Sample records for aminopterin

  1. The effect of aminopterin-induced folic acid deficiency on spermatogenesis. (United States)

    Mathur, U; Datta, S L; Mathur, B B


    Folic acid deficiency was produced by injecting aminopterin into adult male albino rats, resulting in inhibition of spermatogenesis. Bone marrow smears were studied to serve as an index of folic acid deficiency; however, changes in spermatogenesis were noticed earlier than the bone marrow changes. Meiotic division was affected more than mitotic division. The nuclei of spermatogenic cells showed degenerative changes. Chromosomal abnormalities, chiefly sticky chromosomes, were noticed in squashed preparations of seminiferous tubules.

  2. Synthesis and Evaluation of Hydrogen Peroxide Sensitive Prodrugs of Methotrexate and Aminopterin for the Treatment of Rheumatoid Arthritis

    DEFF Research Database (Denmark)

    Peiró Cadahía, Jorge; Bondebjerg, Jon; Hansen, Christian A.


    A series of novel hydrogen peroxide sensitive prodrugs of methotrexate (MTX) and aminopterin (AMT) were synthesized and evaluated for therapeutic efficacy in mice with collagen induced arthritis (CIA) as a model of chronic rheumatoid arthritis (RA). The prodrug strategy selected is based on ROS...... assays. Selected candidates showed moderate to good solubility, high chemical and enzymatic stability, and therapeutic efficacy comparable to the parent drugs in the CIA model. Importantly, the prodrugs displayed the expected safer toxicity profile and increased therapeutic window compared to MTX and AMT...

  3. Chromosomes of older humans are more prone to aminopterine-induced breakage

    International Nuclear Information System (INIS)

    Esposito, D.; Fassina, G.; Szabo, P.; Weksler, M.; De Angelis, P.; Siniscalco, M.; Rodgers, L.


    The authors have adopted a simplified version of the cell hybrid cotransfer method to test the hypothesis that human lymphocytes derived from elderly individuals have a higher chromosome instability. Peripheral blood lymphocytes from old male individuals and young controls were fused with a Chinese hamster cell line (CHO-YH21), yielding 10 HAT-resistant rodent-human clones from the old propositi and 22 from the young controls. Both series of hybrid clones were analyzed with respect to the retention of the enzyme glucose-6-phosphate dehydrogenase and the surface antigen MIC2 identified by monoclonal antibody 12E7, two human X chromosome-linked markers located at opposite ends of the X chromosome. Cell hybrid clones with an X chromosome from a young control retained both markers in about 70% of the cells. In contrast, cell hybrid clones with an X chromosome from an old donor retained the MIC2 marker in only 30% of their cells. Slot-blot hybridization studies have established that the observed loss of the MIC2 marker is due to loss of the coding gene, not to suppression of its expression. T lymphocytes from old donors were also found to have an LD 50 for aminopterine significantly lower than the concentration of this drug in the HAT medium used to grow the hybrids. They speculate that the higher rate of chromosomal breakage and of marker loss observed along the old-age X chromosomes could be the result of molecular scars accumulated with aging at sites of constitutive chromosomal fragility

  4. Loss and stabilization of resistance to aminopterin in mouse cell lines

    International Nuclear Information System (INIS)

    Safronov, V.V.; Kapitsa, O.S.; Sapegina, M.B.; Gorodetskii, S.I.


    Using step-wise selection, lines of mouse L-cells were obtained (clones B-82, TK-), resistance to aminopterin (AP) of which exceeds resistance of parental cells by 10 3 -5 x 10 4 times. Increased resistance is the result of amplification of the gene for dihydrofolate reductase (DHFR), which was established according to increase in enzyme activity by 15-120 times and by cytogenetic methods. Development and disappearance of resistance to AP was studied and karyological analysis of lines obtained was conducted. Two types of karylogical changes were revealed: the presence of double microchromosomes (DM) and of a marker chromosome having homogeneously staining regions (HSR). The localization of the DHFR and HSR genes was demonstrated using in situ hybridization. At early stages of the development of resistance and for a long time, an extrachromosomal localization of amplified genes in the structure of DM, which determine unstable resistance to toxin, is fundamental. The possibility was demonstrated of the long-term existence of cells in which DM and HSR are present simultaneously. Change in number of copies of the DHFR gene in lines of such cells occurs through change in the number of DM, while size and localization of HSR are constant in different conditions of cell culturing. The presence of HSR determines stable resistance to AP. Data were obtained in support of an intermediate relative stabilization of resistance, which is caused by temporary insertion of copies of the DHFR gene into other sections of chromosomes, in addition to HSR dispersed variously through the genome

  5. Syntheses and antifolate activity of 5-methyl-5-deaza analogues of aminopterin, methotrexate, folic acid, and N10-methylfolic acid. (United States)

    Piper, J R; McCaleb, G S; Montgomery, J A; Kisliuk, R L; Gaumont, Y; Sirotnak, F M


    Evidence indicating that modifications at the 5- and 10-positions of classical folic acid antimetabolites lead to compounds with favorable differential membrane transport in tumor vs. normal proliferative tissue prompted an investigation of 5-alkyl-5-deaza analogues. 2-Amino-4-methyl-3,5-pyridinedicarbonitrile, prepared by hydrogenolysis of its known 6-chloro precursor, was treated with guanidine to give 2,4-diamino-5-methylpyrido[2,3-d]pyrimidine-6-carbonitrile which was converted via the corresponding aldehyde and hydroxymethyl compound to 6-(bromomethyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine. Reductive condensation of the nitrile 8 with diethyl N-(4-amino-benzoyl)-L-glutamate followed by ester hydrolysis gave 5-methyl-5-deazaaminopterin. Treatment of 12 with formaldehyde and Na(CN)BH3 afforded 5-methyl-5-deazamethotrexate, which was also prepared from 15 and dimethyl N-[(4-methylamino)benzoyl]-L-glutamate followed by ester hydrolysis. 5-Methyl-10-ethyl-5-deazaaminopterin was similarly prepared from 15. Biological evaluation of the 5-methyl-5-deaza analogues together with previously reported 5-deazaaminopterin and 5-deazamethotrexate for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for their effect on cell growth inhibition, transport characteristics, and net accumulation of polyglutamate forms in L1210 cells revealed the analogues to have essentially the same properties as the appropriate parent compound, aminopterin or methotrexate (MTX), except that 20 and 21 were approximately 10 times more growth inhibitory than MTX. In in vivo tests against P388/0 and P388/MTX leukemia in mice, the analogues showed activity comparable to that of MTX, with the more potent 20 producing the same response in the P388/0 test as MTX but at one-fourth the dose; none showed activity against P388/MTX. Hydrolytic deamination of 12 and 20 produced 5-methyl-5-deazafolic acid and 5,10-dimethyl-5-deazafolic acid, respectively. In bacterial studies on

  6. Synthesis and antifolate activity of 5-methyl-5,10-dideaza analogues of aminopterin and folic acid and an alternative synthesis of 5,10-dideazatetrahydrofolic acid, a potent inhibitor of glycinamide ribonucleotide formyltransferase. (United States)

    Piper, J R; McCaleb, G S; Montgomery, J A; Kisliuk, R L; Gaumont, Y; Thorndike, J; Sirotnak, F M


    The title compounds were prepared in extensions of a general synthetic approach used earlier to prepare 5-alkyl-5-deaza analogues of classical antifolates. Wittig condensation of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carboxaldehyde (2a) and its 5-methyl analogue 2b with [4-(methoxycarbonyl)benzylidene] triphenylphosphorane gave 9,10-ethenyl precursors 3a and 3b. Hydrogenation (DMF, ambient, 5% Pd/C) of the 9,10-ethenyl group of 3b followed by ester hydrolysis led to 4-[2-(2,4-diamino-5-methylpyrido[2,3-d]pyrimidin-6-yl)ethyl]ben zoi c acid (5), which was converted to 5-methyl-5,10-dideazaaminopterin (6) via coupling with dimethyl L-glutamate (mixed-anhydride method using i-BuOCOCl) followed by ester hydrolysis. Standard hydrolytic deamination of 6 gave 5-methyl-5,10-dideazafolic acid (7). Intermediates 3a and 3b were converted through concomitant deamination and ester hydrolysis to 8a and 8b. Peptide coupling of 8a,b (using (EtO)2POCN) with diesters of L-glutamic acid gave intermediate esters 9a and 9b. Hydrogenation of both the 9,10 double bond and the pyrido ring of 9a and 9b (MeOH-0.1 N HCl, 3.5 atm, Pt) was followed by ester hydrolysis to give 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (11a) and the 5-methyl analogue 11b. Biological evaluation of 6, 7, 11a, and 11b for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for growth inhibition and transport characteristics toward L1210 cells revealed 6 to be less potent than methotrexate in the inhibition of DHFR and cell growth. Compounds 6, 11a, and 11b were transported into cells more efficiently than methotrexate. Growth inhibition IC50 values for 11a and 11b were 57 and 490 nM, respectively; the value for 11a is in good agreement with that previously reported (20-50 nM). In tests against other folate-utilizing enzymes, 11a and 11b were found to be inhibitors of glycinamide ribonucleotide formyltransferase (GAR formyltransferase) from one bacterial (Lactobacillus casei) and two mammalian (Manca and L1210) sources with 11a being decidedly more inhibitory than 11b. Neither 11a nor 11b inhibited aminoimidazolecarboxamide ribonucleotide formyltransferase. These results support reported evidence that 11a owes its observed antitumor activity to interference with the purine de novo pathway with the site of action being GAR formyltransferase.

  7. Induction of morphological aberrations by enzyme inhibition in Drosophila melanogaster

    NARCIS (Netherlands)

    Bos, M.; Scharloo, W.; Bijlsma, R.; de Boer, I.M.; den Hollander, J.


    Zusatz zum Futter vonDrosophila melanogaster von 5-Fluoro-2-deoxyuridin oder Aminopterin induziert überzählige Skutellar- und Dorsozentralborsten sowie gekerbte Flügel. Diese Modifikationen wurden als Konsequenz von Enzymhemmung interpretiert.

  8. Tritiated thymidine and deoxycytidine suicide of mouse hemopoietic colony forming cells (CFC)

    International Nuclear Information System (INIS)

    Uyeki, E.M.; Wierzba, K.; Bisel, T.U.


    Significant enhancement of tritiated dCyd suicide occurred when unlabelled dThd was added to cultures of mouse monocytic colony-forming cells. Incorporation experiments supported the suicide experiments in that incorporation of tritiated dCyd into DNA was significantly increased. One hundred micromolar dCyd significantly reduced the radiotoxicity of 0.3 μCi of tritiated dThd; incorporation experiments indicated a dose-related reduction in the incorporation of tritiated dThd into DNA with the addition of 1-100 μM unlabelled dCyd. The addition of 1 μM aminopterin reversed the effect of 100 μM deoxycytidine; viz., incorporation of dThd into DNA was 90% of controls. Aminopterin had a similar effect on deoxyuridine reversal of tritiated dThd incorporation into DNA. Aminopterin had no effect on the reduction of tritiated dThd incorporation into DNA due to the addition of 100 μM unlabelled thymidine. Unlabelled ribonucleosides, Urd and Cyd, did not significantly affect the suicide pattern of tritiated dThd or dCyd when they were added to CFC cultures. Unlabelled deoxyribonucleosides, dThd or dCyd, did not significantly affect the suicide pattern of either tritiated Cyd or Urd when they were added to cultures containing tritiated ribonucleosides. Unlabelled Urd or Cyd was effective in reversing the suicide due to tritiated Urd or Cyd. (author)

  9. Enteroantigen (eAg)-binding B lymphocytes in the mouse - phenotype, distribution, function and eAg-specific antibody secretion

    DEFF Research Database (Denmark)

    Venning, Freja Albjerg; Trempenau, Mette Louise; Schmidt, Esben


    cells in causing proliferation of eAg-specific T cells. In comparison with eAg- B cells, eAg+ B cells secrete a significant amount of IL-10 in vitro, suggesting an anti-inflammatory potential. Compared with wild-type B cells, B cell receptor (BCR) transgenic, hen egg lysozyme-specific B cells show...... inferior eAg binding and T cell stimulatory activity suggesting involvement of the BCR in eAg binding and processing. After activation of CD19(+) B cells by eAg and hybridization with hypoxanthine-aminopterin-thymidine (HAT) sensitive ×63 lymphoma cells followed by cloning at limiting dilution conditions...

  10. Stimulation of cytolytic T lymphocytes by azaguanine-resistant mouse tumor cells in selective hat medium

    International Nuclear Information System (INIS)

    Snick, J. van; Uyttenhove, C.; Pel, A. van; Boon, T.


    Primed syngeneic or umprimed allogeneic mouse spleen cells were stimulated with azaguanine-resistant P815 tumor cells that were killed by the addition of aminopterin to the stimulation medium. The recovery of lymphocytes and their cytolytic activity and specificity were similar to those obtained after stimulation with irradiated cells. This method conveniently replaces the inactivation of stimulatory cells by irradiation or mitomycin treatment. Moreover, it has the advantage of inactivating not only the stimulatory cells but also the tumor cells that often contaminate the spleens of tumor-bearing animals, provided these animals have been inoculated with azaguanine-resistant tumor cell mutants. (Auth.)

  11. Naegleria fowleri and N. lovaniensis: differences in sensitivity to trimethoprim and other antifolates. (United States)

    Cerva, L


    The growth of Naegleria fowleri cultures in a BCS medium was not affected either by trimethoprim at 400 micrograms/ml or by aminopterine, 3,5-diaminopterine and methotrexate at 500 micrograms/ml. N. lovaniensis propagation in the same medium was inhibited with 10 micrograms/ml of trimethoprim, 50 micrograms/ml methotrexate and 100 micrograms/ml 3,5-diaminopteridine. Aminopterine was ineffective at a concentration of 500 micrograms/ml. The inhibitory effect of trimethoprim on N. lovaniensis cultures depended on the medium composition and could be neutralized by an addition of folic or tetrahydrofolic acids and a suspension of heat-killed Enterobacter aerogenes. Thymine, thymidine, hypoxantine and 2-amino-4-hydroxy-6-(tetrahydroxybutyl)-pteridine did not have an adverse effect. Trimethoprim activity in N. fowleri cultures could not be enhanced by the addition of Triton X-100 and Polymyxine B. Cryolyzate of N. fowleri amoebae did not influence the trimethoprim inhibition of N. lovaniensis cultures. Deviation in dihydrofolatereductase chemical structure or thymine dependency seems to be the probable explanation for N. fowleri antifolate resistance.

  12. Analogues of methotrexate in rheumatoid arthritis. 1. Effects of 10-deazaaminopterin analogues on type II collagen-induced arthritis in mice. (United States)

    DeGraw, J I; Colwell, W T; Crase, J; Smith, R L; Piper, J R; Waud, W R; Sirotnak, F M


    Carbonation of the dianions (LDA) of 5-methylthiophene-2-carboxylic, 2-methylpyridine-5-carboxylic, and 3-methylpyridine-6-carboxylic acids provided the respective carboxy heteroarylacetic acids. The crude diacids were directly esterified in MeOH-HCl to afford the diesters. Alkylation of the sodio anions with ethyl iodide yielded the appropriate alpha-ethyl diesters. The anions of the various diester substrates were then alkylated by 2,4-diamino-6-(bromomethyl)-pteridine followed by ester saponification at room temperature to afford the respective 2,4-diamino-4-deoxy-10-carboxy-10-deazapteroic acids. The 10-carboxyl group was readily decarboxylated by heating in DMSO at temperatures of 110-135 degrees C to give the diamino 10-deaza heteropteroic acid intermediates. Coupling with diethyl L-glutamate followed by ester hydrolysis afforded the target aminopterins. The analogues were evaluated for antiinflammatory effect in the mouse type II collagen model. The thiophene analogue of 10-ethyl-10-deazaaminopterin was found to be an effective inhibitor in terms of reduced visual evidence of inflammation and swelling as determined by caliper measurement.

  13. Antivitamins for Medicinal Applications. (United States)

    Zelder, Felix; Sonnay, Marjorie; Prieto, Lucas


    Antivitamins represent a broad class of compounds that counteract the essential effects of vitamins. The symptoms triggered by such antinutritional factors resemble those of vitamin deficiencies, but can be successfully reversed by treating patients with the intact vitamin. Despite being undesirable for healthy organisms, the toxicities of these compounds present considerable interest for biological and medicinal purposes. Indeed, antivitamins played fundamental roles in the development of pioneering antibiotic and antiproliferative drugs, such as prontosil and aminopterin. Their development and optimisation were made possible by the study, throughout the 20th century, of the vitamins' and antivitamins' functions in metabolic processes. However, even with this thorough knowledge, commercialised antivitamin-based drugs are still nowadays limited to antagonists of vitamins B9 and K. The antivitamin field thus still needs to be explored more intensely, in view of the outstanding therapeutic success exhibited by several antivitamin-based medicines. Here we summarise historical achievements and discuss critically recent developments, opportunities and potential limitations of the antivitamin approach, with a special focus on antivitamins K, B9 and B12 . © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Cloning, recombinant expression and inhibitor profiles of dihydrofolate reductase from the Australian sheep blow fly, Lucilia cuprina. (United States)

    Kotze, A C; Bagnall, N H; Ruffell, A P; Pearson, R


    While dihydrofolate reductase (DHFR) is an important drug target in mammals, bacteria and protozoa, no inhibitors of this enzyme have been developed as commercial insecticides. We therefore examined the potential of this enzyme as a drug target in an important ectoparasite of livestock, the Australian sheep blow fly, Lucilia cuprina (Diptera: Calliphoridae) (Wiedemann). The non-specific DHFR inhibitors aminopterin and methotrexate significantly inhibited the growth of L. cuprina larvae, with IC50 values at µg levels. Trimethoprim and pyrimethamine were 5-30-fold less active. Relative IC50 values for the inhibition of recombinant L. cuprina DHFR by various inhibitors were in accordance with their relative effects on larval growth. The active-site amino acid residues of L. cuprina DHFR differed by between 34% and 50% when compared with two mammalian species, as well as two bacteria and two protozoa. There were significant charge and size differences in specific residues between the blow fly and human DHFR enzymes, notably the L. cuprina Asn21, Lys31 and Lys63 residues. This study provides bioassay evidence to highlight the potential of blow fly DHFR as an insecticide target, and describes differences in active site residues between blow flies and other organisms which could be exploited in the design of blow fly control chemicals. © 2014 The Royal Entomological Society.

  15. Molecular Characteristics and Serodiagnostic Potential of Dihydrofolate Reductase from Echinococcus granulosus. (United States)

    Song, Xingju; Hu, Dandan; Yan, Min; Wang, Yu; Wang, Ning; Gu, Xiaobin; Yang, Guangyou


    The larval stage of Echinococcus granulosus causes cystic echinococcosis (CE), a neglected tropical disease that leads to morbidity and mortality in humans and livestock worldwide. Here, we identified and characterized dihydrofolate reductase (Eg-DHFR) from E. granulosus, and evaluated its potential as a diagnostic antigen for sheep CE. Comparison between mammalian (host) DHFR and Eg-DHFR indicates that 45.7% of the 35 active site residues are different. Immunolocalisation analysis showed that native Eg-DHFR was widely distributed in all life-cycle stages of E. granulosus. Recombinant Eg-DHFR (rEg-DHFR) showed typical DHFR enzymatic parameters towards substrate, and was very sensitive to inhibition by methotrexate (IC 50  = 27.75 ± 1.03 nM) and aminopterin (IC 50  = 63.67 ± 6.76 nM). However, inhibition of DHFR exhibited little protoscolicidal effect in vitro. As there is no reliable method to monitor sheep CE, the immunogenicity of rEg-DHFR was detected, and we developed an indirect ELISA (iELISA) for CE serodiagnosis. The iELISA exhibited diagnostic specificity of 89.58%, diagnostic sensitivity of 95.83%, and the diagnostic accuracy was 91.67% compared with necropsy. Cross-reactivity assay showed analytical specificity of 85.7%. These suggest that rEg-DHFR is an effective antigen for the diagnosis of sheep CE.

  16. Targeted transgenesis at the HPRT locus: an efficient strategy to achieve tightly controlled in vivo conditional expression with the tet system. (United States)

    Palais, G; Nguyen Dinh Cat, A; Friedman, H; Panek-Huet, N; Millet, A; Tronche, F; Gellen, B; Mercadier, J-J; Peterson, A; Jaisser, F


    The tet-inducible system has been widely used to achieve conditional gene expression in genetically modified mice. To alleviate the frequent difficulties associated with recovery of relevant transgenic founders, we tested whether a controlled strategy of transgenesis would support reliable cell-specific, doxycycline (Dox)-controlled transgene expression in vivo. Taking advantage of the potent hypoxanthine-aminopterin-thymidine selection strategy and an embryonic stem (ES) cell line supporting efficient germ-line transmission, we used hypoxanthine phosphoribosyltransferase (HPRT) targeting to insert a single copy tet-inducible construct designed to allow both glucocorticoid receptor (GR) and beta-galactosidase (beta-Gal) expression. Conditional, Dox-dependent GR and beta-Gal expression was evidenced in targeted ES cells. Breeding ES-derived single copy transgenic mice with mice bearing appropriate tet transactivators resulted in beta-Gal expression both qualitatively and quantitatively similar to that observed in mice with random integration of the same construct. Interestingly, GR expression in mice was dependent on transgene orientation in the HPRT locus while embryonic stem cell expression was not. Thus, a conditional construct inserted in single copy and in predetermined orientation at the HPRT locus demonstrated a Dox-dependent gene expression phenotype in adult mice suggesting that controlled insertion of tet-inducible constructs at the HPRT locus can provide an efficient alternative strategy to reproducibly generate animal models with tetracycline-induced transgene expression.

  17. Production and Characterization of Monoclonal Antibodies Against the Dimerization Domain of Human HER2

    Directory of Open Access Journals (Sweden)



    Full Text Available Background Human Epidermal Growth factor Receptor 2 (HER2, also known as ErbB2 is a 185 kDa protein belonging to the Human Epidermal Receptor (HER family of tyrosine kinase receptors overexpressed in 20% - 30% of patients with breast cancer. Similar to other members of the HER family, HER2 glycoprotein comprises of multiple domains including an extracellular ligand-binding domain, a single transmembrane domain and a cytoplasmic domain with tyrosine kinase activity. The extracellular domain of HER2 with 632 amino acids is composed of four subdomains (I - IV; subdomains I and III form a ligand binding site, and cysteine-rich subdomains II and IV play an important role in dimerization of the receptor. Objectives In this study we aimed to produce murine Monoclonal Antibodies (MAbs with the ability of specific recognition of the HER2 dimerization arm. Materials and Methods Primarily, BALB/c mice were immunized with a 30-aminoacid peptide as a part of the human HER2 subdomain II. Splenocytes from hyperimmunized mice were fused with myeloma cells (SP2/0, selected in hypoxanthine-aminopterin-thymidine (HAT medium, and screened by indirect Enzyme-Linked Immunosorbent Assay (ELISA. Secreted MAbs were characterized according to isotypes, reactions with the native HER2 in SKBR3 cells by western blotting, and in tissue sections from HER2 positive breast cancer specimens by Immunohistochemistry (IHC. Results Isotype of 1F1 clone was determined to be IgG1, which reacted with native protein in the western blot experiment and stained 20% of the membrane of neoplastic cells overexpressing HER2 with 3+ grade. However, 3L5 clone showed a low reaction (10% with native HER2 in immunohistochemistry. Conclusions The results of both western blotting and Immunohistochemistry showed that native HER2 can be detected with 1F1 monoclonal antibody.

  18. Sandwich enzyme-linked immunosorbent assay for naringin. (United States)

    Qu, Huihua; Wang, Xueqian; Qu, Baoping; Kong, Hui; Zhang, Yue; Shan, Wenchao; Cheng, Jinjun; Wang, Qingguo; Zhao, Yan


    Among the currently used immunoassay techniques, sandwich ELISA exhibits higher specificity, lower cross-reactivity, and a wider working range compared to the corresponding competitive assays. However, it is difficult to obtain a pair of antibodies that can simultaneously bind to two epitopes of a molecule with a molecular weight of less than 1000 Da. Naringin (Nar) is a flavonoid with a molecular mass of 580 Da. The main aim of this study was to develop a sandwich ELISA for detecting Nar. Two hybridomas secreting anti-Nar monoclonal antibodies (mAbs) were produced by fusing splenocytes from a mouse immunised against Nar-bovine serum albumin (BSA) conjugated with a hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line; a sandwich ELISA for detecting Nar was developed using these two well-characterised anti-Nar mAbs. The performance of the sandwich assay was further evaluated by limit of detection (LOD), limit of quantification (LOQ), recovery, and interference analyses. A dose-response curve to Nar was obtained with an LOD of 6.78 ng mL(-1) and an LOQ of 13.47 ng mL(-1). The inter-assay and intra-assay coefficients of variation were 4.32% and 7.48%, respectively. The recovery rate of Nar from concentrated Fructus aurantii granules was 83.63%. A high correlation was obtained between HPLC and sandwich ELISA. These results demonstrate that the sandwich ELISA method has higher specificity for Nar than indirect competitive ELISA. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Xenogeneic gene expression in chimeric mice derived from rat--mouse hybrid cells. (United States)

    Illmensee, K; Croce, C M


    Thymidine kinase-deficient OTT6050 mouse teratocarcinoma cells were fused with hypoxanthine phosphoribosyltransferase-deficient Fu5AH rat hepatoma cells by means of inactivated Sendai virus. The resulting hybrid cells, which were selected in hypoxanthine/aminopterin/thymidine medium, retained almost all of the mouse chromosomes and various numbers of rat chromosomes, and showed many chromosomal rearrangements. The hybrid cells, as well as both parental lines, formed tumors after subcutaneous injection into athymic nude mice. Single rat--mouse hybrid cells from a clonally established subline were transplanted into C57BL6/J mouse blastocysts carrying many genetic markers suitable for the detection of hybrid cell-derived tissue contributions. From 144 blastocysts, each of which was injected with a hybrid cell and then surgically transferred to the uterus of a pseudopregnant foster mother, 62 adult mice developed without any visible coat mosaicism. However, three of these mice showed internal hybrid-cell participation in their livers and a limited number of organs of endomesodermal origin. A tumor classifiable as hemangio endothelioma was found in the liver, the only mosaic tissue, of one of the chimeric mice. Nine different rat-specific enzyme variants were detected in the mosaic organs. A considerable number of variations concerning the presence and quantitative activity of the foreign gene products probably resulted from chromosomal segregation, tissue-specific gene activity, or dosage compensation during differentiation in vivo. Our results demonstrate that cultured malignant rat--mouse hybrid cells differentiate normally and become functionally integrated during development. The appearacne in vivo of certain rat-specific gene products that are not found in the hybrid cells under conditions in vitro indicates differential gene expression of the introduced xenogeneic chromosomes.

  20. Establishment and characterization of mouse bone marrow-derived mast cell hybridomas

    Energy Technology Data Exchange (ETDEWEB)

    Kawahara, Takeshi, E-mail: [Integrated Department of Sciences of Functional Foods, Graduate School of Agriculture, Shinshu University, Nagano (Japan)


    Interleukin (IL)-3-dependent mouse bone marrow-derived mast cells (BMMCs) are an important model for studying the function of mucosal-type mast cells. In the present study, BMMCs were successfully immortalized by cell fusion using a hypoxanthine-aminopterin-thymidine medium-sensitive variant of P815 mouse mastocytoma (P815-6TgR) as a partner cell line. The established mouse mast cell hybridomas (MMCHs) expressed {alpha}, {beta}, and {gamma} subunits of high-affinity immunoglobulin E (IgE) receptor (Fc{epsilon}RI) and possessed cytoplasmic granules devoid of or partially filled with electron-dense material. Four independent MMCH clones continuously proliferated without supplemental exogenous IL-3 and showed a degranulation response on stimulation with IgE+antigen. Furthermore, histamine synthesis and release by degranulation were confirmed in MMCH-D5, a MMCH clone that showed the strongest degranulation response. MMCH-D5 exhibited elevated levels of IL-3, IL-4, IL-13, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF)-{alpha}, and cyclooxygenase 2, and production of prostaglandin D{sub 2} and leukotriene C{sub 4} in response to IgE-induced stimulation. MMCH clones also expressed Toll-like receptors (TLRs) 1, 2, 4, and 6 and showed elevated levels of TNF-{alpha} expression in response to stimulation with TLR2 and TLR4 ligands. The MMCHs established using this method should be suitable for studies on Fc{epsilon}RI- and TLR-mediated effector functions of mast cells.

  1. Dominant mutations confer resistance to the immunosuppressant, rapamycin, in variants of a T cell lymphoma. (United States)

    Dumont, F J; Staruch, M J; Grammer, T; Blenis, J; Kastner, C A; Rupprecht, K M


    Rapamycin (RAP) disrupts signaling events implicated in cytokine-dependent proliferation of lymphocytes and other cells. This action is known to involve the formation of molecular complexes between the drug and intracellular binding proteins, termed FKBPs. However, the biochemical target(s) for the effector RAP-FKBP complexes remain uncharacterized. As an approach to explore the mechanism of action of RAP, we have isolated three independent sets of somatic mutants of the YAC-1 murine T cell line with markedly reduced sensitivity to the drug's inhibitory effects on proliferation and on IL-1-induced IFN-gamma production. These mutants were still fully sensitive to FK-506, an immunosuppressant structurally related to RAP whose mode of action also involves an interaction with FKBPs. Furthermore, the 12-kDa FKBP, FKBP12, was detectable in immunoblots from cytosolic extracts and eluates from RAP-affinity matrix in the mutants as in wild-type cells, suggesting that the resistance to RAP in the mutants is not due to a lack of FKBP12 expression. Cell fusion experiments were conducted to further define the nature of the alterations imparting RAP resistance in these mutants. Clones deficient in either thymidine kinase or hypoxanthine-guanine phosphoribosyltransferase, suitable as fusion partners for aminopterin-based selection of hybrids were generated from the wild-type or mutant lines. In most instances, the hybrids derived from the fusion between RAP-sensitive clones and RAP-resistant clones exhibited a RAP-resistant phenotype. Similar results were obtained with hybrids between RAP-resistant YAC-1 clones and the RAP-sensitive EL-4 cell line. Therefore, the mutations that confer resistance to RAP in the present system are dominant. Altogether, our observations are consistent with a model where pharmacologically relevant targets for the RAP-FKBP complex, rather than FKBP, might be altered in the mutants such that the inactivation of these targets by the effector complex is

  2. Mammalian folylpoly-γ-glutamate synthetase. 2. Substrate specificity and kinetic properties

    International Nuclear Information System (INIS)

    Cichowicz, D.J.; Shane, B.


    The specificity of hog liver folylpolyglutamate synthetase for folate substrates and for nucleotide and L-[ 14 C]glutamate substrates and analogues has been investigated. The kinetic mechanism, determined by using aminopterin as the folate substrate, is ordered Ter-Ter with MgATP binding first, folate second, and glutamate last. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. Folate, dihydrofolate, and tetrahydrofolate possess the optimal configurations for catalysis while 5- and 10-position substitutions of the folate molecule impair catalysis. k/sub cat/ values decrease with increasing glutamate chain length, and the rate of decrease varies depending on the state of reduction and substitution of the folate molecule. Folate binding, as assessed by on rates, is slow. Dihydrofolate exhibits the fastest rate, and the rates are slightly reduced for tetrahydrofolate and 10-formyltetrahydrofolate and greatly reduced for 5-methyltetrahydrofolate and folic acid. Tetrahydrofolate polyglutamates are the only long glutamate chain length folates with detectable substrate activity. The specificity of the L-glutamate binding site is very narrow. L-Homocysteate and 4-threo-fluoroglutamate are alternate substrates and act as chain termination inhibitors in that their addition to the folate molecule prevents or severely retards the further addition of glutamate moieties. The K/sub m/ for glutamate is dependent on the folate substrate used. MgATP is the preferred nucleotide substrate, and β,γ-methylene-ATP, β,γ-imido-ATP, adenosine 5'-O-(3-thiotriphosphate), P 1 ,P 5 -di(adenosine-5') pentaphosphate, and free ATP 4- are potent inhibitors of the reaction

  3. Detailed analysis of X chromosome inactivation in a 49,XXXXX pentasomy

    Directory of Open Access Journals (Sweden)

    Menezes Albert N


    Full Text Available Abstract Background Pentasomy X (49,XXXXX has been associated with a severe clinical condition, presumably resulting from failure or disruption of X chromosome inactivation. Here we report that some human X chromosomes from a patient with 49,XXXXX pentasomy were functionally active following isolation in inter-specific (human-rodent cell hybrids. A comparison with cytogenetic and molecular findings provided evidence that more than one active X chromosome was likely to be present in the cells of this patient, accounting for her abnormal phenotype. Results 5-bromodeoxyuridine (BrdU-pulsed cultures showed different patterns among late replicating X chromosomes suggesting that their replication was asynchronic and likely to result in irregular inactivation. Genotyping of the proband and her mother identified four maternal and one paternal X chromosomes in the proband. It also identified the paternal X chromosome haplotype (P, indicating that origin of this X pentasomy resulted from two maternal, meiotic non-disjunctions. Analysis of the HUMANDREC region of the androgen receptor (AR gene in the patient's mother showed a skewed inactivation pattern, while a similar analysis in the proband showed an active paternal X chromosome and preferentially inactivated X chromosomes carrying the 173 AR allele. Analyses of 33 cell hybrid cell lines selected in medium containing hypoxanthine, aminopterin and thymidine (HAT allowed for the identification of three maternal X haplotypes (M1, M2 and MR and showed that X chromosomes with the M1, M2 and P haplotypes were functionally active. In 27 cell hybrids in which more than one X haplotype were detected, analysis of X inactivation patterns provided evidence of preferential inactivation. Conclusion Our findings indicated that 12% of X chromosomes with the M1 haplotype, 43.5% of X chromosomes with the M2 haplotype, and 100% of the paternal X chromosome (with the P haplotype were likely to be functionally active in the

  4. Initiation of protein synthesis by folate-sufficient and folate-deficient Streptococcus faecalis R: partial purification and properties of methionyl-transfer ribonucleic acid synthetase and methionyl-transfer ribonucleic acid formyltransferase. (United States)

    Samuel, C E; Rabinowitz, J C


    . Tetrahydrofolate and uncharged tRNA(f) (Met) are competitive inhibitors of both plus- and minus-folate S. faecalis formyltransferase; folic acid, pteroic acid, aminopterin, and Met-tRNA(m) (Met) are not inhibitory. These results indicate that the presence or absence of folic acid in the culture medium of S. faecalis has no apparent effect on either methionyl-tRNA synthetase or methionyl-tRNA formyltransferase, the two enzymes directly involved in the formation of formylmethionyl-tRNA(f) (Met). Therefóre, the lack of N-formylation of Met-tRNA(f) (Met) in minus-folate S. faecalis is due to the absence of the formyl donor, a 10-formyl-tetrahydropteroyl derivative. Although the general properties of S. faecalis methionyl-tRNA synthetase are similar to those of other aminoacyl-tRNA synthetases, S. faecalis methionyl-tRNA formyltransferase differs from other previously described transformylases in certain kinetic parameters.