Sample records for aminopterin

  1. Teratogenicity studies with methotrexate, aminopterin, and acetylsalicylic acid in domestic cats. (United States)

    Khera, K S


    Pregnancy was timed in cats following induced ovulation. Methotrexate, (0.5 mg/kg), aminopterin, (0.1 mg/kg), and acetylsalicylic acid, (25 or 50 mg/kg) were administered orally in gelatin capsules in single daily doses on different days of gestation, methotrexate (MTX) on days 11-14, 14-17, or 17-20, aminopterin on day 12, 14, or 16, and acetylsalicylic acid (ASA) on days 10-15 or 15-20. Maternal toxicity was produced only by MTX. MTX given on days 11-14 and 14-17 produced high frequencies of malformations including umbilical hernia. Aminopterin caused no conclusive teratogenic response. An overall increased frequency of anomalies occurred after 50 mg/kg ASA but no single anomaly predominated.

  2. Screening for inhibitors of dihydrofolate reductase using pulsed ultrafiltration mass spectrometry. (United States)

    Nikolic, D; van Breemen, R B


    A method of screening combinatorial libraries for inhibitors of eukaryotic dihydrofolate reductase has been developed using pulsed ultra-filtration electrospray mass spectrometry, which is a continuous-flow affinity separation system for extracting and identifying high affinity ligands in combinatorial libraries. In this application, pulsed ultrafiltration conditions were optimized for the isolation and identification of inhibitors of dihydrofolate reductase from a 22 compound library containing six known inhibitors of the enzyme including trimethoprim, aminopterin, methotrexate, pyrimethamine, folic acid, and folinic acid, and 16 compounds without known affinity. In order to optimize the screening method, sources of non-specific binding were identified and minimized. A significant source of non-specific binding for this set of library compounds was hydrophobic interaction with the surfaces of the ultrafiltration chamber. After affinity separation of bound (high affinity) versus free (low affinity) library compounds during pulsed ultrafiltration, receptor-bound ligands were released and eluted using either organic solvent or acidified mobile phase. Although 80% methanol easily disrupted the receptor-ligand complexes, organic solvent had the undesirable effect of releasing non-specifically bound compounds from the chamber and thereby increasing the background noise. Interference from non-specific binding was minimized by releasing bound ligands using a low pH mobile phase eluent instead of organic solvent. Under the conditions used, pulsed ultrafiltration mass spectrometry selectively identified the two library compounds with the highest affinity for dihydrofolate reductase, methotrexate and aminopterin.

  3. Comparison of Substrate Specificity of Escherichia Coli p-Aminobenzoyl-Glutamate Hydrolase with Pseudomonas Carboxypeptidase G (United States)

    Larimer, Cassandra M.; Slavnic, Dejan; Pitstick, Lenore D.; Green, Jacalyn M.


    Reduced folic acid derivatives support biosynthesis of DNA, RNA and amino acids in bacteria as well as in eukaryotes, including humans. While the genes and steps for bacterial folic acid synthesis are known, those associated with folic acid catabolism are not well understood. A folate catabolite found in both humans and bacteria is p-aminobenzoyl-glutamate (PABA-GLU). The enzyme p-aminobenzoyl-glutamate hydrolase (PGH) breaks down PABA-GLU and is part of an apparent operon, the abg region, in E. coli. The subunits of PGH possess sequence and catalytic similarities to carboxypeptidase enzymes from Pseudomonas species. A comparison of the subunit sequences and activity of PGH, relative to carboxypeptidase enzymes, may lead to a better understanding of bacterial physiology and pathway evolution. We first compared the amino acid sequences of AbgA, AbgB and carboxypeptidase G2 from Pseudomonas sp. RS-16, which has been crystallized. Then we compared the enzyme activities of E. coli PGH and commercially available Pseudomonas carboxypeptidase G using spectrophotometric assays measuring cleavage of PABA-GLU, folate, aminopterin, methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate. The Km and Vmax values for the folate and anti-folate substrates of PGH could not be determined, because the instrument reached its limit before the enzyme was saturated. Therefore, activity of PGH was compared to the activity of CPG, or normalized to PABA-GLU (nmole/min/µg). Relative to its activity with 10 µM PABA-GLU (100%), PGH cleaved glutamate from methotrexate (48%), aminopterin (45%) and folate (9%). Reduced folates leucovorin (5-formyltetrahydrofolate) and 5-methyltetrahydrofolate were not cleaved by PGH. Our data suggest that E. coli PGH is specific for PABA-GLU as its activity with natural folates (folate, 5-methyltetrahydrofolate, and leucovorin) was very poor. It does, however, have some ability to cleave anti-folates which may have clinical applications in

  4. Methotrexate use in rheumatoid arthritis. A Clinician's perspective. (United States)

    Alarcón, G S


    Aminopterine, a precursor of methotrexate (MTX), was first used for the treatment of rheumatoid arthritis (RA) in 1951 [Gubner, R., 1951. Therapeutic suppression of tissue reactivity: I. Comparison of the effects of cortisone and aminopterin. Am. J. Med. Sci. 221, 169-175; Gubner, R., August, S., Ginsberg, V., 1951. Therapeutic suppression of tissue reactivity: II. Effect of aminopterin in rheumatoid arthritis and psoriasis. Am. J. Med. Sci. 221, 176-182.]. Corticosteroids, and to some extent cyclophosphamide, took MTX out of the rheumatologist's armamentarium until the late 1970s-early 1980s when the toxic profile of these compounds became apparent. By the mid 1980s, four randomized clinical trials (RCTs) had proven beyond doubt the beneficial effects of MTX when administered to patients with established disease who had failed to respond to other compounds such as gold salts and D-penicillamine [Thompson, R.N., Watts, C., Edelman, J., Esdaile, J., and Russell, A.S., 1984. A controlled two-centre trial of parenteral methotrexate therapy for refractory rheumatoid arthritis. J. Rheumatol. 11, 760-763; Andersen, P.A., West, S.G., O'Dell, J.R., Via, C.S., Claypool, R.G., and Kotzin, B.L., 1985. Weekly pulse methotrexate in rheumatoid arthritis. Clinical and immunologic effects in a randomized, double-blind study. Ann. Intern. Med. 103, 489-496; Weinblatt, M.E., Coblyn, J.S., Fox, D.A., Fraser, P.A., Holdsworth, D.E., Glass, D.N., and Trentham, D.E., 1985. Efficacy of low-dose methotrexate in rheumatoid arthritis. N. Engl. J. Med. 312, 818-822; Williams, H.J., Willkens, R.F., Samuelson, C.O.J., Alarcón, G.S., Guttadauria, M., Yarboro, C., Polisson, R.P., Weiner, S.R., Luggen, M.E., Billingsley, L.M., Dahl, S.L., Egger, M.J., Reading, J.C., and Ward, J.R., 1985. Comparison of low-dose oral pulse methotrexate and placebo in the treatment of rheumatoid arthritis. A controlled clinical trial. Arthritis Rheum. 28, 721-730.]. Subsequently, these four trials were included in

  5. Biochemical transformation of deoxythymidine kinase-deficient mouse cells with uv-irradiated equine herpesvirus type 1

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    Allen, G.P.; McGowan, J.J.; Gentry, G.A.; Randall, C.C.


    A line of 3T3 mouse cells lacking deoxythymidine kinase (dTK/sup -/) was stably transformed to the dTK/sup +/ phenotype after exposure to uv-irradiated equine herpesvirus type 1 (EHV-1). Biochemical transformants were isolated in a system selective for the dTK/sup +/ phenotype (Eagle minimal essential medium containing 10/sup -4/ M hypoxanthine, 6 x 10/sup -7/ M aminopterin, and 2 x 10/sup -5/ M deoxythymidine). Transformation was accompanied by the acquisition of a dTK activity with immunological, electrophoretic, and biochemical characteristics identical to those of the dTK induced by EHV-1 during productive infection. The transformed cells have been maintained in selective culture medium for more than 50 passages and have retained the capacity to express EHV-1-specific antigens. Spontaneous release of infectious virus has not been detected in the transformed lines, and the cells were not oncogenic for athymic nude mice. In contrast to normal dTK/sup +/ 3T3 cells, EHV-1 transformants were unable to grow in the presence of arabinosylthymine, a drug selectively phosphorylated by herpesvirus-coded dTK's. These results indicate that a portion of the EHV-1 genome is able to persist in the transformed cells for many generations and be expressed as an enzymatically active viral gene product.

  6. Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition. (United States)

    Wibowo, Ardian S; Singh, Mirage; Reeder, Kristen M; Carter, Joshua J; Kovach, Alexander R; Meng, Wuyi; Ratnam, Manohar; Zhang, Faming; Dann, Charles E


    Antifolates, folate analogs that inhibit vitamin B9 (folic acid)-using cellular enzymes, have been used over several decades for the treatment of cancer and inflammatory diseases. Cellular uptake of the antifolates in clinical use occurs primarily via widely expressed facilitative membrane transporters. More recently, human folate receptors (FRs), high affinity receptors that transport folate via endocytosis, have been proposed as targets for the specific delivery of new classes of antifolates or folate conjugates to tumors or sites of inflammation. The development of specific, FR-targeted antifolates would be accelerated if additional biophysical data, particularly structural models of the receptors, were available. Here we describe six distinct crystallographic models that provide insight into biological trafficking of FRs and distinct binding modes of folate and antifolates to these receptors. From comparison of the structures, we delineate discrete structural conformations representative of key stages in the endocytic trafficking of FRs and propose models for pH-dependent conformational changes. Additionally, we describe the molecular details of human FR in complex with three clinically prevalent antifolates, pemetrexed (also Alimta), aminopterin, and methotrexate. On the whole, our data form the basis for rapid design and implementation of unique, FR-targeted, folate-based drugs for the treatment of cancer and inflammatory diseases.

  7. Preparation and Preliminary Biological Evaluation of {sup 177}Lu-DOTA folate as Potential Folate Receptor Targeting Therapeutic Agent

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    Choi, Kang-Hyuk; Hong, Young-Don; Pyun, Mi-Sun; Lee, So-Young; Felipe, Fenelope; Yoon, Sun-Ha; Choi, Sun-Ju [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)


    Folic Acid (FA) and FA derivatives are overexpressed on several tumor cells. The cell-membrane folic acid receptors are known to be responsible for the cellular accumulation of FA and FA analogs, such as methotrexate and folic acid. Folate has been characterized to have high affinity for the folate-receptor positive cells and tissues and considered to be useful as diagnostic imaging and therapeutic agent. In 1940s, Folate analogue, aminopterin, was first used for treatment of leukemia and recently, many folate derivatives were tried for cancer-treatment agent as well as visualization of folate receptor. Many researchers tried to conjugate folic acid with macromolecules or low molecular weight chelators through its alpha or gamma carboxylate. However, despite the reduced binding affinity, FAs are still recognized by the folate receptor. Therefore, we focused to develop folate-based radiopharmaceutical that has the potential to be used as a therapeutic agent. We report here the synthesis and the radiolabeling of {sup 177}Lu-DOTA as well as the biodistribution data of our developed compound.

  8. Genetic stability of murine pluripotent and somatic hybrid cells may be affected by conditions of their cultivation. (United States)

    Ivanovna, Shramova Elena; Alekseevich, Larionov Oleg; Mikhailovich, Khodarovich Yurii; Vladimirovna, Zatsepina Olga


    Using mouse pluripotent teratocarcinoma PCC4azal cells and proliferating spleen lymphocytes we obtained a new type of hybrids, in which marker lymphocyte genes were suppressed, but expression the Oct-4 gene was not effected; the hybrid cells were able to differentiate to cardiomyocytes. In order to specify the environmental factors which may affect the genetic stability and other hybrid properties, we analyzed the total chromosome number and differentiation potencies of hybrids respectively to conditions of their cultivation. Particular attention was paid to the number and transcription activity of chromosomal nucleolus organizing regions (NORs), which harbor the most actively transcribed - ribosomal - genes. The results showed that the hybrids obtained are characterized by a relatively stable chromosome number which diminished less than in 5% during 27 passages. However, a long-term cultivation of hybrid cells in non-selective conditions resulted in preferential elimination of some NO- chromosomes, whereas the number of active NORs per cell was increased due to activation of latent NORs. On the contrary, in selective conditions, i.e. in the presence of hypoxantine, aminopterin and thymidine, the total number of NOR-bearing chromosomes was not changed, but a partial inactivation of remaining NORs was observed. The higher number of active NORs directly correlated with the capability of hybrid cells for differentiation to cardiomyocytes.

  9. Characterization of an amber suppressor in Pneumococcus. (United States)

    Gasc, A M; Vacher, J; Buckingham, R; Sicard, A M


    Partial revertant has been isolated, with resistance to aminopterin intermediate between wild type and mutant. This phenotype is the result of a mutation at a gene unlinked to the amiA locus. This suppressor mutation (su+) has no phenotypic characteristics by itself except a slow growth. 9 amiA mutants (belonging to 6 sites) are affected by su+ out of the 30 investigated mutants (i.e. 22 sites). The efficiency of suppression is site dependent. Two sites out of 14 mutants belonging to the thymidylate synthetase gene are suppressible. Thymidylate synthetase activity is partially restored by su+. Optochin mutants can also be suppressed. Thus su+ is not gene specific but site specific. Moreover when the str-41 allele conferring resistance to streptomycin is introduced by transformation, the suppression effect is restricted. All these properties are characteristic of an informational suppressor. The t-RNA extracted from the suppressor strain su+ but not the wild type restored the synthesis of coat protein coded by RNA from an amber mutant of bacteriophage f2. Attempts to detect ochre suppression activity gave negative results. It is suggested that the su+ gene is amber specific. Thus su+ can provide insight into the nature of suppressible mutations which should be point mutations. Both low efficiency and high efficiency mutants are affected by su+; this is additional evidence that both categories contain point mutations.

  10. Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus

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    L.C. Kreutz


    Full Text Available Three Brazilian isolates of bovine viral diarrhea virus (BVDV, antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs. Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11, were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid and 1:100 (hybridoma culture supernatant in IFA and immunoperoxidase (IPX staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes.

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

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    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.

  12. An anti-hapten camelid antibody reveals a cryptic binding site with significant energetic contributions from a nonhypervariable loop

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    Fanning, Sean W.; Horn, James R. (NIU)


    Conventional anti-hapten antibodies typically bind low-molecular weight compounds (haptens) in the crevice between the variable heavy and light chains. Conversely, heavy chain-only camelid antibodies, which lack a light chain, must rely entirely on a single variable domain to recognize haptens. While several anti-hapten VHHs have been generated, little is known regarding the underlying structural and thermodynamic basis for hapten recognition. Here, an anti-methotrexate VHH (anti-MTX VHH) was generated using grafting methods whereby the three complementarity determining regions (CDRs) were inserted onto an existing VHH framework. Thermodynamic analysis of the anti-MTX VHH CDR1-3 Graft revealed a micromolar binding affinity, while the crystal structure of the complex revealed a somewhat surprising noncanonical binding site which involved MTX tunneling under the CDR1 loop. Due to the close proximity of MTX to CDR4, a nonhypervariable loop, the CDR4 loop sequence was subsequently introduced into the CDR1-3 graft, which resulted in a dramatic 1000-fold increase in the binding affinity. Crystal structure analysis of both the free and complex anti-MTX CDR1-4 graft revealed CDR4 plays a significant role in both intermolecular contacts and binding site conformation that appear to contribute toward high affinity binding. Additionally, the anti-MTX VHH possessed relatively high specificity for MTX over closely related compounds aminopterin and folate, demonstrating that VHH domains are capable of binding low-molecular weight ligands with high affinity and specificity, despite their reduced interface.

  13. Generation of human hybridomas producing migration inhibitory factor (MIF) and of murine hybridomas secreting monoclonal antibodies to human MIF. (United States)

    Weiser, W Y; Remold, H G; David, J R


    Human T-cell hybridomas were established by hybridization of concanavalin A (Con A)-stimulated human peripheral blood T lymphocytes with cells from a 6-thioguanine-resistant, aminopterin-sensitive mutant line designated CEM-WH4, derived from the continuously growing human T cell line, CEM. High levels of MIF activity were demonstrated in the supernatants of two hybridoma lines, T-CEMA and T-CEMB but not of CEM-WH4 when stimulated with phorbol myristate acetate and phytohemagglutinin. In comparison, MIF derived from Con A-stimulated peripheral blood mononuclear cells showed 100 times less activity. Upon isoelectrofocusing, MIF activity of T-CEMB was found exclusively between pH 4.6 and 5.3 whereas MIF derived from T-CEMA showed heterogeneity with a major peak of MIF recovered at pH 4.6-5.3 and a minor peak at pH 2.4-3.3. These molecules, however, were all found to have an apparent MW of 68,000 and were resistant to trypsin. Most of these characteristics are in accordance with second day pH 3- and pH 5-MIF derived from peripheral blood mononuclear cells. When spleen cells from BALB/c mice immunized with T-CEMB-MIF were used to fuse with NS-1 mouse myeloma cells, nine hybridomas secreting antibodies to human MIF were obtained. Clone D112 which demonstrated the highest MIF-neutralizing activity was found to neutralize MIF derived from T-CEMA, peripheral blood mononuclear cells, and a T cell line, Mo.

  14. p53 ser249突变对小鼠EF细胞周期和凋亡的影响%Effect of p53 249 Arg to Ser mutation on cell cycle and apoptosis in mouse EF cells

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    吴智群; 李庆霞; 李臻; 于翠娟; 罗亚宁


    Objective To explore the effect of p53 249 Arg to Ser mutation on function of p53 in mouse EF cells.Methods Arg to Ser mutation was introduced into the p53 249 code by knock-in method.The ES cells with p53 249 Arg to Ser mutation were selected according to the homologues-recombination with RCR and Southern blot.The positive ES cells without a selection marker were injected into blastocysts recovered from Hprt-/-mice,which were derived from Hprt-deficient ES cells.The injected blastocysts then were implanted into pseudopregnant females.At embryonic day 14,MEFs were recovered from the embryos and cultured under the selection of HAT (0.016 mg of hypoxanthine/ml,0.01 mM aminopterin,0.0048 mg of thymidine/ml) and the cell cycle,apoptosis,and the relative protein expression were analyzed by flow cytometry and Western blot.Results 1) The EF cells with 249 Arg to Ser mutation were more resistant to the UV induced cell cycle arrest compared to the wild type EF cell (P<0.05).2) The EF cells with 249 Arg to Ser mutation were more resistant to the UV induced apoptosis compared to the wild type ES cell (P<0.05).3) The mutation did not affect the expression of p53 after UV but decrease the expression of BAX and p21 after UV.Conclusion Thep53 249 Arg to Ser mutation can disable the function of p53 but do not change the expression of p53 in EF cells.%目的 研究p53 249编码子突变对小鼠EF细胞p53功能和信号传导的影响.方法 利用基因打靶技术在小鼠ES细胞p53基因249编码子中引入点突变.使编码子249由Arg变成Ser,根据同源重组规律采用PCR或Southern方法筛选带有p53 249突变的阳性ES细胞,并通过测序确定该ES细胞的p53 249编码子已经由Arg变成Ser,然后将含突变而不含筛选标记的ES细胞微注射到从Hprt-/-小鼠收集的囊胚中,将注射过的囊胚植入假孕的雌性小鼠子宫,到第14天取小鼠胚胎EF细胞,用含HAT的培养液[DMEM含10%FCS,glutamine,antibiotics,50 mM mecaptoethanol