G.


    We described a radioimmunoassay for pantothenic acid in biological tissues. D-Pantothenic acid was conjugated with bovine serum albumin by use of a bromoacetyl derivative of pantothenic acid, and antibody to this antigen was raised by injecting it into the foot pads of rabbits. For the radioimmunoassay, a 100-fold dilution of the resulting antiserum was incubated with radiolabeled panthothentic acid. The antibodies were precipitated and dissolved, and the radioactivity of the solution was measured in a liquid scintillation counter. Between 5 and 125 ng of pantothenic acid can be detected in 75 of tissue extract. Validation included recovery and precision studies, parallelism with tissue extracts, and competitive binding studies. Results of the radioimmunoassay and those of microbiological assay with use of Lactobacillus plantarum correlated well (r = 0.80).

  9. The expression of the proteins of equine herpesvirus 1 which share homology with herpes simplex virus 1 glycoproteins H and L. (United States)

    Stokes, A; Alber, D G; Greensill, J; Amellal, B; Carvalho, R; Taylor, L A; Doel, T R; Killington, R A; Halliburton, I W; Meredith, D M


    Several expression systems were used in studies aimed at characterizing the equine herpesvirus 1 (EHV-1) glycoprotein H and L homologues of HSV-1 (EHV-1 gH and gL) and the products were compared to the authentic proteins synthesized in virus infected cells. Using an in vitro transcription/translation system two gH species were detected (an unprocessed 89 kDa and a processed 116 kDa product). Three low molecular weight proteins were found in the case of gL (21.8 kDa, 22.9 kDa and 26.9 kDa) and these showed a slight reduction in mobility on the addition of microsomal membranes to the reactions. A gL fusion protein was produced in pGEX-2T, expression being confirmed by Western blotting using a gL-specific antiserum raised against a peptide incorporating the 13 carboxyl terminal amino acids of the protein. A gH specific peptide antiserum precipitated both gH and two smaller proteins from EHV-1 infected cells thought to be two forms of gL. Insect cells infected with gH or gL baculovirus recombinants were used to vaccinate C3H (H-2k) mice. Some protection against EHV-1 infection was conferred to the gH inoculated mice. The results will enable further studies on the importance of the gH and gL interaction in the pathogenesis of EHV-1 to be evaluated and their potential in contributing to a subunit vaccine to be assessed.

  10. Roles of substance P and somatostatin on transmission of nociceptive information induced by formalin in spinal cord

    Energy Technology Data Exchange (ETDEWEB)

    Ohkubo, T.; Shibata, M.; Takahashi, H.; Inoki, R. (Fukuoka Dental College (Japan))


    Nociceptive response induced by 0.5% Formalin in the hindpaw of mice had two peaks, 0-5 min (first phase) and 15-20 min (second phase). By using the distinct biphasic response, the nature of the transmitter systems activated by Formalin in the spinal cord was studied for the purpose of determining the difference of the role of substance P (SP) and somatostatin (SST). The injection of (D-Pro2, D-Trp7,9)SP, (D-Arg1, D-Pro2, D-Trp7,9, Leu11)SP and SP antiserum inhibited only the first phase response. The i.t. injection of -Aminoheptanoyl-Phe-D-Trp-Lys-(OBz)-Thr- (an SST antagonist), SST antiserum and cysteamine (an SST depletor) inhibited only the second phase. This result indicates that SP is involved in the transmission of the first phase, and SST is involved in the transmission of the second phase of the Formalin-induced nociceptive response. With regard to other nociceptive stimuli, two i.t. SP antagonists produced a significant analgesia in the hot plate and tail pinch tests but had no effect in the acetic acid writhing test. However, i.t. SST antagonist and cysteamine produced a significant analgesia in the writhing test but had no effect in the hot plate and tail pinch test. These results suggest that SP participates in the transient pain induced by such acute stimuli as hot plate, tail pinch and the first phase of Formalin response and that SST participates in the prolonged and inflammatory pain induced by stimuli such as acetic acid and the second phase response.

  11. Analysis Atrazine Residues in Water Using Radioimmunoassay%用放射免疫测定法分析水中莠去津残留量的研究

    Institute of Scientific and Technical Information of China (English)

    徐德武; 朱永清; 杨根海; 楚慧民; 阎美玉


    The determination of atrazine residue in water with radioimmunoassay method was studied. The High bioactive antigen was synthesized and the high titer anti-atrazine antibody was prepared. The antiserum was specific for atrazine, the cross-reactivity of the antiserum to propazine and simazine was 93 % and 8 %, respectively. The RIA for atrazine labeled by 3H was established and showed that the rate of recovery by adding atrazine in drink water and yunhe water were 88.5% -107.5 % and 87.4 %-110.9 % respectively.%以莠去津均三氮苯类结构特征,合成连有活性羧基的莠去津半抗原,通过活性酯法制备出具有生物活性的人工抗原,以此免疫兔子获得抗莠去津的多克隆抗体。抗体的特异性强,与扑灭津的交叉反应性为93%,与西玛津的交叉反应性为8%。以3H标记莠去津建立的莠去津放射免疫测定法对水中莠去津添加回收率的测定表明,自来水的添加回收率为88.5%~107.5%,运河水的添加回收率为87.4%~110.9%。

  12. Serological relationships among subgroups in bovine viral diarrhea virus genotype 1 (BVDV-1). (United States)

    Alpay, Gizem; Yeşilbağ, Kadir


    Bovine viral diarrhea virus (BVDV) has various economic impacts associated with diarrhea, poor performance, an increase in the frequency of other infections and lethal outcomes. Both genotypes, namely BVDV-1 and BVDV-2, as well as different subgroups within these genotypes have been reported worldwide. Understanding the serological differences among the BVDV subgroups is important for disease epidemiology and prevention as well as vaccination programs. The aim of this study was to determine the serological relatedness among the subgroups in BVDV-1. For that purpose, sheep hyperimmune sera were collected against representative strains from 6 of the subgroups of BVDV-1 (BVDV-1a, -1b, -1d, -1f, -1h and -1l). The serum samples that gave the peak antibody titer to the homologous strains were used to perform cross neutralization assays. The highest homologous antibody titer (1:5160) was obtained against BVDV-1h. Regarding the cross neutralizing (heterologous) antibodies, the lowest titer (1:20) was produced by the BVDV-1f antiserum against the BVDV-1a and BVDV1-b viruses. The highest cross neutralizing titer (1:2580) achieved by the BVDV-1h antiserum was against the BVDV-1b strain. The cross neutralization results indicated particular serological differences between the recently described subgroup (BVDV-1l) and BVDV-1a/-1b, which are widely used in commercial vaccines. Considering the cross neutralization titers, it is concluded that selected BVDV-1l and BVDV-1h strains can be used for the development of diagnostic and control tools.

  13. The differential hepatic uptake of chylomicron remnants of different fatty acid composition is not mediated by hepatic lipase. (United States)

    Lambert, M S; Avella, M A; Berhane, Y; Shervill, E; Botham, K M


    The hypothesis that hepatic lipase mediates the differential hepatic uptake of chylomicron remnants of different fatty acid composition, demonstrated in previous work from our laboratory, was tested by investigating the effect of antibodies to the enzyme on the uptake of remnants enriched with saturated or n-3 polyunsaturated fatty acids by the perfused rat liver. After perfusion of rat livers with polyclonal antibodies to rat hepatic lipase raised in rabbits or with rabbit non-immune serum for 15 min, [3H]oleate-labelled chylomicron remnants, derived from chylomicrons of rats given a bolus of either palm (rich in saturated fatty acids) oil or fish (rich in n-3 polyunsaturated fatty acids) oil, were added. The disappearance of radioactivity from the perfusate during 120 min and its recovery in the liver at the end of the experiments were then measured. Although the rabbit anti-rat hepatic lipase antiserum was shown to inhibit hepatic lipase activity by up to 90%, and to bind extensively to hepatic sinusoidal surfaces when added to the perfusate, radioactivity from remnants of chylomicrons from rats given a bolus of fish oil as compared with palm oil disappeared from the perfusate and appeared in the liver more rapidly in the presence both the antiserum and the non-immune serum, and the differences between the uptake of the two types of remnants were similar. We conclude, therefore, that differential interaction with hepatic lipase is not responsible for the differences in the rate of removal of chylomicron remnants of different fatty acid composition from the blood.

  14. Properties of hemolysin and protease produced by Aeromonas trota.

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    Eizo Takahashi

    Full Text Available We examined the properties of exotoxins produced by Aeromonas trota (A. enteropelogenes, one of the diarrheagenic species of Aeromonadaceae. Nine of 19 A. trota isolates that grew on solid media containing erythrocytes showed hemolytic activity. However, the hemolytic activities of the culture supernatants of these hemolytic strains of A. trota were markedly lower than those of A. sobria when cultured in liquid medium, and the amount of hemolysin detected by immunoblotting using antiserum against the hemolysin produced by A. sobria was also low. A mouse intestine loop assay using living bacterial cells showed that A. trota 701 caused the significant accumulation of fluid, and antiserum against the hemolysin produced suppressed the enterotoxic action of A. trota 701. These results indicated that A. trota 701 was diarrheagenic and the hemolysin produced was the causative agent of the enterotoxic activity of A. trota. The hemolysin in A. sobria was previously shown to be secreted in a preform (inactive form and be activated when the carboxy-terminal domain was cleaved off by proteases in the culture supernatant. Since mature hemolysin was detected in the culture supernatants of A. trota, we analyzed the extracellular protease produced by A. trota. Fifteen of 19 A. trota isolates that grew on solid media containing skim milk showed proteolytic activity. We subsequently found that most A. trota isolates possessed the serine protease gene, but not the metalloprotease gene. Therefore, we determined the nucleotide sequence of the serine protease gene and its chaperone A. trota gene. The results obtained revealed that the deduced amino acid sequences of serine protease and the chaperone were homologous to those of A. sobria with identities of 83.0% and 75.8%, respectively.

  15. Effect of gamma irradiation on the behavioral properties of crotoxin

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    E.G. Moreira


    Full Text Available Crotoxin has been detoxified with gamma radiation in order to improve crotalic antiserum production. Nevertheless, present knowledge of the biological characteristics of irradiated crotoxin is insufficient to propose it as an immunizing agent. Crotoxin is known to increase the emotional state of rats and to decrease their exploratory behavior (Moreira EG, Nascimento N, Rosa GJM, Rogero JR and Vassilieff VS (1996 Brazilian Journal of Medical and Biological Research, 29: 629-632. Therefore, we decided 1 to evaluate the effects of crotoxin in the social interaction test, which has been widely used for the evaluation of anxiogenic drugs, and 2 to determine if irradiated crotoxin induces behavioral alterations similar to those of crotoxin in the social interaction, open-field and hole-board tests. Male Wistar rats (180-220 g were used. Crotoxin (100, 250, and 500 µg/kg was injected intraperitoneally 2 h before the social interaction test. Similarly, irradiated crotoxin (2000 Gy gamma radiation from a 60Co source was administered at the doses of 100, 250, and 500 µg/kg for the hole-board test, and at the doses of 1000 and 2500 µg/kg for the open-field and social interaction tests. ANOVA complemented with the Dunnett test was used for statistical analysis (P<0.05. Crotoxin decreased the social interaction time (s at the doses of 100, 250 and 500 µg/kg (means ± SEM from 51.6 ± 4.4 to 32.6 ± 3.7, 28.0 ± 3.6 and 31.6 ± 4.4, respectively. Irradiated crotoxin did not induce behavioral alterations. These results indicate that 1 crotoxin may be an anxiogenic compound, and 2 in contrast to crotoxin, irradiated crotoxin was unable to induce behavioral alterations, which makes it a promising compound for the production of crotalic antiserum

  16. Conservation and antigenicity of N-terminal sequences of GP185 from different Plasmodium falciparum isolates. (United States)

    Howard, R F; Ardeshir, F; Reese, R T


    Complementary DNA (cDNA) clones for GP185, a major antigenically diverse glycoprotein of Plasmodium falciparum, were isolated from a cDNA library of the Honduras I/CDC (Honduras I) isolate, and 1052 bp were sequenced. The expression of cDNA fragments in Escherichia coli using the vector pCQV2 allowed verification of the reading frame. This GP185 cDNA sequence, like the cDNA sequence for a homologous gene of the K1 isolate [Hall et al., Nature 311 (1984) 379-382], codes for a polypeptide which is truncated due to multiple, in-frame stop codons. This polypeptide corresponds to the N-terminal 15% of the proposed coding region of the GP185 gene [Holder et al., Nature 317 (1985) 270-273]. Comparison of the nucleotide sequences for the GP185 gene of Honduras I and five other isolates indicated that there are two areas of conserved DNA sequence, one of 310 bp (beginning 181 bp upstream from the proposed initiation codon) and the other of greater than or equal to 360 bp (located entirely within the coding region), separated by a region encoding isolate-specific tandem amino acid repeats. Rat antiserum was raised to a fusion protein derived from the conserved regions and the intervening repeat region of this Honduras I protein. This antiserum bound GP185 on immunoblots of the homologous Honduras I isolate and the heterologous K1 isolate, which has different tandem repeats. Serum from owl monkeys and humans previously infected with P. falciparum reacted with the fusion protein on immunoblots demonstrating that determinants in the N-terminal 15% of GP185 were immunogenic in infected individuals and suggesting that some of these sites are conserved among isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Production and assay of forskolin antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Ho, L.T.; Ho, R.J.


    Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using /sup 3/H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added /sup 3/H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC/sub 50/ was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound.

  18. Dynamics of virus shedding and in situ confirmation of chelonid herpesvirus 5 in Hawaiian green turtles with Fibropapillomatosis (United States)

    Work, Thierry M.; Dagenais, Julie; Balazs, George H.; Schettle, Nelli; Ackermann, Mathias


    Cancers in humans and animals can be caused by viruses, but virus-induced tumors are considered to be poor sites for replication of intact virions (lytic replication). Fibropapillomatosis (FP) is a neoplastic disease associated with a herpesvirus, chelonid herpesvirus 5 (ChHV5), that affects green turtles globally. ChHV5 probably replicates in epidermal cells of tumors, because epidermal intranuclear inclusions (EIIs) contain herpesvirus-like particles. However, although EIIs are a sign of herpesvirus replication, they have not yet been firmly linked to ChHV5. Moreover, the dynamics of viral shedding in turtles are unknown, and there are no serological reagents to confirm actual presence of the specific ChHV5 virus in tissues. The investigators analyzed 381 FP tumors for the presence of EIIs and found that overall, about 35% of green turtles had lytic replication in skin tumors with 7% of tumors showing lytic replication. A few (11%) turtles accounted for more than 30% cases having lytic viral replication, and lytic replication was more likely in smaller tumors. To confirm that turtles were actively replicating ChHV5, a prerequisite for shedding, the investigators used antiserum raised against F-VP26, a predicted capsid protein of ChHV5 that localizes to the host cell nucleus during viral replication. This antiserum revealed F-VP26 in EIIs of tumors, thus confirming the presence of replicating ChHV5. In this light, it is proposed that unlike other virus-induced neoplastic diseases, FP is a disease that may depend on superspreaders, a few highly infectious individuals growing numerous small tumors permissive to viral production, for transmission of ChHV5.

  19. Characterization of Melon necrotic spot virus Occurring on Watermelon in Korea

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    Hae-Ryun Kwak


    Full Text Available Melon necrotic spot virus (MNSV was recently identified on watermelon (Citrullus vulgaris in Korea, displaying as large necrotic spots and vein necrosis on the leaves and stems. The average occurrence of MNSV on watermelon was found to be 30–65% in Hapcheon and Andong City, respectively. Four isolates of the virus (MNSV-HW, MNSV-AW, MNSV-YW, and MNSV-SW obtained from watermelon plants in different areas were non-pathogenic on ten general indicator plants, including Chenopodium quinoa, while they infected systemically six varieties of Cucurbitaceae. The virus particles purified by 10–40% sucrose density gradient centrifugation had a typical ultraviolet spectrum, with a minimum at 245 nm and a maximum at 260 nm. The morphology of the virus was spherical with a diameter of 28–30 nm. Virus particles were observed scattered throughout the cytoplasm of watermelon cells, but no crystals were detected. An ELISA was conducted using antiserum against MNSV-HW; the optimum concentrations of IgG and conjugated IgG for the assay were 1 μl/ml and a 1:8,000–1:10,000 dilutions, respectively. Antiserum against MNSV-HW could capture specifically both MNSV-MN from melon and MNSV-HW from watermelon by IC/RT-PCR, and they were effectively detected with the same specific primer to produce product of 1,172 bp. The dsRNA of MNSV-HW had the same profile (4.5, 1.8, and 1.6 kb as that of MNSV-MN from melon. The nucleotide sequence of the coat protein of MNSV-HW gave a different phylogenetic tree, having 17.2% difference in nucleotide sequence compared with MNSV isolates from melon.

  20. Characterization of Melon necrotic spot virus Occurring on Watermelon in Korea. (United States)

    Kwak, Hae-Ryun; Kim, Jeong-Soo; Cho, Jeom-Deog; Lee, Joong-Hwan; Kim, Tae-Sung; Kim, Mi-Kyeong; Choi, Hong-Soo


    Melon necrotic spot virus (MNSV) was recently identified on watermelon (Citrullus vulgaris) in Korea, displaying as large necrotic spots and vein necrosis on the leaves and stems. The average occurrence of MNSV on watermelon was found to be 30-65% in Hapcheon and Andong City, respectively. Four isolates of the virus (MNSV-HW, MNSV-AW, MNSV-YW, and MNSV-SW) obtained from watermelon plants in different areas were non-pathogenic on ten general indicator plants, including Chenopodium quinoa, while they infected systemically six varieties of Cucurbitaceae. The virus particles purified by 10-40% sucrose density gradient centrifugation had a typical ultraviolet spectrum, with a minimum at 245 nm and a maximum at 260 nm. The morphology of the virus was spherical with a diameter of 28-30 nm. Virus particles were observed scattered throughout the cytoplasm of watermelon cells, but no crystals were detected. An ELISA was conducted using antiserum against MNSV-HW; the optimum concentrations of IgG and conjugated IgG for the assay were 1 μl/ml and a 1:8,000-1:10,000 dilutions, respectively. Antiserum against MNSV-HW could capture specifically both MNSV-MN from melon and MNSV-HW from watermelon by IC/RT-PCR, and they were effectively detected with the same specific primer to produce product of 1,172 bp. The dsRNA of MNSV-HW had the same profile (4.5, 1.8, and 1.6 kb) as that of MNSV-MN from melon. The nucleotide sequence of the coat protein of MNSV-HW gave a different phylogenetic tree, having 17.2% difference in nucleotide sequence compared with MNSV isolates from melon.

  1. A juvenile hormone-repressible transferrin-like protein from the bean bug, Riptortus clavatus: cDNA sequence analysis and protein identification during diapause and vitellogenesis. (United States)

    Hirai, M; Watanabe, D; Chinzei, Y


    We found several juvenile hormone-responsive cDNAs in the bean bug, Riptortus clavatus, by using mRNA differential display (Hirai et al., 1998). One of them, a juvenile hormone-repressible cDNA, JR-3, was cloned, sequenced, characterized and identified as a transferrin (RcTf). RcTf cDNA encoded 652 amino acids with a calculated molecular weight of 71,453 Da. The deduced amino acid sequence showed significant homology with the transferin genes of several insects, Manduca sexta (43% identity), Blaberus discoidalis (43%), Aedes aegypti (43%), Drosophila melanogaster (36%), Sarcophaga peregrina (36%) and the human (25%). Antiserum was prepared by using recombinant RcTf protein expressed in Escherichia coli as an antigen. The antiserum reacted specifically with both the recombinant protein and the native protein from the bugs, with sizes of 70 and 75 kDa, respectively. The 75 kDa protein was partially purified from hemolymph of diapausing female bugs and the first ten amino acids were found to be identical to that of RcTf cDNA, indicating that the 75 kDa protein is RcTf. The tissue distribution of RcTf in the bug was examined by Western blot analysis. In diapausing animals, RcTf was detected in the fat body, hemolymph and ovary but not in the gut. In the post-diapause stage, RcTf was also detected in eggs, in addition to the fat body and ovary. These results indicate that RcTf is incorporated into the oocytes during vitellogenesis, and suggest that it may provide iron for the developing embryos.

  2. The oxidation of dicarboxylic acid CoA esters via peroxisomal fatty acyl-CoA oxidase. (United States)

    Poosch, M S; Yamazaki, R K


    Evidence supporting a common peroxisomal beta-oxidation pathway for the coenzyme A thioesters of medium-chain-length dicarboxylic acids (DCn-CoA) and monocarboxylic acids (MCn-CoA) has been obtained. Using the mono-CoA esters of dodecanedioic acid (DC12-CoA) and lauroyl-CoA (MC12-CoA) as substrates, parallel inductions of activities and parallel increases in specific activities during purification of peroxisomal fatty acyl-CoA oxidase (EC from rat liver after di(2-ethylhexyl)phthalate treatment were seen. The purified enzyme was used for antiserum production in rabbits; antiserum specificity was verified by immunoblot analysis. Coincident losses of oxidase activities with MC12-CoA and DC12-CoA were found in immunotitration experiments with rat liver homogenates, supporting the hypothesis that peroxisomal fatty acyl-CoA oxidase is solely responsible for the oxidation of medium-chain length dicarboxylic acid substrates. Kinetic studies with purified enzyme using the mono-CoA esters of sebacic (DC10-CoA), suberic (DC8-CoA), and adipic (DC6-CoA) acids along with DC12-CoA revealed substrate inhibition. Although these substrates exhibited similar calculated Vmax values, with decreasing chain length, the combination of increasing Km values and decreasing substrate inhibition constant (Ki) caused the maximum obtainable velocity to decrease. These studies offer an explanation for the previously observed limit of the ability of peroxisomes to chain-shorten dicarboxylates and increased urinary excretion of adipic acid when peroxisomal oxidation of dicarboxylic acids is enhanced.

  3. Synthesis and Posttranslational Activation of Sulfhydryl-Endopeptidase in Cotyledons of Germinating Vigna mungo Seeds. (United States)

    Mitsuhashi, W; Minamikawa, T


    A sulfhydryl-endopeptidase was purified as a 33 kilodalton (kD) mass polypeptide from cotyledons of Vigna mungo seedlings. Immunoblot analysis with antiserum made against the purified enzyme showed that the sulfhydryl-endopeptidase was synthesized only in the cotyledons during germination and that the amount of the enzyme increased until 4 days after imbibition and decreased thereafter. Next, an RNA fraction was prepared from cotyledons of 3 day old seedlings and translated in a wheat germ system. The synthesis of a 45 kD polypeptide was shown by the analysis of its translation products by immunoprecipitation with the antiserum to the endopeptidase and gel electrophoresis. When the RNA fraction was translated in the presence of canine microsomal membranes, a smaller polypeptide, having a 43 kD molecular mass, was detected as the translation product. When membrane-bound polysomes, but not free polysomes, prepared from cotyledons were used for translation in the wheat germ system, both the 43 and 45 kD polypeptides were synthesized. By incubation of a crude enzyme extract from cotyledons at 5 +/- 1 degrees C at neutral pH, the 43 kD polypeptide was sequentially cleaved to the 33 kD polypeptide via 39 and 36 kD intermediate polypeptides. The endopeptidase was activated simultaneously with the processing. Two-dimensional polyacrylamide gel electrophoresis showed that the 33 kD polypeptide was the fully activated form of the enzyme, whereas little or no activity was detected in other forms. From the present results, we postulate that the sulfhydryl-endopeptidase is first synthesized as the 45 kD precursor with a 2 kD signal peptide being cleaved, and that the 43 kD polypeptide is further cleaved to give the 33kD mature enzyme.

  4. Synthesis and Posttranslational Activation of Sulfhydryl-Endopeptidase in Cotyledons of Germinating Vigna mungo Seeds 1 (United States)

    Mitsuhashi, Wataru; Minamikawa, Takao


    A sulfhydryl-endopeptidase was purified as a 33 kilodalton (kD) mass polypeptide from cotyledons of Vigna mungo seedlings. Immunoblot analysis with antiserum made against the purified enzyme showed that the sulfhydryl-endopeptidase was synthesized only in the cotyledons during germination and that the amount of the enzyme increased until 4 days after imbibition and decreased thereafter. Next, an RNA fraction was prepared from cotyledons of 3 day old seedlings and translated in a wheat germ system. The synthesis of a 45 kD polypeptide was shown by the analysis of its translation products by immunoprecipitation with the antiserum to the endopeptidase and gel electrophoresis. When the RNA fraction was translated in the presence of canine microsomal membranes, a smaller polypeptide, having a 43 kD molecular mass, was detected as the translation product. When membrane-bound polysomes, but not free polysomes, prepared from cotyledons were used for translation in the wheat germ system, both the 43 and 45 kD polypeptides were synthesized. By incubation of a crude enzyme extract from cotyledons at 5 ± 1°C at neutral pH, the 43 kD polypeptide was sequentially cleaved to the 33 kD polypeptide via 39 and 36 kD intermediate polypeptides. The endopeptidase was activated simultaneously with the processing. Two-dimensional polyacrylamide gel electrophoresis showed that the 33 kD polypeptide was the fully activated form of the enzyme, whereas little or no activity was detected in other forms. From the present results, we postulate that the sulfhydryl-endopeptidase is first synthesized as the 45 kD precursor with a 2 kD signal peptide being cleaved, and that the 43 kD polypeptide is further cleaved to give the 33kD mature enzyme. Images Figure 1 Figure 2 Figure 4 Figure 5 Figure 6 PMID:16666526

  5. Preparation of hemocyanin polyclonal antibody and the identification of 35 kDa hemocyanin fragment in HaHotis diversicolor Reeve%杂色鲍血蓝蛋白多克隆抗体的制备与血蓝蛋白35kDa片段鉴定

    Institute of Scientific and Technical Information of China (English)

    姜敬哲; 韩焘; 王江勇; 杨慧英; 刘金叶


    以杂色鲍(Haliotis diversicolor Reeve)为研究对象,通过密度梯度离心方法纯化得到高纯度血蓝蛋白,以其作为抗原皮下注射免疫新西兰大白兔,从而获得高效价的兔源多克隆抗血清。进一步通过ProteinA抗原亲和纯化的方法对该抗血清纯化,最终获得效价更高、检测特异性更好的血蓝蛋白多克隆抗体。应用该抗体进行Western检测发现,鲍血淋巴中存在着多样的血蓝蛋白衍生产物;进一步结合质谱技术对其中35kDa条带进行鉴定发现,其来源于血蓝蛋白I型亚基的H结构域。%Haliotis diversicolor Reeve, was taken as research material. High purity hemocyanin proteins were puri- fied from the hemolymph of abalone using CsC1 density gradient centrifugation method. By injection of the purified antigen to a rabbit, high titer polyclonal anti-serum was acquired. The anti-serum was further processed with the protein A affinity purification to produce purified antibodies. Finally, the purified polyclonal antibodies with higher titer and specificity were acquired. Further Western blotting with these antibodies, plenty of hemocyanin was found as derivates of various molecular weights in abalone hemolymph.

  6. Immunohistochemical study on effects of [sup 60]Co [gamma] irradiation on salivary gland ducts of rat

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    Saitoh, Kazuhiko (Meikai Univ., Sakato, Saitama (Japan). School of Dentistry)


    Cytokeratin distribution in salivary glands was detected by use of polyclonal antikeratin antiserum (TK) and monoclonal antibodies (KL 1, RGE 53, and RPN 1164). The salivary glands of male rats received either 17.82 Gy or 27.97 Gy [sup 60]Co [gamma] irradiation in a single exposure and were then compared immunohistochemically with those of normal rats. Polyclonal anti-keratin antiserum (TK), which reacts with 41-65 KD keratins, stained almost all ducts in normal glands. RPN 1164 (no. 8 keratin) staining was negative in intercalated ducts of normal parotid and submandibular glands, but strongly positive in both striated and excretory ducts of these glands. Monoclonal antibody KL 1 (55-57 KD keratins) and RGE 53 (no. 18 keratin) did not bind to any ductal or acinar epithelia. Only in the sublingual gland were acini positive for TK staining, possibly indicating myoepithelial cells. No effects of [sup 60]Co [gamma] irradiation were apparent regarding keratin distribution in the intercalated in apical cytoplasm by [sup 60]Co [gamma] irradiation. Also, in the excretory duct, the basal side of the cells exhibited weakened staining following [sup 60]Co [gamma] irradiation were the most significant in the parotid and the least in the sublingual gland. Also this reaction depended upon the doses of [sup 60]Co used. The present findings suggested that negative or weakened staining at the basal and perinuclear portions of striated duct cells specifically reflects the primary or secondary cell damage produced by [sup 60]Co [gamma] irradiation. Since the distribution of cytoskeletal proteins in the cytoplasm reflects certain pathological conditions, immunohistochemical detection of these proteins seem to have a diagnostic value with respect to cellular injury. (J.P.N.) 77 refs.

  7. Requirement for p34cdc2 kinase is restricted to mitosis in the mammalian cdc2 mutant FT210. (United States)

    Hamaguchi, J R; Tobey, R A; Pines, J; Crissman, H A; Hunter, T; Bradbury, E M


    The mouse FT210 cell line is a temperature-sensitive cdc2 mutant. FT210 cells are found to arrest specifically in G2 phase and unlike many alleles of cdc2 and cdc28 mutants of yeasts, loss of p34cdc2 at the nonpermissive temperature has no apparent effect on cell cycle progression through the G1 and S phases of the division cycle. FT210 cells and the parent wild-type FM3A cell line each possess at least three distinct histone H1 kinases. H1 kinase activities in chromatography fractions were identified using a synthetic peptide substrate containing the consensus phosphorylation site of histone H1 and the kinase subunit compositions were determined immunochemically with antisera prepared against the "PSTAIR" peptide, the COOH-terminus of mammalian p34cdc2 and the human cyclins A and B1. The results show that p34cdc2 forms two separate complexes with cyclin A and with cyclin B1, both of which exhibit thermal lability at the non-permissive temperature in vitro and in vivo. A third H1 kinase with stable activity at the nonpermissive temperature is comprised of cyclin A and a cdc2-like 34-kD subunit, which is immunoreactive with anti-"PSTAIR" antiserum but is not recognized with antiserum specific for the COOH-terminus of p34cdc2. The cyclin A-associated kinases are active during S and G2 phases and earlier in the division cycle than the p34cdc2-cyclin B1 kinase. We show that mouse cells possess at least two cdc2-related gene products which form cell cycle regulated histone H1 kinases and we propose that the murine homolog of yeast p34cdc/CDC28 is essential only during the G2-to-M transition in FT210 cells.

  8. Peripherally-derived BDNF promotes regeneration of ascending sensory neurons after spinal cord injury.

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    Xing-Yun Song

    Full Text Available BACKGROUND: The blood brain barrier (BBB and truncated trkB receptor on astrocytes prevent the penetration of brain derived neurotrophic factor (BDNF applied into the peripheral (PNS and central nervous system (CNS thus restrict its application in the treatment of nervous diseases. As BDNF is anterogradely transported by axons, we propose that peripherally derived and/or applied BDNF may act on the regeneration of central axons of ascending sensory neurons. METHODOLOGY/PRINCIPAL FINDINGS: The present study aimed to test the hypothesis by using conditioning lesion of the sciatic nerve as a model to increase the expression of endogenous BDNF in sensory neurons and by injecting exogenous BDNF into the peripheral nerve or tissues. Here we showed that most of regenerating sensory neurons expressed BDNF and p-CREB but not p75NTR. Conditioning-lesion induced regeneration of ascending sensory neuron and the increase in the number of p-Erk positive and GAP-43 positive neurons was blocked by the injection of the BDNF antiserum in the periphery. Enhanced neurite outgrowth of dorsal root ganglia (DRG neurons in vitro by conditioning lesion was also inhibited by the neutralization with the BDNF antiserum. The delivery of exogenous BDNF into the sciatic nerve or the footpad significantly increased the number of regenerating DRG neurons and regenerating sensory axons in the injured spinal cord. In a contusion injury model, an injection of BDNF into the footpad promoted recovery of motor functions. CONCLUSIONS/SIGNIFICANCE: Our data suggest that endogenous BDNF in DRG and spinal cord is required for the enhanced regeneration of ascending sensory neurons after conditioning lesion of sciatic nerve and peripherally applied BDNF may have therapeutic effects on the spinal cord injury.

  9. Activity and Tissue Expression of Tyrosine Phosphatase PTP-MEG2

    Institute of Scientific and Technical Information of China (English)

    DONG Hong-bo; LI Guo-dong; WANG Shao-feng; FU Xue-qi; ZHAO Zhi-zhuang Joe


    Protein tyrosine phosphatases(PTPs) are crucial regulators of signal transduction. Among them,PTP-MEG2 is an intracellular enzyme of 593 amino acid residues with a putative lipid-binding domain at the N-terminus. In the present study, we cloned the full-length form of the enzyme and expressed it in E. coli cells as a 6xHis-tagged protein. The majority of the expressed enzyme was found in the inclusion body of E. coli cell extracts.Upon extraction with a buffer containing urea, the recombinant enzyme was purified to near homogeneity on a single Ni-NTA-agarose column. This procedure resulted in the production of over 100 mg of purified recombinant PTP-MEG2 from 1 L E. coli cell culture. The purified protein displayed a single polypeptide band with expected molecular size on SDS-polyacrylamide gel electrophoresis under reducing conditions. Isolated under denatured conditions in urea, the purified enzyme was re-natured by dialyzing against a refolding buffer. The re-natured enzyme effectively dephosphorylated the common PTP substrate para-nitrophenylphosphate with a specific activity of 2000 units/mg. Meanwhile, the denatured enzyme was used to immunize a rabbit to produce antibodies. The resulting antiserum had extremely high sensitivity and specificity. When used for Western blot analysis, the anti-serum revealed a wide expression of PTP-MEG2 in many tissues of mice. Together, we developed a highly effective way to purify a large amount of PTP-MEG2 and generated highly sensitive antibodies that can specifically detect endogenous expression of the enzyme in tissues.

  10. Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, C.I.; Lennick, M.; Lehar, S.M.; Beltz, G.A.; Young, E. (Cambridge Bioscience Corporation, Worcester, MA (USA))


    Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4.

  11. G(o) transduces GABAB-receptor modulation of N-type calcium channels in cultured dorsal root ganglion neurons. (United States)

    Menon-Johansson, A S; Berrow, N; Dolphin, A C


    High-voltage-activated (HVA) calcium channel currents (IBa) were recorded from acutely replated cultured dorsal root ganglion (DRG) neurons. IBa was irreversibly inhibited by 56.9 +/- 2.7% by 1 microM omega-conotoxin-GVIA (omega-CTx-GVIA), whereas the 1,4-dihydropyridine antagonist nicardipine was ineffective. The selective gamma-aminobutyric acidB (GABAB) agonist, (-)-baclofen (50 microM), inhibited the HVA IBa by 30.7 +/- 5.4%. Prior application of omega-CTx-GVIA completely occluded inhibition of the HVA IBa by (-)-baclofen, indicating that in this preparation (-)-baclofen inhibits N-type current. To investigate which G protein subtype was involved, cells were replated in the presence of anti-G protein antisera. Under these conditions the antibodies were shown to enter the cells through transient pores created during the replating procedure. Replating DRGs in the presence of anti-G(o) antiserum, raised against the C-terminal decapeptide of the G alpha o subunit, reduced (-)-baclofen inhibition of the HVA IBa, whereas replating DRGs in the presence of the anti-Gi antiserum did not. Using anti-G alpha o antisera (1:2000) and confocal laser microscopy, G alpha o localisation was investigated in both unreplated and replated neurons. G alpha o immunoreactivity was observed at the plasma membrane, neurites, attachment plaques and perinuclear region, and was particularly pronounced at points of cell-to-cell contact. The plasma membrane G alpha o immunoreactivity was completely blocked by preincubation with the immunising G alpha o undecapeptide (1 for 1 h at 37 degrees C. A similar treatment also blocked recognition of G alpha o in brain membranes on immunoblots.(ABSTRACT TRUNCATED AT 250 WORDS)

  12. Biochemical characterization of Drosophila glutathione S-transferases D1 and D21. (United States)

    Tang, A H; Tu, C P


    The genomic DNA for the two Drosophila genes, gstD1 and gstD21, were engineered for expression in Escherichia coli by polymerase chain reaction using a pair of specially designed primers. This newly designed expression system produced consistently high yields of the recombinant glutathione S-transferases (GSTs), which were purified to electrophoretic homogeneity by S-hexyl-GSH affinity chromatography. Consistent with their differences in size, GST D1 and GST D21 displayed different mobilities on SDS-polyacrylamide gel electrophoresis. Circular dichroism spectrometry revealed some differences in the protein secondary structural organization between the two GST D isozymes. Polyclonal antibodies against GST D1 and GST D21 revealed that they are immunologically distinct from each other. The GST D1 antiserum cross-reacted weakly with GST D21, but the GST D21 antiserum had no detectable cross-reactivity with GST D1. The amino acid sequences of GST D1 and GST D21 have 70% identity. GST D1 is active toward CDNB with 17% of the catalytic efficiency of the human alpha GST121, whereas CDNB is a poor substrate for GST D21. Both GST D1 and GST D21 have similar levels of GSH peroxidase activity against cumene hydroperoxide. Another major difference in substrate specificities between GST D1 and GST D21 is in the activity of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) dehydrochlorinase, which exists only in the GST D1 isozyme. This is the first definitive demonstration that DDT dehydrochlorinase activity is an intrinsic property of a Drosophila GST. Our results suggest that GST D1 may play a role in DDT metabolism in Drosophila.

  13. Regulatory interaction of the Galpha protein with phospholipase A2 in the plasma membrane of Eschscholzia californica. (United States)

    Heinze, Michael; Steighardt, Jörg; Gesell, Andreas; Schwartze, Wieland; Roos, Werner


    Plant heterotrimeric G-proteins are involved in a variety of signaling pathways, though only one alpha and a few betagamma isoforms of their subunits exist. In isolated plasma membranes of California poppy (Eschscholzia californica), the plant-specific Galpha subunit was isolated and identified immunologically and by homology of the cloned gene with that of several plants. In the same membrane, phospholipase A(2) (PLA(2)) was activated by yeast elicitor only if GTPgammaS (an activator of Galpha) was present. From the cholate-solubilized membrane proteins, PLA(2) was co-precipitated together with Galpha by a polyclonal antiserum raised against the recombinant Galpha. In this immunoprecipitate and in the plasma membrane (but not in the Galpha-free supernatant) PLA(2) was stimulated by GTPgammaS. Plasma membranes and immunoprecipitates obtained from antisense transformants with a low Galpha content allowed no such stimulation. An antiserum raised against the C-terminus (which in animal Galphas is located near the target coupling site) precipitated Galpha without any PLA(2) activity. Using non-denaturing PAGE, complexes of solubilized plasma membrane proteins were visualized that contained Galpha plus PLA(2) activity and dissociated at pH 9.5. At this pH, PLA(2) was no longer stimulated by GTPgammaS. It is concluded that a distinct fraction of the plasma membrane-bound PLA(2) exists in a detergent-resistant complex with Galpha that can be dissociated at pH 9.5. This complex allows the Galpha-mediated activation of PLA(2).

  14. An alpha-crystallin protein cognate in germ cells of the moth, Plodia interpunctella. (United States)

    Shirk, P D; Zimowska, G


    Previously we had reported the production of an antiserum to an antigen found primarily in germ cells of the Indianmeal moth, Plodia interpunctella (Zimowska et al., 1991). The antigen, molecular weight 25,000 kDa, and a related protein, molecular weight 21,000 kDa, co-purified with the follicular epithelium yolk protein. Antisera to the two proteins were raised, and they both reacted with the same four small polypeptides, which had molecular weights of 20,000, 21,000, 25,000 and 28,000 kDa, that were present in the eggs throughout embryogenesis. A 30 amino acid sequence of an internal fragment of the 25,000 kDa molecular weight polypeptide showed sequence similarity with the alpha-crystallin A chain polypeptides from the lenses of vertebrate eyes and, to a lesser extent, with small heat shock proteins. Based on the sequence similarity with the alpha-crystallins, we suggest that this family of polypeptides from the germ cells of this moth be considered as cognates of the alpha-crystallins, and the 25,000 molecular weight polypeptide described here be given the designation ac25. Using immuno-gold labeling with antiserum to ac25, the alpha-crystallins were shown to be distributed throughout the cytoplasm and nucleoplasm of the oocyte and nurse cells, but not present within yolk spheres or other organelles of the oocyte or nurse cells. Immunofluorescent staining of males showed antigenic material in the sperm bundles within the testes. Oenocytes of the pupal and adult stages also contained cross-reactive material.

  15. Bacteroides gingivalis antigens and bone resorbing activity in root surface fractions of periodontally involved teeth

    Energy Technology Data Exchange (ETDEWEB)

    Patters, M.R.; Landsberg, R.L.; Johansson, L.A.; Trummel, C.L.; Robertson, P.R. (Department of Periodontology, University of Connecticut, School of Dental Medicine, Farmington, Connecticut, U.S.A.)


    Bone resorbing activity and the presence of antigens of Bacteroides gingivalis were assessed in plaque, calculus, cementum, and dentin obtained from roots of teeth previously exposed to periodontitis. Each fraction was obtained by scaling the root surface. The fraction were extracted by stirring and sonication, and the soluble centrifuged, sterilized, dialyzed, and adjusted to equivalent protein concentrations. Cementum and dentin extracts from impacted teeth were prepared similarly and served as controls. Stimulation of bone resorption by each extract was assessed in organ cultures of fetal rat bones by measurement of release of previously-incorporated /sup 45/Ca from the bone into the medium. In some groups of teeth, calculus and cementum were treated with acid prior to scaling. Citric acid washes were recovered and dialyzed. An enzyme-linked immunosorbent assay (ELISA) was used to assess the extracts for the presence of antigens reactive with an antiserum to B. gingivalis. Significant stimulation of bone resorption was found in all calculus and periodontally-involved cementum preparations. ELISA showed significant levels of B.gingivalis antigens in plaque, calculus, and cementum of periodontally-involved teeth, but not in involved dentin nor in cementum or dentin of impact teeth. Treatment with citric acid removed essentially all B.gingivalis antigens from cementum but not calculus. The results suggest that substances which stimulate bone resorption and substances which react with B. gingivalis antiserum are present in surface plaque, calculus, and cementum or periodontally-involved teeth. These substances are not present in cementum and dentin of impacted teeth nor in dentin of periodontally-involved teeth. Treatment by both scaling and citric demineralization will remove most of these substances from cementum of teeth previously exposed to periodontitis.

  16. Investigations on the radioimmunological determination of stilbenes in tissue samples from pigs

    Energy Technology Data Exchange (ETDEWEB)

    Hoffmann, B.; Weiler, S.

    Based on the extraction procedures described for tissue of veal calves and following the introduction of reversed-phase-column chromatography as an alternative purification step, a radioimmunoassay is described for the determination of DES in pork-tissue. When using the DES-specific antiserum AS 254 and depending on the tissue examined, the lower limits of detection were between 29-69 pg. A mean enor of 10.8% (CV between 13-17%) for the ecovery added to tissue samples and a CV between 3.6 and 10.5% for the reproducibility demonstrate a good reliability. Testing the stilbene-specific antiserum AS 6139 by using /sup 3/H-DES as tracer revealed, the DES - and to a lesser extent also HEX - could be quantified with an acceptable degree of reliability when using the homologous RIA-system (use of DES and HEX resp. as calibration-standard), differently to DIEN; the possibility of a transformation of DIEN during the extraction into a compound exhibiting a higher cross reactivity is discussed. Application of the heterologous test system (quantification of the sample-stilbene by using each of the other two stilbenes for calibration) yielded the expected over- and underestimations. Furtheron, in respect to total stilbenes, i.e. not knowing whether DES, HEX or DIEN are present in the sample, it has been shown, that the assay - though qualitative - is highly sensitive (59-86 pg lower level of detection) when DES is used as tracer and for calibration purposes.

  17. Alloantigens of human lymphoid cell lines; `human Ia-like antigens' (United States)

    Koyama, K.; Nakamuro, K.; Tanigaki, N.; Pressman, D.


    Membrane glycoproteins that appear to belong to a new alloantigen system were partially purified by gel filtration and lentil-lectin affinity chromatography from a non-ionic detergent (Renex 30) solubilized membrane fraction of each of two Burkitt lymphoma cell lines, B46M and Daudi. The preparations were radioiodinated and further purified by gel filtration, lentil—lectin affinity chromatography and anti-HLA antibody affinity chromatography. The labelled preparations thus obtained did not have binding activity with any of rabbit anti-HLA, rabbit anti-human β2-microglobulin and rabbit anti-human IgG-Fab antisera, but did have a high level of binding activity with rabbit anti-B-cell membrane antiserum. Moreover, the labelled preparations showed relatively high binding activity with some conventional HLA typing sera. Out of sixty-eight human tissue typing alloantisera tested, three (Berlin 373, Betz and TO-29-01) gave especially high binding with both of the labeled preparations. The antigens involved in reaction with these alloantisera and also with the rabbit anti-B-cell membrane antiserum contained two components, one 31,000 ∼ 32,000 daltons and another 24,000 ∼ 25,000 daltons, bound non-covalently. The alloantigens were specific to B cell type cell lines (B-lymphoid cell lines and Burkitt-lymphoma cell lines) in cultured cell lines and also to B lymphocytes in peripheral blood. In organs and tissues, however, they were found to be present widely distributed in lymphoid organs (thymus as well as spleen and lymph node) and in non-lymphoid organs (including liver, kidney, testis and heart). PMID:24587

  18. Tuberal hypothalamic neurons secreting the satiety molecule Nesfatin-1 are critically involved in paradoxical (REM sleep homeostasis.

    Directory of Open Access Journals (Sweden)

    Sonia Jego

    Full Text Available The recently discovered Nesfatin-1 plays a role in appetite regulation as a satiety factor through hypothalamic leptin-independent mechanisms. Nesfatin-1 is co-expressed with Melanin-Concentrating Hormone (MCH in neurons from the tuberal hypothalamic area (THA which are recruited during sleep states, especially paradoxical sleep (PS. To help decipher the contribution of this contingent of THA neurons to sleep regulatory mechanisms, we thus investigated in rats whether the co-factor Nesfatin-1 is also endowed with sleep-modulating properties. Here, we found that the disruption of the brain Nesfatin-1 signaling achieved by icv administration of Nesfatin-1 antiserum or antisense against the nucleobindin2 (NUCB2 prohormone suppressed PS with little, if any alteration of slow wave sleep (SWS. Further, the infusion of Nesfatin-1 antiserum after a selective PS deprivation, designed for elevating PS needs, severely prevented the ensuing expected PS recovery. Strengthening these pharmacological data, we finally demonstrated by using c-Fos as an index of neuronal activation that the recruitment of Nesfatin-1-immunoreactive neurons within THA is positively correlated to PS but not to SWS amounts experienced by rats prior to sacrifice. In conclusion, this work supports a functional contribution of the Nesfatin-1 signaling, operated by THA neurons, to PS regulatory mechanisms. We propose that these neurons, likely releasing MCH as a synergistic factor, constitute an appropriate lever by which the hypothalamus may integrate endogenous signals to adapt the ultradian rhythm and maintenance of PS in a manner dictated by homeostatic needs. This could be done through the inhibition of downstream targets comprised primarily of the local hypothalamic wake-active orexin- and histamine-containing neurons.

  19. Quantitative immunobinding assay for vitamin D-dependent calcium-binding protein (calbindin-D28k) using nitrocellulose filters

    Energy Technology Data Exchange (ETDEWEB)

    Varghese, S.; Christakos, S.


    A sensitive dot immunobinding assay has been developed for the quantitative determination of vitamin D-dependent calcium-binding protein (calbindin-D28k; CaBP) in rat and human kidney and brain. Protein samples are spotted onto nitrocellulose sheets, fixed, and then rinsed with Tris-buffered saline. The remaining protein binding sites are blocked with bovine serum albumin, gelatin, or nonfat dry milk protein and the filters are then incubated sequentially with antiserum to calbindin-D28k (1:500 dilution) and /sup 125/I-protein A (200,000 cpm/ml). After washing, the radioactivity bound to each sample is quantitated by counting in a gamma counter. The sensitivity of the assay is such that 10 ng calbindin-D28k can be accurately quantitated. The highest levels of CaBP were detected in kidney (7.8 +/- 0.5 micrograms/mg protein) and cerebellum (22.1 +/- 1.4 micrograms/mg protein). Ten micrograms calmodulin, lactalbumin, or parvalbumin and 100 micrograms liver extract showed no reactivity in the assay. The assay is precise (intraassay variability, 4.0%) and reproducible (interassay variability, 8.8%). There was good agreement between the data in this assay and the data we obtained using radioimmunoassay (RIA). The assay has several advantages over the RIA. Iodination of pure antigen is not required and it is possible to detect membrane-bound and insoluble antigens using this assay. Also, the antiserum and /sup 125/I-protein A solutions can be saved and reused. This assay represents a major modification of the original immunobinding assays which used the less sensitive peroxidase stain. It is also an improvement over previous /sup 125/I immunobinding assays which were not quantitative but were used as antigen spot tests or which required iodination of the antibody.

  20. Circulating immune complexes of Hodgkin's disease contain an antigen that is present in Hodgkin and Reed-Sternberg cells. (United States)

    Bepler, G; Zhen, Q Y; Havemann, K


    Circulating immune complexes (CIC), isolated from the serum of a patient with Hodgkin's disease (HD) and from control serum (CS) of healthy adults, were used to generate heterologous antisera in rabbits. The antiserum directed against CIC from HD (AS-HD) and the antiserum directed against CIC from CS (AS-CS) were used to identify immunoglobulins, complement factors and alpha2-macroglobulin as immune complex components. After adsorbing both antisera with normal human sera, we found that the adsorbed AS-HD was immunoreactive with radio-labelled CIC from HD serum but not with radiolabelled CIC from CS. Sera of patients with different diseases and sera of healthy adults were assessed for the occurrence of this Hodgkin immune complex-associated antigen (HIC-Ag). The HIC-Ag was present in 37% (12/33) of sera from patients with HD, 8% (8/101) of sera from patients with nonmalignant diseases, and 0% (0/6) of sera from healthy adults. This antigen was equally distributed among HD patients with and without symptoms, but its occurrence correlated with an advanced clinical stage of the disease. Using the adsorbed AS-HD in the immunoperoxidase technique, we identified the HIC-Ag as a cytoplasmic antigen in Hodgkin and Reed-Sternberg cells; whereas, the adsorbed AS-CS did not reveal any staining. These data indicate the presence of an HIC-Ag in the sera of patients with HD and suggest that the adsorbed AS-HD might be useful for isolation and characterization of this antigen for future use as a tumour marker.

  1. Characterization and comparison of mycobacterial antigens by two-dimensional immunoelectrophoresis. (United States)

    Roberts, D B; Wright, G L; Affronti, L F; Reich, M


    Two-dimensional immunoelectrophoresis (2D-IEP), in which a complex of antigens is subjected to electrophoresis first through an agarose matrix in one direction and secondly through an antiserum-agarose matrix at right angles to the first direction, was evaluated as a tool for analysis of mycobacterial antigens. Cell extracts from four species of mycobacteria, Mycobacterium tuberculosis (four strains), M. bovis strain BCG, M. scrofulaceum, and M. phlei, were assayed by 2D-IEP with four anti-mycobacterial antisera. Besides displaying the precipitin curves in a more easily interpreted format than did conventional immunoelectrophoresis (IEP), 2D-IEP offered greater sensitivity in terms of numbers of precipitin curves when like reactions were compared with IEP patterns. As many as 60 immunoprecipitates were observed on 2D-IEP slides compared to 18 on comparable IEP plates. Technical reproducibility of patterns from run to run was excellent. Other parameters, such as the influence of using different batches of antigen on the pattern, are discussed. Each of the cell extract antigens gave a unique pattern of precipitin peaks which could be easily differentiated from the patterns given by the other mycobacterial cell extracts when reacted with any of the antisera in 2D-IEP. Since both the species and strains of mycobacteria could be easily and reproducibly differentiated solely on the basis of two-dimensional immunoelectrophoretic patterns obtained with any of the antisera employed in this study, it may be possible, by using IEP, to differentiate and identify all species and strains of mycobacteria with one standard, highly sensitive antiserum, rather than a battery of antisera.

  2. Preparation of polycolonal antibody against goose prolactin receptor and the receptor expression in goose tissues%鹅催乳素受体多克隆抗体制备及组织表达分析

    Institute of Scientific and Technical Information of China (English)

    邢光东; 杨廷桂; 段修军; 宦海琳; 闫俊书; 王根林


    To study the functions of prolactin receptor( PRLR) in goose,we constructed a prokaryotic expression vector exPRLR-Pet-28a( +) that contains exPRLR, the coding sequences of the whole extracellular domain of goose PRLR. Transformed into host bacteria E. Coli Roster and induced by IPTG,ex:PRLR-Pet-28a( +)expressed recombinant protein exPRLR. By immunizing rabbit with the re-combinant protein, we obtained rabbit anti-goose exPRLR polycolonal serum. Western blot assay revealed that the anti-serum recognized the recombinant protein expressed by the transformed bacteria specifically. Analyzing the proteins isolated from adult female goose tissues with the anti-serum, we found that adult geese might have at least three isoforms of PRLR with different relative molecular mass.%为深入研究鹅催乳素受体(PRLR)的功能,构建了含鹅PRLR胞外域编码序列exPRLR的原核表达质粒exPRLR-pET-28a(+),通过转化E coli Roster建立了稳定的原核表达系统,在IPTG诱导下获得了PRLR胞外域的重组exPRLR.以重组exPRLR为抗原免疫家兔,获得了兔抗鹅exPRLR的抗血清.Western blot分析证明该抗血清可特异识别由原核生物表达的重组exPRLR.进一步分析成年母鹅的组织蛋白,发现成年鹅中可能至少存在3种相对分子质量不同的PRLR蛋白异形体.

  3. Exposure to environmental tobacco smoke measured by cotinine sup 125 I-radioimmunoassay

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    Knight, G.J.; Palomaki, G.E.; Lea, D.H.; Haddow, J.E. (Foundation for Blood Research, Scarborough, ME (USA))


    We describe a polyclonal-antiserum-based {sup 125}I-radioimmunoassay for cotinine that is suitable for measuring nonsmokers' passive exposure to tobacco smoke in the environment. The standard curve ranged from 0.25 to 12.0 micrograms/L, with an estimated lower limit of sensitivity of 0.2 microgram/L (95% B/Bo = 0.2 microgram/L; 50% B/Bo = 4.0 micrograms/L). The median within-assay CVs for patients' samples with cotinine values from 0.4 to 1.3, 1.4 to 2.4, 2.5 to 4.6, and 4.7 to 15.6 micrograms/L were 13.9%, 7.2%, 5.1%, and 5.7%, respectively. Between-assay CVs for two quality-control sera with average values of 1.53 and 3.68 micrograms/L were 14.3% and 7.8%, respectively. Analytical recoveries of cotinine from smokers' sera diluted in zero calibrant ranged from 91% to 116%. Cotinine values determined on 79 paired sera and urines from nonsmokers showed significant correlation with self-reported exposure to environmental tobacco smoke (r = 0.49, P less than 0.001 for sera; r = 0.57, P less than 0.001 for urine). The log of the values for serum and urine cotinine were also significantly correlated (r = 0.85, P less than 0.001). Evidently, polyclonal antiserum can be used to develop a cotinine assay for measuring exposure to environmental tobacco smoke that compares well with that described for monoclonal-based assays.

  4. Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine. (United States)

    Mizrachi Nebenzahl, Yaffa; Blau, Karin; Kushnir, Tatyana; Shagan, Marilou; Portnoi, Maxim; Cohen, Aviad; Azriel, Shalhevet; Malka, Itai; Adawi, Asad; Kafka, Daniel; Dotan, Shahar; Guterman, Gali; Troib, Shany; Fishilevich, Tali; Gershoni, Jonathan M; Braiman, Alex; Mitchell, Andrea M; Mitchell, Timothy J; Porat, Nurith; Goliand, Inna; Chalifa Caspi, Vered; Swiatlo, Edwin; Tal, Michael; Ellis, Ronald; Elia, Natalie; Dagan, Ron


    In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA) is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinβ4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development.

  5. Immunization with the Haemophilus ducreyi hemoglobin receptor HgbA with adjuvant monophosphoryl lipid A protects swine from a homologous but not a heterologous challenge. (United States)

    Fusco, William G; Afonina, Galyna; Nepluev, Igor; Cholon, Deborah M; Choudhary, Neelima; Routh, Patricia A; Almond, Glenn W; Orndorff, Paul E; Staats, Herman; Hobbs, Marcia M; Leduc, Isabelle; Elkins, Christopher


    Haemophilus ducreyi, the etiological agent of chancroid, has a strict requirement for heme, which it acquires from its only natural host, humans. Previously, we showed that a vaccine preparation containing the native hemoglobin receptor HgbA purified from H. ducreyi class I strain 35000HP (nHgbAI) and administered with Freund's adjuvant provided complete protection against a homologous challenge. In the current study, we investigated whether nHgbAI dispensed with monophosphoryl lipid A (MPL), an adjuvant approved for use in humans, offered protection against a challenge with H. ducreyi strain 35000HP expressing either class I or class II HgbA (35000HPhgbAI and 35000HPhgbAII, respectively). Pigs immunized with the nHgbAI/MPL vaccine were protected against a challenge from homologous H. ducreyi strain 35000HPhgbAI but not heterologous strain 35000HPhgbAII, as evidenced by the isolation of only strain 35000HPhgbAII from nHgbAI-immunized pigs. Furthermore, histological analysis of the lesions showed striking differences between mock-immunized and nHgbAI-immunized animals challenged with strains 35000HPhgbAI but not those challenged with strain 35000HPhgbAII. Mock-immunized pigs were not protected from a challenge by either strain. The enzyme-linked immunosorbent assay (ELISA) activity of the nHgbAI/MPL antiserum was lower than the activity of antiserum from animals immunized with the nHgbAI/Freund's vaccine; however, anti-nHgbAI from both studies bound whole cells of 35000HPhgbAI better than 35000HPhgbAII and partially blocked hemoglobin binding to nHgbAI. In conclusion, despite eliciting lower antibody ELISA activity than the nHgbAI/Freund's, the nHgbAI/MPL vaccine provided protection against a challenge with homologous but not heterologous H. ducreyi, suggesting that a bivalent HgbA vaccine may be needed.

  6. Revisiting the Concept of Targeting Only Bacillus anthracis Toxins as a Treatment for Anthrax. (United States)

    Glinert, Itai; Bar-David, Elad; Sittner, Assa; Weiss, Shay; Schlomovitz, Josef; Ben-Shmuel, Amir; Mechaly, Adva; Altboum, Zeev; Kobiler, David; Levy, Haim


    Protective antigen (PA)-based vaccines are effective in preventing the development of fatal anthrax disease both in humans and in relevant animal models. The Bacillus anthracis toxins lethal toxin (lethal factor [LF] plus PA) and edema toxin (edema factor [EF] plus PA) are essential for the establishment of the infection, as inactivation of these toxins results in attenuation of the pathogen. Since the toxins reach high toxemia levels at the bacteremic stages of the disease, the CDC's recommendations include combining antibiotic treatment with antitoxin (anti-PA) immunotherapy. We demonstrate here that while treatment with a highly potent neutralizing monoclonal antibody was highly efficient as postexposure prophylaxis treatment, it failed to protect rabbits with any detectable bacteremia (≥10 CFU/ml). In addition, we show that while PA vaccination was effective against a subcutaneous spore challenge, it failed to protect rabbits against systemic challenges (intravenous injection of vegetative bacteria) with the wild-type Vollum strain or a toxin-deficient mutant. To test the possibility that additional proteins, which are secreted by the bacteria under pathogenicity-stimulating conditions in vitro, may contribute to the vaccine's potency, we immunized rabbits with a secreted protein fraction from a toxin-null mutant. The antiserum raised against the secreted fraction reacts with the bacteria in an immunofluorescence assay. Immunization with the secreted protein fraction did not protect the rabbits against a systemic challenge with the fully pathogenic bacteria. Full protection was obtained only by a combined vaccination with PA and the secreted protein fraction. Therefore, these results indicate that an effective antiserum treatment in advanced stages of anthrax must include toxin-neutralizing antibodies in combination with antibodies against bacterial cell targets.

  7. The soluble 'low-Km' 5'-nucleotidase of rat kidney represents solubilized ecto-5'-nucleotidase. (United States)

    Piec, G; Le Hir, M


    A soluble 'low-Km' 5'-nucleotidase has been described previously in several organs. It has been presumed to be of cytosolic origin and thus to play a role in the intracellular production of adenosine. Its catalytic properties are similar to those of the ecto-5'-nucleotidase of cell membranes. In the present study we compared molecular properties of the two enzymes in the kidney of the rat. The Mr of the main peak of soluble 'low-Km' 5'-nucleotidase in gel-filtration chromatography was similar to that of the ecto-5'-nucleotidase solubilized by a phosphatidylinositol-specific phospholipase C from renal brush-border membranes. In phase-partition experiments using Triton X-114, the soluble enzyme appeared to be hydrophobic. Its hydrophobicity was decreased on treatment with a phosphatidylinositol-specific phospholipase C, suggesting that the soluble 'low-Km' 5'-nucleotidase contains the phosphatidylinositol anchor which is characteristic for the ecto-enzyme. An anti-ecto-5'-nucleotidase antiserum provoked an almost complete inhibition of the soluble enzyme. Immunoblotting using anti-ecto-5'-nucleotidase antiserum revealed in the high-speed supernatants a polypeptide with a similar Mr to the subunit of the ecto-5'-nucleotidase. The soluble 'low-Km' 5'-nucleotidase, like the ecto-5'-nucleotidase, bound specifically to concanavalin A. We conclude that the soluble 'low-Km' 5'-nucleotidase is not a cytosolic enzyme, but that it most probably originates from the solubilization of the ecto-5'-nucleotidase, and that it therefore cannot participate in the intracellular production of adenosine.

  8. Identification and characterization of two temperature-induced surface-associated proteins of Streptococcus suis with high homologies to members of the Arginine Deiminase system of Streptococcus pyogenes. (United States)

    Winterhoff, Nora; Goethe, Ralph; Gruening, Petra; Rohde, Manfred; Kalisz, Henryk; Smith, Hilde E; Valentin-Weigand, Peter


    The present study was performed to identify stress-induced putative virulence proteins of Streptococcus suis. For this, protein expression patterns of streptococci grown at 32, 37, and 42 degrees C were compared by one- and two-dimensional gel electrophoresis. Temperature shifts from 32 and 37 to 42 degrees C induced expression of two cell wall-associated proteins with apparent molecular masses of approximately 47 and 53 kDa. Amino-terminal sequence analysis of the two proteins indicated homologies of the 47-kDa protein with an ornithine carbamoyltransferase (OCT) from Streptococcus pyogenes and of the 53-kDa protein with the streptococcal acid glycoprotein (SAGP) from S. pyogenes, an arginine deiminase (AD) recently proposed as a putative virulence factor. Cloning and sequencing the genes encoding the putative OCT and AD of S. suis, octS and adiS, respectively, revealed that they had 81.2 (octS) and 80.2% (adiS) identity with the respective genes of S. pyogenes. Both genes belong to the AD system, also found in other bacteria. Southern hybridization analysis demonstrated the presence of the adiS gene in all 42 serotype 2 and 9 S. suis strains tested. In 9 of these 42 strains, selected randomly, we confirmed expression of the AdiS protein, homologous to SAGP, by immunoblot analysis using a specific antiserum against the SAGP of S. pyogenes. In all strains AD activity was detected. Furthermore, by immunoelectron microscopy using the anti-S. pyogenes SAGP antiserum we were able to demonstrate that the AdiS protein is expressed on the streptococcal surface in association with the capsular polysaccharides but is not coexpressed with them.

  9. Cholinergic urethral brush cells are widespread throughout placental mammals. (United States)

    Deckmann, Klaus; Krasteva-Christ, Gabriela; Rafiq, Amir; Herden, Christine; Wichmann, Judy; Knauf, Sascha; Nassenstein, Christina; Grevelding, Christoph G; Dorresteijn, Adriaan; Chubanov, Vladimir; Gudermann, Thomas; Bschleipfer, Thomas; Kummer, Wolfgang


    We previously identified a population of cholinergic epithelial cells in murine, human and rat urethrae that exhibits a structural marker of brush cells (villin) and expresses components of the canonical taste transduction signaling cascade (α-gustducin, phospholipase Cβ2 (PLCβ2), transient receptor potential cation channel melanostatin 5 (TRPM5)). These cells serve as sentinels, monitoring the chemical composition of the luminal content for potentially hazardous compounds such as bacteria, and initiate protective reflexes counteracting further ingression. In order to elucidate cross-species conservation of the urethral chemosensory pathway we investigated the occurrence and molecular make-up of urethral brush cells in placental mammals. We screened 11 additional species, at least one in each of the five mammalian taxonomic units primates, carnivora, perissodactyla, artiodactyla and rodentia, for immunohistochemical labeling of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), villin, and taste cascade components (α-gustducin, PLCβ2, TRPM5). Corresponding to findings in previously investigated species, urethral epithelial cells with brush cell shape were immunolabeled in all 11 mammals. In 8 species, immunoreactivities against all marker proteins and ChAT were observed, and double-labeling immunofluorescence confirmed the cholinergic nature of villin-positive and chemosensory (TRPM5-positive) cells. In cat and horse, these cells were not labeled by the ChAT antiserum used in this study, and unspecific reactions of the secondary antiserum precluded conclusions about ChAT-expression in the bovine epithelium. These data indicate that urethral brush cells are widespread throughout the mammalian kingdom and evolved not later than about 64.5millionyears ago.

  10. 3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus). (United States)

    Sheldon, P S; Kekwick, R G; Smith, C G; Sidebottom, C; Slabas, A R


    3-Oxoacyl-[ACP] reductase (E.C., alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.

  11. Non-polio enteroviruses associated with acute flaccid paralysis (AFP) and facial paralysis (FP) cases in Romania, 2001-2008. (United States)

    Persu, Ana; Băicuş, Anda; Stavri, Simona; Combiescu, Mariana


    Acute flaccid paralysis is a complex clinical syndrome, with a wide variety of possible etiologies and with clinical manifestations that can vary according to age or geographical region. Enteroviruses (polioviruses and non-polio enteroviruses) are among the viral agents that can cause AFP. AFP surveillance is important for public health through its use in monitoring poliomyelitis, in the context of the Global Initiative to eradicate this disease. The current paper aims to assess the non-polio enteroviruses (NPEV) association with AFP and FP cases registered in Romania in the period 2001-2008 and to identify prevalent serotypes. Within the framework of Surveillance of AFP Cases Program, were collected samples from 579 children with AFP or FP (3.069 samples). The samples were processed and inoculated onto two types of cell culture (RD and L20B), according to WHO protocol. The identification of isolated viruses has been done by the reaction of seroneutralization with pools of specific antiserum and then with monospecific antiserum for confirmation. NPEV were isolated from 58 cases (123 positive samples). During the analyzed period, 23 NPEV serotypes have circulated (15 Echo serotypes and 8 coxsackie serotypes). The most frequently identified were the Echoviruses 13 and 11 and the coxsackie A viruses. 88% of positive cases have occurred in children between 1 and 5 years. As seasonal distribution, the peak of NPEV circulation was in the months August-September (36.2%). The paper provides information about NPEV circulation in Romania in the past 8 years, about its association with the AFP and FP and it indicates the need for monitoring NPEV circulation even after the eradication of poliomyelitis.

  12. Purification and Immunity Analysis of Recombinant 6His- HPT Protein Expressed in E.coli

    Institute of Scientific and Technical Information of China (English)



    Objective To obtain HPT protein (Hygromycin B Phosphotransferase), a kind of plantselective maker gene product expressed from E. coli and to prepare the polyclonal antibody (pAbs)against it. Methods HPT cDNA fragment was obtained by PCR and was inserted into theprokaryotic expressing vector pBV222. Then the constructed recombinant plasmid pBV222-HPT wastransfered into E. coli DH5α for HPT expression. The recombinant expressing system was confirmedby restriction endonuclease digestion, DNA sequencing and protein expression. E. coli cells were lysedby sonication and detergent dissolution. After cell membrane was extracted, the inclusion bodies weredenatured by 8 mol/L Urea and purified with metal chelate affinity chromatography on Ni-NTAagarose under denaturing condition. The purified 6His-HPT was characterized by SDS-PAGE, andused to immunize rabbit. The titer and specificity of antisera were detected by ELISA and Westernblot respecitively. Results Analysis of DNA sequence and restricted enzymes showed that thesequence of PBV222-HPT plasmid was correct. The amount of recombinant HPT expressed in E. coliaccounted for 30% of total cellular proteins. From 1 liter of fermentative bacteria about 22 milligramsof pure recombinant HPT was isolated with purity above 95%. The recombinant HPT protein couldproduce high titer antiserum in rabbits and show good immunity activity. Western blot showedspecific binding reaction between the antiserum to the purified 6His-HPT protein and their expressedproducts (plants protein and bacterial protein). Conclusion HPT protein can be expressed andpurified from E. coli by a relatively simple method, which has high immunity activity.

  13. IgE and IgG cross-reactivity among Lol p I and Lol p II/III. Identification of the C-termini of Lol p I, II, and III as cross-reactive structures. (United States)

    van Ree, R; van Leeuwen, W A; van den Berg, M; Weller, H H; Aalberse, R C


    In this study, the homologous C-termini of Lol p I, Lol p II, and Lol p III were shown to contain cross-reactive B-cell epitopes. This was demonstrated by inhibition studies with purified Lol p I, II, and III and synthetic peptides of their C-termini. It was ruled out that the observed cross-reactivity was caused by cross-contamination of the purified allergens. Both human IgE and IgG bound to the C-terminus of Lol p I. These antibodies were cross-reactive with Lol p II and, more specifically, with its C-terminus. Within a small panel of allergic patients, no cross-reactivity with Lol p III was found. A hyperimmune polyclonal rabbit antiserum against Lol p I also recognized the Lol p I C-terminus. As for human antibodies, cross-reactivity with Lol p II and its C-terminus was demonstrated. Cross-reactivity with Lol p III was demonstrated with C-terminal peptides, but not with native Lol p III. A polyclonal rabbit antiserum against Lol p II bound to the C-terminal peptides of both Lol p II and III. This binding was inhibited with Lol p I, confirming that cross-reactive structures exist not only on the C-termini of Lol p II and Lol p I, but also of Lol p III and Lol p I. The existence of cross-reactivity between Lol p I and Lol p II and III possibly contributes to the frequently observed cosensitization for these allergens in grass-pollen-allergic patients.

  14. Determining Specificity of Fab Antibody against Mouse Serologically Detected Male Antigen%血清学检测的雄性特异性抗原Fab抗体的特异性鉴定

    Institute of Scientific and Technical Information of China (English)

    王乃东; 袁安文; 邓治邦; 屠迪; 许道军; 夏南松; 杨大为; 薛立群


    旨在研究血清学检测的雄性特异性抗原噬菌体Fab抗体的特异性.以从噬菌体Fab抗体库中筛选到具有较高雄性特异性结合活性的重组噬菌体克隆为基础,构建Fab抗体的可溶性表达噬质粒,并诱导和纯化该抗体片段,利用免疫荧光FITC/DAPI共染技术和ELISA分析其特异性.结果表明,该抗体在约49 ku处有条带出现.在Fab抗体和H-Y抗血清的细胞免疫荧光比较分析试验中发现,从雌、雄鼠脾细胞的阳性细胞数量和平均荧光强度的角度分析,Fab抗体的雄性特异性强于抗血清.石蜡切片免疫荧光定量分析显示,Fab抗体与雄鼠肝脏的结合活性明显高于对应雌鼠,差异极显著(t=20.73,P=0.002 3<0.01),而H-Y抗血清与雄鼠肝脏的结合活性略高于对应雌鼠,差异显著(t=7.11,P=0.019 2<0.05).以C57BL/6雄鼠脾细胞、睾丸细胞作为抗原的ELISA分析显示,Fab抗体具有雄性特异性,但Fab抗体的OD值低于抗血清.结果提示,Fab抗体的雄性特异性高于H-Y抗血清,但其雄性特异性结合活性低于H-Y抗血清,Fab抗体在具备雄性特异性的同时,仍有少量雌性非特异性结合,Fab抗体的体外亲和力成熟可作为后续研究工作,以获得高亲和力、高雄性特异性的可溶性Fab抗体分子.%To study specificity of Fab antibody against mouse serologically detected male antigen, based on recombinant phage clone with male specific activity screened from phage Fab antibody library, expression plasmid of soluble Fab antibody was constructed, antibody fragment was induced and purified, the antibody specificity was determined by immunofluorescence staining with FITC/DAPI and ELISA. The results showed that an about 49 ku band appeared in the SDS-PAGE, comparison of cell immunofluorescence staining of Fab antibody and H-Y antiserum showed that male specificity and binding activity of Fab antibody was higher than that of antiserum, based on number of positive cells and mean

  15. 红笛鲷主要组织相容性复合物Ⅰα抗原基因的克隆与表达分析%Cloning and expression analysis of histocompatibility complex Ⅰ α antigen (MHC I α)from humphead snapper Lutjanus sanguineus

    Institute of Scientific and Technical Information of China (English)

    张新中; 鲁义善; 吴灶和; 简纪常


    In the present study, full-length cDNA sequences of histocompatibility complex Ⅰ a antigen (MHC Ⅰ α was cloned by rapid amplification of cDNA ends technique(RACE)from humphead snapper( Lutjanus sanguineus). Full-length cDNA sequence of MHC Ⅰ α is 1 369 bp, encoding 354 amino acids. BLAST analysis revealed that the amino acids sequence of MHC Ⅰ a shared high identity (84% ) with other MHC I . Phylogenetic tree was constructed by the Neighbor-Joining method, and the results suggested that MHC I a of humphead snapper shared the closest genetic relationship with the MHC Ⅰ of Epinephelus coioides. The results of fluorescent real-time quantitative RT-PCR showed that the expression of MHC Ⅰ a mRNA could be detected in head kidney, and the maximum expression appeared in 6 - 15 h post infection. MHC I a was subcloned into pET32a( + )to construct expression plasmids pET32-MHC Ⅰ α. SDS-PAGE and Western blot analysis indicated that the recombinant proteins were successfully expressed in Escherichia coli BL21 ( DE3). Then the recombinant proteins were purified and the antiserum was obtained by immunizing rabbits with the purified recombinant proteins emulsified with adjuvant. ELISA analysis showed that the titer of the antiserum prepared in this study was 1:40 000. The results of the Western blot revealed that specific antigen-antibody reaction occurred between the antiserum and the recombinant proteins.%为研究红笛鲷免疫防御相关基因的作用机理和调控机制,实验应用cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)成功克隆了红笛鲷组织相容性复合物Ⅰ α(MHC Ⅰ α)抗原基因的全长cDNA序列,MHC Ⅰα的全长序列为1 369 bp,编码354个氨基酸残基.BLAST分析显示,红笛鲷MHC Ⅰ α与其他已知物种MHC Ⅰ α基因的最高同源性为84%.构建的系统进化树显示,红笛鲷MHC Ⅰα与石斑鱼等MHC Ⅰ α亲缘关系较近.Real-time PCR分析表明,MHC Ⅰ α在头肾中的最大

  16. Preparation of Polyclonal Antibodies Against MHC Ⅱα and MHC Ⅱβ of Mangrove Red Snapper (Lutjanus argentimaculatus)%紫红笛鲷MHC Ⅱα和MHC Ⅱβ多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    王天燕; 常虹; 余时琛; 陈璐; 蔡中华


    目的:制备紫红笛鲷主要组织相溶性复合体MHC Ⅱα和MHC Ⅱβ多克隆抗体,为蛋白水平研究紫红笛鲷MHCⅡ分子提供理论和实践依据.方法:从已有的紫红笛鲷cDNA文库菌中分别克隆其MHC Ⅱα和MHC Ⅱ3分子的部分开放阅读框,与PQE-30构建表达载体,转入大肠杆菌E.coli M15以IPTG诱导表达;纯化得到的重组蛋白与弗氏佐剂混合乳化后注射新西兰大白兔制备多克隆抗体,再以酶联免疫吸附(ELISA)和免疫印迹(Western blot)检测所获抗血清的效价及效果.结果:①重组表达和纯化得到紫红笛鲷MHC Ⅱα和MHC Ⅱβ部分肽链.②制备的紫红笛鲷MHC Ⅱα和MHC Ⅱβ兔抗血清效价都大于1:25600,达到预期水平.③以获得的紫红笛鲷MHC Hα和MHC Ⅱβ兔抗血清分别与紫红笛鲷头肾巨噬细胞蛋白进行免疫印迹,显示两种抗血清能分别杂合出各自的目标蛋白,说明制备的多克隆抗体实际应用效果良好.结论:紫红笛鲷MHC Ⅱα和MHC Ⅱβ多克隆抗血清制备成功.%Objective: To prepare the polyclonal antibodies against MHC Ⅱα and MHC Ⅱβ of mangrove red snapper. Methods: A partial of MHC Ⅱα and MHC Up chain was cloned from the cDNA library of mangrove red snapper, respectively. The PCR products were inserted into the expression vectors pQE30 and transformed into the E. coli M15. By inducing of IPTG, the recombinant proteins of MHC Ⅱα and MHC Ⅱβ fragments were purified, respectively. The proteins were thoroughly mixed with Freund's adjuvant, and injected rabbit. The antiserums were detected by ELISA and Western blot. Results: ① The recombinant proteins of MHC Ⅱα and MHC Ⅱβ fragments were purified successfully. ② The antiserums against MHC Ⅱα and MHC Ⅱβ both had a high titer above 1:25600. ③The western blot of head kidney macrophages showed the specification of MHC Ⅱα and MHC Ⅱβ antiserums, respectively. Conclusions: The high titer and specific polyclonal

  17. 香蕉束顶病毒Haikou 2分离物DNA2编码框原核表达及抗血清制备%Prokaryotic Expression of DNA2 ORF of BBTV Haikou2 Isolate and Preparation of Polyclonal Antibody

    Institute of Scientific and Technical Information of China (English)

    谭老喜; 王洪星; 张雨良; 王健华; 章绍延; 周朋; 余乃通; 刘志昕


    Banana bunchy top disease caused by Banana bunchy top virus which is one of the worst diseases of banana. It is a serious threat to banana production. BBTV genome is composed of at least six circular ssDNA components, and the component DNA2 with less research is the largest variation. About 114 bp of Haikou2 BBTV DNA2 ORF was amplified by regular PCR in this research. The PCR product, digested with restriction enzymes, was inserted into a prokaryotic expression vector pET32a of the same digestion, then recombinant plasmid pET32a-DNA2 ORF was identified and was transferred into E.coli Trasetta(DE3). 12% SDS-PAGE analysis showed that a fusion protein band about 21 ku was detected after induced with IPTG. By the ultrasonic broken instrument, the analysis results showed that the fusion protein was expressed steadily in LB culture and the. optimal expression conditions was 30 ℃, 1.0 mmol/L IPTG with 3 h. The fusion protein was further purified via Ni2+-NTA agarose affinity chromatography, and was used to immune rabbits for antiserum preparation. 0.954 μg/mL purified protein in banana juice was detected and the optimal titer of the antiserum was determined to be 1∶ 25 000 diluted by indirect enzyme -linked immunosorbent assay (ID -ELISA). Western blot analysis- revealed that antiserum was specifically binded the fused protein.%香蕉束顶病毒(Banana bunchy top virus,BBTV)是危害香蕉的主要病毒之一,严重威胁着香蕉的生产.BBTV基因组至少由6个环状ssDNA组分组成,其中DNA2组分变异最大,是否编码蛋白及其功能缺乏研究.本研究通过PCR方法克隆了BBTV Haikou2 DNA2ORF,将其构建到pET32a载体中,进行原核表达和抗血清制备.结果表明重组表达载体pET32a-DNA2ORF在表达菌Transetta(DE3)中表达一个约21 ku的特异融合蛋白,通过超声波破碎仪破碎细胞和诱导条件优化,分析表明,该蛋白主要以可溶形式存在,最佳表达条件为30℃、1.0 mmol/L IPTG诱导3h

  18. Characterization of gene expression on genomic segment 7 of infectious salmon anaemia virus

    Directory of Open Access Journals (Sweden)

    Qian Biao


    Full Text Available Abstract Background Infectious salmon anaemia (ISA virus (ISAV, an important pathogen of fish that causes disease accompanied by high mortality in marine-farmed Atlantic salmon, is the only species in the genus Isavirus, one of the five genera of the Orthomyxoviridae family. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; haemagglutinin-esterase (HE protein encoded on segment 6 and fusion (F protein encoded on segment 5. Based on the initial demonstration of two 5'-coterminal mRNA transcripts by RT-PCR, ISAV genomic segment 7 was suggested to share a similar coding strategy with segment 7 of influenza A virus, encoding two proteins. However, there appears to be confusion as to the protein sizes predicted from the two open reading frames (ORFs of ISAV segment 7 which has in turn led to confusion of the predicted protein functions. The primary goal of the present work was to clone and express these two ORFs in order to assess whether the predicted protein sizes match those of the expressed proteins so as to clarify the coding assignments, and thereby identify any additional structural proteins of ISAV. Results In the present study we show that ISAV segment 7 encodes 3 proteins with estimated molecular masses of 32, 18, and 9.5 kDa. The 18-kDa and 9.5-kDa products are based on removal of an intron each from the primary transcript (7-ORF1 so that the translation continues in the +2 and +3 reading frames, respectively. The segment 7-ORF1/3 product is variably truncated in the sequence of ISAV isolates of the European genotype. All three proteins are recognized by rabbit antiserum against the 32-kDa product of the primary transcript, as they all share the N-terminal 22 amino acids. This antiserum detected a single 35-kDa protein in Western blots of purified virus, and immunoprecipitated a 32-kDa protein in ISAV-infected TO cells. Immunofluorescence staining of infected cells with the

  19. 被动皮肤过敏性评价中阳性对照的建立%Establishment of Positive Control in Passive Cutaneous Anaphylaxis Evaluation

    Institute of Scientific and Technical Information of China (English)

    孙鸽; 李炜宾; 岳娟; 尹晶晶; 安全


    Objective: To establish an accurate and repeatable method for positive control of passive cutaneous anaphylaxis(PCA) test.Methods:30 SD rats were randomly divided into 1 negative control group( sodium chloride injection plus Freund′s adjuvant incom-plete) and 4 positive treatment groups(the positive 1 group:ovalbumin, the positive 2 group:ovalbumin plus diphtheria-pertussis-tetanus vaccine, the positive 3 group: ovalbumin plus Freund′s adjuvant incomplete, the positive 4 group: ovalbumin plus Freund′s adjuvant complete and Freund′s adjuvant incomplete ) were set in the experiment.Firstly, antiserum was prepared with 5 subcutaneous injections.Secondly, passive sensitization was conducted by intradermal injection of antiserum.After 48h, the immuno-stimulation was fol-lowed by intravenous injection of equal amount of antigen.Finally,the result was evaluated by inspecting the amount and diameter of intra-dermal blue spots.Results:The blue spot was not found in the negative control group, the positive 1 group and the positive 2 group.While both the positive 3 group and the positive 4 group produced intradermal blue spots.The average diameter of blue spots of the 1∶2 and 1∶8 antiserum dilution injection was larger than 5 mm.Conclusion:In PCA test, by using different sensitive treatment, we found that sensi-tization with Freund′s adjuvant could carry out 100%positive results.It is an accurate and reliable method for establishment of positive control in PCA test.%目的:建立被动皮肤过敏性试验中准确且具可重复的阳性对照。方法:30只SD雄性大鼠随机分为1个阴性对照组(氯化钠注射液+弗氏不完全佐剂)和4个阳性处理组(阳性1组:卵白蛋白,阳性2组:卵白蛋白+百白破疫苗,阳性3组:卵白蛋白+弗氏不完全佐剂,阳性4组:卵白蛋白+弗氏完全佐剂及弗氏不完全佐剂),分别致敏5次制备抗血清,通过皮内注射被动致敏、48 h后激发,以

  20. 抗草胺膦bar基因原核表达和纯化及其免疫反应性分析%Prokaryotic expression, purification and immunoreactivity analysis of glufosinate resistant gene(bar)

    Institute of Scientific and Technical Information of China (English)

    吕莉琨; 苏旭; 王静; 李闻; 刘洪亮; 卢长明


    Objective To obtain high expression of bar gene in Escherichia coli and purify its expressed protein, then analyze its immunoreactivity. Methods The recombinant vector pET28a-bar was transformed into BL21 and its expression condition was optimized through temperature and IPTG, Ni-NTA affinity chromatography was used to purify the recombinant protein. The purified protein was used to immunize BALB/c mice, the titer of antiserum was tested by ELISA. The equivalence of BAR protein expressed in Escherichia coli and in transgenic bar rape was analyzed by Western blot Results The prokaryotic expression system was used to express BAR recombinant protein. The optimum condition of expressing BAR was 0.5 mmol/L IPTG, 20 ℃ and 120 r/min, overnight. The titer of the antiserum from immunized BALB/c was 1:102 400 by indirect ELISA. Western blot showed that antiserum could bond specially with BAR recombinant protein and transgenic bar rape. Conclusion Specific polyclonal antibody with high purified BAR recombinant protein has been prepared in the present study and the results will provide the basis for food safety assessment of genetically modified products with bar gene.%目的 实现抗草胺膦bar基因在原核表达系统中的高效表达及纯化,并分析其免疫反应性.方法 将携带bar基因的重组质粒pET28a-bar转化到大肠杆菌BL21(DE3)中诱导表达,确定最佳诱导表达条件.获得的重组蛋白经镍亲和层析纯化后,免疫BALB/c小鼠,测定抗体效价,以获得的抗BAR的抗血清作为一抗进行Western blot反应,检测蛋白的免疫反应性,分析该原核表达蛋白与转bar基因油菜中蛋白的等同性.结果 利用原核表达系统成功实现了BAR重组蛋白的高效表达,其最适表达条件为:0.5 mmol/L异丙基-β-D-硫代吡喃半乳糖苷(IPTG)、20℃和120 r/min摇床转速诱导过夜.以纯化后的目的蛋白作为抗原免疫小鼠,得到抗BAR的抗血清.以间接ELISA法测定抗血清效价达1∶102

  1. Using antibodies against ATPase and microarray immunoassays for the search for potential extraterrestrial life in saline environments on Mars. (United States)

    Weigl, Andreas; Gruber, Claudia; Blanco-López, Yolanda; Rivas, Luis A.; Parro, Victor; Stan-Lotter, Helga


    For the search for extraterrestrial life it is proposed to use receptors such as labelled antibodies for the detection of organic biomarkers. One of these organic molecules to be tested is the universal enzyme ATP synthase which is present in highly conserved forms in all organisms on earth. Therefore it is necessary to evaluate antibodies against ATPase respectively ATP synthase and their subunits. As it is known, that there are halite deposits on Mars the experiments in this study have been carried out with regard to halophile microorganisms and saline environments. Standard F1F0 ATPase from Escherichia coli LE 392 and Bacillus megaterium as well as haloarchaeal A-ATPase from Halorubrum saccharovorum and Halobacterium salinarum NRC-1 were used. The cultivated cells, except Bacillus, were broken by passage through a French Pressure Cell. Separation of enzyme subunits was performed by polyacrylamide gel electrophoresis. Western Blotting with antisera produced in rabbit against A-ATPase subunits A (85 kD) and subunits B (60 kD) from Halorubrrum saccharovorum (1) showed positive reactions with the membrane fraction, which should be enriched with ATPase from Halorubrum saccharovorum, Halobacterium salinarum NRC-1 and Escherichia coli LE 392. Particular attention was given to the question if ATPase subunits can be detected in whole cells. Therefore whole cell preparations of all cells and spore suspensions from Geobacillus stearothermophilus were tested against the antiserum as well as against protein-A-purified antibody against A-ATPase subunit A from Halorubrum saccharovorum. A positive immuno reaction of all cell preparations with the antiserum as well as with the purified antibody was detected. The spores of Geobacillus stearothermophilus reacted positively with the antiserum against subunit A of the A-ATPase from Hrr. saccharovorum. A commercial antibody Rabbit Anti-V-ATPase subunit A polyclonal antibody from the GenScript Corporation reacted positively with

  2. Influence of bovine LH tracer quality on levels of LH in GnRH-treated cows

    Energy Technology Data Exchange (ETDEWEB)

    Madej, A.; Hallin, P.; Madej, M.; Seguin, B.; Edqvist, L.E.


    Chromatography of 125I-bovine LH (LER-1716-2 and USDA-I-1) by means of anion exchange high performance liquid chromatography (HPLC) revealed two main peaks of radioactivity regardless as to whether or not the tracer was initially purified on cellulose CF11. The content of radioactivity in the first peak tended to increase as the storage time of the bLH preparation, either before or after iodination, increased. The first peak of radioactivity after HPLC fractionation either with or without cellulose adsorption consisted of material with low binding ability to bLH antiserum (6.9% +/- 0.5 and 13.0% +/- 1.0, respectively) and high binding ability to ovine LH alpha antiserum (51.0% +/- 2.7 and 35.2% +/- 3.6, respectively). The average ratio of alpha-subunit immuno-reactivity to 125I-bLH immunoreactivity in this material was 7.4 +/- 0.1 and 2.7 +/- 0.2, respectively (P less than 0.001). Peaks in 125I-bLH radioactivity and 125I-bLH immunoreactivity had different elution times. Radioimmunoassays with tracers obtained from fractions derived from the first radioactive peak after HPLC chromatography (i.e. 125I-bLH-LER-1716-2) both with and without cellulose adsorption, yielded significantly lower mean plasma LH levels in GnRH-treated cows compared with the control tracer routinely purified only on cellulose CF11 (e.g. 5.7 vs. 8.2 micrograms/; 4.6 vs. 8.2 micrograms/l). Plasma LH levels in GnRH-treated cows were significantly (P less than 0.001) lower as measured by radioimmunoassay utilizing 125I-USDA-blH-I-1 tracers than by radioimmunoassays utilizing 125I-blH-LER-1716-2 tracers (i.e. either Y = 0.17 + 0.75X or Y = 1.18 + 0.60X).

  3. Design, Expression and Identification of Specific Multi-Epitope Antigen of Campylobacter Jejuni Surface Proteins%空肠弯曲菌表面蛋白特异性多表位抗原的设计、表达与鉴定

    Institute of Scientific and Technical Information of China (English)

    欧瑜; 鄢方兵; 唐泰山; 祝长青; 蒋原; 张常印


    构建空肠弯曲菌表面蛋白特异性多表位抗原原核表达载体,并在大肠杆菌中诱导表达,免疫动物后检测其抗血清与空肠弯曲菌菌体的反应性.从空肠弯曲菌的6个表面蛋白中分析到特异性抗原表位,人工合成其串联基因片段,将其插入原核表达载体,获得重组质粒pET-32a(+ )-CJMEA-A,经IPTG诱导后表达出25k的目标蛋白,纯化后免疫新西兰大白兔,Western blot和间接ELISA检测重组蛋白抗原性和抗血清反应性.结果表明目标蛋白具有良好的反应原和免疫原性,抗血清能与菌体发生反应.其抗体可用于C.jejuni免疫磁珠和免疫胶体金等检测方法的建立.%To construct and to express the gene of specific multi-epitope antigen from Campylobacter jejuni and to detect the reactivity of its antibody to this bacteria. Firstly ,some specific antigenic determinants were obtained after 6 surface proteins of Campytobacter jejuni were analyzed. Secondly these epitopes were linked in series and its gene was synthesized. Thirdly, the recombinant plasmid pET-32a( + )-CJMEA-A was obtained when the gene was inserted into pET-32a( + ) vector. Then,after being induced by IPTG,the 25ku of fusion protein was expressed,purified and used for the immunization of rabbits. Finally,SDS-PAGE and indirect ELISA were used to detect the antigenicity of the protein and the reactivity of the antiserum. The results showed that this protein was expressed successfully;it possesses good unnuinogem'city and reactogenicity;its antiserum can react to thalli of C. Jejuni . A conclusion can be made that the antibody of this protein could be used to detect C. Jejuni by inununomagnetic beads or immune colloidal gold technique.

  4. [A sensitive and specific radioimmunoassay for arginine vasopressin and its validation]. (United States)

    Oki, Y; Ohgo, S; Yoshimi, T


    A sensitive and specific radioimmunoassay (RIA) for arginine vasopressin (AVP) has been developed and validated. Synthetic AVP was coupled to bovine serum albumin (BSA) with glutaraldehyde. Antisera against AVP were raised in three rabbits immunized with AVP-BSA complex. After 6 months, at the 16th injection, one of the antisera had a titer high enough to be utilizable for RIA at a final dilution of 1:400,000. The labeling of AVP with 125I Na was performed with the modified chloramine T method, and the purification of iodinated AVP was done with gel filtration chromatography on a Sephadex G-25 fine column (1 X 20 cm) with an elution buffer of 0.01 M acetic acid containing 0.1% BSA. Radioactivities from the Sephadex G-25 were eluted in three peaks. 125I-AVP, which was reactive to the antiserum, was contained in the third peak, and 125I-AVP in the fractions on the down slope of the peak was used for the radioligand in the amount of 1000 cpm. The specific activity of purified 125I-AVP was about 400 muCi/microgram. Diluted antiserum and samples, unlabeled AVP or related peptides were preincubated at 4 degrees C for 24 hr, and then 125I-AVP was added to the mixture and incubated for a further 72 hr. Separation of B and F was done with polyethyleneglycol. The minimal detection limit of AVP, which was 95% of the confidence limit of the mean value of B0, was 0.4 pg/tube. The cross-reactivities with lysine vasopressin, arginine vasotocin, DDAVP and oxytocin were 0.1%, 30%, 1% and 0%, respectively. AVP in plasma was extracted with cold acetone and petroleum ether. The recoveries of synthetic AVP from plasma which was added (2-16 pg) were more than 94%. The intra and inter-assay coefficients of variation determined by plasma of AVP concentration of about 4.8 pg/ml were 8.7% and 11.3%, respectively. The RIA detected AVP of concentration as low as 1 pg/ml following the extraction procedure. AVP immunoreactivity was detected without extraction in urine, and the lyophilized

  5. [Serotypes and antimicrobial susceptibilities of invasive group A streptococci identified in eastern Black Sea region of Turkey]. (United States)

    Bayramoğlu, Gülçin; Topkaya, Aynur E; Balıkcı, Ahmet; Aydın, Faruk


    Frequency of invasive group A streptococcus (GAS) infections is increasing worldwide in recent 20 years. Serotypes responsible for these clinical manifestations and their antibiotic susceptibilities should be known in order to establish preventive measures and initiate appropriate treatment. This study was aimed to determine the serotypes, antibiotic susceptibilities and inducible clindamycin resistance among invasive GAS isolated between 2006-2009 period. A total of 22 GAS strains isolated from clinical samples [sterile body fluids (peritoneal, pleural, pericardial, joint and cerebrospinal fluids), blood, tissue biopsy] of the patients (14 male, 8 female; age range: 3-82 years, median age: 59) who admitted to Karadeniz Technical University Faculty of Medicine, Farabi Hospital located in Trabzon province (Eastern Black Sea Region of Turkey), between March 2006 and March 2009 were included in the study. GAS serotypes were determined by the investigation of serum opacity factors (SOF), T proteins and M proteins. SOF production was investigated by microplate method using human serum and SOF types were determined by SOF-inhibition test using specific antisera. T protein types were detected by agglutination method using polyvalent anti-T sera, and M serotypes were detected by capillary precipitation method using M antisera. Antimicrobial susceptibility tests were performed by disk-diffusion method according to CLSI recommendations. SOF were positive in 9 (41%) samples. Use of T antiserum yielded T (n= 8) and U (n= 7) types and M antiserum M1 (n= 4) and M2 (n= 3) types. The overall antibiotic susceptibility rate of the isolates was 68% (15/22) and overall resistance rate was 32% (7/22). All of the GAS strains were found susceptible to benzylpenicillin, ceftriaxone, vancomycin, levofloxacine and linezolid, however 9 (41%) were intermediate susceptible to tetracycline and 1 (4.5%) was intermediate susceptible to erythromycin. Four (18%) strains were found resistant to

  6. Effect of exogenous circulating anti-bPL antibodies on bovine placental lactogen measurements in foetal samples

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    Taverne Marcel AM


    Full Text Available Abstract Background The involvement of placental lactogen (PL in the regulation of foetal growth has been investigated in different species by in vivo immunomodulation techniques. However, when circulating antibodies are present together with the hormone, the procedure for hormonal measurement becomes considerably complex. The aim of this study was the immunoneutralization of bovine placental lactogen (bPL concentrations in bovine foetal circulation by direct infusion of rabbit anti-bPL purified immunoglobulins (IgG via a foetal catheter (in vivo study. The ability of a RIA based on guinea pig anti-bPL antiserum, for the measurement of bPL concentrations in samples containing exogenous rabbit anti-bPL immunoglobulins, was also analyzed in in vitro and in vivo conditions. Methods Six bovine foetuses were chronic cannulated on the aorta via the medial tarsal artery. Infusion of rabbit anti-bPL IgG was performed during late gestation. Pooled rabbit anti-bPL antisera had a maximal neutralization capacity of 25 μg bPL/mL of immunoglobulin. Interference of rabbit anti-bPL immunoglobulin with radioimmunoassay measurement using guinea pig anti-bPL as primary antibody was first evaluated in vitro. Polyclonal anti-bPL antibodies raised in rabbit were added in foetal sera to produce 100 samples with known antibodies titers (dilutions ranging from 1:2,500 till 1:1,280,000. Result(s Assessment of the interference of rabbit anti-bPL antibody showed that bPL concentrations were significantly lower (P Conclusion(s The use of a bPL RIA using a guinea pig anti-bPL as primary antiserum allowed for the measurement of bPL concentrations in foetal plasma in presence of rabbit anti-bPL IgG into the foetal circulation. Long-term foetal catheterization allowed for the study of the influence of direct infusion of anti-bPL IgG on peripheral bPL concentrations in bovine foetuses.

  7. Expression and characterization of the UL31 protein from duck enteritis virus

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    Zhu Dekang


    Full Text Available Abstract Background Previous studies indicate that the UL31 protein and its homology play similar roles in nuclear egress of all herpesviruses. However, there is no report on the UL31 gene product of DEV. In this study, we expressed and presented the basic properties of the DEV UL31 product. Results The entire ORF of the UL31 was cloned into pET 32a (+ prokaryotic expression vector. Escherichia coli BL21(DE3 competent cells were transformed with the construct followed by the induction of protein expression by the addition of IPTG. Band corresponding to the predicted sizes (55 kDa was produced on the SDS-PAGE. Over expressed 6×His-UL31 fusion protein was purified by nickel affinity chromatography. The DEV UL31 gene product has been identified by using a rabbit polyclonal antiserum raised against the purified protein. A protein of approximate 35 kDa that reacted with the antiserum was detected in immunoblots of DEV-infected cellular lysates, suggesting that the 35 kDa protein was the primary translation product of the UL31 gene. RT-PCR analyses revealed that the UL31 gene was transcribed most abundantly during the late phase of replication. Subsequently, Immunofluorescence analysis revealed that the protein was widespread speckled structures in the nuclei of infected cells. Western blotting of purified virion preparations showed that UL31 was a component of intracellular virions but was absent from mature extracellular virions. Finally, an Immunofluorescence assay was established to study the distribution of the UL31 antigen in tissues of artificially DEV infected ducks. The results showed that the UL31 antigen was primarily located in the cells of digestive organs and immunological organs. Conclusion In this work, we present the basic properties of the DEV UL31 product. The results indicate that DEV UL31 shares many similarities with its HSV or PRV homolog UL31 and suggest that functional cross-complementation is possible between members of the

  8. Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts. (United States)

    Wilson, I B; Harthill, J E; Mullin, N P; Ashford, D A; Altmann, F


    Carbohydrates have been suggested to account for some IgE cross-reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti-horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose

  9. Cubilin and megalin expression and their interaction in the rat intestine: effect of thyroidectomy. (United States)

    Yammani, R R; Seetharam, S; Seetharam, B


    Cubilin is a 460-kDa multipurpose, multidomain receptor that contains an NH(2)-terminal 110-residue segment followed by 8 epidermal growth factor (EGF)-like repeats and a contiguous stretch (representing nearly 88% of its mass) of 27 CUB (initially found in complement components C1r/C1s, Uegf, and bone morphogenic protein-1) domains. Cubilin binds to intrinsic factor (IF)-cobalamin (cbl, vitamin B(12)) complex and promotes the ileal transport of cbl. The 460-kDa form of cubilin is the predominant form present in the apical brush-border membranes of rat intestine, kidney, and yolk sac, but a 230-kDa form of cubilin is also noted in the intestinal membranes. In thyroidectomized (TDX) rats, levels of intestinal brush-border IF-[(57)Co]-labeled cbl binding, 460-kDa cubilin protein levels and tissue (kidney) accumulation of cbl were reduced by approximately 70%. Immunoblot analysis using cubilin antiserum of intestinal total membranes from TDX rats revealed cubilin fragments with molecular masses of 200 and 300 kDa. Both of these bands, along with the 230-kDa band detected in the total membranes of control rats and unlike the 460-kDa form, failed to react with antiserum to EGF. Mucosal membrane cubilin associated with megalin was reduced from approximately 12% in control to approximately 4% in TDX rats, and this decreased association was not due to altered megalin levels. Thyroxine treatment of TDX rats resulted in reversal of all of these effects, including an increase to nearly 24% of cubilin associated with megalin. In vitro, megalin binding to cubilin occurred with the NH(2)-terminal region that contained the EGF-like repeats and CUB domains 1 and 2 but not with a downstream region that contained CUB domains 2-10. These studies indicate that thyroxine deficiency in rats results in decreased uptake and tissue accumulation of cbl caused mainly by destabilization and deficit of cubilin in the intestinal brush border.

  10. The antenna-specific odorant-binding protein AlinOBP13 of the alfalfa plant bug Adelphocoris lineolatus is expressed specifically in basiconic sensilla and has high binding affinity to terpenoids. (United States)

    Sun, L; Xiao, H-J; Gu, S-H; Zhou, J-J; Guo, Y-Y; Liu, Z-W; Zhang, Y-J


    Odorant-binding proteins (OBPs) are crucial in the olfactory pathway of insects. In the present study, the antenna-enriched OBP AlinOBP13 was investigated because of its potential contribution to the peripheral olfactory perception in the alfalfa plant bug Adelphocoris lineolatus. The results of quantitative reverse transcriptase-PCR showed that the transcript level of AlinOBP13 was higher in the adult stage than in the nymph stages. The transcript levels of AlinOBP13 in the male and female antennae significantly increased after 4 and 8 h of starvation, respectively. Fine ultrastructures of different types of chemosensilla in both female and male antennae were investigated using transmission electron microscopy and immunocytochemical labelling. The results revealed that the anti-AlinOBP13 antiserum strongly and specifically labelled short basiconic sensilla; this antiserum was restricted to the inner lumen and the cavities below the sensillum base of the sensilla. By contrast, multiporous sensilla trichodea, medium long sensilla basiconica, and aporous sensilla chaetica were not labelled. The present study is the first to report an OBP showing specific expression in the short basiconic sensilla of a member of the Hemipteran species. The results of a fluorescence displacement binding assay indicated that recombinant AlinOBP13 showed a more specific binding preference to terpenoids than to sex pheromones and other classes of chemicals. This binding ability was dramatically affected by pH; higher binding affinities were displayed at pH 10.0 than at pH 7.4 and 5.0. In addition, the results of dose-dependent electroantennogram recordings from the antennae showed that both female and male adult bugs responded to the terpenoids tested, suggesting an apparent physiological relevance of AlinOBP13 in A. lineolatus chemoreception. The results of this study suggest that AlinOBP13 functions as a specific carrier of terpenoids and provide insights into the mechanism of A

  11. Differential distribution of G-protein beta-subunits in brain: an immunocytochemical analysis. (United States)

    Brunk, I; Pahner, I; Maier, U; Jenner, B; Veh, R W; Nürnberg, B; Ahnert-Hilger, G


    Heterotrimeric G proteins play central roles in signal transduction of neurons and other cells. The variety of their alpha-, beta-, and gamma-subunits allows numerous combinations thereby confering specificity to receptor-G-protein-effector interactions. Using antisera against individual G-protein beta-subunits we here present a regional and subcellular distribution of Gbeta1, Gbeta2, and Gbeta5 in rat brain. Immunocytochemical specificity of the subtype-specific antisera is revealed in Sf9 cells infected with various G-protein beta-subunits. Since Gbeta-subunits together with a G-protein gamma-subunit affect signal cascades we include a distribution of the neuron-specific Ggamma2- and Ggamma3-subunits in selected brain areas. Gbeta1, Gbeta2, and Gbeta5 are preferentially distributed in the neuropil of hippocampus, cerebellum and spinal cord. Gbeta2 is highly concentrated in the mossy fibres of dentate gyrus neurons ending in the stratum lucidum of hippocampal CA3-area. High amounts of Gbeta2 also occur in interneurons innervating spinal cord alpha-motoneurons. Gbeta5 is differentially distributed in all brain areas studied. It is found in the pyramidal cells of hippocampal CA1-CA3 as well as in the granule cell layer of dentate gyrus and in some interneurons. In the spinal cord Gbeta5 in contrast to Gbeta2 concentrates around alpha-motoneurons. In cultivated mouse hippocampal and hypothalamic neurons Gbeta2 and Gbeta5 are found in different subcellular compartments. Whereas Gbeta5 is restricted to the perikarya, Gbeta2 is also found in processes and synaptic contacts where it partially colocalizes with the synaptic vesicle protein synaptobrevin. An antiserum recognizing Ggamma2 and Ggamma3 reveals that these subunits are less expressed in hippocampus and cerebellum. Presumably this antiserum specifically recognizes Ggamma2 and Ggamma3 in combinations with certain G alphas and/or Gbetas. The widespread but regionally and cellularly rather different distribution of

  12. Selection of gonadotrophin surge attenuating factor phage antibodies by bioassay

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    Mason Helen D


    Full Text Available Abstract Background We aimed to combine the generation of "artificial" antibodies with a rat pituitary bioassay as a new strategy to overcome 20 years of difficulties in the purification of gonadotrophin surge-attenuating factor (GnSAF. Methods A synthetic single-chain antibody (Tomlinson J phage display library was bio-panned with partially purified GnSAF produced by cultured human granulosa/luteal cells. The initial screening with a simple binding immunoassay resulted in 8 clones that were further screened using our in-vitro rat monolayer bioassay for GnSAF. Initially the antibodies were screened as pooled phage forms and subsequently as individual, soluble, single-chain antibody (scAbs forms. Then, in order to improve the stability of the scAbs for immunopurification purposes, and to widen the range of labelled secondary antibodies available, these were engineered into full-length human immunoglobulins. The immunoglobulin with the highest affinity for GnSAF and a previously described rat anti-GnSAF polyclonal antiserum was then used to immunopurify bioactive GnSAF protein. The two purified preparations were electrophoresed on 1-D gels and on 7 cm 2-D gels (pH 4–7. The candidate GnSAF protein bands and spots were then excised for peptide mass mapping. Results Three of the scAbs recognised GnSAF bioactivity and subsequently one clone of the purified scAb-derived immunoglobulin demonstrated high affinity for GnSAF bioactivity, also binding the molecule in such as way as to block its bioactivity. When used for repeated immunopurification cycles and then Western blot, this antibody enabled the isolation of a GnSAF-bioactive protein band at around 66 kDa. Similar results were achieved using the rat anti-GnSAF polyclonal antiserum. The main candidate molecules identified from the immunopurified material by excision of 2-D gel protein spots was human serum albumin precursor and variants. Conclusion This study demonstrates that the combination of

  13. Construction of hpaA gene from a clinical isolate of Helicobacter pyloriand identification of fusion protein

    Institute of Scientific and Technical Information of China (English)

    Ya-Fei Mao; Jie Yan; Li-Wei Li; Shu-Ping Li


    AIM: To clone hpaA gene from a clinical strain of Helicobacter pylori and to construct the expression vector of the gene and to identify immunity of the fusion protein.METHODS: The hpaA gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The recombinant expression vector inserted with hpaA gene was constructed. The expression of HpaA fusion protein in E. coli BL21DE3 induced by IPTG at different dosages was examined by SDS-PAGE.Western blot with commercial antibody against whole cell of H. pylorias well as immunodiffusion assay with selfprepared rabbit antiserum against HpaA fusion protein were applied to determine immunity of the fusion protein. ELISA was used to detect the antibody against HpaA in sera of 125 patients infected with H. pylori and to examine HpaA expression of 109 clinical isolates of H. pylori.RESULTS: In comparison with the reported corresponding sequences, the homologies of nucleotide and putative amino acid sequences of the cloned hpaA gene were from 94.25-97.32 % and 95.38-98.46 %, respectively. The output of HpaA fusion protein in its expression system of pET32ahpaA-BL21DE3 was approximately 40 % of the total bacterial proteins. HpaA fusion protein was able to combine with the commercial antibody against whole cell of H. pyloriand to induce rabbit producing specific antiserum with 1:4immunodiffusion titer after the animal was immunized with the fusion protein. 81.6 % of the serum samples from 125patients infected with H.pylori(102/125) were positive for HpaA antibody and all of the tested isolates of H.pylori(109/109) were detectable for HpaA.CONCLUSION: A prokaryotic expression system with high efficiency of H.pylorihpaA gene was successfully established.The HpaA expressing fusion protein showed satisfactory immunoreactivity and antigenicity. High frequencies of HpaA expression in different H. pyloriclinicalstrains

  14. Expression of Galpha14 in sweet-transducing taste cells of the posterior tongue

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    Kim Soochong


    Full Text Available Abstract Background "Type II"/Receptor cells express G protein-coupled receptors (GPCRs for sweet, umami (T1Rs and mGluRs or bitter (T2Rs, as well as the proteins for downstream signalling cascades. Transduction downstream of T1Rs and T2Rs relies on G-protein and PLCβ2-mediated release of stored Ca2+. Whereas Gαgus (gustducin couples to the T2R (bitter receptors, which Gα-subunit couples to the sweet (T1R2 + T1R3 receptor is presently not known. We utilized RT-PCR, immunocytochemistry and single-cell gene expression profiling to examine the expression of the Gαq family (q, 11, 14 in mouse taste buds. Results By RT-PCR, Gα14 is expressed strongly and in a taste selective manner in posterior (vallate and foliate, but not anterior (fungiform and palate taste fields. Gαq and Gα11, although detectable, are not expressed in a taste-selective fashion. Further, expression of Gα14 mRNA is limited to Type II/Receptor cells in taste buds. Immunocytochemistry on vallate papillae using a broad Gαq family antiserum reveals specific staining only in Type II taste cells (i.e. those expressing TrpM5 and PLCβ2. This staining persists in Gαq knockout mice and immunostaining with a Gα11-specific antiserum shows no immunoreactivity in taste buds. Taken together, these data show that Gα14 is the dominant Gαq family member detected. Immunoreactivity for Gα14 strongly correlates with expression of T1R3, the taste receptor subunit present in taste cells responsive to either umami or sweet. Single cell gene expression profiling confirms a tight correlation between the expression of Gα14 and both T1R2 and T1R3, the receptor combination that forms sweet taste receptors. Conclusion Gα14 is co-expressed with the sweet taste receptor in posterior tongue, although not in anterior tongue. Thus, sweet taste transduction may rely on different downstream transduction elements in posterior and anterior taste fields.

  15. 江阴市2010年狂犬病门诊暴露者流行病学分析%Epidemiological analysis of population exposed to rabies in Jiangyin city in 2010

    Institute of Scientific and Technical Information of China (English)

    姚建香; 马焰


    Objective To summarize epidemiological characteristics of population exposed to rabies and treatment sta-tion in rabies - clinics in Jiangyin city. Method Collecting and analyzing the monitoring monthly reports of population ex-posed to rabies of rabies - climes. Results Males were more than females in population exposed to rabies. Most of the expo-sure occurred in summer and autumn, lower and upper limbs were the main exposure sites, most were single and shallow wounds. Most of population could treatment after espousing to rabies and all taking vaccines, but not many population injec-ted antiserum. Conclusions In order to reduce the rabies, we need to attach weight to health education and improve the sense of protection, treatment wounds, inoculate vaccines and inject antiserum timely after exposing. At the same time strengthen dogs management and decrease exposure occurrence.%目的 掌握江阴市狂犬病门诊监测点暴露人群流行病学特征及暴露后处理情况.方法 对江阴市狂犬病门诊暴露人群监测月报表进行统计分析.结果 暴露人群分布上男性略多于女性,时间分布上夏秋季节较为集中,暴露部位主要为下肢和上肢,以浅表单处伤居多.暴露后绝大多数人能及时处理伤口,到门诊就医者能坚持全程疫苗接种,注射人抗狂犬病免疫球蛋白的为数不多.结论 为减少狂犬病的发生,要重视全民健康教育,增强其防范意识,做到暴露后及时处理伤口、接种疫苗和抗血清注射,同时加强犬只管理,减少暴露发生.

  16. Botulism: a laboratory investigation on biological and food samples from cases and outbreaks in Brazil (1982-2001 Botulismo: investigação laboratorial de amostras biológicas e de alimentos de casos e surtos no Brasil (1982-2001

    Directory of Open Access Journals (Sweden)

    Dilma Scala GELLI


    Full Text Available Laboratory investigation of botulism from 1982 to 2001 confirmed the occurrence of eight positive outbreaks/cases of botulism in Brazil. From those, type A botulism was observed in seven of them. Biological material of one case (serum and feces was positive in the first step of the bioassay, but the amount of sample was not sufficient for typification. One of the outbreaks that occurred in 2001 was negative for botulinum toxin in samples of serum, gastric washing and feces, collected eight days before the onset of the symptoms in the affected person who was clinically diagnosed as presenting the disease. Other two cases presenting compatible clinical diagnoses presented negative results. However, in those cases, the collection of samples was (1 after antiserum administration or (2 later than eight days of the onset of symptoms. Investigation was performed by mouse bioassay, as described in the Compendium of Methods for the Microbiological Examination of Foods (compiled by American Public Health Association - APHA11, using specific antiserum from Centers for Disease Control (CDC, USA.A investigação laboratorial de botulismo durante 1982-2001, confirma a ocorrência de surtos/casos de botulismo no Brasil. Dentre estes, a toxina botulínica tipo A foi encontrada em 7. O material biológico de 1 caso (soro e fezes foi positivo para a primeira etapa do bioensaio, mas a quantidade do material não foi suficiente para a sua tipificação. Um surto, ocorrido em 2001, foi negativo para a presença de toxina botulínica em amostras de soro, lavado gástrico e fezes coletadas antes de 8 dias da instalação dos sintomas na pessoa afetada com diagnóstico clínico compatível com a doença. Outros 2 casos com diagnóstico clínico compatível foram negativos, porém nestes a coleta de amostras foi (1 depois da administração de anti-soro ao paciente e (2 em período superior a 8 dias do início dos sintomas. A investigação laboratorial foi realizada

  17. Serological differences between the multiple amine oxidases of yeasts and comparison of the specificities of the purified enzymes from Candida utilis and Pichia pastoris. (United States)

    Green, J; Haywood, G W; Large, P J


    1. Antiserum to purified methylamine oxidase of Candida boidinii formed precipitin lines (with spurs) in double-diffusion tests with crude extracts of methylamine-grown cells of the following yeast species: Candida nagoyaensis, Candida nemodendra, Hansenula minuta, Hansenula polymorpha and Pichia pinus. No cross-reaction was observed with extracts of Candida lipolytica, Candida steatolytica, Candida tropicalis, Candida utilis, Pichia pastoris, Sporobolomyces albo-rubescens, Sporopachydermia cereana or Trigonopsis variabilis. Quantitative enzyme assays enabled the relative titre of antiserum against the various methylamine oxidases to be determined. 2. The amine oxidases from two non-cross-reacting species, C. utilis and P. pastoris, were purified to near homogeneity. 3. The methylamine oxidases, despite their serological non-similarity, showed very similar catalytic properties to methylamine oxidase from C. boidinii. Their heat-stability, pH optima, molecular weights, substrate specificities and sensitivity to inhibitors are reported. 4. The benzylamine oxidases of C. utilis and P. pastoris both oxidized putrescine, and the latter enzyme failed to show any cross-reaction with antibody to C. boidinii methylamine oxidase. Benzylamine oxidase from C. boidinii itself also did not cross-react with antibody to methylamine oxidase. The heat-stability, molecular weights, substrate specificities and sensitivity to inhibitors of the benzylamine/putrescine oxidases are reported. 5. The benzylamine/putrescine oxidase of C. utilis differed only slightly from that of C. boidinii. 6. Benzylamine/putrescine oxidase from P. pastoris differed from the Candida enzymes in heat-stability, subunit molecular weight and substrate specificity. In particular it catalysed the oxidation of the primary amino groups of spermine, spermidine, lysine, ornithine and 1,2-diaminoethane, which are not substrates for either of the Candida benzylamine oxidases that have been purified. 7. Spermine and

  18. C-peptide of preproinsulin-like peptide 7: localization in the rat brain and activity in vitro. (United States)

    Brailoiu, E; Dun, S L; Gao, X; Brailoiu, G C; Li, J-G; Luo, J J; Yang, J; Chang, J K; Liu-Chen, L-Y; Dun, N J


    With the use of a rabbit polyclonal antiserum against a conserved region (54-118) of C-peptide of human preproinsulin-like peptide 7, referred to herein as C-INSL7, neurons expressing C-INSL7-immunoreactivity (irC-INSL7) were detected in the pontine nucleus incertus, the lateral or ventrolateral periaqueductal gray, dorsal raphe nuclei and dorsal substantia nigra. Immunoreactive fibers were present in numerous forebrain areas, with a high density in the septum, hypothalamus and thalamus. Pre-absorption of C-INSL7 antiserum with the peptide C-INSL7 (1 microg/ml), but not the insulin-like peptide 7 (INSL7; 1 microg/ml), also known as relaxin 3, abolished the immunoreactivity. Optical imaging with a voltage-sensitive dye bis-[1,3-dibutylbarbituric acid] trimethineoxonol (DiSBAC4(3)) showed that C-INSL7 (100 nM) depolarized or hyperpolarized a small population of cultured rat hypothalamic neurons studied. Ratiometric imaging studies with calcium-sensitive dye fura-2 showed that C-INSL7 (10-1000 nM) produced a dose-dependent increase in cytosolic calcium concentrations [Ca2+]i in cultured hypothalamic neurons with two distinct patterns: (1) a sustained elevation lasting for minutes; and (2) a fast, transitory rise followed by oscillations. In a Ca2+-free Hanks' solution, C-INSL7 again elicited two types of calcium transients: (1) a fast, transitory increase not followed by a plateau phase, and (2) a transitory rise followed by oscillations. INSL7 (100 nM) elicited a depolarization or hyperpolarization in a small population of hypothalamic neurons, and an increase of [Ca2+]i with two patterns that were dissimilar from that of C-INSL7. [125I]C-INSL7 bindings to rat brain membranes were inhibited by C-INSL7 in a dose-dependent manner; the Kd and Bmax. values were 17.7 +/- 8.2 nM and 45.4 +/- 20.5 fmol/mg protein. INSL7 did not inhibit [125I]C-INSL7 binding to rat brain membranes, indicating that C-INSL7 and INSL7 bind to distinct binding sites. Collectively, our result

  19. Cellular expression, trafficking, and function of two isoforms of human ULBP5/RAET1G.

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    Robert A Eagle

    Full Text Available BACKGROUND: The activating immunoreceptor NKG2D is expressed on Natural Killer (NK cells and subsets of T cells. NKG2D contributes to anti-tumour and anti-viral immune responses in vitro and in vivo. The ligands for NKG2D in humans are diverse proteins of the MIC and ULBP/RAET families that are upregulated on the surface of virally infected cells and tumours. Two splicing variants of ULBP5/RAET1G have been cloned previously, but not extensively characterised. METHODOLOGY/PRINCIPAL FINDINGS: We pursue a number of approaches to characterise the expression, trafficking, and function of the two isoforms of ULBP5/RAET1G. We show that both transcripts are frequently expressed in cell lines derived from epithelial cancers, and in primary breast cancers. The full-length transcript, RAET1G1, is predicted to encode a molecule with transmembrane and cytoplasmic domains that are unique amongst NKG2D ligands. Using specific anti-RAET1G1 antiserum to stain tissue microarrays we show that RAET1G1 expression is highly restricted in normal tissues. RAET1G1 was expressed at a low level in normal gastrointestinal epithelial cells in a similar pattern to MICA. Both RAET1G1 and MICA showed increased expression in the gut of patients with celiac disease. In contrast to healthy tissues the RAET1G1 antiserum stained a wide variety or different primary tumour sections. Both endogenously expressed and transfected RAET1G1 was mainly found inside the cell, with a minority of the protein reaching the cell surface. Conversely the truncated splicing variant of RAET1G2 was shown to encode a soluble molecule that could be secreted from cells. Secreted RAET1G2 was shown to downregulate NKG2D receptor expression on NK cells and hence may represent a novel tumour immune evasion strategy. CONCLUSIONS/SIGNIFICANCE: We demonstrate that the expression patterns of ULBP5RAET1G are very similar to the well-characterised NKG2D ligand, MICA. However the two isoforms of ULBP5/RAET1G have


    Directory of Open Access Journals (Sweden)

    T.R. Neyestani


    Full Text Available Antigenicity of proteins found in cow's milk is age dependent. This is primarily due to infants possessing a more permeable intestinal wall than that in adults. Thus infants may acquire cow's milk allergy during their first year of life. While milk antigen specific IgE may cause allergy in susceptible subjects, there is some evidence indicating that milk antigen specific IgG may play some role in chronic disease development. The puropose of this study was to determine the antigenicity of cow's milk proteins in two animal models and to recommend the more sensitivie one, as an evaluation tool, to assess the antigenicity of a poteintial hypoallergenic formula. A crude extract of cow's milk was injected either to young male rabbits or BALB/C mice in four doses. Pure standard proteins of cow's milk were also injected to separate groups of animals to use their anti sera in later stages. The polyclonal pooled serum was then used to evaluate the antigenicity of the extract by indirect enzyme-linked immunossorbeni assay (LEISA. and Western blotting. Both the rabbit and BALB/C murine mode! demonstrated strong ELISA titres against casein and BSA proteins. However, the rabbit model also had a high antibody response against beta-lactoglobulin (/Mg. The lowest antibody response was found against alpha-kictalbumin («-la in both animal models and no response against immunoglobulins (Igs in either model. In Western blotting, rabbit antiserum showed four bands («-la, /Mg, caseins and BSA compared to two bands (caseins and BSA for mouse antiserum. Considering the allergenicity of these proteins in genetically prone subjects, it may be wise to exclude food sources of caseins as well as major whey proteins (BSA, from the diet of infants with a family history of atopy during the first year of life. The rabbit hyperimmunization model was more sensitive than the murine mode! in detecting antibodies against milk proteins. Thus, the rabbii model should be employed when

  1. Genotypic Analysis, Construction of the Expression System and Immunological Identification of the Recombinant Proteins of the LipL32 Gene in the Dominant Serogroups of Leptospira interrogans in China

    Institute of Scientific and Technical Information of China (English)

    范兴丽; 严杰; 毛亚飞; 李立伟; 李淑萍


    To investigate the existence of the major outer membrane pmtein (MOMP) gene Lip132 in 15 dominant Chineses trains of 15 semgroups of Leptospira iraerrogans and 2 intemational strains of 2 semgroups of Leptospira biflexa, and to clone and construct the expression system as well as to identify the recombinant pmteins, genomic DNAs from strains of leptospira were prepared by mutine phenol-chloroform method, and the fragments of the LipL32 gene with the whole length from the strains were amplified with high fidelity PCR. The target amplification products were, sequenced after T-A cloning, and the expression system for the genes were thereby constructed, Expression of the recombinant proteins was identified by using SDSPAGE after induction with IPTG at different dosages. Western blot assays with rabbit antiserum against the whole cell of TR/Patoc Ⅰ of Leptospira and immunized serum with rMOMPs were used to determine the immunoreactivity and immunogenicity of the recombinant proteins. Microscopic agglutination test was used to determine the cross- agglutination titres in rabbit sera immunized with rMOMPs, and the cell adherence model of Leptospira was used to examine the blocking effects of rabbit antisera against these rMOMPs. It was found that the LipL32 gene could be found in all the 17 strains of Leptospira mentioned above with two different genotypes, i.e.LipL32/1 and LipL32/2. Amounts of expressions of rMOMP1 and rMOMP2 after IPTG accounted for 40% and 10% of the total bacterial pmteins respectively. Both rMOMP1 and rMOMP2 could combine with the rab-bit antiserum against leptospiral TR/Patoc Ⅰ, and could induce the production of agglutination antibodies to these 17 strains of Leptospira with 1:2 to 1:64 MAT titres. The rabbit anti-rMOMP1 and anti-MOMP2 antibodies at 1:2 to 1 : 16 dilutions could efficiently block adherence of Leptospira. It concludes that all the Leptospira tested in the present study possess LipL32/1 or LipL32/2 genes, and the constructed


    Institute of Scientific and Technical Information of China (English)

    王欣欣; 绳秀珍; 战文斌


    采用菌体吸附法对海豚链球菌(Streptococcus iniae)、迟缓爱德华氏菌(Edwardsiella tarda)、鳗弧菌(Vibrio anguillarum)、杀鲑气单胞菌(Aeromonas salmonicida)、荧光假单胞菌(Psedomonas fluorescens)和海洋分支杆菌(Mycobacterium marinum)6种养殖鱼类病原菌的兔抗血清进行吸附,以减小抗血清与其它菌株的交叉反应;吸附纯化后的抗体作为捕获抗体构建了病原菌检测抗体芯片.实验结果显示,与未经过吸附的纯化抗血清构建的抗体芯片相比,血清吸附实验能有效减少交叉反应,降低抗体的非特异性吸附,提高细菌检测的特异性.同时,以迟缓爱德华氏菌为例,采用吸附纯化后抗体制备的抗体芯片检测人工感染迟缓爱德华氏菌和自然发病的牙鲆,均观察到迟缓爱德华民菌的特异性显色反应,得到了较理想的检测结果.本文结果说明优化后的病原菌检测抗体芯片可用于水产养殖鱼类常见的上述6种细菌性疾病的有效检测.%Six kinds of bacterias from marine cultured fish, including Streptococcus iniae , Edwardsiella tarda, Vibrio anguillarum , Aeromonas salmonicida , Psedomonas fluorescens and Mycobacterium ma-rinum , were chosen, and their rabbit antiserum were adsorbed to reduce cross reaction by serum adsorption experiment. And then antibody microarrays were developed using the purified rabbit antiserum as capture antibodies. The results showed that the cross reaction of the antibody microarray was reduced effectively, and the specificity and sensibility of bacteria detection were improved greatly after serum adsorption. Meanwhile, taking E. tarda for example, the antibody microarray was applied to detect E. tarda in Paralichthys olivaceus infected artificially and naturally with E. tarda, and specific positive staining were observed, suggesting the developed antibody microarrays could effectively detect the six kinds of bacteria above-mentioned in fish.

  3. 耐甲氧西林金黄色葡萄球菌微量凝集试验方法的建立及初步应用%Establishment and preliminary application of the microagglutination assay for detecting methicillin-resistant Staphylococcus aureus

    Institute of Scientific and Technical Information of China (English)

    周顺; 刘长浩; 张灿; 邹玲; 刘文华; 任慧英


    The objective of the study is to establish a method for rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) in animals.The microagglutination assay was developed with the antiserum prepared from rabbits immunized by the purified recombinant MRSA PBP2a protein expressed in E.coli.Under the optimized conditions of rabbit antiserum dilution,bacteria concentration and reaction temperature,the results showed that the assay was specific and reproductive,with a detection limit of 4×109 cfu/mL of MRSA,but no cross-reactions with other related bacteria.In addition,Application of this method to 75 isolates of S.aureus isolated from chickens,pigs,rabbits and mice indicated that 57.3% of the isolates were resistant to methicillin,which was 92% accordant with the disk diffusion method.Therefore,the rapid detection method is suitable for clinical or on-site application.%为建立一种快速检测动物源耐甲氧西林金黄色葡萄球菌(MRSA)的方法,本研究以MRSA耐药性决定蛋白PBP2a的抗血清为检测抗体,通过对抗原浓度及反应温度等进行优化,确定最佳的反应条件后,建立了快速检测MRSA的微量凝集试验方法,并进行了敏感性、特异性、重复性试验以及药敏纸片法比较试验.其中,微量凝集试验最低检出MRSA的浓度为40亿/mL,该方法对MRSA的凝集反应具有良好的特异性,试验结果具有较好的重复性.采用该检测方法对分离自鸡、猪、家兔及小鼠的75株金黄色葡萄球菌进行检测,检出MRSA占57.3%,经过与药敏纸片法检测进行比较,符合率为92%.该方法快速、准确、简单易行,适于临床或基层应用.

  4. 牛初乳中sIgA双抗夹心ELISA检测方法的建立%Establishment of a sandwich ELISA for quantitive measurement of sIgA

    Institute of Scientific and Technical Information of China (English)

    朱洪艳; 赵红宇; 周盛华; 高学军


    为定量检测牛初乳中sIgA,建立双抗夹ELISA方法.以牛初乳中sIgA为抗原,免疫新西兰白兔,制得抗血清,经纯化后作为包被抗体;利用简易过碘酸钠法对兔抗牛sIgA标记辣根过氧化物酶(HTP),制得酶标抗体;通过方阵滴定法确定ELISA最佳工作条件;进行特异性、重复性、稳定性实验并且检测牛初乳粉样本中的sIgA含量.结果:成功建立了一种定量检测牛初乳中sIgA的双抗夹心ELISA方法.该方法线性范围:15.625~1 000μg/L;最低检测限(LOD)为15.625μg/L;精密度为:批内CV=4.8%;批间CV=6.5%.%To establish an effective ELISA mathod for the determination of bovine colostrum sIgA.Rabbit was immunized by bovine colostrum sIgA to obtain the antiserum.Useing the purified antiserum as coating antibody.All the anti- sIgA were labeled with horseradish peroxidase by sodium oxidation method, and take the HRP labeled anti- sIgA for labeled antibody.The optimal concentrations of ELISA were defined by titration.For speciticity reproducibility and sensitivity experiment and measured the sIgA levels of bovine colostrum with this assay.Results a sandwich ELISA assay for detecting sIgA in bovine colostrum was established successfully.The assay result was linear over a concentration range of 15.625~1 000 μg/L with a limit of detection(LOD) of 15.625 μg/L.The coefficients of variation was 4.8% within assay and 6.5% between assay.

  5. Comparative analysis of Haemophilus influenzae hifA (pilin) genes. (United States)

    Clemans, D L; Marrs, C F; Patel, M; Duncan, M; Gilsdorf, J R


    Adherence of Haemophilus influenzae to epithelial cells plays a central role in colonization and is the first step in infection with this organism. Pili, which are large polymorphic surface proteins, have been shown to mediate the binding of H. influenzae to cells of the human respiratory tract. Earlier experiments have demonstrated that the major epitopes of H. influenzae pili are highly conformational and immunologically heterogenous; their subunit pilins are, however, immunologically homogenous. To define the extent of structural variation in pilins, which polymerize to form pili, the pilin genes (hifA) of 26 type a to f and 16 nontypeable strains of H. influenzae were amplified by PCR and subjected to restriction fragment length polymorphism (RFLP) analysis with AluI and RsaI. Six different RFLP patterns were identified. Four further RFLP patterns were identified from published hifA sequences from five nontypeable H. influenzae strains. Two patterns contained only nontypeable isolates; one of these contained H. influenzae biotype aegyptius strains F3031 and F3037. Another pattern contained predominantly H. influenzae type f strains. All other patterns were displayed by a variety of capsular and noncapsular types. Sequence analysis of selected hifA genes confirmed the 10 RFLP patterns and showed strong identity among representatives displaying the same RFLP patterns. In addition, the immunologic reactivity of pili with antipilus antisera correlated with the groupings of strains based on hifA RFLP patterns. Those strains that show greater reactivity with antiserum directed against H. influenzae type b strain M43 pili tend to fall into one RFLP pattern (pattern 3); while those strains that show equal or greater reactivity with antiserum directed against H. influenzae type b strain Eagan pili tend to fall in a different RFLP pattern (pattern 1). Sequence analysis of representative HifA pilins from typeable and nontypeable H. influenzae identified several highly

  6. Mitis group streptococci express variable pilus islet 2 pili.

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    Dorothea Zähner

    Full Text Available BACKGROUND: Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguinis are members of the Mitis group of streptococci and agents of oral biofilm, dental plaque and infective endocarditis, disease processes that involve bacteria-bacteria and bacteria-host interactions. Their close relative, the human pathogen S. pneumoniae uses pilus-islet 2 (PI-2-encoded pili to facilitate adhesion to eukaryotic cells. METHODOLOGY/PRINCIPAL FINDINGS: PI-2 pilus-encoding genetic islets were identified in S. oralis, S. mitis, and S. sanguinis, but were absent from other isolates of these species. The PI-2 islets resembled the genetic organization of the PI-2 islet of S. pneumoniae, but differed in the genes encoding the structural pilus proteins PitA and PitB. Two and three variants of pitA (a pseudogene in S. pneumoniae and pitB, respectively, were identified that showed ≈20% difference in nucleotide as well as corresponding protein sequence. Species-independent combinations of pitA and pitB variants indicated prior intra- and interspecies horizontal gene transfer events. Polyclonal antisera developed against PitA and PitB of S. oralis type strain ATCC35037 revealed that PI-2 pili in oral streptococci were composed of PitA and PitB. Electronmicrographs showed pilus structures radiating >700 nm from the bacterial surface in the wild type strain, but not in an isogenic PI-2 deletion mutant. Anti-PitB-antiserum only reacted with pili containing the same PitB variant, whereas anti-PitA antiserum was cross-reactive with the other PitA variant. Electronic multilocus sequence analysis revealed that all PI-2-encoding oral streptococci were closely-related and cluster with non-PI-2-encoding S. oralis strains. CONCLUSIONS/SIGNIFICANCE: This is the first identification of PI-2 pili in Mitis group oral streptococci. The findings provide a striking example of intra- and interspecies horizontal gene transfer. The PI-2 pilus diversity provides a possible key to

  7. [Immunocytochemical studies on the phase of differentiation of hatching gland cells in brine shrimp, Artemia salina]. (United States)

    Li, Ling; Fan, Ting Jun; Wang, Xiao Feng; Cong, Ri Shan; Yu, Qiu Tao; Zhong, Qi Wang


    Hatching enzyme (HE), synthesized in hatching gland cells (HGCs), plays vital roles in animal hatching. Immunocytochemical techniques employing anti-GST-UVS.2 antiserum, prepared from Xenopus HE and with specificity to brine shrimp HE, were first used to investigate the differentiation and variability of hatching gland cells (HGCs) in the hatching process of embryos of brine shrimp, Artemia salina, in this study. HGCs with immunoreactivity to anti-GST-UVS.2 antiserum were identified, for the first time, in brine shrimp embryos during hatching process. Immunocytochemical staining results showed that, (1) HE-positive immunoreactivity is really specific to Artemia HE, and its appearance and disappearance are closely correlated with the hatching process of Artemia salina. (2) Artemia HGCs, first appeared in embryos 5 hours before hatching and disappeared 4 hours after hatching, were also a transient type of cells, with an existence period of 9 hours. (3) The head portion of Artemia embryo is probably the initial position of HE secretion, and likely to be the main position of HE secretion as well. The detailed process and mechanism need to be studied. (4) The appearance of HGCs is in a synchronous mode from places all over the embryos, and their disappearance is also in a synchronous mode. (5) The number of HGCs increased gradually along with embryo development process and reached a maximum number at hatching. Contrarily, the number of HGCs decreased gradually after hatching, and HGCs disappeared 5 hours after hatching. However, the intensity of HE-positive reaction was almost at the same level at the period of HGCs'presence. (6) Artemia HGCs were distributed throughout the body of embryos at all time during their presence. Therefore, it can concluded that Artemia HGCs, as a transient type of cells, first appeared in embryos 4 hours before hatching and disappeared in embryos 5 hours after hatching, and with distinguished patterns of appearance, disappearance and

  8. Use of silver nanoparticles increased inhibition of cell-associated HIV-1 infection by neutralizing antibodies developed against HIV-1 envelope proteins

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    Garza Treviño Elsa N


    Full Text Available Abstract Background HIV/AIDS pandemic is a worldwide public health issue. There is a need for new approaches to develop new antiviral compounds or other therapeutic strategies to limit viral transmission. The envelope glycoproteins gp120 and gp41 of HIV are the main targets for both silver nanoparticles (AgNPs and neutralizing antibodies. There is an urgency to optimize the efficiency of the neutralizing antibodies (NABs. In this study, we demonstrated that there is an additive effect between the four NABs and AgNPs when combined against cell-associated HIV-1 infection in vitro Results Four NABs (Monoclonal antibody to HIV-1 gp41 126-7, HIV-1 gp120 Antiserum PB1 Sub 2, HIV-1 gp120 Antiserum PB1, HIV-1 gp120 Monoclonal Antibody F425 B4e8 with or without AgNPs of 30-50 nm in size were tested against cell free and cell-associated HIVIIIB virus. All NABs inhibited HIV-1 cell free infection at a dose response manner, but with AgNPs an antiviral additive effect was not achieved Although there was no inhibition of infection with cell-associated virus by the NABs itself, AgNPs alone were able to inhibit cell associated virus infection and more importantly, when mixed together with NABs they inhibited the HIV-1 cell associated infection in an additive manner. Discussion The most attractive strategies to deal with the HIV problem are the development of a prophylactic vaccine and the development of effective topical vaginal microbicide. For two decades a potent vaccine that inhibits transmission of infection of HIV has been searched. There are vaccines that elicit NABs but none of them has the efficacy to stop transmission of HIV-1 infection. We propose that with the addition of AgNPs, NABs will have an additive effect and become more potent to inhibit cell-associated HIV-1 transmission/infection. Conclusions The addition of AgNPs to NABs has significantly increased the neutralizing potency of NABs in prevention of cell-associated HIV-1 transmission

  9. Interaction forces between red cells agglutinated by antibody. IV. Time and force dependence of break-up. (United States)

    Tees, D F; Coenen, O; Goldsmith, H L


    We report on an extension of a previously described method to measure the hydrodynamic force to separate doublets of fixed, sphered and swollen red cells cross-linked by antibody (S. P. Tha, J. Shuster, and H. L. Goldsmith. 1986. Biophys. J. 50:1117-1126). With a traveling microtube apparatus, doublets are tracked and videotaped in a slowly accelerating Poiseuille flow in 150-microns-diameter tubes, and the hydrodynamic normal force at break-up, Fn, is computed from the measured doublet velocity and radial position. Previous results showed a large range of Fn, the mean of which increased with [antiserum], and an absence of clustering at discrete values of Fn. Since it was assumed that the cells separate the instant a critical force to break all crossbridges was reached, lack of clustering could have been due to the use of a polyclonal antiserum. We therefore studied the effect of monoclonal IgM or IgA antibody on the distribution of Fn. The results showed that the data are as scattered as ever, with Fn varying from 2 to 200 pN, and exhibit no evidence of clustering. However, the scatter in Fn could be due to the stochastic nature of intercellular bonds (E. Evans, D. Berk, and A. Leung. 1991a. Biophys. J. 59:838-848). We therefore studied the force dependence of the time to break-up under constant shear stress (Fn from 30 to 200 pN), both in Poiseuille and Couette flow, the latter by using a counter-rotating cone and plate rheoscope. When 280 doublets were rapidly accelerated in the traveling microtube and then allowed to coast in steady flow for up to 180 s, 91% survived into the constant force region; 16% of these broke up after time intervals, tP, of 2-30s. Of 340 doublets immediately exposed to constant shear in the rheoscope, 37% broke after time intervals, tc, from 5. In the rheoscope, the time intervals and number of rotations to break-up, tc, were quite well reproduced assuming (Nb) = 4. The similarity of (Fn) for monoclonal IgM and IgA for doublet break

  10. The development of a radioimmunoassay for reverse triiodothyronine sulfate in human serum and amniotic fluid

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Sing-Yung (Veterans Administration Medical Center, Long Beach, CA (United States)); Huang, Wen-Sheng; Chen, Wei-Lian (Tri-Service General Hospital, Taipei (Taiwan, Province of China)); Polk, D.; Reviczky, A.; Williams, J. III; Chopra, I.J.; Fisher, D.A. (Univ. of California, Los Angeles (United States))


    Sulfated iodothyronines including T[sub 4]-sulfate (T[sub 4]S) and T[sub 3]-sulfate (T[sub 3]S) have been identified in human serum and amniotic fluid. Little is know, however, about the existence of sulfate conjugation of reverse T[sub 3] (rT[sub 3]S) in man. In this report, the authors employed a novel, sensitive, and specific rT[sub 3]S RIA to address this question. The rabbit antiserum to rT[sub 3]S was highly specific; T[sub 4], T[sub 3], rT[sub 3], and 3,3'-T[sub 2] showed less than 0.002% cross-reaction with the antiserum. Only T[sub 4]S and T[sub 3]S cross-reacted significantly (0.3% and 0.01%, respectively); other analogs cross-reacted less than 0.0001%. The detection threshold of the RIA was 14 pmol/L (1.0 ng/dL). The mean serum rT[sub 3]S concentration (pmol/L) was 40 in euthyroid subjects. Values were similar in hypothyroid patients (38) and pregnant women (52) but significantly (P < 0.01) elevated to 176 in hyperthyroid patient, 74 in patients with nonthyroid illnesses, and 684 in cord sera of newborns. Serum rT[sub 3]S increased significantly in hyperthyroid patients 1 day after administration of 1 g sodium ipodate orally. Reverse T[sub 3]S was detected consistently in amniotic fluid at 14 to 22 weeks of gestation and showed a marked rise 1-3 weeks after intraamniotic administration of 500-1000 [mu]g T[sub 4]. The various data suggest that : (1) rT[sub 3]S is a normal component of human serum and amniotic fluid; (2) it is derived from metabolism of T[sub 4] or rT[sub 3]; (3) circulating rT[sub 3]S increases in hyperthyroidism and in circumstances where type I 5'-monodeiodinating activity is low, e.g. nonthyroid illnesses, fetal life, and after administration of ipodate. 20 refs., 4 figs.

  11. A potentiometric biosensor for rapid on-site disease diagnostics. (United States)

    Tarasov, Alexey; Gray, Darren W; Tsai, Meng-Yen; Shields, Niall; Montrose, Armelle; Creedon, Niamh; Lovera, Pierre; O'Riordan, Alan; Mooney, Mark H; Vogel, Eric M


    Quantitative point-of-care (POC) devices are the next generation for serological disease diagnosis. Whilst pathogen serology is typically performed by centralized laboratories using Enzyme-Linked ImmunoSorbent Assay (ELISA), faster on-site diagnosis would infer improved disease management and treatment decisions. Using the model pathogen Bovine Herpes Virus-1 (BHV-1) this study employs an extended-gate field-effect transistor (FET) for direct potentiometric serological diagnosis. BHV-1 is a major viral pathogen of Bovine Respiratory Disease (BRD), the leading cause of economic loss ($2 billion annually in the US only) to the cattle and dairy industry. To demonstrate the sensor capabilities as a diagnostic tool, BHV-1 viral protein gE was expressed and immobilized on the sensor surface to serve as a capture antigen for a BHV-1-specific antibody (anti-gE), produced in cattle in response to viral infection. The gE-coated immunosensor was shown to be highly sensitive and selective to anti-gE present in commercially available anti-BHV-1 antiserum and in real serum samples from cattle with results being in excellent agreement with Surface Plasmon Resonance (SPR) and ELISA. The FET sensor is significantly faster than ELISA (disease intervention. This sensor technology is versatile, amenable to multiplexing, easily integrated to POC devices, and has the potential to impact a wide range of human and animal diseases.

  12. Intraspecific variation in protein pattern of red scorpion (Mesobuthus tamulus, coconsis, pocock venoms from Western and Southern India

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    R. V. Badhe


    Full Text Available Red scorpions Mesobuthus tamulus (Coconsis, Pocock were obtained from different regions of West and South India (Ratnagiri, Chiplun and Ahmednagar from Maharashtra and Chennai from Tamil Nadu, respectively. Their venoms composition was analyzed using gel electrophoresis (SDS-PAGE. All venom samples shared six bands of 170, 80, 60, 57, 43, and 38 kDa molecular weights. Bands of 115 kDa and 51.5 kDa were characteristic of venoms obtained from red scorpions of Chiplun region, and the 26kDa band was absent in scorpion venom from Tamil Nadu. The separated protein band patterns suggest that the venoms from Ratnagiri, Ahmednagar and Tamil Nadu had high similarities in their biochemical composition but differed from that of Chiplun region. These data were also supported by the Jaccard (J index. The J value was 0.33 for venom obtained from Ratnagiri-Ahmednagar, 0.31 for venom from Ratnagiri-Tamil Nadu, and 0.3 for venom from Ratnagiri-Chiplun region. This suggests the existence of genetic variation among the different strains of red scorpion in western and southern India. The antiserum produced by Haffkine Biopharmaceuticals Corporation Ltd. completely neutralized proteins of venoms from all the regions studied.

  13. Development of a blocking latex agglutination test for the detection of antibodies to chicken anemia virus. (United States)

    Trinh, Dai Quang; Ogawa, Haruko; Bui, Vuong Nghia; Nguyen, Tham Thi Hong; Gronsang, Dulyatad; Baatartsogt, Tugsbaatar; Kizito, Mugimba Kahoza; AboElkhair, Mohammed; Yamaguchi, Shigeo; Nguyen, Viet Khong; Imai, Kunitoshi


    A blocking latex agglutination test (b-LAT) developed in this study was evaluated for the detection of antibodies against chicken anemia virus (CAV) in chickens. Polystyrene latex beads were coupled with a neutralizing monoclonal antibody (mAb) to CAV (mAb-beads). When mAb-beads were mixed with antigens prepared from the lysate of MDCC-MSB1 cells infected with CAV, agglutination occurred. A short pre-incubation of CAV antigens with CAV-specific antiserum inhibited the agglutination of mAb-beads. The test results were obtained within 5min. The specificity of b-LAT was evaluated using sera from specific pathogen-free chickens and sera containing antibodies to avian influenza virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus; nonspecific agglutination and cross-reactivity with antibodies to unrelated viruses were not observed. The examination of 94 serum samples collected from commercial breeder chickens of various ages (17-63 weeks) revealed good agreement (93.6%, Kappa value=0.82) between b-LAT and a virus neutralization test, known to be most sensitive and specific in the detection of antibodies to CAV. These results indicate that b-LAT, a simple and rapid test, is a useful and reliable tool in CAV serology.

  14. Dodecamer is required for agglutination of Litopenaeus vannamei hemocyanin with bacterial cells and red blood cells. (United States)

    Pan, Jian-yi; Zhang, Yue-ling; Wang, San-ying; Peng, Xuan-xian


    Hemocyanins are multi-functional proteins, although they are well known to be respiratory proteins of invertebrate to date. In the present study, the agglutination ability of two oligomers of hemocyanin, hexamer and dodecamer, with pathogenic bacteria and red blood cells (RBCs) is investigated in pacific white shrimp, Litopenaeus vannamei. Hexameric hemocyanin exhibits an extremely high stability even in the absence of Ca(2+) and in alkaline pH. Dodecamer (di-hexamer) is easily dissociated into hexamers in unphysiological conditions. Hexamer and dodecamer are interchanged reciprocally with environmental conditions. Both oligomers can bind to bacteria and RBCs, but agglutination is observed only using dodecamer but not using hexamer in agglutination assay. However, the agglutination is detected when hexamer is utilized in the presence of antiserum against hemocyanin. These results indicate that dodecamer of hemocyanin is required for agglutination with bacteria and RBCs. It can be logically inferred that there is only one carbohydrate-binding site to bacterial cells and RBCs in the hexamer, while at least two sites in the dodecamer. Our finding has provided new insights into structural-functional relationship of hemocyanin.

  15. Prevalence of equine herpesvirus type 2 (EHV-2) DNA in ocular swabs and its cell tropism in equine conjunctiva. (United States)

    Borchers, K; Ebert, M; Fetsch, A; Hammond, T; Sterner-Kock, A


    Equine herpes virus 2 (EHV-2), a gamma(2)-herpesvirus, is common in horses of all ages. Its role as a primary pathogen is unclear but there is an association between EHV-2, respiratory disease and keratoconjunctivitis. The purpose of this study was to gain more information on the prevalence of EHV-2 DNA in conjunctival swabs from horses with and without ocular disease and to define the anatomical site and cell type harbouring viral genome or antigen. By polymerase chain reaction (PCR) 22 out of 77 (28.6%) ocular swabs of clinically healthy and only 4 out of 48 (8.3%) samples from diseased horses were positive. To define the main virus reservoir ocular tissue from 13 randomly selected horses without pathological evidence of ocular disease were analysed by nested PCR. In two horses optic nerve, lacrimal gland and conjunctiva, in further two cases lacrimal gland and conjunctiva and in four horses the conjunctiva only were EHV-2 PCR positive. For specifying the target cell we focused on conjunctivae and selected 3 out of 15 clinically healthy slaughterhouse horses positive for EHV-2 by PCR. In situ hybridisation on sections of these paraffin embedded conjunctivae localized viral genome in histiocyte-like cells of the submucosa. Immunohistochemical staining with an EHV-2 or S100 specific polyclonal antiserum demonstrated that Langerhans cells were co-localized in the same region of the sample section where virus positive cells were detected. Furthermore, we concluded that detection of viral antigen revealed a productive virus infection.

  16. Structural and antigenic identification of the ORF12 protein (alpha TIF) of equine herpesvirus 1. (United States)

    Lewis, J B; Thompson, Y G; Feng, X; Holden, V R; O'Callaghan, D; Caughman, G B


    The equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, alpha TIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12) and shown to transactivate immediate early (IE) gene promoters. However, a specific virion protein corresponding to the ORF12 product has not been identified definitively. In the present study the ORF12 protein, designated ETIF, was identified as a 60-kDa virion component on the basis of protein fingerprint analyses in which the limited proteolysis profiles of the major 60-kDa in vitro transcription/ translation product of an ORF12 expression vector (pT7-12) were compared to those of purified virion proteins of similar size. ETIF was localized to the viral tegument in Western blot assays of EHV-1 virions and subvirion fractions using polyclonal antiserum and monoclonal antibodies generated against a glutathione-S-transferase-ETIF fusion protein. Northern and Western blot analyses of EHV-1-infected cell lysates prepared under various metabolic blocks indicated that ORF12 is expressed as a late gene, and cross reaction of polyclonal anti-GST-ETIF with a 63.5-kDa HSV-1 protein species suggested that ETIF and HSV-1 alpha TIF are antigenically related. Last, DNA band shift assays used to assess ETIF-specific complex formation indicated that ETIF participates in an infected cell protein complex with the EHV-1 IE promoter TAATGARAT motif.

  17. Identification and possible roles of three types of endopeptidase from germinated wheat seeds. (United States)

    Sutoh, K; Kato, H; Minamikawa, T


    Little or no endopeptidase activity was detected in extracts of dry mature wheat seeds, but when they were allowed to imbibe water in darkness, the activity expressed per seedling increased notably after d 1, reached a maximum on d 3 and then decreased. Two major endopeptidases, named WEP-1 and WEP-2, were present in the 50-70% saturated ammonium sulfate fraction of d-3 seedlings, and could be separated by hydrophobic column chromatography. WEP-1 was further purified and identified as a 31-kDa polypeptide that was immunoreactive to antiserum raised against REP-1, a major rice cysteine endopeptidase. Experiments with proteinase inhibitors revealed that WEP-1 and WEP-2 are cysteine and serine endopeptidases, respectively. The two enzymes differed in substrate specificity, pH dependence, and the ability to digest major wheat seed proteins. Determination of its amino-terminal amino acid sequence indicated the similarity of WEP-1 to other cereal cysteine endopeptidases which are involved in the digestion of seed storage proteins. The expression of WEP-1 in de-embryonated seeds was induced in the presence of gibberellic acid and its effect was eliminated by abscisic acid. In addition to WEP-1 and WEP-2, a legumain-like asparaginyl endopeptidase was identified in the extract of seedlings on hydrophobic chromatography. The asparaginyl endopeptidase may function in the early step of mobilization of wheat storage proteins in germinated seeds.

  18. Grizzly bear corticosteroid binding globulin: Cloning and serum protein expression. (United States)

    Chow, Brian A; Hamilton, Jason; Alsop, Derek; Cattet, Marc R L; Stenhouse, Gordon; Vijayan, Mathilakath M


    Serum corticosteroid levels are routinely measured as markers of stress in wild animals. However, corticosteroid levels rise rapidly in response to the acute stress of capture and restraint for sampling, limiting its use as an indicator of chronic stress. We hypothesized that serum corticosteroid binding globulin (CBG), the primary transport protein for corticosteroids in circulation, may be a better marker of the stress status prior to capture in grizzly bears (Ursus arctos). To test this, a full-length CBG cDNA was cloned and sequenced from grizzly bear testis and polyclonal antibodies were generated for detection of this protein in bear sera. The deduced nucleotide and protein sequences were 1218 bp and 405 amino acids, respectively. Multiple sequence alignments showed that grizzly bear CBG (gbCBG) was 90% and 83% identical to the dog CBG nucleotide and amino acid sequences, respectively. The affinity purified rabbit gbCBG antiserum detected grizzly bear but not human CBG. There were no sex differences in serum total cortisol concentration, while CBG expression was significantly higher in adult females compared to males. Serum cortisol levels were significantly higher in bears captured by leg-hold snare compared to those captured by remote drug delivery from helicopter. However, serum CBG expression between these two groups did not differ significantly. Overall, serum CBG levels may be a better marker of chronic stress, especially because this protein is not modulated by the stress of capture and restraint in grizzly bears.

  19. Effects of postnatal anti-NGF on the development of CGRP-IR neurons in the dorsal root ganglion. (United States)

    Tonra, J R; Mendell, L M


    Experiments were undertaken to examine anatomical correlates of physiological effects of rabbit sera raised against nerve growth factor (anti-NGF) on nociceptive afferents. This antiserum has been shown to deplete the population of A-delta high threshold mechanoreceptors and to reduce neurogenic vasodilatation. Because numerous studies implicate calcitonin gene related peptide (CGRP)-containing sensory neurons in these effects, immunocytochemical and anatomical techniques were used to examine the normal development of CGRP-immunoreactive (-IR) neurons in the dorsal root ganglion (DRG) of rats from 13 days to 19 weeks of age, and to compare this to the development in rats treated neonatally (postnatal days 2-14) with anti-NGF. In controls the rate of increase in the mean diameter of CGRP-IR cells was substantially greater between 13 days and 5 weeks of age than it was between 5 weeks and 19 weeks, in contrast to CGRP-negative neurons whose rate of growth remained relatively constant. Anti-NGF had no significant effect on growth rate, but rats treated with anti-NGF exhibited a reduced proportion of CGRP-IR neurons at 5 weeks. This deficit was reversed by 19 weeks unlike the physiological changes. These results indicate independent regulation of CGRP expression and nociceptor physiology by NGF.


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    I Wayan Suardana


    Full Text Available Escherichia coli O157:H7 with the ability to produce shiga-like toxin was isolated from beef, cattle, chicken, and human feces. Due to its importance to human health, it is necessary to identify the genes encoding the production of shiga-like toxin, stx1 and stx2 respectively to further understand the pathogenesis. Isolation of E. coli was done on Eosin Methylene Blue Agar (EMBA, followed by identification on Sorbitol MacConkey Agar (SMAC, latex agglutination test, and H7 antiserum test, respectivelly. The existence of genes stx1 and stx2 in E. coli O157:H7 was confirmed molecularly using PCR method with specific primers LP 30/31 and LP 43/44, Stx2 (F/Stx2 (R respectively. Escherichia coli O157:H7 was isolated from 22 out of 344 samples (6,4%. Some isolates showed gene stx1 and stx2 was detected in two isolates as indicated by a 384 bp band (stx1 gene, 584 bp and 1588 bp bands (stx2 gene respectivelly. The results indicated that local isolates E. coli O157:H7 are potential as a zoonoses agent.

  1. Extracellular enolase of Candida albicans is involved in colonization of mammalian intestinal epithelium

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    Richard Cardoso Silva


    Full Text Available Enolase is secreted by C. albicans and is present in its biofilms although its extracellular function is unknown. Here we show that extracellular enolase mediates the colonization of small intestine mucosa by C. albicans. Assays using intestinal mucosa disks show that C. albicans adhesion is inhibited, in a dose dependent mode, either by pretreatment of intestinal epithelium mucosa disks with recombinant C. albicans enolase (70% at 0.5 mg/ml enolase or by pretreatment of C. albicans yeasts with anti-enolase antibodies (48% with 20 µg antiserum. Also using flow cytometry, immunoblots of conditioned media and confocal microscopy we demonstrate that enolase is present in biofilms and that the extracellular enolase is not an artifact due to cell lysis, but must represent functional secretion of a stable form. This is the first direct evidence that C. albicans extracellular enolase mediates colonization on its primary translocation site. Also, because enolase is encoded by a single locus in C. albicans, its dual role peptide, as glycolytic enzyme and extracellular peptide, is a remarkable example of gene sharing in fungi.

  2. Immunofluorescent Localization of Tobacco Ringspot Nepovirus in the Vector Nematode Xiphinema americanum. (United States)

    Wang, S; Gererich, R C


    ABSTRACT An indirect immunofluorescent technique was developed to localize tobacco ringspot nepovirus (TRSV) in the vector nematode Xiphinema americanum sensu stricto. A population of this nematode that efficiently transmitted TRSV was given an acquisition access period of 10 days on TRSV-infected cucumber. Treatment of fragments of viruliferous nematodes with a polyclonal antiserum against TRSV followed by fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G resulted in virus-specific bright fluorescence only in the lumen of the stylet extension and esophagus. Virus-specific fluorescent signals were observed in the virus-retention region of 44% of the nematode fragments examined. The percentage of nematodes labeled with virus-specific fluorescence increased as the acquisition access period increased from 0 to 22 days; the increase paralleled the increase in the transmission efficiency of the nematode population. Visualization of the entire virus-retention region of individual nematodes within a population of vector or nonvector nematodes provides a rapid and simple means of monitoring specific attachment of plant viruses.

  3. The conserved Tarp actin binding domain is important for chlamydial invasion.

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    Travis J Jewett

    Full Text Available The translocated actin recruiting phosphoprotein (Tarp is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells.

  4. Restricted expression of Ovol2/MOVO in XY body of mouse spermatocytes at the pachytene stage. (United States)

    Chizaki, Ryusuke; Yao, Ikuko; Katano, Tayo; Matsuda, Tadashi; Ito, Seiji


    The development of multicellular organisms is controlled by sequential activation of a hierarchy of regulatory genes, which encode transcription factors having DNA-binding motifs. We previously identified a testis-specific zinc finger transcriptional factor, Ovol2/MOVO, as a mouse homologue of Drosophila Ovo. Because mice deficient in Ovol2/Movo die during early embryogenesis, its function in male germ cells has remained unknown. We have recently succeeded in preparing anti-Ovol2/MOVO antiserum for immunohistochemical use. In the present study, we demonstrated that Ovol2/MOVO protein started to be expressed in male germ cells at 2 weeks after birth and that Ovol2/MOVO expression was restricted to the XY body in spermatocytes at the pachytene stage. In a reporter assay, Ovol2/MOVO repressed the histone H1t promoter activity in the spermatogenic cell line GC-2spd. These results suggest that Ovol2/MOVO may play an important role in the XY body during spermatogenesis, possibly in the processes of XY body formation and meiotic sex chromosome inactivation.

  5. Mechanistic characterization and inhibition of sphingomyelinase C over substituted Iron Schiff bases of chitosan adsorbed on glassy carbon electrode. (United States)

    Caro, Claudia A; Lillo, Luis; Valenzuela, Francisco J; Cabello, Gerardo


    The medical treatment of laxoscelisms is based solely on supportive measures. Although equine antiserum for Sphingomyelinase C (SMASE) and D isomers are available, it is not used due to the risk of an anaphylactic reaction and its unproven efficacy. As potential enzyme inhibitors, derivatives of Iron chitosan complexes were studied (Shiff base having -R = -H, -Cl, -Br, -F, -OCH3, -CH3, -NO2). These chitosan complexes were chosen because they have revealed good results in medicine and catalysis due to their biodegradable characteristics and bioavailability. Besides considering that these complexes have not been studied in relation to this toxin. The mechanisms underlying the catalytic and catcher effects of Iron chitosan complexes were studied using electrochemistry, UV-Vis spectroscopy and microscopic assay at physiological pH. The electrochemical studies showed that one of seven Schiff bases of chitosan adsorbed on glassy carbon electrode was electrocatalytically active for the oxidation of sphingomyelinase at 1.27 V, and that allowed proposing a reaction scheme for SMASE oxidation by adsorbed Iron complexes. On the other hand, even though the spectroscopic studies indicated that there was no chemical bond formation between the complex and SMASE in solution, the microscopic studies showed that this complex proved to be a remarkable cellular protector in presence of the enzyme. In conclusion, Shiff base of chitosan with R = -CH3 was the only active complex in front of sphingomyelinase C, protecting red blood cells, according to our electrochemical and microscopic studies.

  6. Observations on white and yellow venoms from an individual southern Pacific rattlesnake (Crotalus viridis helleri). (United States)

    Johnson, E K; Kardong, K V; Ownby, C L


    Biochemical differences in white and yellow venoms produced in the separate venom glands of an individual southern Pacific rattlesnake (Crotalus viridis helleri) were investigated. Compared to the yellow venom, the white venom contained fewer low molecular weight components and was considerably less toxic. Although the exact LD50 was not determined, the white venom did not produce toxic effects in mice when injected i.v. at concentrations up to 10 mg/kg. The i.v. LD50 of the yellow venom was approximately 1.6 mg/kg. Both white and yellow venoms had hemorrhagic activity, but the white venom caused less intradermal hemorrhage in mice. No L-amino acid oxidase activity was measured in the white venom and protease and phospholipase A2 activities of the white venom were much less than in the yellow venom. The white and yellow venoms both produced myonecrosis at 1, 3 and 24 hr after i.m. injection into mice, however, there were some qualitative differences in the myonecrosis produced. When the venom samples were reacted against Wyeth's polyvalent (Crotalidae) antivenom using immunodiffusion, three precipitin bands formed against the yellow venom, whereas only one formed against the white venom. When reacted against an antiserum to myotoxin alpha from C. viridis viridis venom, both the white and yellow venoms produced one precipitin band each.

  7. Autophosphorylation, electrophoretic mobility and immunoreaction of oat phototropin 1 under UV and blue Light. (United States)

    Knieb, Elke; Salomon, Michael; Rüdiger, Wolfhart


    Phototropins are UV-A/blue light photoreceptors containing two flavin mononucleotide (FMN)-binding domains, light, oxygen and voltage (LOV)1 and LOV2, of which LOV2 is more sensitive toward light and more important for the physiological response compared with LOV1. Some physiological responses are plant phototropism, chloroplast migration and stomatal opening. Oat phototropin 1 together with light-dependent autophosphorylation shows a reduced electrophoretic mobility and reduced immunoreaction against a heterologous antiserum; both effects were suggested to be caused by phosphorylation at the same sites (M. Salomon, E. Knieb, T. von Zeppelin and W. Rudiger [2003] Biochemistry 42, 4217-4225). In this study, we show that both effects can be separated from each other: at low temperature, reduced immunoreaction preceded the mobility shift, and irradiation with UV-C light led to the mobility shift without the loss of immunoreactivity. We demonstrated that UV-C light at 280 nm, which does not match any absorption maximum of FMN, leads to autophosphorylation of phototropin. It is hypothesized that UV-C light causes differential activation of the LOV domains via energy transfer from aromatic amino acids.

  8. Purification and polyclonal antibody preparation of Cd-MT from Pteria penguin%企鹅珍珠贝镉金属硫蛋白的分离纯化及其多克隆抗体的制备

    Institute of Scientific and Technical Information of China (English)

    吴晓萍; 廖爱琳; 章超桦; 廖艳; 卢虹玉; 杨捷


    Heavy metals in three wastes are discharged into water by a variety of means with the industry development such as mine, smelting and electroplating, which lead to pollution of aquatic environments and threaten human health and biological survival seriously. Cadmium(Cd) is one of the main marine pollutants. As Cd can accumulate in different tissues of shellfish through food chains, which threaten the quality and safety of shellfish foods, it is extremely necessary to research and monitor conditions of shellfish contamination with Cd. Cadmium-metallothioneins (Cd-MTs) are important biomarker used for indicating cadmium pollution in aquatic environment. The research on these Cd-MTs not only is propitious to establish Cd-MT immunoassay method, but also provides the theoretical basis for technology development in monitoring heavy metal pollution. Cd-MT was induced to synthesize by injecting different concentrations of CdCl2(0 -0.8 mg/L)in Pteria penguin and was isolated and purified from whole tissues of P. penguin by tissue homogenization, freezing centrifugation, heat treatment and Sephadex G-50 gel column chromatography. The characteristic absorption of Cd-MT in crude extract by ultraviolet spectrophotometry was the strongest at 0.8 mg/L Cd2 +. The results showed that the content of Cd-MT in P. penguin increased with concentration of Cd2 +. The results from SDS-PAGE assay showed that molecular weight of purified Cd-MT and its dimer were 9 ku and 18 ku respectively. Then Cd-MT was coupled to bovine serum albumin (BSA) with glutaraldehyde and polyclonal antiserum by immunizing rabbits with Cd-MT-BSA was prepared. The titer of antiserum was measured by indirect ELISA assay and reached 1:12 800. IgG ( molecular weight is about 35 ku) in the antiserum was obtained by methods including saturated ammonium persulfate precipitation and DEAE-Sephadex A50 column chromatography. ELISA assay showed that the IgG from antiserum could react to Cd-MT of P. martensi as well as Cd

  9. Experiment K-6-18. Study of muscarinic and gaba (benzodiazepine) receptors in the sensory-motor cortex, hippcampus and spinal code (United States)

    Daunton, N.; Damelio, F.; Krasnov, I.


    Frontal lobe samples of rat brains flown aboard Cosmos 1887 were processed for the study of muscarinic (cholinergic) and GABA (benzodiazepine) receptors and for immunocytochemical localization of the neurotransmitter gamma-aminobutyric acid (GABA) and glial fibrillary acidic protein (GFAP). Although radioactive labeling of both muscarinic cholinergic and GABA (benzodiazepine) receptors proved to be successful with the techniques employed, distinct receptor localization of individual laminae of the frontal neocortex was not possible since the sampling of the area was different in the various groups of animals. In spite of efforts made for proper orientation and regional identification of laminae, it was found that a densitometric (quantitation of autoradiograms) analysis of the tissue did not contribute to the final interpretation of the effects of weightlessness on these receptors. As to the immunocytochemical studies the use of both markers, GFAP and GABA antiserum, confirmed the suitability of the techniques for use in frozen material. However, similar problems to those encountered in the receptor studies prevented an adequate interpretation of the effects of micro-G exposure on the localization and distribution of GABA and GFAP. This study did, however, confirm the feasibility of investigating neurotransmitters and their receptors in future space flight experiments.

  10. Prevalence of Salmonella spp. in Imported Powered Infant Formula (PIF

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    Full Text Available Salmonella species are well-known long-standing foodborne human pathogen that demonstrate long-term survival in/on dry or low-water activity (aw in foods. Salmonellosis caused by ingestion of contaminated powdered infant formula has been reported nationwide. In recent years, 8 reported outbreaks of Salmonella infection in infants have been linked to the consumption of powdered infant formula. Outbreaks of Salmonellosis due to contaminated PIF are likely to be under-reported nationwide even in Albania. The aim of this study was to investigate the potential existence of Salmonella spp. in canned powdered infant formula in Albania. During two years investigation Salmonella spp was on the focus and was detected in 1 out of 70 analysed samples (1.43%. The strain of Salmonella spp. was biochemically identified by the analytical profile index (API 20 E system and poly A, H, and Vi antiserum. Food safety criteria are laid down in EU regulation “EC No. 2073/2005” for Salmonella spp. in dried infant formula and dried dietary foods for special medical purposes intended for infants. These criterias are transposed to Albanian Legislation. A laboratory-based on food-borne disease surveillance systems is needed in terms of strethening control and reducing the risk of exposure.

  11. Immunoglobulin Cmu RNA in T lymphoma cells is not translated. (United States)

    Walker, I D; Harris, A W


    It is widely believed that immunoglobulin genes might encode at least part of the receptor for antigen on the T lymphocyte. Evidence supporting this comes from the effects of anti-immunoglobulin idiotype antibodies on cellular immune networks and from the presence of idiotypes on immunologically active factors from T cells. Detailed molecular characterization of the receptors, however, has been seriously hampered by the lack of a suitable cellular source from which it might be isolated. The recent demonstration of Kemp et al. that thymocytes and certain cultured lines of mouse T lymphoma cells contain polyadenylated RNA molecules encoded by the immunoglobulin Cmu gene (Cmu RNA) prompted us to identify the corresponding protein molecules in those cells. As the haploid mouse genome contains a single Cmu gene, any polypeptide encoded by this gene should react with at least some of the antibodies present in rabbit anti-mouse IgM antiserum. In this letter we report that a number of T lymphoma lines, regardless of whether they contain Cmu RNA, synthesize no detectable mu polypeptides.

  12. Neonatal citalopram exposure decreases serotonergic fiber density in the olfactory bulb of male but not female adult rats

    Directory of Open Access Journals (Sweden)

    Junlin eZhang


    Full Text Available Manipulation of serotonin (5HT during early development has been shown to induce long-lasting morphological changes within the raphe nuclear complex and serotonergic circuitry throughout the brain. Recent studies have demonstrated altered raphe-derived 5HT transporter (SERT immunoreactive axonal expression in several cortical target sites after brief perinatal exposure to selective 5HT reuptake inhibitors such as citalopram (CTM. Since the serotonergic raphe nuclear complex projects to the olfactory bulb (OB and perinatal 5HT disruption has been shown to disrupt olfactory behaviors, the goal of this study was to further investigate such developmental effects in the OB of CTM exposed animals. Male and female rat pups were exposed to CTM from postnatal day 8-21. After animals reach adulthood (>90 days, OB tissue sections were processed immunohistochemically for SERT antiserum. Our data revealed that the density of the SERT immunoreactive fibers decreased ~40% in the OB of CTM exposed male rats, but not female rats. Our findings support a broad and long-lasting change throughout most of the 5HT system, including the OB, after early manipulation of 5HT. Because dysfunction of the early 5HT system has been implicated in the etiology of neurodevelopmental disorders such as autism spectrum disorders (ASDs, these new findings may offer insight into the abnormal olfactory perception often noted in patients with ASD.

  13. A 24-kDa cloned zinc metalloprotease from Actinobacillus pleuropneumoniae is common to all serotypes and cleaves actin in vitro. (United States)

    García-Cuéllar, C; Montañez, C; Tenorio, V; Reyes-Esparza, J; Durán, M J; Negrete, E; Guerrero, A; de la Garza, M


    Actinobacillus pleuropneumoniae causes pleuropneumonia in swine. This bacterium secretes proteases that degrade porcine hemoglobin and IgA in vitro. To further characterize A. pleuropneumoniae proteases, we constructed a genomic library expressed in Escherichia coli DH5alpha, and selected a clone that showed proteolytic activity. The recombinant plasmid carries an 800-base pair A. pleuropneumoniae gene sequence for a 24-kDa polypeptide. A 350-base pair PstI fragment from the sequence hybridized at high stringency with DNA from 12 serotypes of A. pleuropneumoniae, but not with DNA from Actinobacillus suis, Haemophilus parasuis, Pasteurella haemolytica, Pasteurella multocida A or D, or E. coli DH5alpha, thus showing specificity for A. pleuropneumoniae. The expressed polypeptide was recognized as an antigen by convalescent-phase pig sera. Furthermore, a polyclonal antiserum developed against the purified polypeptide recognized an A. pleuropneumoniae oligomeric protein in both crude-extract and cell-free culture media. This recombinant polypeptide cleaved azocoll, gelatin, and actin. Inhibition of the proteolytic activity by diethylpyrocarbonate suggests that this polypeptide is a zinc metalloprotease. Images Figure 1. Figure 2. Figure 3. Figure 4. Figure 6. Figure 7. PMID:10805246

  14. Application of Current Hapten in the Production of Broad Specificity Antibodies Against Organophosphorus Pesticides

    Institute of Scientific and Technical Information of China (English)

    LIU Xian-jin; YAN Chun-rong; LIU Yuan; YU Xiang-yang; ZHANG Cun-zheng


    Diethylphosphono acetic acid (DPA) was used as a current hapten to generate broad specificity polycolonal antibodies against a group of organophosphorus pesticides. Six New Zealand white rabbits were immunized with immunogens synthesized by the active ester method (AEM) or 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide method (EDC). The titers of antisera reached 25 600 by AEM and 6 400 by EDC, respectively. Polyclonal antibodies raised against DPA were screened and selected for the competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). A CI-ELISA for DPA was developed with a detection limit of 3.536 ng mL-1 and an I50 value of 0.182 ug mL-1. The assay specificity was evaluated by obtaining competitive curves for several structurally related compounds as competitors. The antiserum showed high affinities to chlorpyrifos, diazinon, omethoate, parathion-ethyl and profenofos with I50 of 0.12, 0.15, 0.21, 0.88, 0.97 and 2.5 ug mL-1, respectively. The results indicate that the assay could be a screening tool for quantitation and semi-quantitation determination of the above former five organophosphorus pesticides.

  15. Lipoprotein NMB0928 from Neisseria meningitidis serogroup B as a novel vaccine candidate. (United States)

    Delgado, Maité; Yero, Daniel; Niebla, Olivia; González, Sonia; Climent, Yanet; Pérez, Yusleydis; Cobas, Karem; Caballero, Evelín; García, Darien; Pajón, Rolando


    Polysaccharide-based vaccines for serogroup B Neisseria meningitidis have failed to induce protective immunity. As a result, efforts to develop vaccines for serogroup B meningococcal disease have mostly focused on outer membrane proteins (OMP). Vaccine candidates based on meningococcal OMP have emerged in the form of outer membrane vesicles (OMVs) or, more recently, purified recombinant proteins, as alternative strategies for serogroup B vaccine development. In our group, the protein composition of the Cuban OMVs-based vaccine VA-MENGOC-BC was elucidated using two-dimensional gel electrophoresis and mass spectrometry. The proteomic map of this product allowed the identification of new putative protective proteins not previously reported as components of an antimeningococcal vaccine. In the present study, we have determined the immunogenicity and protective capacity of NMB0928, one of those proteins present in the OMVs. The antigen was obtained as a recombinant protein in Escherichia coli, purified and used to immunize mice. The antiserum produced against the protein was capable to recognize the natural protein in different meningococcal strains by whole-cell ELISA and Western blotting. After immunization, recombinant NMB0928 induced bactericidal antibodies, and when the protein was administered inserted into liposomes, the elicited antibodies were protective in the infant rat model. These results suggest that NMB0928 is a novel antigen worth to be included in a broadly protective meningococcal vaccine.

  16. Outer membrane protein A, peptidoglycan-associated lipoprotein, and murein lipoprotein are released by Escherichia coli bacteria into serum. (United States)

    Hellman, J; Loiselle, P M; Tehan, M M; Allaire, J E; Boyle, L A; Kurnick, J T; Andrews, D M; Sik Kim, K; Warren, H S


    Complexes containing lipopolysaccharide (LPS) and three outer membrane proteins (OMPs) are released by gram-negative bacteria incubated in human serum and into the circulation in an experimental model of sepsis. The same OMPs are bound by immunoglobulin G (IgG) in the cross-protective antiserum raised to Escherichia coli J5 (anti-J5 IgG). This study was performed to identify the three OMPs. The 35-kDa OMP was identified as outer membrane protein A (OmpA) by immunoblotting studies using OmpA-deficient bacteria and recombinant OmpA protein. The 18-kDa OMP was identified as peptidoglycan-associated lipoprotein (PAL) based on peptide sequences from the purified protein and immunoblotting studies using PAL-deficient bacteria. The 5- to 9-kDa OMP was identified as murein lipoprotein (MLP) based on immunoblotting studies using MLP-deficient bacteria. The studies identify the OMPs released into human serum and into the circulation in an experimental model of sepsis as OmpA, PAL, and MLP.

  17. Isolation, cDNA cloning, and growth promoting activity of rabbitfish (Siganus guttatus) growth hormone. (United States)

    Ayson, F G; de Jesus, E G; Amemiya, Y; Moriyama, S; Hirano, T; Kawauchi, H


    We report the isolation, cDNA cloning, and growth promoting activity of rabbitfish (Siganus guttatus; Teleostei; Perciformes; Siganidae) growth hormone (GH). Rabbitfish GH was extracted from pituitary glands under alkaline conditions, fractionated by gel filtration chromatography on Sephadex G-100, and purified by high-performance liquid chromatography. The fractions containing GH were identified by immunoblotting with bonito GH antiserum. Under nonreducing conditions, the molecular weight of rabbitfish GH is about 19 kDa as estimated by SDS-PAGE. The purified hormone was potent in promoting growth in rabbitfish fry. Weekly intraperitoneal injections of the hormone significantly accelerated growth. This was evident 3 weeks after the start of the treatment, and its effect was still significant 2 weeks after the treatment was terminated. Rabbitfish GH cDNA was cloned to determine its nucleotide sequence. Excluding the poly (A) tail, rabbitfish GH cDNA is 860 base pairs (bp) long. It contained untranslated regions of 94 and 175 bp in the 5' and 3' ends, respectively. It has an open reading frame of 588 bp coding for a signal peptide of 18 amino acids and a mature protein of 178 amino acid residues. Rabbitfish GH has 4 cysteine residues. On the amino acid level, rabbitfish GH shows high identity (71-74%) with GHs of other perciforms, such as tuna, sea bass, yellow tail, bonito, and tilapia, and less (47-49%) identity with salmonid and carp GHs.

  18. Isolation and cDNA cloning of somatolactin in rabbitfish (Siganus guttatus). (United States)

    Ayson, F G; de Jesus, E G; Amemiya, Y; Moriyama, S; Hirano, T; Kawauchi, H


    We report the isolation and cDNA cloning of somatolactin (SL) from rabbitfish, Siganus guttatus. Rabbitfish SL was isolated from an alkaline extract of the pituitary glands by gel filtration chromatography on Sephadex G-100 and reversed-phase high-performance liquid chromatography. SL was monitored by immunoblotting with flounder SL antiserum. The preparation (yield: 0.86 mg/g wet tissues) contained two immunoreactive bands of 24 and 28 kDa on SDS-PAGE. Overlapping partial cDNA clones corresponding to teleost SLs were amplified by PCR from single-strand cDNA from pituitary glands. Excluding the poly(A) tail, rabbitfish SL cDNA is 1605 bp long. It contains a 693-bp open reading frame encoding a signal peptide of 24 amino acids (aa) and a mature protein of 207 aa. Rabbitfish SL has two possible N-glycosylation sites at positions 11 and 121 and seven half Cys residues. The deduced amino acid sequence shows over 80% identity with those of advanced teleosts like sea bream, red drum, and flounder, 76% with the salmonids, 57% with the eel, and 46% with the goldfish SL.

  19. The presence of 19-norandrostenedione and its sulphate form in yolk-sac fluid of the early equine conceptus. (United States)

    Raeside, J I; Christie, H L


    C(18) neutral steroid formation by cytochrome P450 aromatase has been recorded for several equine and porcine tissues. High activity of P450 aromatase is reflected in the quantities of estrogens in yolk-sac (y-s) fluid of early equine conceptuses. In a previous study of y-s fluid we detected large amounts of androgens by radioimmunoassay (RIA), using an antiserum for androstenedione (A(4)). Here, we report that RIA, following chromatography, gave tentative identification of the major peak as norandrostenedione (19-norA) not as A(4). Furthermore, even greater quantities of 19-norA seemed to be present in y-s fluid as a sulphoconjugate, as noted from extraction, solvolysis, HPLC, followed by RIA. Confirmation of these unusual findings was attained after further purification with two HPLC systems and definitive identification by LC-MS with an authentic standard of 19-norA. Initial extraction of the steroid sulphate as a methylene-blue complex also yielded 19-norA suggesting that the 3-enol form had enabled sulphoconjugation. The biological significance of retention mainly as a sulphate is not known; however, the large amounts of 19-norA found in the fluid accords well with reports on the catalytic activity shown in vitro by the blastocyst isozyme of P450 aromatase in the pig and horse.

  20. Salmonella typhi O:9,12 polysaccharide-protein conjugates: characterization and immunoreactivity with pooled and individual normal human sera, sera from patients with paratyphoid A and B and typhoid fever, and animal sera. (United States)

    Aron, L; Di Fabio, J; Cabello, F C


    Polysaccharide of O:9,12 specificity purified from Salmonella typhi was conjugated to tetanus toxoid or bovine serum albumin in order to obtain defined antigenic material that would contain O chain free of other S. typhi antigens and that would be suitable for characterizing host humoral response to only S. typhi O-chain antigens. These artificial conjugates were strongly reactive in immunodots with 18 pooled and 3 individual serum samples from patients with typhoid fever and with rabbit anti-Salmonella O antiserum (group D, factors 1, 9, and 12). They reacted weakly with one serum sample from one human with paratyphoid A. These results suggest that the periodate oxidation and the reductive amination used in the conjugation conserved the immunogenicity of the O chain and allowed its absorption to nitrocellulose. They also suggest that the bovine serum albumin conjugate could be used in the diagnosis of S. typhi infections as normal sera may react with the protein molecule of the tetanus toxoid conjugate.

  1. Oral DNA Vaccine in Chickens

    Directory of Open Access Journals (Sweden)

    Seyed Davoud Jazayeri


    Full Text Available Attenuated Salmonella has been used as a carrier for DNA vaccine. However, in vitro and in vivo studies on the bacteria following transfection of plasmid DNA were poorly studied. In this paper, eukaryotic expression plasmids encoding avian influenza virus (AIV subtype H5N1 genes, pcDNA3.1/HA, NA, and NP, were transfected into an attenuated Salmonella enteric typhimurium SV4089. In vitro stability of the transfected plasmids into Salmonella were over 90% after 100 generations. The attenuated Salmonella were able to invade MCF-7 (1.2% and MCF-10A (0.5% human breast cancer cells. Newly hatched specific-pathogen-free (SPF chicks were inoculated once by oral gavage with 109 colony-forming unit (CFU of the attenuated Salmonella. No abnormal clinical signs or deaths were recorded after inoculation. Viable bacteria were detected 3 days after inoculation by plating from spleen, liver, and cecum. Fluorescent in situ hybridization (FISH and polymerase chain reaction (PCR were carried out for confirmation. Salmonella was not detected in blood cultures although serum antibody immune responses to Salmonella O antiserum group D1 factor 1, 9, and 12 antigens were observed in all the inoculated chickens after 7 days up to 35 days. Our results showed that live attenuated S. typhimurium SV4089 harboring pcDNA3.1/HA, NA, and NP may provide a unique alternative as a carrier for DNA oral vaccine in chickens.

  2. Genotyping of human rhinovirus in adult patients with acute respiratory infections identified predominant infections of genotype A21 (United States)

    Ren, Lili; Yang, Donghong; Ren, Xianwen; Li, Mingkun; Mu, Xinlin; Wang, Qi; Cao, Jie; Hu, Ke; Yan, Chunliang; Fan, Hongwei; Li, Xiangxin; Chen, Yusheng; Wang, Ruiqin; An, Fucheng; An, Shuchang; Luo, Ming; Wang, Ying; Xiao, Yan; Xiang, Zichun; Xiao, Yan; Li, Li; Huang, Fang; Jin, Qi; Gao, Zhancheng; Wang, Jianwei


    Human rhinovirus (HRV) is an important causative agent of acute respiratory tract infections (ARTIs). The roles of specific HRV genotypes in patients suffering from ARTIs have not been well established. We recruited 147 adult inpatients with community-acquired pneumonia (CAP) and 291 adult outpatients with upper ARTIs (URTIs). Respiratory pathogens were screened via PCR assays. HRV was detected in 42 patients, with 35 species A, five B and two C. Seventeen genotypes were identified, and HRV-A21 ranked the highest (9/42, 21.4%). The HRV-A21-positive infections were detected in four patients with CAP and in five with URTIs, all without co-infections. The HRV-A21 genome sequenced in this study contained 12 novel coding polymorphisms in viral protein (VP) 1, VP2 EF loop, VP3 knob and 3D regions. The infections of HRV-A21 virus obtained in this study could not be neutralized by antiserum of HRV-A21 prototype strain (VR-1131), indicating remarkable antigenic variation. Metagenomic analysis showed the HRV-A21 reads were dominant in bronchoalveolar lavage fluid of the three HRV-A21-positive patients with severe CAP, in which two dead. Our results highlight an unexpected infection of genotype HRV-A21 in the clinic, indicating the necessity of precise genotyping and surveillance of HRVs to improve the clinical management of ARTIs. PMID:28128353

  3. Radioimmunoassay for 3,3'-L-diiodothyronine (3,3'T/sub 2/)

    Energy Technology Data Exchange (ETDEWEB)

    Burman, K.D.; Strum, D.; Dimond, R.C.; Djuh, Y.Y.; Wright, F.D.; Earll, J.M.; Wartofsky, L.


    The report describes the development of a radioimmunoassay for 3,3'-L-diiodothyronine (3,3'T/sub 2/) which may be performed on unextracted serum. Utilizing a specific antiserum to 3,3'-L-T/sub 2/-bovine serum albumin conjugates developed in rabbits, cross-reactivity was less than 0.5% with 3,5,3'-triiodothyronine (T/sub 3/) and 3,3',5'-triiodothyronine (reverse T/sub 3/) and less than 0.01% with thyroxine (T/sub 4/). Intra-assay variation averaged 2.9% and inter-assay variation was 7.8% and 18.5% when serum samples with 3,3'T/sub 2/ concentrations of 8 ng/dl and 12 ng/dl, respectively, were analyzed. Assay sensitivity was considered to be 6 ng/dl by statistical criteria. The data suggest that 3,3'T/sub 2/ circulates in the serum of normal individuals and tends to parallel serum concentrations of T/sub 3/, T/sub 4/ and rT/sub 3/ in various states of thyroid function.

  4. Employing Escherichia coli-derived outer membrane vesicles as an antigen delivery platform elicits protective immunity against Acinetobacter baumannii infection (United States)

    Huang, Weiwei; Wang, Shijie; Yao, Yufeng; Xia, Ye; Yang, Xu; Li, Kui; Sun, Pengyan; Liu, Cunbao; Sun, Wenjia; Bai, Hongmei; Chu, Xiaojie; Li, Yang; Ma, Yanbing


    Outer membrane vesicles (OMVs) have proven to be highly immunogenic and induced an immune response against bacterial infection in human clinics and animal models. We sought to investigate whether engineered OMVs can be a feasible antigen-delivery platform for efficiently inducing specific antibody responses. In this study, Omp22 (an outer membrane protein of A. baumannii) was displayed on E. coli DH5α-derived OMVs (Omp22-OMVs) using recombinant gene technology. The morphological features of Omp22-OMVs were similar to those of wild-type OMVs (wtOMVs). Immunization with Omp22-OMVs induced high titers of Omp22-specific antibodies. In a murine sepsis model, Omp22-OMV immunization significantly protected mice from lethal challenge with a clinically isolated A. baumannii strain, which was evidenced by the increased survival rate of the mice, the reduced bacterial burdens in the lung, spleen, liver, kidney, and blood, and the suppressed serum levels of inflammatory cytokines. In vitro opsonophagocytosis assays showed that antiserum collected from Omp22-OMV-immunized mice had bactericidal activity against clinical isolates, which was partly specific antibody-dependent. These results strongly indicated that engineered OMVs could display a whole heterologous protein (~22 kDa) on the surface and effectively induce specific antibody responses, and thus OMVs have the potential to be a feasible vaccine platform.

  5. Multiple lineages of antigenically and genetically diverse influenza A virus co-circulate in the United States swine population. (United States)

    Webby, R J; Rossow, K; Erickson, G; Sims, Y; Webster, R


    Before the isolation of H3N2 viruses in 1998, swine influenza in the United States was an endemic disease caused exclusively by classical-swine H1N1 viruses. In this study we determined the antigenic and phylogenetic composition of a selection of currently circulating strains and revealed that, in contrast to the situation pre-1998, the swine population in the United States is now a dynamic viral reservoir containing multiple viral lineages. H3N2 viruses still circulate and representatives of each of two previously identified phylogenetic groups were isolated. H1N1 and H1N2 viruses were also identified. In addition to the genotypic diversity present, there was also considerable antigenic diversity seen. At least three antigenic profiles of H1 viruses were noted and all of the recent H3N2 viruses reacted poorly, if at all, to the index A/swine/Texas/4199-2/98 H3N2 antiserum in hemagglutination inhibition assays. The influenza reservoir in the United States swine population has thus gone from a stable single viral lineage to one where genetically and antigenically heterogenic viruses co-circulate. The growing complexity of influenza at this animal-human interface and the presence of viruses with a seemingly high affinity for reassortment makes the United States swine population an increasingly important reservoir of viruses with human pandemic potential.

  6. Human rhinovirus VPg uridylylation AlphaScreen for high-throughput screening. (United States)

    Gingras, Rock; Mekhssian, Kevork; Fenwick, Craig; White, Peter W; Thibeault, Diane


    As an obligate step for picornaviruses to replicate their genome, the small viral peptide VPg must first be specifically conjugated with uridine nucleotides at a conserved tyrosine hydroxyl group. The resulting VPg-pUpU serves as the primer for genome replication. The uridylylation reaction requires the coordinated activity of many components, including the viral polymerase, a conserved internal RNA stem loop structure, and additional viral proteins. Formation of this complex and the resulting conjugation reaction catalyzed by the polymerase, offers a number of biochemical targets for inhibition of an essential process in the viral life cycle. Therefore, an assay recapitulating uridylylation would provide multiple opportunities for discovering potential antiviral agents. Our goal was to identify inhibitors of human rhinovirus (HRV) VPg uridylylation, which might ultimately be useful to reduce or prevent HRV-induced lower airway immunologic inflammatory responses, a major cause of asthma and chronic obstructive pulmonary disease exacerbations. We have reconstituted the complex uridylylation reaction in an AlphaScreen suitable for high-throughput screening, in which a rabbit polyclonal antiserum specific for uridylylated VPg serves as a key reagent. Assay results were validated by quantitative mass spectrometric detection of uridylylation.

  7. Identification of a macromolecule containing an anticarcinoembryonic antigen-reactive substance and immunoglobulin M in human pancreatic cancer. (United States)

    Harvey, S R; Van Dusen, L R; Douglass, H O; Holyoke, E D; Chu, T M


    Ascitic fluid from a patient with carcinoma of the pancreas was fractionated by ammonium sulfate precipitation. The fraction precipitated between 25 and 50% saturation of ammonium sulfate was sequentially chromatographed on Sephadex G-200 and Sepharose 6B. A macromolecular fraction (greater than 10(6) daltons) obtained was found to react with both antihuman IgM and antiserum to carcinoembryonic antigen (CEA). This fraction was further purified by adsorption with protein A-Sepharose CL-4B and chromatography on DEAE-Sephacel. The purified macromolecular fraction had a sedimentation value of 28S as determined by ultracentrifugation. Upon dissociation of the purified macromolecule at pH 2.3 and purification of the dissociated components on Sepharose CL-2B and BioGel A 1.5M, a 19S protein and a 5S protein were recovered. The 19S protein showed a complete line of identity with a reference human IgM when reacted with antihuman IgM in gel diffusion, whereas the 5S protein showed a partial immunologic identity with colon CEA against anti-CEA. These results indicated the existence of an IgM-containing macromolecular complex with an anti-CEA cross-reactive substance in the extracellular fluid of human pancreatic cancer.

  8. Biological characterization of purified macrophage-derived neutrophil chemotactic factor

    Directory of Open Access Journals (Sweden)

    M. Dias-Baruffi


    Full Text Available We have recently described the purification of a 54 kDa acidic protein, identified as macrophage-derived neutrophil chemotactic factor (MNCF. This protein causes in vitro chemotaxis as well as in vivo neutrophil migration even in animals treated with dexamethasone. This in vivo chemotactic activity of MNCF in animals pretreated with dexamethasone is an uncommon characteristic which discriminates MNCF from known chemotactic cytokines. MNCF is released in the supernatant by macrophage monolayers stimulated with lipopolysaccharide (LPS. In the present study, we describe some biological characteristics of homogenous purified MNCF. When assayed in vitro, MNCF gave a bell-shaped dose–response curve. This in vitro activity was shown to be caused by haptotaxis. Unlike N-formyl-methionylleucyl- phenylalanine (FMLP or interleukin 8 (IL-8, the chemotactic activity of MNCF in vivo and in vitro, was inhibited by preincubation with D-galactose but not with D-mannose. In contrast with IL-8, MNCF did not bind to heparin and antiserum against IL-8 was ineffective in inhibiting its chemotactic activity. These data indicate that MNCF induces neutrophil migration through a carbohydrate recognition property, but by a mechanism different from that of the known chemokines. It is suggested that MNCF may be an important mediator in the recruitment of neutrophils via the formation of a substrate bound chemotactic gradient (haptotaxis in the inflamed tissues.

  9. Re-expression by Candida albicans germ tubes of antigens lost during subculture of blastospores. (United States)

    Hernando, F L; Calvo, E; Rodriguez, J A; Barea, P L; Rementeria, A; Sevilla, M J; Ponton, J


    The effect of germ tube induction on the antigenic variability in C.albicans was studied in strains from blood cultures (Group I) and superficial candidiasis (Group II). When compared by immunoblotting with a rabbit antiserum, antigenic extracts from Group I strains grown as blastospores showed a higher reactivity than that of Group II strains. Major bands in Group I strains (45-47, 33, 30 kDa) were continuously expressed through the subcultures in vitro but, with the exception of the 45 kDa band, the reactivity of all of them decreased or disappeared after the tenth subculture in Group II strains. The induction of the germ tubes produced the re-expression of the antigens lost during subculture in the yeast form, the effect being very clear in Group II strains. The re-expression by C. albicans germ tubes of antigens lost during subculture of blastospores in vitro and the higher reactivity shown by Group I strains grown in mycelial phase should be taken into consideration when a test to detect anti-C. albicans antibodies is to be developed.

  10. A G-Box-Binding Protein from Soybean Binds to the E1 Auxin-Response Element in the Soybean GH3 Promoter and Contains a Proline-Rich Repression Domain. (United States)

    Liu, Z. B.; Hagen, G.; Guilfoyle, T. J.


    The E1 promoter fragment (-249 to -203) is one of three auxin-response elements (AuxREs) in the soybean (Glycine max L.) GH3 promoter (Z.-B. Liu, T. Ulmasov, X. Shi, G. Hagen, T.J. Guilfoyle [1994] Plant Cell 6: 645-657). Results presented here further characterize and delimit the AuxRE within the E1 fragment. The E1 fragment functioned as an AuxRE in transgenic tobacco (Nicotiana tabacum L.) plants, as well as in transfected protoplasts. The AuxRE within E1 contains a G-box, and this G-box was used to clone a G-box-binding factor (GBF) from soybean (SGBF-2). This 45-kD GBF contains an N-terminal proline-rich domain and a C-terminal basic/leucine zipper DNA-binding domain. Gel-mobility shift assays were used to characterize the binding specificity of SGBF-2. Antiserum raised against recombinant SGBF-2 was used to further characterize SGBF-2 and antigenically related GBFs in soybean nuclear extracts. Co-transfection assays with effector and reporter plasmids in carrot (Daucus carota L.) protoplasts indicated that the N-terminal proline-rich domain of SGBF-2 functioned as a repression domain in both basal and auxin-inducible transcription.

  11. cDNA cloning, tissue distribution, and chromosomal localization of Ocp2, a gene encoding a putative transcription-associated factor predominantly expressed in the auditory organs

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Hong; Thalmann, I.; Thalmann, R. [Washington Univ., St. Louis, MO (United States)] [and others


    We report the cloning of the Ocp2 gene encoding OCP-II from a guinea pig organ-of-Corti cDNA library. The predicted open reading frame encodes a protein of 163 amino acids with an estimated molecular mass of 18.6 kDa. A homology search revealed that Ocp2 shares significant sequence similarity with p15, a sub-unit of transcription factor SIII that regulates the activity of the RNA polymerase II elongation complex. The Ocp2 messenger RNA is expressed abundantly in the cochlea while not significantly in any other tissues examined, including brain, eye, heart, intestine, kidney, liver, lung, thigh muscle, and testis, demonstrating that the expression of this gene may be restricted to auditory organs. A polyclonal antiserum was raised against the N-terminal region of OCP-II. Immunohistochemical staining of paraffin-embedded sections of the cochlea showed that OCP-II is localized abundantly in nonsensory cells in the organ of Corti; in addition, it was also detected, at a lower concentration, in vestibular sensory organs, as well as auditory and vestibular brain stem nuclei. The Ocp2 gene was mapped to mouse chromosome 4 as well as 11. Our results suggest that OCP-II may be involved in transcription regulation for the development or maintenance of specialized functions of the inner ear. 40 refs., 5 figs.

  12. Expression and ontogeny of growth hormone (Gh) in the protogynous hermaphroditic ricefield eel (Monopterus albus). (United States)

    Chen, Dong; Liu, Jiang; Chen, Wanping; Shi, Shuxia; Zhang, Weimin; Zhang, Lihong


    Growth hormone (GH) is a single-chain polypeptide hormone mainly secreted by somatotropes of the anterior pituitary gland and is an important regulator of somatic growth in vertebrates including teleosts. In this study, a polyclonal antiserum against ricefield eel Gh was generated and the expression of Gh at the mRNA and protein levels was analyzed. Both RT-PCR and western blot analysis showed that Gh was predominantly expressed in the pituitary glands of ricefield eels. The immunoreactive Gh signals were localized to the multicellular layers of the adenohypophysis adjacent to the neurohypophysis in ricefield eels. Ontogenetic analysis showed that immunoreactive Gh signals could be detected in the pituitary glands of ricefield eel embryos as early as 3 days post-fertilization. During the sex change from female to male, the levels of the immunoreactive Gh signals in the pituitary glands of the ricefield eels peaked at the intersexual stage. These results suggest that Gh in the pituitary glands may be associated with embryonic development before hatching, as well as with the sex change in the adult ricefield eels, possibly via the classical endocrine manner.

  13. Degranulation of mast cells located in median eminence in response to compound 48/80 evokes adrenocortical secretion via histamine and CRF in dogs. (United States)

    Matsumoto, Itsuro; Inoue, Yasuhisa; Tsuchiya, Katsuhiko; Shimada, Toshio; Aikawa, Tadaomi


    The effect of intracerebroventricular infusion of compound 48/80 (C48/80), a mast cell secretagogue, on adrenal cortisol secretion was investigated in dogs under pentobarbital sodium anesthesia. A marked increase in adrenal cortisol secretion was elicited by C48/80 along with a concomitant increase in the plasma levels of cortisol and immunoreactive ACTH, but neither arterial blood pressure and heart rate nor the plasma histamine level altered significantly. Pretreatment with either anti-CRF antiserum or pyrilamine maleate (H(1) histamine-receptor antagonist) significantly attenuated the C48/80-evoked increase in cortisol secretion, but pretreatment with metiamide (H(2)-receptor antagonist) significantly potentiated it. Significant attenuation of the C48/80-evoked increase in cortisol also occurred in dogs given ketotifen, a mast cell stabilizing drug, before pharmacologic challenge. In the pars tuberalis and median eminence (ME), mast cells were highly concentrated in close association with the primary plexus of the hypophysial portal system. Degranulated mast cells were extensively found in the ME of C48/80-treated animals. These results suggest that mast cells located in these regions liberated histamine within the brain as a result of degranulation induced by C48/80 and that this led to activation of the hypothalamic-pituitary-adrenocortical axis.

  14. Hypothalamic L-Histidine Decarboxylase Is Up-Regulated During Chronic REM Sleep Deprivation of Rats (United States)

    Hoffman, Gloria E.; Koban, Michael


    A competition of neurobehavioral drives of sleep and wakefulness occurs during sleep deprivation. When enforced chronically, subjects must remain awake. This study examines histaminergic neurons of the tuberomammillary nucleus of the posterior hypothalamus in response to enforced wakefulness in rats. We tested the hypothesis that the rate-limiting enzyme for histamine biosynthesis, L-histidine decarboxylase (HDC), would be up-regulated during chronic rapid eye movement sleep deprivation (REM-SD) because histamine plays a major role in maintaining wakefulness. Archived brain tissues of male Sprague Dawley rats from a previous study were used. Rats had been subjected to REM-SD by the flowerpot paradigm for 5, 10, or 15 days. For immunocytochemistry, rats were transcardially perfused with acrolein-paraformaldehyde for immunodetection of L-HDC; separate controls used carbodiimide-paraformaldehyde for immunodetection of histamine. Immunolocalization of histamine within the tuberomammillary nucleus was validated using carbodiimide. Because HDC antiserum has cross-reactivity with other decarboxylases at high antibody concentrations, titrations localized L-HDC to only tuberomammillary nucleus at a dilution of ≥ 1:300,000. REM-SD increased immunoreactive HDC by day 5 and it remained elevated in both dorsal and ventral aspects of the tuberomammillary complex. Our results suggest that up-regulation of L-HDC within the tuberomammillary complex during chronic REM-SD may be responsible for maintaining wakefulness. PMID:27997552

  15. Development of plasma 21-deoxycortisol radioimmunoassay and application to the diagnosis of patients with 21-hydroxylase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Milewicz, A.; Vecsei, P.; Korth-Schuetz, S.; Haack, D.; Lichtwald, K.; Lewicka, S. (Heidelberg Univ. (Germany, F.R.)); Roesler, A. (Hadassah Medical School, Jerusalem (Israel)); Mittelstaedt, G.V. (Heidelberg Univ. (Germany, F.R.). Dept of Nephrology, Medical Faculty)


    Specific 21-deoxycortisol (21-DF) antiserum was raised in rabbits using a 21-DF-3,20-oxime-bovine serum albumin complex. Plasma radioimmunoassay of 21-DF was developed and used together with a radioimmunoassay of 17-hydroxyprogesterone (17-OH-P) for diagnosis of patients with 21-hydroxylase deficiency of congenital and postpubertal forms. The response of plasma 21-DF and 17-OH-P to iv.v. ACTH (25 IU) was studied in 15 adult controls and compared to 8 women with the late onset form of 21-hydroxylase deficiency and 23 women with idiopathic hirsutism. Normal 21-DF values for women were 6.9 +- 3.6 ng/dl and for men 9.71 +- 2.73 ng/dl. Newborn children (age: 3-10 days) had a value of 8.3 +- 4.8 ng/dl. During the menstrual cycle the 21-DF values did not change. The baseline and post-stimulated concentrations of hormone were similar in controls and women with hirsutism but significantly higher in women with the late onset form of 21-hydroxylase deficiency. In the congenital form of 21-hydroxylase deficiency, 21-DF values (baseline) were high. In general, the 21-DF and 17-OH-P values have shown parallel changes. However, one case of 21-hydroxylase deficiency with elevated 21-DF but normal 17-OH-P was observed. The use of 21-DF for the diagnosis of 21-hydroxylase deficiency is suggested.

  16. The involvement of luteinizing hormone (LH) and Pregnancy-Associated Glycoprotein family (PAG) in pregnancy maintenance in the pig. (United States)

    Panasiewicz, Grzegorz; Majewska, Marta; Szafrańska, Bozena


    The paper presents the effect of in vivo immuno-neutralization of porcine luteinizing hormone (pLH) by species-homologous porcine antiserum (anti-pLH) administrations on pregnancy maintenance and immunodetection of the PAG proteins in precipitated plasma proteins of pregnant gilts. Pregnant gilts were passively immunized with 100 ml of porcine anti-pLH (titer 1:10 000) by multiple intravenous infusions performed from 37(th) to 42(nd) day post coitum (dpc; 12-h intervals). Blood samples of pregnant gilts were taken 12 times daily from 35 until 50 dpc. Concentrations of progesterone (P(4)) and pLH were determined by radioimmunoassays in systemic blood plasma of treated gilts and control pregnant gilts. The immuno-neutralization of peripheral pLH with the use of homologous anti-pLH serum resulted in a significant reduction (ppLH) did not affect the pregnancy maintenance. Thus, the maintenance of mid-pregnancy in gilts may depend also on other than LH luteotrophic factors. In addition, Western analysis of precipitated plasma proteins of pregnant pigs suggests a role of the PAG family during pregnancy in the pig.

  17. A cyanobacterial protein with similarity to phytochelatin synthases catalyzes the conversion of glutathione to gamma-glutamylcysteine and lacks phytochelatin synthase activity. (United States)

    Harada, Emiko; von Roepenack-Lahaye, Edda; Clemens, Stephan


    Phytochelatins are glutathione-derived, non-translationally synthesized peptides essential for cadmium and arsenic detoxification in plant, fungal and nematode model systems. Recent sequencing programs have revealed the existence of phytochelatin synthase-related genes in a wide range of organisms that have not been reported yet to produce phytochelatins. Among those are several cyanobacteria. We have studied one of the encoded proteins (alr0975 from Nostoc sp. strain PCC 7120) and demonstrate here that it does not possess phytochelatin synthase activity. Instead, this protein catalyzes the conversion of glutathione to gamma-glutamylcysteine. The thiol spectrum of yeast cells expressing alr0975 shows the disappearance of glutathione and the formation of a compound that by LC-MSMS analysis was unequivocally identified as gamma-glutamylcysteine. Purified recombinant protein catalyzes the respective reaction. Unlike phytochelatin synthesis, the conversion of glutathione to gamma-glutamylcysteine is not dependent on activation by metal cations. No evidence was found for the accumulation of phytochelatins in cyanobacteria even after prolonged exposure to toxic Cd2+ concentrations. Expression of alr0975 was detected in Nostoc sp. cells with an antiserum raised against the protein. No indication for a responsiveness of expression to toxic metal exposure was found. Taken together, these data provide further evidence for possible additional functions of phytochelatin synthase-related proteins in glutathione metabolism and provide a lead as to the evolutionary history of phytochelatin synthesis.

  18. Nalmefene: radioimmunoassay for a new opioid antagonist. (United States)

    Dixon, R; Hsiao, J; Taaffe, W; Hahn, E; Tuttle, R


    A specific radioimmunoassay (RIA) has been developed for the quantitation of a new opioid antagonist, nalmefene, in human plasma. The method employs a rabbit antiserum to an albumin conjugate of naltrexone-6-(O-carboxymethyl)oxime and [3H]naltrexone as the radioligand. Assay specificity was achieved by extraction of nalmefene from plasma at pH 9 into ether prior to RIA. The procedure has a limit of sensitivity of 0.2 ng/mL of nalmefene using a 0.5-mL sample of plasma for analysis. The intra- and interassay coefficients of variation did not exceed 5.6 and 11%, respectively. The specificity of the RIA was established by demonstrating excellent agreement (r = 0.99) with a less sensitive and more time consuming HPLC procedure in the analysis of clinical plasma samples. The use of the RIA for the pharmacokinetic evaluation of nalmefene is illustrated with plasma concentration profiles of the drug in humans following intravenous and oral administration.

  19. HLA-DR-positive cells in large plaque (atrophic) parapsoriasis. (United States)

    McMillan, E M; Wasik, R; Everett, M A


    The development of a monoclonal antibody directed against HLA DR (Ia-like) antigens of B cells and monocytes but not against normal peripheral human T cells suggested that this antibody might be used as a marker of B cells and monocytes in tissue sections. The T cell nature of large plaque (atrophic) parapsoriasis has recently been demonstrated by the immunoperoxidase technic. Immunoperoxidase examination of serial sections of tissues from two cases of large plaque parapsoriasis with one T cell antiserum, two monoclonal T cell antibodies, and one monoclonal reagent directed against HLA DR indicated that T cells in the cutaneous infiltrates were also HLA DR-positive. Evidence is accumulating that HLA DR positivity may be expressed by activated T cells. The findings here therefore suggest that many of the T lymphoid cells in two cases of large plaque (atrophic) parapsoriasis examined were activated in nature, and that HLA DR may not be a specific marker for B cells and monocytes in certain pathologic conditions. Caution should therefore presently be exercised in attempting to use this marker for the specific identification of B cells and monocytes in pathologic specimens, without simultaneous testing for T cell markers.

  20. New sensitive direct radioimmunoassay for human plasma renin and its clinical application

    Energy Technology Data Exchange (ETDEWEB)

    Higaki, J.; Ogihara, T.; Imai, N.; Kumahara, Y.; Hontani, S.; Nishiura, M.; Ogawa, H.; Hirose, S.; Murakami, K.


    A new sensitive direct radioimmunoassay for human plasma renin has been developed. Renin was purified from Haas' preparation utilizing a pepstatin-C/sub 6/-Sepharose affinity chromatography. Antiserum, prepared by immunizing rabbits with the purified renin, was used for the direct radioimmunoassay at a final dilution of 1:30,000. The antibody was specific for human renal and plasma renin, but did not cross-react with cathepsin D, trypsin, or renins of mouse, dog, and rat. Radioimmunoassay was performed by the double antibody technique using the delayed tracer addition method. In this method, a standard curve was obtained over a range from 0.2 to 8.0 ng/ml. The values from this assay correlated well with total renin activity measured as the generation rate of angiotensin I after trypsin activation, but correlated weakly with active renin activity. This finding disclosed that both active and inactive renin were detected by this method. In normal participants, plasma renin concentration determined by direct radioimmunoassay was increased by standing and furosemide injection. The plasma renin concentration determined by direct radioimmunoassay of patients with essential hypertension was not significantly different from values in normal controls. The values were higher in patients with renovascular hypertension, malignant hypertension and Bartter's syndrome, but lower in patients with primary aldosteronism than in normal controls. 20 references, 7 figures.