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Sample records for human hypoxanthine phosphoribosyltransferase

  1. Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells

    SciTech Connect

    Palella, T.D.; Silverman, L.J.; Schroll, C.T.; Homa, F.L.; Levine, M.; Kelley, W.N.

    1988-01-01

    The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.

  2. Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency: Activity in Normal, Mutant, and Heterozygote-Cultured Human Skin Fibroblasts

    PubMed Central

    Fujimoto, Wilfred Y.; Seegmiller, J. Edwin

    1970-01-01

    Cultured skin fibroblasts from patients deficient for the enzyme hypoxanthine-guanine phosphoribosyltransferase (PRT) activity show very low but nevertheless significant levels of apparent PRT enzyme despite absence of detectable activity (<0.004% of normal) in erythrocytes of the same patients. In fibroblasts this mutant enzyme is more heat labile than the normal enzyme. These findings indicate that PRT deficiency in this disorder is not due to a deletion mutation of the PRT locus. Individual cultured skin fibroblasts from heterozygote females for PRT deficiency show normal, intermediate, or very low levels of PRT activity. The mosaicism demonstrated in the heterozygotes for this X-linked disorder accounts for the cells with normal and very low activities of PRT. Intermediate activity can best be explained by the phenomenon of metabolic cooperation presumably from the transfer of either PRT enzyme or messenger RNA, from normal to mutant cells. Images PMID:5267139

  3. Molecular and clonal analysis of in vivo hprt (hypoxanthine-guanine phosphoribosyl-transferase) mutations in human cells

    SciTech Connect

    Albertini, R.J.; O'Neill, J.P.; Nicklas, J.A.; Allegretta, M. . Genetics Lab.); Recio, L.; Skopek, T.R. )

    1989-08-08

    There is no longer doubt that gene mutations occur in vivo in human somatic cells, and that methods can be developed to detect, quantify and study them. Four assays are now available for such purpose; two detecting mutations that arise in bone marrow erythroid stem cells and two defining mutations that occur in T-lymphocytes. The red cell assays measure changes in mature red blood cells that involve either the blood group glycophorin-A locus or the hemoglobin loci; the lymphocyte assays score for genetic events at either the X-chromosomal hypoxanthine-guanine phosphoribosyl-transferase (hprt) locus. We describe here our attempts in studying in vivo gene mutations in human T-lymphocytes. 35 refs., 3 figs., 3 tabs.

  4. Differential Distortion of Purine Substrates by Human and Plasmodium falciparum Hypoxanthine-Guanine Phosphoribosyltransferase to Catalyse the Formation of Mononucleotides.

    PubMed

    Karnawat, Vishakha; Gogia, Spriha; Balaram, Hemalatha; Puranik, Mrinalini

    2015-07-20

    Plasmodium falciparum (Pf) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a potential therapeutic target. Compared to structurally homologous human enzymes, it has expanded substrate specificity. In this study, 9-deazapurines are used as in situ probes of the active sites of human and Pf HGPRTs. Through the use of these probes it is found that non-covalent interactions stabilise the pre-transition state of the HGPRT-catalysed reaction. Vibrational spectra reveal that the bound substrates are extensively distorted, the carbonyl bond of nucleobase moiety is weakened and the substrate is destabilised along the reaction coordinate. Raman shifts of the human and Pf enzymes are used to quantify the differing degrees of hydrogen bonding in the homologues. A decreased Raman cross-section in enzyme-bound 9-deazaguanine (9DAG) shows that the phenylalanine residue (Phe186 in human and Phe197 in Pf) of HGPRT stacks with the nucleobase. Differential loss of the Raman cross-section suggests that the active site is more compact in human HGPRT as compared to the Pf enzyme, and is more so in the phosphoribosyl pyrophosphate (PRPP) complex 9DAG-PRPP-HGPRT than in 9-deazahypoxanthine (9DAH)-PRPP-HGPRT. PMID:25944719

  5. Ubiquitous and neuronal DNA-binding proteins interact with a negative regulatory element of the human hypoxanthine phosphoribosyltransferase gene.

    PubMed Central

    Rincón-Limas, D E; Amaya-Manzanares, F; Niño-Rosales, M L; Yu, Y; Yang, T P; Patel, P I

    1995-01-01

    The hypoxanthine phosphoribosyltransferase (HPRT) gene is constitutively expressed at low levels in all tissues but at higher levels in the brain; the significance and mechanism of this differential expression are unknown. We previously identified a 182-bp element (hHPRT-NE) within the 5'-flanking region of the human HPRT (hHPRT) gene, which is involved not only in conferring neuronal specificity but also in repressing gene expression in nonneuronal tissues. Here we report that this element interacts with different nuclear proteins, some of which are present specifically in neuronal cells (complex I) and others of which are present in cells showing constitutive expression of the gene (complex II). In addition, we found that complex I factors are expressed in human NT2/D1 cells following induction of neuronal differentiation by retinoic acid. This finding correlates with an increase of HPRT gene transcription following neuronal differentiation. We also mapped the binding sites for both complexes to a 60-bp region (Ff; positions -510 to -451) which, when analyzed in transfection assays, functioned as a repressor element analogous to the full-length hHPRT-NE sequence. Methylation interference footprintings revealed a minimal unique DNA motif, 5'-GGAAGCC-3', as the binding site for nuclear proteins from both neuronal and nonneuronal sources. However, site-directed mutagenesis of the footprinted region indicated that different nucleotides are essential for the associations of these two complexes. Moreover, UV cross-linking experiments showed that both complexes are formed by the association of several different proteins. Taken together, these data suggest that differential interaction of DNA-binding factors with this regulatory element plays a crucial role in the brain-preferential expression of the gene, and they should lead to the isolation of transcriptional regulators important in neuronal expression of the HPRT gene. PMID:8524221

  6. Fine structure mapping of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene region of the human X chromosome (Xq26).

    PubMed Central

    Nicklas, J A; Hunter, T C; O'Neill, J P; Albertini, R J

    1991-01-01

    The Xq26-q27 region of the X chromosome is interesting, as an unusually large number of genes and anonymous RFLP probes have been mapped in this area. A number of studies have used classical linkage analysis in families to map this region. Here, we use mutant human T-lymphocyte clones known to be deleted for all or part of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene, to order anonymous probes known to map to Xq26. Fifty-seven T-cell clones were studied, including 44 derived from in vivo mutation and 13 from in vitro irradiated T-lymphocyte cultures. Twenty anonymous probes (DXS10, DXS11, DXS19, DXS37, DXS42, DXS51, DXS53, DXS59, DXS79, DXS86, DXS92, DXS99, DXS100d, DXS102, DXS107, DXS144, DXS172, DXS174, DXS177, and DNF1) were tested for codeletion with the hprt gene by Southern blotting methods. Five of these probes (DXS10, DXS53, DXS79, DXS86 and DXS177) showed codeletion with hprt in some mutants. The mutants established the following unambiguous ordering of the probes relative to the hprt gene: DXS53-DXS79-5'hprt3'-DXS86-DXS10-DXS177 . The centromere appears to map proximal to DXS53. These mappings order several closely linked but previously unordered probes. In addition, these studies indicate that rather large deletions of the functionally haploid X chromosome can occur while still retaining T-cell viability. Images Figure 1 PMID:1678246

  7. Absence of hypoxanthine:guanine phosphoribosyltransferase activity in murine Dunn osteosarcoma

    SciTech Connect

    Abelson, H.T.; Gorka, C.

    1983-09-01

    The transplantable murine Dunn osteosarcoma has no detectable hypoxanthine:guanine phosphoribosyltransferase (EC 2.4.2.8) activity. This was established from the tumors directly and from tissue culture cell lines derived from the tumor using a variety of assays: e.g., no (3H)hypoxanthine uptake into tumor or tissue culture cells, no conversion of (3H)hypoxanthine to (3H)IMP by cell extracts from tumors or tissue culture cells, no growth of tissue culture cells in hypoxanthine:aminopterin:thymidine medium, and normal growth of these cells in 10 microM 6-mercaptopurine. Ten human osteosarcomas have been assayed, and two have no apparent hypoxanthine:guanine phosphoribosyltransferase enzyme activity. After high-dose methotrexate treatment in vivo, murine tumors could be selectively killed and normal tissues could be spared by using a rescue regimen of hypoxanthine-thymidine-allopurinol.

  8. Kinetic mechanism of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase.

    PubMed

    Roy, Sourav; Nagappa, Lakshmeesha K; Prahladarao, Vasudeva S; Balaram, Hemalatha

    2015-12-01

    Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT) exhibits a kinetic mechanism that differs from that of the human homolog. Human HGPRT follows a steady-state ordered mechanism, wherein PRPP binding precedes the binding of hypoxanthine/guanine and release of product IMP/GMP is the rate limiting step. In the current study, initial velocity kinetics with PfHGXPRT indicates a steady-state ordered mechanism, wherein xanthine binding is conditional to the binding of PRPP. The value of the rate constant for IMP dissociation is greater by 183-fold than the kcat for hypoxanthine phosphoribosylation and this results in the absence of burst in progress curves from pre-steady-state kinetics. Further, IMP binding is 1000 times faster (4s(-1) at 0.5μM IMP) when compared to the kcat (3.9±0.2×10(-3)s(-1)) for the reverse IMP pyrophosphorolysis reaction. These results lend support to the fact that in both forward and reverse reactions, the process of chemical conversion (formation of IMP/hypoxanthine) is slow and the events of ligand association and dissociation are faster. PMID:26902413

  9. Human Adenovirus Type 2 but Not Adenovirus Type 12 Is Mutagenic at the Hypoxanthine Phosphoribosyltransferase Locus of Cloned Rat Liver Epithelial Cells

    PubMed Central

    Paraskeva, Christos; Roberts, Carl; Biggs, Paul; Gallimore, Phillip H.

    1983-01-01

    Using resistance to the base analog 8-azaguanine as a genetic marker, we showed that adenovirus type 2, but not adenovirus type 12, is mutagenic at the hypoxanthine phosphoribosyltransferase locus of cloned diploid rat liver epithelial cells. Adenovirus type 2 increased the frequency of 8-azaguanine-resistant colonies by up to ninefold over the spontaneous frequency, depending on expression time and virus dose. PMID:6572280

  10. Acyclic phosph(on)ate inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase

    PubMed Central

    Clinch, Keith; Crump, Douglas R.; Evans, Gary B.; Hazleton, Keith Z.; Mason, Jennifer M.; Schramm, Vern L.

    2013-01-01

    The pathogenic protozoa responsible for malaria lack enzymes for the de novo synthesis of purines and rely on purine salvage from the host. In Plasmodium falciparum (Pf), hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) converts hypoxanthine to inosine monophosphate and is essential for purine salvage making the enzyme an anti-malarial drug target. We have synthesized a number of simple acyclic aza-C- nucleosides and shown that some are potent inhibitors of Pf HGXPRT while showing excellent selectivity for the Pf versus the human enzyme. PMID:23810424

  11. Mosaic mice with teratocarcinoma-derived mutant cells deficient in hypoxanthine phosphoribosyltransferase.

    PubMed

    Dewey, M J; Martin, D W; Martin, G R; Mintz, B

    1977-12-01

    Mutagenized stem cells of a cultured mouse teratocarcinoma cell line were selected for resistance to the purine base analog 6-thioguanine. Cells of a resistant clone were completely deficient in activity of the enzyme hypoxanthine phosphoribosyltransferase (HPRT, IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), the same X-linked lesion as occurs in human Lesch-Nyhan disease. After microinjection into blastocysts of another genetic strain, the previously malignant cells successfully participated in normal embryogenesis and tumor-free, viable mosaic mice were obtained. Cells of tumor lineage were identified by strain markers in virtually all tissues of some individuals. Mature function of those cells was evident from their tissue-specific products (e.g., melanins, liver proteins). These mutagenized teratocarcinoma cells are therefore developmentally totipotent. Retention of the severe HPRT deficiency in the differentiated state was documented in extracts of mosaic tissues by depressed specific activity of the enzyme, and also by presence of unlabeled clones in autoradiographs of explanted cells incubated in [(3)H]hypoxanthine. Some mosaic individuals had mutant-strain cells in only one or a few tissues. Such animals may provide unique opportunities to identify the tissue sources of particular aspects of the complex disease syndrome. The tissue distribution of HPRT-deficient cells suggests that selection against them is particularly strong in blood of the mosaic mice, as is already known to be the case in human heterozygotes. This phenotypic parallelism supports the expectation that afflicted F(1) male mice that might be obtained from mutant germ cells can serve as a model of the human disease. PMID:271982

  12. Acyclic Immucillin Phosphonates. Second-Generation Inhibitors of Plasmodium falciparum Hypoxanthine- Guanine-Xanthine Phosphoribosyltransferase

    SciTech Connect

    Hazelton, Keith Z.; Ho, Meng-Chaio; Cassera, Maria B.; Clinch, Keith; Crump, Douglas R.; Rosario Jr., Irving; Merino, Emilio F.; Almo, Steve C.; Tyler, Peter C.; Schramm, Vern L.

    2012-06-22

    We found that Plasmodium falciparum is the primary cause of deaths from malaria. It is a purine auxotroph and relies on hypoxanthine salvage from the host purine pool. Purine starvation as an antimalarial target has been validated by inhibition of purine nucleoside phosphorylase. Hypoxanthine depletion kills Plasmodium falciparum in cell culture and in Aotus monkey infections. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) from P. falciparum is required for hypoxanthine salvage by forming inosine 5'-monophosphate, a branchpoint for all purine nucleotide synthesis in the parasite. We present a class of HGXPRT inhibitors, the acyclic immucillin phosphonates (AIPs), and cell permeable AIP prodrugs. The AIPs are simple, potent, selective, and biologically stable inhibitors. The AIP prodrugs block proliferation of cultured parasites by inhibiting the incorporation of hypoxanthine into the parasite nucleotide pool and validates HGXPRT as a target in malaria.

  13. Crystal structures of Apo and GMP bound hypoxanthine-guanine phosphoribosyltransferase from Legionella pneumophila and the implications in gouty arthritis.

    PubMed

    Zhang, Nannan; Gong, Xiaojian; Lu, Min; Chen, Xiaofang; Qin, Ximing; Ge, Honghua

    2016-06-01

    Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) reversibly catalyzes the transfer of the 5-phophoribosyl group from 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) to hypoxanthine or guanine to form inosine monophosphate (IMP) or guanosine monophosphate (GMP) in the purine salvage pathway. To investigate the catalytic mechanism of this enzyme in the intracellular pathogen Legionella pneumophila, we determined the crystal structures of the L. pneumophila HGPRT (LpHGPRT) both in its apo-form and in complex with GMP. The structures reveal that LpHGPRT comprises a core domain and a hood domain which are packed together to create a cavity for GMP-binding and the enzymatic catalysis. The binding of GMP induces conformational changes of the stable loop II. This new binding site is closely related to the Gout arthritis-linked human HGPRT mutation site (Ser103Arg). Finally, these structures of LpHGPRT provide insights into the catalytic mechanism of HGPRT. PMID:26968365

  14. Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases*

    PubMed Central

    Boitz, Jan M.; Ullman, Buddy

    2010-01-01

    Leishmania donovani cannot synthesize purines de novo and obligatorily scavenge purines from the host. Previously, we described a conditional lethal Δhgprt/Δxprt mutant of L. donovani (Boitz, J. M., and Ullman, B. (2006) J. Biol. Chem. 281, 16084–16089) that establishes that L. donovani salvages purines primarily through hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT). Unlike wild type L. donovani, the Δhgprt/Δxprt knock-out cannot grow on 6-oxypurines and displays an absolute requirement for adenine or adenosine and 2′-deoxycoformycin, an inhibitor of parasite adenine aminohydrolase activity. Here, we demonstrate that the ability of Δhgprt/Δxprt parasites to infect mice was profoundly compromised. Surprisingly, mutant parasites that survived the initial passage through mice partially regained their virulence properties, exhibiting a >10-fold increase in parasite burden in a subsequent mouse infection. To dissect the mechanism by which Δhgprt/Δxprt parasites persisted in vivo, suppressor strains that had regained their capacity to grow under restrictive conditions were cloned from cultured Δhgprt/Δxprt parasites. The ability of these suppressor clones to grow in and metabolize 6-oxypurines could be ascribed to a marked amplification and overexpression of the adenine phosphoribosyltransferase (APRT) gene. Moreover, transfection of Δhgprt/Δxprt cells with an APRT episome recapitulated the suppressor phenotype in vitro and enabled growth on 6-oxypurines. Biochemical studies further showed that hypoxanthine, unexpectedly, was an inefficient substrate for APRT, evidence that could account for the ability of the suppressors to metabolize hypoxanthine. Subsequent analysis implied that APRT amplification was also a potential contributory mechanism by which Δhgprt/Δxprt parasites displayed persistence and increased virulence in mice. PMID:20363738

  15. Nicotinamide Phosphoribosyltransferase in Human Diseases

    PubMed Central

    Zhang, Li Qin; Heruth, Daniel P.; Ye, Shui Qing

    2011-01-01

    Nicotinamide phosphoribosyltransferase (NAMPT) was first reported as a pre-B-cell colony enhancing factor in 1994 with little notice, but it has received increasing attention in recent years due to accumulating evidence indicating that NAMPT is a pleiotropic protein such as a growth factor, a cytokine, an enzyme and a visfatin. Now, NAMPT has been accepted as an official name of this protein. Because of NAMPT’s multiple functions in a variety of physiological processes, their dysregulations have been implicated in the pathogenesis of a number of human diseases or conditions such as acute lung injury, aging, atherosclerosis, cancer, diabetes, rheumatoid arthritis and sepsis. This review will cover the current understanding of NAMPT’s structure and functions with an emphasis on recent progress of nicotinamide phosphoribosyltransferase’s pathological roles in various human diseases and conditions. Future directions on exploring its Terra incognita will be offered in the end. PMID:22140607

  16. The Housekeeping Gene Hypoxanthine Guanine Phosphoribosyltransferase (HPRT) Regulates Multiple Developmental and Metabolic Pathways of Murine Embryonic Stem Cell Neuronal Differentiation

    PubMed Central

    Bader, Joel S.; Friedmann, Theodore

    2013-01-01

    The mechanisms by which mutations of the purinergic housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT) cause the severe neurodevelopmental Lesch Nyhan Disease (LND) are poorly understood. The best recognized neural consequences of HPRT deficiency are defective basal ganglia expression of the neurotransmitter dopamine (DA) and aberrant DA neuronal function. We have reported that HPRT deficiency leads to dysregulated expression of multiple DA-related developmental functions and cellular signaling defects in a variety of HPRT-deficient cells, including human induced pluripotent stem (iPS) cells. We now describe results of gene expression studies during neuronal differentiation of HPRT-deficient murine ESD3 embryonic stem cells and report that HPRT knockdown causes a marked switch from neuronal to glial gene expression and dysregulates expression of Sox2 and its regulator, genes vital for stem cell pluripotency and for the neuronal/glial cell fate decision. In addition, HPRT deficiency dysregulates many cellular functions controlling cell cycle and proliferation mechanisms, RNA metabolism, DNA replication and repair, replication stress, lysosome function, membrane trafficking, signaling pathway for platelet activation (SPPA) multiple neurotransmission systems and sphingolipid, sulfur and glycan metabolism. We propose that the neural aberrations of HPRT deficiency result from combinatorial effects of these multi-system metabolic errors. Since some of these aberrations are also found in forms of Alzheimer's and Huntington's disease, we predict that some of these systems defects play similar neuropathogenic roles in diverse neurodevelopmental and neurodegenerative diseases in common and may therefore provide new experimental opportunities for clarifying pathogenesis and for devising new potential therapeutic targets in developmental and genetic disease. PMID:24130677

  17. Cotransfer of linked eukaryotic genes and efficient transfer of hypoxanthine phosphoribosyltransferase by DNA-mediated gene transfer.

    PubMed Central

    Peterson, J L; McBride, O W

    1980-01-01

    The efficiency of DNA-mediated transfer of the gene (hprt) for hypoxanthine phosphoribosyltransferase (HPRT; IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) is dependent upon the recipient cell used. hprt has been transferred into mouse TG8 or Chinese hamster CHTG49 cells at a high frequency, similar to the frequency of the gene (tk) for thymidine kinase (TK; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) transfer into mouse LMTK- cells (i.e., 10(-6)). In contrast, the frequency of transfer of hprt into mouse A9 cells was about two orders of magnitude less. The identification of efficient recipient cells for hprt transfer permits the use of DNA-mediated transfer as a bioassay for the gene. Cotransfer of the linked tk gene and the gene (galk) for galactokinase (ATP: D-galactose 1-phosphotransferase, EC 2.7.1.6) to LMTK- cells has been detected once among 87 tk transferrents. This suggests that the distance between the tk and galk genes in the Chinese hamster genome may be smaller than was previously thought. Significant differences between chromosome-mediated and DNA-mediated gene transfer were observed with respect to both the size of the transferred functional genetic fragment and the recipient cell specificity. Images PMID:6929511

  18. Crystal structure of human nicotinic acid phosphoribosyltransferase.

    PubMed

    Marletta, Ada Serena; Massarotti, Alberto; Orsomando, Giuseppe; Magni, Giulio; Rizzi, Menico; Garavaglia, Silvia

    2015-01-01

    Nicotinic acid phosphoribosyltransferase (EC 2.4.2.11) (NaPRTase) is the rate-limiting enzyme in the three-step Preiss-Handler pathway for the biosynthesis of NAD. The enzyme catalyzes the conversion of nicotinic acid (Na) and 5-phosphoribosyl-1-pyrophosphate (PRPP) to nicotinic acid mononucleotide (NaMN) and pyrophosphate (PPi). Several studies have underlined the importance of NaPRTase for NAD homeostasis in mammals, but no crystallographic data are available for this enzyme from higher eukaryotes. Here, we report the crystal structure of human NaPRTase that was solved by molecular replacement at a resolution of 2.9 Å in its ligand-free form. Our structural data allow the assignment of human NaPRTase to the type II phosphoribosyltransferase subfamily and reveal that the enzyme consists of two domains and functions as a dimer with the active site located at the interface of the monomers. The substrate-binding mode was analyzed by molecular docking simulation and provides hints into the catalytic mechanism. Moreover, structural comparison of human NaPRTase with the other two human type II phosphoribosyltransferases involved in NAD biosynthesis, quinolinate phosphoribosyltransferase and nicotinamide phosphoribosyltransferase, reveals that while the three enzymes share a conserved overall structure, a few distinctive structural traits can be identified. In particular, we show that NaPRTase lacks a tunnel that, in nicotinamide phosphoribosiltransferase, represents the binding site of its potent and selective inhibitor FK866, currently used in clinical trials as an antitumoral agent. PMID:26042198

  19. Crystal structure of human nicotinic acid phosphoribosyltransferase

    PubMed Central

    Marletta, Ada Serena; Massarotti, Alberto; Orsomando, Giuseppe; Magni, Giulio; Rizzi, Menico; Garavaglia, Silvia

    2015-01-01

    Nicotinic acid phosphoribosyltransferase (EC 2.4.2.11) (NaPRTase) is the rate-limiting enzyme in the three-step Preiss–Handler pathway for the biosynthesis of NAD. The enzyme catalyzes the conversion of nicotinic acid (Na) and 5-phosphoribosyl-1-pyrophosphate (PRPP) to nicotinic acid mononucleotide (NaMN) and pyrophosphate (PPi). Several studies have underlined the importance of NaPRTase for NAD homeostasis in mammals, but no crystallographic data are available for this enzyme from higher eukaryotes. Here, we report the crystal structure of human NaPRTase that was solved by molecular replacement at a resolution of 2.9 Å in its ligand-free form. Our structural data allow the assignment of human NaPRTase to the type II phosphoribosyltransferase subfamily and reveal that the enzyme consists of two domains and functions as a dimer with the active site located at the interface of the monomers. The substrate-binding mode was analyzed by molecular docking simulation and provides hints into the catalytic mechanism. Moreover, structural comparison of human NaPRTase with the other two human type II phosphoribosyltransferases involved in NAD biosynthesis, quinolinate phosphoribosyltransferase and nicotinamide phosphoribosyltransferase, reveals that while the three enzymes share a conserved overall structure, a few distinctive structural traits can be identified. In particular, we show that NaPRTase lacks a tunnel that, in nicotinamide phosphoribosiltransferase, represents the binding site of its potent and selective inhibitor FK866, currently used in clinical trials as an antitumoral agent. PMID:26042198

  20. Mouse model for somatic mutation at the HPRT (hypoxanthine phosphoribosyl-transferase) gene: Molecular and cellular analyses

    SciTech Connect

    Burkhart-Schultz, K.; Strout, C.L.; Jones, I.M.

    1989-07-11

    Our goal is to use the mouse to model the organismal, cellular and molecular factors that affect somatic mutagenesis in vivo. A fundamental tenet of genetic toxicology is that the principles of mutagenesis identified in one system can be used to predict the principles of mutagenesis in another system. The validity of this tenet depends upon the comparability of the systems involved. To begin to achieve an understanding of somatic mutagenesis in vivo, we have been studying mutations that occur in the hypoxanthine phosphoribosyl-transferase (HPRT) gene of lymphocytes of mice. Our in vivo model for somatic mutation allows us to analyse factors that affect somatic mutation. Having chosen the mouse, we are working with cells in which the karyotype is normal, and metabolic and DNA repair capacity are defined by the mouse strain chosen. At the organismal level, we can vary sex, age, the exposure history, and the tissue source of cells analysed. (All studies reported here have, however, used male mice.) At the cellular level, T lymphocytes and their precursors are the targets and reporters of mutation. 26 refs., 1 fig., 1 tab.

  1. Pre-thymic somatic mutation leads to high mutant frequency at hypoxanthine-guanine phosphoribosyltransferase gene

    SciTech Connect

    Jett, J.

    1994-12-01

    While characterizing the background mutation spectrum of the Hypoxathine-guanine phosphoribosyltransferase (HPRT) gene in a healthy population, an outlier with a high mutant frequency of thioguanine resistant lymphocytes was found. When studied at the age of 46, this individual had been smoking 60 cigarettes per day for 38 years. His mutant frequency was calculated at 3.6 and 4.2x10{sup {minus}4} for two sampling periods eight months apart. Sequencing analysis of the HPRT gene in his mutant thioguanine resistant T lymphocytes was done to find whether the cells had a high rate of mutation, or if the mutation was due to a single occurrence of mutation and, if so, when in the T lymphocyte development the mutation occurred. By T-cell receptor analysis it has been found that out of 35 thioguanine resistant clones there was no dominant gamma T cell receptor gene rearrangement. During my appointment in the Science & Engineering Research Semester, I found that 34 of those clones have the same base substitution of G{yields}T at cDNA position 197. Due to the consistent mutant frequency from both sampling periods and the varying T cell receptors, the high mutant frequency cannot be due to recent proliferation of a mature mutant T lymphocyte. From the TCR and DNA sequence analysis we conclude that the G{yields}T mutation must have occurred in a T lymphocyte precursor before thymic differentiation so that the thioguanine resistant clones share the same base substitution but not the same gamma T cell receptor gene.

  2. Analysis of HeLa cell hypoxanthine phosphoribosyltransferase mutants and revertants by two-dimensional polyacrylamide gel electrophoresis: evidence for silent gene activation.

    PubMed Central

    Milman, G; Lee, E; Ghangas, G S; McLaughlin, J R; George, M

    1976-01-01

    The spot corresponding to hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) has been identified in two-dimensional polyacrylamide gels of HeLa cell extracts. This spot is absent in gels of 24 HPRT dificient mutants. A missense mutant displays a new HPRT spot at the same molecular weight but different isoelectric focusing position. Five independently isolated revertants of the missense mutant display spots corresponding to both the wild-type and mutant proteins indicating that they synthesize HPRT from two separate genes. If the missense protein is synthesized from a mutated form of the initially active HPRT gene, then wild-type HPRT protein in the revertants must be snythesized from a newly activated but prevously silent wild-type gene. The newly activated gene in the revertants of the missense mutation appears unstable producing a high frequency of spontaneous HPRT mutants. Images PMID:63948

  3. Product Release Pathways in Human and Plasmodium falciparum Phosphoribosyltransferase.

    PubMed

    Karmakar, Tarak; Roy, Sourav; Balaram, Hemalatha; Prakash, Meher K; Balasubramanian, Sundaram

    2016-08-22

    Atomistic molecular dynamics (MD) simulations coupled with the metadynamics technique were carried out to delineate the product (PPi.2Mg and IMP) release mechanisms from the active site of both human (Hs) and Plasmodium falciparum (Pf) hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT). An early movement of PPi.2Mg from its binding site has been observed. The swinging motion of the Asp side chain (D134/D145) in the binding pocket facilitates the detachment of IMP, which triggers the opening of flexible loop II, the gateway to the bulk solvent. In PfHGXPRT, PPi.2Mg and IMP are seen to be released via the same path in all of the biased MD simulations. In HsHGPRT too, the product molecules follow similar routes from the active site; however, an alternate but minor escape route for PPi.2Mg has been observed in the human enzyme. Tyr 104 and Phe 186 in HsHGPRT and Tyr 116 and Phe 197 in PfHGXPRT are the key residues that mediate the release of IMP, whereas the motion of PPi.2Mg away from the reaction center is guided by the negatively charged Asp and Glu and a few positively charged residues (Lys and Arg) that line the product release channels. Mutations of a few key residues present in loop II of Trypanosoma cruzi (Tc) HGPRT have been shown to reduce the catalytic efficiency of the enzyme. Herein, in silico mutation of corresponding residues in loop II of HsHGPRT and PfHGXPRT resulted in partial opening of the flexible loop (loop II), thus exposing the active site to bulk water, which offers a rationale for the reduced catalytic activity of these two mutant enzymes. Investigations of the product release from these HsHGPRT and PfHGXPRT mutants delineate the role of these important residues in the enzymatic turnover. PMID:27404508

  4. Allopurinol enhances the activity of hypoxanthine-guanine phosphoribosyltransferase in inflammatory bowel disease patients during low-dose thiopurine therapy: preliminary data of an ongoing series.

    PubMed

    Seinen, Margien L; de Boer, Nanne K H; Smid, Kees; van Asseldonk, Dirk P; Bouma, Gerd; van Bodegraven, Adriaan A; Peters, Godefridus J

    2011-12-01

    Thiopurines are crucial in the treatment of inflammatory bowel disease. The phenotype of pivotal metabolic enzymes determines whether thioguanine nucleotides (6-TGN) are generated in clinically sufficiently high levels. The first step in activation of thiopurine prodrugs to 6-TGN is catalysis by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Often, patients exhibit a clinically unfavorable metabolism, leading to discontinuation of conventional thiopurine therapy. The combination of allopurinol and low-dose thiopurine therapy may optimize this variant metabolism, presumably by affecting enzyme activities. We performed a prospective pharmacodynamic study to determine the effect of combination therapy on the activity of HGPRT. The activity of HGPRT and 6-TGN concentrations was measured in red blood cells during thiopurine monotherapy and after 4 weeks of combination therapy. The activity of HGPRT was also measured after 12 weeks of combination therapy. From the results, we conclude that combination therapy increases the activity of HGPRT and subsequently 6-TGN concentrations. PMID:22132961

  5. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5'-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa).

    PubMed

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E; Gallo-Reynoso, Juan P

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5'-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5'-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5'-monophosphate (IMP), adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), ATP, guanosine 5'-diphosphate (GDP), guanosine 5'-triphosphate (GTP), and xanthosine 5'-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts. PMID:26283971

  6. Identification and characterization of human uracil phosphoribosyltransferase (UPRTase).

    PubMed

    Li, Jixi; Huang, Shengdong; Chen, Jinzhong; Yang, Zhenxing; Fei, Xiangwei; Zheng, Mei; Ji, Chaoneng; Xie, Yi; Mao, Yumin

    2007-01-01

    Uracil phosphoribosyltransferase, which catalyzes the conversion of uracil and 5-phosphoribosyl-1-R-diphosphate to uridine monophosphate, is important in the pyrimidine salvage pathway and is an attractive target for rational drug design by incorporation of prodrugs that are lethal to many parasitic organisms specifically. So far, uracil phosphoribosyltransferase has been reported in Arabidopsis thaliana only, not in mammals. In this study, a novel uracil phosphoribosyltransferase family cDNA encoding a 309 amino acid protein with a putative uracil phosphoribosyltransferase domain was isolated from the human fetal brain library. It was named human UPRTase (uracil phosphoribosyltransferase). The ORF of human UPRTase gene was cloned into pQE30 and expressed in Escherichia coli M15. The protein was purified by Ni-NTA affinity chromatography, but UPRTase activity could not be detected by spectrophotometry. RT-PCR analysis showed that human UPRTase was strongly expressed in blood leukocytes, liver, spleen, and thymus, with lower levels of expression in the prostate, heart, brain, lung, and skeletal muscle. Subcellular location of UPRTase-EGFP fusion protein revealed that human UPRTase was distributed in the nucleus and cytoplasm of AD293 cells. Evolutional tree analyses of UPRTases or UPRTase-domain-containing proteins showed that UPRTase was conserved in organisms. UPRTases of archaebacteria or eubacterium had UPRTase activity whereas those higher than Caenorhabditis elegans, which lacked two amino acids in the uracil-binding region, had no UPRTase activity. This means that human UPRTase may have enzymatic activity with another, unknown, factor or have other activity in pyrimidine metabolism. PMID:17384901

  7. Purine salvage in the apicomplexan Sarcocystis neurona, and generation of hypoxanthine-xanthine-guanine phosphoribosyltransferase-deficient clones for positive-negative selection of transgenic parasites.

    PubMed

    Dangoudoubiyam, Sriveny; Zhang, Zijing; Howe, Daniel K

    2014-09-01

    Sarcocystis neurona is an apicomplexan parasite that causes severe neurological disease in horses and marine mammals. The Apicomplexa are all obligate intracellular parasites that lack purine biosynthesis pathways and rely on the host cell for their purine requirements. Hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) and adenosine kinase (AK) are key enzymes that function in two complementary purine salvage pathways in apicomplexans. Bioinformatic searches of the S. neurona genome revealed genes encoding HXGPRT, AK and all of the major purine salvage enzymes except purine nucleoside phosphorylase. Wild-type S. neurona were able to grow in the presence of mycophenolic acid (MPA) but were inhibited by 6-thioxanthine (6-TX), suggesting that the pathways involving either HXGPRT or AK are functional in this parasite. Prior work with Toxoplasma gondii demonstrated the utility of HXGPRT as a positive-negative selection marker. To enable the use of HXGPRT in S. neurona, the SnHXGPRT gene sequence was determined and a gene-targeting plasmid was transfected into S. neurona. SnHXGPRT-deficient mutants were selected with 6-TX, and single-cell clones were obtained. These Sn∆HXG parasites were susceptible to MPA and could be complemented using the heterologous T. gondii HXGPRT gene. In summary, S. neurona possesses both purine salvage pathways described in apicomplexans, thus allowing the use of HXGPRT as a positive-negative drug selection marker in this parasite. PMID:24923662

  8. Hydrophilic-interaction liquid chromatography-tandem mass spectrometric determination of erythrocyte 5-phosphoribosyl 1-pyrophosphate in patients with hypoxanthine-guanine phosphoribosyltransferase deficiency.

    PubMed

    Hasegawa, Hiroshi; Shinohara, Yoshihiko; Nozaki, Sayako; Nakamura, Makiko; Oh, Koei; Namiki, Osamu; Suzuki, Kiyotaka; Nakahara, Akihiko; Miyazawa, Mari; Ishikawa, Ken; Himeno, Takahiro; Yoshida, Sayaka; Ueda, Takanori; Yamada, Yasukazu; Ichida, Kimiyoshi

    2015-01-22

    Mutations in the gene encoding hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause Lesch-Nyhan disease (LND) and its variants (LNV). Due to the technical problems for measuring the HPRT activity in vitro, discordances between the residual HPRT activity and the clinical severity were found. 5-Phosphoribosyl 1-pyrophosphate (PRPP) is a substrate for HPRT. Since increased PRPP concentrations were observed in erythrocytes from patients with LND and LNV, we have turned our attention to erythrocyte PRPP as a biomarker for the phenotype classification. In the present work, a method for determination of PRPP concentration in erythrocyte was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with multiple reaction monitoring (MRM). Packed erythrocyte samples were deproteinized by heating and the supernatants were injected into the LC-MS/MS system. All measurement results showed good precision with RSD <6%. PRPP concentrations of nine normal male subjects, four male patents with LND and six male patients with LNV were compared. The PRPP concentrations in erythrocyte from patients with LND were markedly increased compared with those from normal subjects, and those from patients with LNV were also increased but the degree was smaller than those with LND. The increase pattern of PRPP concentration in erythrocyte from patients with HPRT deficiency was consistent with the respective phenotypes and was correlated with the disease severity. PRPP concentration was suggested to give us supportive information for the diagnosis and the phenotype classification of LND and LNV. PMID:25482009

  9. Synthesis and Evaluation of Novel Acyclic Nucleoside Phosphonates as Inhibitors of Plasmodium falciparum and Human 6-Oxopurine Phosphoribosyltransferases.

    PubMed

    Kaiser, Martin M; Hocková, Dana; Wang, Tzu-Hsuan; Dračínský, Martin; Poštová-Slavětínská, Lenka; Procházková, Eliška; Edstein, Michael D; Chavchich, Marina; Keough, Dianne T; Guddat, Luke W; Janeba, Zlatko

    2015-10-01

    Acyclic nucleoside phosphonates (ANPs) are a promising class of antimalarial therapeutic drug leads that exhibit a wide variety of Ki values for Plasmodium falciparum (Pf) and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases [HG(X)PRTs]. A novel series of ANPs, analogues of previously reported 2-(phosphonoethoxy)ethyl (PEE) and (R,S)-3-hydroxy-2-(phosphonomethoxy)propyl (HPMP) derivatives, were designed and synthesized to evaluate their ability to act as inhibitors of these enzymes and to extend our ongoing antimalarial structure-activity relationship studies. In this series, (S)-3-hydroxy-2-(phosphonoethoxy)propyl (HPEP), (S)-2-(phosphonomethoxy)propanoic acid (CPME), or (S)-2-(phosphonoethoxy)propanoic acid (CPEE) are the acyclic moieties. Of this group, (S)-3-hydroxy-2-(phosphonoethoxy)propylguanine (HPEPG) exhibits the highest potency for PfHGXPRT, with a Ki value of 0.1 μM and a Ki value for human HGPRT of 0.6 μM. The crystal structures of HPEPG and HPEPHx (where Hx=hypoxanthine) in complex with human HGPRT were obtained, showing specific interactions with active site residues. Prodrugs for the HPEP and CPEE analogues were synthesized and tested for in vitro antimalarial activity. The lowest IC50 value (22 μM) in a chloroquine-resistant strain was observed for the bis-amidate prodrug of HPEPG. PMID:26368337

  10. Hypoxanthine enters human vascular endothelial cells (ECV 304) via the nitrobenzylthioinosine-insensitive equilibrative nucleoside transporter.

    PubMed Central

    Osses, N; Pearson, J D; Yudilevich, D L; Jarvis, S M

    1996-01-01

    The transport properties of the nucleobase hypoxanthine were examined in the human umbilical vein endothelial cell line ECV 304. Initial rates of hypoxanthine influx were independent of extracellular cations: replacement of Na+ with Li+, Rb+, N-methyl-D-glucamine or choline had no significant effect on hypoxanthine uptake by ECV 304 cells. Kinetic analysis demonstrated the presence of a single saturable system for the transport of hypoxanthine in ECV 304 cells with an apparent K(m) of 320 +/- 10 microM and a Vmax of 5.6 +/- 0.9 pmol/10(6) cells per s. Hypoxanthine uptake was inhibited by the nucleosides adenosine, uridine and thymidine (apparent Ki 41 +/- 6, 240 +/- 27 and 59 +/- 8 microM respectively) and the nucleoside transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and dipyridamole (apparent Ki 2.5 +/- 0.3, 11 +/- 3 and 0.16 +/- 0.006 microM respectively), whereas the nucleobases adenine, guanine and thymine had little effect (50% inhibition at > 1 mM). ECV 304 cells were also shown to transport adenosine via both the NBMPR-sensitive and -insensitive nucleoside carriers. Hypoxanthine specifically inhibited adenosine transport via the NBMPR-insensitive system in a competitive manner (apparent Ki 290 +/- 14 microM). These results indicate that hypoxanthine entry into ECV 304 endothelial cells is mediated by the NBMPR-insensitive nucleoside carrier present in these cells. PMID:8760371

  11. Hypoxanthine-guanine phosphoribosyltransferase and inosine 5′-monophosphate dehydrogenase activities in three mammalian species: aquatic (Mirounga angustirostris), semi-aquatic (Lontra longicaudis annectens) and terrestrial (Sus scrofa)

    PubMed Central

    Barjau Pérez-Milicua, Myrna; Zenteno-Savín, Tania; Crocker, Daniel E.; Gallo-Reynoso, Juan P.

    2015-01-01

    Aquatic and semiaquatic mammals have the capacity of breath hold (apnea) diving. Northern elephant seals (Mirounga angustirostris) have the ability to perform deep and long duration dives; during a routine dive, adults can hold their breath for 25 min. Neotropical river otters (Lontra longicaudis annectens) can hold their breath for about 30 s. Such periods of apnea may result in reduced oxygen concentration (hypoxia) and reduced blood supply (ischemia) to tissues. Production of adenosine 5′-triphosphate (ATP) requires oxygen, and most mammalian species, like the domestic pig (Sus scrofa), are not adapted to tolerate hypoxia and ischemia, conditions that result in ATP degradation. The objective of this study was to explore the differences in purine synthesis and recycling in erythrocytes and plasma of three mammalian species adapted to different environments: aquatic (northern elephant seal) (n = 11), semiaquatic (neotropical river otter) (n = 4), and terrestrial (domestic pig) (n = 11). Enzymatic activity of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) was determined by spectrophotometry, and activity of inosine 5′-monophosphate dehydrogenase (IMPDH) and the concentration of hypoxanthine (HX), inosine 5′-monophosphate (IMP), adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), ATP, guanosine 5′-diphosphate (GDP), guanosine 5′-triphosphate (GTP), and xanthosine 5′-monophosphate (XMP) were determined by high-performance liquid chromatography (HPLC). The activities of HGPRT and IMPDH and the concentration of HX, IMP, AMP, ADP, ATP, GTP, and XMP in erythrocytes of domestic pigs were higher than in erythrocytes of northern elephant seals and river otters. These results suggest that under basal conditions (no diving, sleep apnea or exercise), aquatic, and semiaquatic mammals have less purine mobilization than their terrestrial counterparts. PMID:26283971

  12. Extensive regulation of nicotinate phosphoribosyltransferase (NAPRT) expression in human tissues and tumors

    PubMed Central

    Duarte-Pereira, Sara; Pereira-Castro, Isabel; Silva, Sarah S.; Correia, Mariana Gonçalves; Neto, Célia; da Costa, Luís Teixeira; Amorim, António; Silva, Raquel M.

    2016-01-01

    Nicotinamide adenine dinucleotide (NAD) is a cofactor in redox reactions and a substrate for NAD-consuming enzymes, such as PARPs and sirtuins. As cancer cells have increased NAD requirements, the main NAD salvage enzymes in humans, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinate phosphoribosyltransferase (NAPRT), are involved in the development of novel anti-cancer therapies. Knowledge of the expression patterns of both genes in tissues and tumors is critical for the use of nicotinic acid (NA) as cytoprotective in therapies using NAMPT inhibitors. Herein, we provide a comprehensive study of NAPRT and NAMPT expression across human tissues and tumor cell lines. We show that both genes are widely expressed under normal conditions and describe the occurrence of novel NAPRT transcripts. Also, we explore some of the NAPRT gene expression mechanisms. Our findings underline that the efficiency of NA in treatments with NAMPT inhibitors is dependent on the knowledge of the expression profiles and regulation of both NAMPT and NAPRT. PMID:26675378

  13. Synergistic action of tiazofurin with hypoxanthine and allopurinol in human neuroectodermal tumor cell lines.

    PubMed

    Szekeres, T; Schuchter, K; Chiba, P; Ressmann, G; Lhotka, C; Gharehbaghi, K; Szalay, S M; Pillwein, K

    1993-12-01

    The activity of IMP dehydrogenase (EC 1.2.1.14), the key enzyme of de novo guanylate biosynthesis, was shown to be increased in tumor cells. Tiazofurin (TR), a potent and specific inhibitor of this enzyme, proved to be effective in the treatment of refractory granulocytic leukemia in blast crisis. We examined the effects of tiazofurin as a single agent and in combination with hypoxanthine and allopurinol in six different neuroectodermal tumor cell lines, the STA-BT-3 and 146-18 human glioblastoma cell lines, the SK-N-SH, LA-N-1 and LA-N-5 human neuroblastoma cell lines, and the STA-ET-1 Ewing tumor cell line. Tiazofurin inhibited tumor cell growth with IC50 values between 2.2 microM (LA-N-1 cell line) and 550 microM (LA-N-5 cells) and caused a significant decrease of intracellular GTP pools (GTP concentrations decreased to 39-79% of control). Incorporation of [8-14C]guanine into GTP pools was determined as a measure of guanylate salvage activity; incubation with 100 microM hypoxanthine caused a 62-96% inhibition of the salvage pathway. Incubation with tiazofurin (100 microM) and hypoxanthine (100 microM) synergistically inhibited tumor cell growth, and the addition of allopurinol (100 microM) strengthened these effects. Therefore, this drug combination, inhibiting guanylate de novo and salvage pathways, may prove useful in the treatment of human neuroectodermal tumors. PMID:7903533

  14. Assignment of the Gene for Adenine Phosphoribosyltransferase to Human Chromosome 16 by Mouse-Human Somatic Cell Hybridization

    PubMed Central

    Tischfield, Jay A.; Ruddle, Frank H.

    1974-01-01

    A series of mouse-human hybrids was prepared from mouse cells deficient in adenine phosphoribosyltransferase (EC 2.4.2.7) and normal human cells. The hybrids were made in medium containing adenine and alanosine, an antimetabolite known to inhibit de novo adenylic acid biosynthesis. The mouse cells, unable to utilize exogenous adenine, were killed in this medium, but the hybrids proliferated as a consequence of their retaining the human aprt gene. The hybrids were then exposed to the adenine analogs 2,6-diaminopurine and 2-fluoroadenine to select for cells that had lost this gene. Before exposure to the adenine analogs, the expression of human adenine phosphoribosyltransferase by the hybrids was strongly associated only with the presence of human chromosome 16, and afterwards this was the only human chromosome consistently lost. This observation suggests that the human aprt gene can be assigned to chromosome 16. Images PMID:4129802

  15. Hypoxanthine deregulates genes involved in early neuronal development. Implications in Lesch-Nyhan disease pathogenesis.

    PubMed

    Torres, R J; Puig, J G

    2015-11-01

    Neurological manifestations in Lesch-Nyhan disease (LND) are attributed to the effect of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency on the nervous system development. HPRT deficiency causes the excretion of increased amounts of hypoxanthine into the extracellular medium and we hypothesized that HPRT deficiency related to hypoxanthine excess may then lead, directly or indirectly, to transcriptional aberrations in a variety of genes essential for the function and development of striatal progenitor cells. We have examined the effect of hypoxanthine excess on the differentiation of neurons in the well-established human NTERA-2 cl.D1 (NT2/D1) embryonic carcinoma neurogenesis model. NT2/D1 cells differentiate along neuroectodermal lineages after exposure to retinoic acid (RA). Hypoxanthine effects on RA-differentiation were examined by the changes on the expression of various transcription factor genes essential to neuronal differentiation and by the changes in tyrosine hydroxylase (TH), dopamine, adenosine and serotonin receptors (DRD, ADORA, HTR). We report that hypoxanthine excess deregulate WNT4, from Wnt/β-catenin pathway, and engrailed homeobox 1 gene and increased TH and dopamine DRD1, adenosine ADORA2A and serotonin HTR7 receptors, whose over expression characterize early neuro-developmental processes. PMID:25940910

  16. Interaction of the recombinant human methylpurine-DNA glycosylase (MPG protein) with oligodeoxyribonucleotides containing either hypoxanthine or abasic sites.

    PubMed Central

    Miao, F; Bouziane, M; O'Connor, T R

    1998-01-01

    Methylpurine-DNA glycosylases (MPG proteins, 3-methyladenine-DNA glycosylases) excise numerous damaged bases from DNA during the first step of base excision repair. The damaged bases removed by these proteins include those induced by both alkylating agents and/or oxidizing agents. The intrinsic kinetic parameters (k(cat) and K(m)) for the excision of hypoxanthine by the recombinant human MPG protein from a 39 bp oligodeoxyribonucleotide harboring a unique hypoxanthine were determined. Comparison with other reactions catalyzed by the human MPG protein suggests that the differences in specificity are primarily in product release and not binding. Analysis of MPG protein binding to the 39 bp oligodeoxyribonucleotide revealed that the apparent dissociation constant is of the same order of magnitude as the K(m) and that a 1:1 complex is formed. The MPG protein also forms a strong complex with the product of excision, an abasic site, as well as with a reduced abasic site. DNase I footprinting experiments with the MPG protein on an oligodeoxyribonucleotide with a unique hypoxanthine at a defined position indicate that the protein protects 11 bases on the strand with the hypoxanthine and 12 bases on the complementary strand. Competition experiments with different length, double-stranded, hypoxanthine-containing oligodeoxyribonucleotides show that the footprinted region is relatively small. Despite the small footprint, however, oligodeoxyribonucleotides comprising <15 bp with a hypoxanthine have a 10-fold reduced binding capacity compared with hypoxanthine-containing oligodeoxyribonucleotides >20 bp in length. These results provide a basis for other structural studies of the MPG protein with its targets. PMID:9705516

  17. Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases.

    PubMed

    Saparbaev, M; Laval, J

    1994-06-21

    The deamination of adenine residues in DNA generates hypoxanthine, which is mutagenic since it gives rise to an A.T to G.C transition. Hypoxanthine is removed by hypoxanthine DNA glycosylase activity present in Escherichia coli and mammalian cells. Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residues, we show that this activity in E. coli is associated with the 3-methyladenine DNA glycosylase II coded for by the alkA gene. This conclusion is based on the following facts: (i) the two enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight, (ii) both are induced during the adaptive response, (iii) a multicopy plasmid bearing the alkA gene overproduces both activities, (iv) homogeneous preparation of AlkA has both enzymatic activities, (v) the E. coli alkA- mutant does not show any detectable hypoxanthine DNA glycosylase activity. Under the same experimental conditions, but using different substrates, the same amount of AlkA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA. The Km for the latter substrate is 420 x 10(-9) M as compared to 5 x 10(-9) M for alkylated DNA. Hypoxanthine is released as a free base during the reaction. Duplex oligodeoxynucleotides containing hypoxanthine positioned opposite T, G, C, and A were cleaved efficiently. ANPG protein, APDG protein, and MAG protein--the 3-methyladenine DNA glycosylases of human, rat, and yeast origin, respectively--were also able to release hypoxanthine from various DNA substrates containing dIMP residues. The mammalian enzyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested. PMID:8016081

  18. Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases.

    PubMed Central

    Saparbaev, M; Laval, J

    1994-01-01

    The deamination of adenine residues in DNA generates hypoxanthine, which is mutagenic since it gives rise to an A.T to G.C transition. Hypoxanthine is removed by hypoxanthine DNA glycosylase activity present in Escherichia coli and mammalian cells. Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residues, we show that this activity in E. coli is associated with the 3-methyladenine DNA glycosylase II coded for by the alkA gene. This conclusion is based on the following facts: (i) the two enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight, (ii) both are induced during the adaptive response, (iii) a multicopy plasmid bearing the alkA gene overproduces both activities, (iv) homogeneous preparation of AlkA has both enzymatic activities, (v) the E. coli alkA- mutant does not show any detectable hypoxanthine DNA glycosylase activity. Under the same experimental conditions, but using different substrates, the same amount of AlkA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA. The Km for the latter substrate is 420 x 10(-9) M as compared to 5 x 10(-9) M for alkylated DNA. Hypoxanthine is released as a free base during the reaction. Duplex oligodeoxynucleotides containing hypoxanthine positioned opposite T, G, C, and A were cleaved efficiently. ANPG protein, APDG protein, and MAG protein--the 3-methyladenine DNA glycosylases of human, rat, and yeast origin, respectively--were also able to release hypoxanthine from various DNA substrates containing dIMP residues. The mammalian enzyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested. Images PMID:8016081

  19. Interaction of Human DNA Polymerase α and DNA Polymerase I from Bacillus stearothermophilus with Hypoxanthine and 8-Oxoguanine Nucleotides †

    PubMed Central

    Patro, Jennifer N.; Urban, Milan; Kuchta, Robert D.

    2009-01-01

    To better understand how DNA polymerases interact with mutagenic bases, we examined how human DNA polymerase α (pol α), a B family enzyme, and DNA polymerase from Bacillus stearothermophilus (BF), an A family enzyme, generate adenine:hypoxanthine and adenine:8-oxo-7,8-dihydroguanine (8-oxoG) base pairs. Pol α strongly discriminated against polymerizing dATP opposite 8-oxoG, and removing N1, N6, or N7 further inhibited incorporation, whereas removing N3 from dATP dramatically increased incorporation (32-fold). Eliminating N6 from 3-deaza-dATP now greatly reduced incorporation, suggesting that incorporation of dATP (analogues) opposite 8-oxoguanine proceeds via a Hoogsteen base-pair and that pol α uses N3 of a purine dNTP to block this incorporation. Pol α also polymerized 8-oxo-dGTP across from a templating A, and removing N6 from the template adenine inhibited incorporation of 8-oxoG. The effects of N1, N6, and N7 demonstrated a strong interdependence during formation of adenine:hypoxanthine base-pairs by pol α and N3 of dATP again helps prevent polymerization opposite a templating hypoxanthine. BF very efficiently polymerized 8-oxo-dGTP opposite adenine, and N1 and N7 of adenine appear to play important roles. BF incorporates dATP opposite 8-oxoG less efficiently, and modifying N1, N6, or N7 greatly inhibits incorporation. N6, and to a lesser extent N1, help drive hypoxanthine:adenine base pair formation by BF. The mechanistic implications of these results showing that different polymerases interact very differently with base lesions are discussed. PMID:19642651

  20. Metabolomics Analysis of Metabolic Effects of Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibition on Human Cancer Cells

    PubMed Central

    Tolstikov, Vladimir; Nikolayev, Alexander; Dong, Sucai; Zhao, Genshi; Kuo, Ming-Shang

    2014-01-01

    Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in cellular bioenergetics. It is responsible for converting nicotinamide to nicotinamide adenine dinucleotide, an essential molecule in cellular metabolism. NAMPT has been extensively studied over the past decade due to its role as a key regulator of nicotinamide adenine dinucleotide–consuming enzymes. NAMPT is also known as a potential target for therapeutic intervention due to its involvement in disease. In the current study, we used a global mass spectrometry–based metabolomic approach to investigate the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on metabolic perturbations in human cancer cells. We treated A2780 (ovarian cancer) and HCT-116 (colorectal cancer) cell lines with FK866 in the presence and absence of nicotinic acid. Significant changes were observed in the amino acids metabolism and the purine and pyrimidine metabolism. We also observed metabolic alterations in glycolysis, the citric acid cycle (TCA), and the pentose phosphate pathway. To expand the range of the detected polar metabolites and improve data confidence, we applied a global metabolomics profiling platform by using both non-targeted and targeted hydrophilic (HILIC)-LC-MS and GC-MS analysis. We used Ingenuity Knowledge Base to facilitate the projection of metabolomics data onto metabolic pathways. Several metabolic pathways showed differential responses to FK866 based on several matches to the list of annotated metabolites. This study suggests that global metabolomics can be a useful tool in pharmacological studies of the mechanism of action of drugs at a cellular level. PMID:25486521

  1. Metabolomics analysis of metabolic effects of nicotinamide phosphoribosyltransferase (NAMPT) inhibition on human cancer cells.

    PubMed

    Tolstikov, Vladimir; Nikolayev, Alexander; Dong, Sucai; Zhao, Genshi; Kuo, Ming-Shang

    2014-01-01

    Nicotinamide phosphoribosyltransferase (NAMPT) plays an important role in cellular bioenergetics. It is responsible for converting nicotinamide to nicotinamide adenine dinucleotide, an essential molecule in cellular metabolism. NAMPT has been extensively studied over the past decade due to its role as a key regulator of nicotinamide adenine dinucleotide-consuming enzymes. NAMPT is also known as a potential target for therapeutic intervention due to its involvement in disease. In the current study, we used a global mass spectrometry-based metabolomic approach to investigate the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on metabolic perturbations in human cancer cells. We treated A2780 (ovarian cancer) and HCT-116 (colorectal cancer) cell lines with FK866 in the presence and absence of nicotinic acid. Significant changes were observed in the amino acids metabolism and the purine and pyrimidine metabolism. We also observed metabolic alterations in glycolysis, the citric acid cycle (TCA), and the pentose phosphate pathway. To expand the range of the detected polar metabolites and improve data confidence, we applied a global metabolomics profiling platform by using both non-targeted and targeted hydrophilic (HILIC)-LC-MS and GC-MS analysis. We used Ingenuity Knowledge Base to facilitate the projection of metabolomics data onto metabolic pathways. Several metabolic pathways showed differential responses to FK866 based on several matches to the list of annotated metabolites. This study suggests that global metabolomics can be a useful tool in pharmacological studies of the mechanism of action of drugs at a cellular level. PMID:25486521

  2. GENETIC ASSAY FOR ANEUPLOIDY: QUANTITATION OF CHROMOSOME LOSS USING A MOUSE/HUMAN MONOCHROMOSOMAL HYBRID CELL LINE (JOURNAL VERSION)

    EPA Science Inventory

    A genetic assay is described in which a mouse/human hybrid cell line R3-5 containing a single human chromosome (a monochromosomal hybrid) is used to detect chemically induced aneuploidy. The hybrid cells are deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT) and ...

  3. Determination of Activity of the Enzymes Hypoxanthine Phosphoribosyl Transferase (HPRT) and Adenine Phosphoribosyl Transferase (APRT) in Blood Spots on Filter Paper.

    PubMed

    Auler, Kasie; Broock, Robyn; Nyhan, William L

    2015-01-01

    Hypoxanthine-guanine phosphoribosyl-transferase (HPRT) deficiency is the cause of Lesch-Nyhan disease. Adenine phosphoribosyl-transferase (APRT) deficiency causes renal calculi. The activity of each enzyme is readily determined on spots of whole blood on filter paper. This unit describes a method for detecting deficiencies of HPRT and APRT. PMID:26132002

  4. A critical role of nicotinamide phosphoribosyltransferase in human telomerase reverse transcriptase induction by resveratrol in aortic smooth muscle cells

    PubMed Central

    Huang, Peixin; Riordan, Sean M.; Heruth, Daniel P.; Grigoryev, Dmitry N.; Zhang, Li Qin; Ye, Shui Qing

    2015-01-01

    Aging is the predominant risk factor for cardiovascular diseases and contributes to a considerably more severe outcome in patients with acute myocardial infarction. Resveratrol, a polyphenol found in red wine, is a caloric restriction mimetic with potential anti-aging properties which has emerged as a beneficial nutraceutical for patients with cardiovascular disease. Although resveratrol is widely consumed as a nutritional supplement, its mechanism of action remains to be elucidated fully. Here, we report that resveratrol activates human nicotinamide phosphoribosyltransferase (NAMPT), SIRT4 and telomerase reverse transcriptase (hTERT) in human aortic smooth muscle cells. Similar observations were obtained in resveratrol treated C57BL/6J mouse heart and liver tissues. Resverotrol can also augment telomerase activity in both human pulmonary microvascular endothelial cells and A549 cells. Blocking NAMPT and SIRT4 expression prevents induction of hTERT in human aortic smooth muscle cells while overexpression of NAMPT elevates the telomerase activity induced by resveratrol in A549 cells. Together, these results identify a NAMPT-SIRT4-hTERT axis as a novel mechanism by which resveratrol may affect the anti-aging process in human aortic smooth muscle cells, mouse hearts and other cells. These findings enrich our understanding of the positive effects of resveratrol in human cardiovascular diseases. PMID:25926556

  5. DELETION MUTATIONS IN THE HPRT GENE OF T-LYMPHOCYTES AS A BIOMARKER FOR GENOMIC REARRANGEMENTS IMPORTANT IN HUMAN CANCERS

    EPA Science Inventory

    The DNA sequence of 11 in vivo-arising intragenic deletion breaksite junctions occurring in the hypoxanthine guanine phosphoribosyltransferase gene of human T-lymphocytes was determined and deletions ranged in size from 16 bp to 4057 bp. o extensive homology was found at the dele...

  6. MULTIPLEX PCR ANALYSIS OF IN VIVO-ARISING DELETION MUTATIONS IN THE HPRT GENE OF HUMAN T-LYMPHOCYTES

    EPA Science Inventory

    A multiplex polymerase chain reaction (PCR) procedure was adapted for the rapid and efficient evaluation of the hypoxanthine guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes for deletions. he hprt clonal assay was used to isolate in-vivo-arising hprt-deficient...

  7. Structural Insights into the Quaternary Catalytic Mechanism of Hexameric Human Quinolinate Phosphoribosyltransferase, a Key Enzyme in de novo NAD Biosynthesis

    PubMed Central

    Youn, Hyung-Seop; Gyun Kim, Tae; Kim, Mun-Kyoung; Bu Kang, Gil; Youn Kang, Jung; Lee, Jung-Gyu; Yop An, Jun; Ryoung Park, Kyoung; Lee, Youngjin; Jun Im, Young; Hyuck Lee, Jun; Hyun Eom, Soo

    2016-01-01

    Quinolinate phosphoribosyltransferase (QPRT) catalyses the production of nicotinic acid mononucleotide, a precursor of de novo biosynthesis of the ubiquitous coenzyme nicotinamide adenine dinucleotide. QPRT is also essential for maintaining the homeostasis of quinolinic acid in the brain, a possible neurotoxin causing various neurodegenerative diseases. Although QPRT has been extensively analysed, the molecular basis of the reaction catalysed by human QPRT remains unclear. Here, we present the crystal structures of hexameric human QPRT in the apo form and its complexes with reactant or product. We found that the interaction between dimeric subunits was dramatically altered during the reaction process by conformational changes of two flexible loops in the active site at the dimer-dimer interface. In addition, the N-terminal short helix α1 was identified as a critical hexamer stabilizer. The structural features, size distribution, heat aggregation and ITC studies of the full-length enzyme and the enzyme lacking helix α1 strongly suggest that human QPRT acts as a hexamer for cooperative reactant binding via three dimeric subunits and maintaining stability. Based on our comparison of human QPRT structures in the apo and complex forms, we propose a drug design strategy targeting malignant glioma. PMID:26805589

  8. Structural Insights into the Quaternary Catalytic Mechanism of Hexameric Human Quinolinate Phosphoribosyltransferase, a Key Enzyme in de novo NAD Biosynthesis.

    PubMed

    Youn, Hyung-Seop; Kim, Tae Gyun; Kim, Mun-Kyoung; Kang, Gil Bu; Kang, Jung Youn; Lee, Jung-Gyu; An, Jun Yop; Park, Kyoung Ryoung; Lee, Youngjin; Im, Young Jun; Lee, Jun Hyuck; Eom, Soo Hyun

    2016-01-01

    Quinolinate phosphoribosyltransferase (QPRT) catalyses the production of nicotinic acid mononucleotide, a precursor of de novo biosynthesis of the ubiquitous coenzyme nicotinamide adenine dinucleotide. QPRT is also essential for maintaining the homeostasis of quinolinic acid in the brain, a possible neurotoxin causing various neurodegenerative diseases. Although QPRT has been extensively analysed, the molecular basis of the reaction catalysed by human QPRT remains unclear. Here, we present the crystal structures of hexameric human QPRT in the apo form and its complexes with reactant or product. We found that the interaction between dimeric subunits was dramatically altered during the reaction process by conformational changes of two flexible loops in the active site at the dimer-dimer interface. In addition, the N-terminal short helix α1 was identified as a critical hexamer stabilizer. The structural features, size distribution, heat aggregation and ITC studies of the full-length enzyme and the enzyme lacking helix α1 strongly suggest that human QPRT acts as a hexamer for cooperative reactant binding via three dimeric subunits and maintaining stability. Based on our comparison of human QPRT structures in the apo and complex forms, we propose a drug design strategy targeting malignant glioma. PMID:26805589

  9. Pharmacological Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis, in Human Cancer Cells

    PubMed Central

    Tan, Bo; Young, Debra A.; Lu, Zhao-Hai; Wang, Tao; Meier, Timothy I.; Shepard, Robert L.; Roth, Kenneth; Zhai, Yan; Huss, Karen; Kuo, Ming-Shang; Gillig, James; Parthasarathy, Saravanan; Burkholder, Timothy P.; Smith, Michele C.; Geeganage, Sandaruwan; Zhao, Genshi

    2013-01-01

    Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD+, essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD+ for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic. PMID:23239881

  10. Fragment-based design of 3-aminopyridine-derived amides as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT).

    PubMed

    Dragovich, Peter S; Zhao, Guiling; Baumeister, Timm; Bravo, Brandon; Giannetti, Anthony M; Ho, Yen-Ching; Hua, Rongbao; Li, Guangkun; Liang, Xiaorong; Ma, Xiaolei; O'Brien, Thomas; Oh, Angela; Skelton, Nicholas J; Wang, Chengcheng; Wang, Weiru; Wang, Yunli; Xiao, Yang; Yuen, Po-wai; Zak, Mark; Zhao, Qiang; Zheng, Xiaozhang

    2014-02-01

    The fragment-based identification of two novel and potent biochemical inhibitors of the nicotinamide phosphoribosyltransferase (NAMPT) enzyme is described. These compounds (51 and 63) incorporate an amide moiety derived from 3-aminopyridine, and are thus structurally distinct from other known anti-NAMPT agents. Each exhibits potent inhibition of NAMPT biochemical activity (IC50=19 and 15 nM, respectively) as well as robust antiproliferative properties in A2780 cell culture experiments (IC50=121 and 99 nM, respectively). However, additional biological studies indicate that only inhibitor 51 exerts its A2780 cell culture effects via a NAMPT-mediated mechanism. The crystal structures of both 51 and 63 in complex with NAMPT are also independently described. PMID:24433859

  11. Structural and biochemical analyses of the catalysis and potency impact of inhibitor phosphoribosylation by human nicotinamide phosphoribosyltransferase.

    PubMed

    Oh, Angela; Ho, Yen-Ching; Zak, Mark; Liu, Yongbo; Chen, Xukun; Yuen, Po-Wai; Zheng, Xiaozhang; Liu, Yichin; Dragovich, Peter S; Wang, Weiru

    2014-05-26

    Prolonged inhibition of nicotinamide phosphoribosyltransferase (NAMPT) is a strategy for targeting cancer metabolism. Many NAMPT inhibitors undergo NAMPT-catalyzed phosphoribosylation (pRib), a property often correlated with their cellular potency. To understand this phenomenon and facilitate drug design, we analyzed a potent cellularly active NAMPT inhibitor (GNE-617). A crystal structure of pRib-GNE-617 in complex with NAMPT protein revealed a relaxed binding mode. Consistently, the adduct formation resulted in tight binding and strong product inhibition. In contrast, a biochemically equipotent isomer of GNE-617 (GNE-643) also formed pRib adducts but displayed significantly weaker cytotoxicity. Structural analysis revealed an altered ligand conformation of GNE-643, thus suggesting weak association of the adducts with NAMPT. Our data support a model for cellularly active NAMPT inhibitors that undergo NAMPT-catalyzed phosphoribosylation to produce pRib adducts that retain efficient binding to the enzyme. PMID:24797455

  12. A survey of splice variants of the human hypoxanthine phosphoribosyl transferase and DNA polymerase beta genes: products of alternative or aberrant splicing?

    PubMed Central

    Skandalis, Adonis; Uribe, Elke

    2004-01-01

    Errors during the pre-mRNA splicing of metazoan genes can degrade the transmission of genetic information, and have been associated with a variety of human diseases. In order to characterize the mutagenic and pathogenic potential of mis-splicing, we have surveyed and quantified the aberrant splice variants in the human hypoxanthine phosphoribosyl transferase (HPRT) and DNA polymerase β (POLB) in the presence and the absence of the Nonsense Mediated Decay (NMD) pathway, which removes transcripts with premature termination codons. POLB exhibits a high frequency of splice variants (40–60%), whereas the frequency of HPRT splice variants is considerably lower (∼1%). Treatment of cells with emetine to inactivate NMD alters both the spectrum and frequency of splice variants of POLB and HPRT. It is not certain at this point, whether POLB and HPRT splice variants are the result of regulated alternative splicing processes or the result of aberrant splicing, but it appears likely that at least some of the variants are the result of splicing errors. Several mechanisms that may contribute to aberrant splicing are discussed. PMID:15601998

  13. Selective potentiation of lometrexol growth inhibition by dipyridamole through cell-specific inhibition of hypoxanthine salvage.

    PubMed Central

    Turner, R. N.; Aherne, G. W.; Curtin, N. J.

    1997-01-01

    The novel antifolate lometrexol (5,10-dideazatetrahydrofolate) inhibits de novo purine biosynthesis, and co-incubation with hypoxanthine abolishes its cytotoxicity. The prevention of hypoxanthine rescue from an antipurine antifolate by the nucleoside transport inhibitor dipyridamole was investigated for the first time in nine human and rodent cell lines from seven different tissues of origin. In A549, HeLa and CHO cells, dipyridamole prevented hypoxanthine rescue and so growth was inhibited by the combination of lometrexol, dipyridamole and hypoxanthine, but in HT29, HCT116, KK47, MDA231, CCRF CEM and L1210 cells dipyridamole had no effect and the combination did not inhibit growth. Dipyridamole inhibited hypoxanthine uptake in A549 but not in CCRF CEM cells. Dipyridamole prevented the hypoxanthine-induced repletion of dGTP pools, depleted by lometrexol, in A549 but not in CCRF CEM cells. Thus, the selective growth-inhibitory effect of the combination of lometrexol, dipyridamole and hypoxanthine is apparently due to the dipyridamole sensitivity (ds) or insensitivity (di) of hypoxanthine transport. Both the human and murine leukaemic cells are of the di phenotype. If this reflects the transport phenotype of normal bone marrow it would suggest that the combination of lometrexol, dipyridamole and hypoxanthine might be selectively toxic to certain tumour types and have reduced toxicity to the bone marrow. PMID:9374375

  14. Temporal order of replication of genes responsible for hypoxanthine phosphoribosyl transferase and Na/sup +//K/sup +/ ATPase in chemically transformed human fibroblasts

    SciTech Connect

    Tsutsui, T.; Suzuki, N.; Elmore, E.; Maizumi, H.

    1986-06-01

    The cytotoxic and mutagenic effects of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of a chemically induced, transformed line (Hut-11A cells) derived from diploid human skin fibroblasts were examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2-h periods during the S phase with 5-bromodeoxyuridine (BrdU) followed by irradiation with near-ultraviolet (UV). The BrdU-plus-irradiation treatment was cytotoxic and mutagenic, while treatment with BrdU alone or irradiation alone was neither cytotoxic nor mutagenic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 h of S phase, whereas scare lethality was observed in late S phase. The BrdU-plus-irradiation treatment induced ouabain- and 6-thioguanine-resistant mutants, while BrdU alone or irradiation alone was not mutagenic. Ouabain-resistant mutants were induced during early S phase by the BrdU-plus-irradiation treatment. 6-Thioguanine-resistant mutants, however, were induced during middle to late S phase. These results suggest that a certain region or regions in the DNA of Hut-11A cells, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by BrdU treatment plus near-UV irradiation for cell survival and that gene(s) responsible for Na/sup +//K/sup +/ ATPase is replicated during early S phase and gene(s) for hypoxanthine phosphoribosyl transferase is replicated during middle to late S phase.

  15. INDUCTION OF MUTATIONS BY CHEMICAL AGENTS AT THE HYPOXANTHINE-GUANINE PHOSPHORIBOSYL TRANSFERASE LOCUS IN HUMAN EPITHELIAL TERATOMA CELLS

    EPA Science Inventory

    Induction of 6-thioguanine (TG) resistance by chemical mutagens was examined in a line of cells derived from a human epithelial teratocarcinoma cell clone. The cells, designated as P3 cells, have a stable diploid karyotype with 46(XX) chromosomes, including a translocation betwee...

  16. Prospects for cellular mutational assays in human populations

    SciTech Connect

    Mendelsohn, M.L.

    1984-06-29

    Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references.

  17. New hypoxanthine nucleosides with RNA antiviral activity.

    PubMed

    Nair, V; Ussery, M A

    1992-08-01

    A series of novel C-2 functionalized hypoxanthine and purine ribonucleosides have been synthesized and evaluated against exotic RNA viruses of the family or genus alpha, arena, flavi, and rhabdo. Both specific and broad-spectrum antiviral activities were discovered but only with hypoxanthine nucleosides. PMID:1444325

  18. Anti-oxidant effects of the extracts from the leaves of Chromolaena odorata on human dermal fibroblasts and epidermal keratinocytes against hydrogen peroxide and hypoxanthine-xanthine oxidase induced damage.

    PubMed

    Thang, P T; Patrick, S; Teik, L S; Yung, C S

    2001-06-01

    In cutaneous tissue repair, oxidants and antioxidants play very important roles. In local acute and chronic wounds, oxidants are known to have the ability to cause as cell damage and may function as inhibitory factors to wound healing. The administration of anti-oxidants or free radical scavengers is reportedly helpful, notably in order to limit the delayed sequelae of thermal trauma and to enhance the healing process. Extracts from the leaves of Chromolaena odorata have been shown to be beneficial for treatment of wounds. Studies in vitro of these extracts demonstrated enhanced proliferation of fibroblasts, endothelial cells and keratinocytes, stimulation of keratinocyte migration in an in vitro wound assay, up-regulation of production by keratinocytes of extracellular matrix proteins and basement membrane components, and inhibition of collagen lattice contraction by fibroblasts. In this study, the anti-oxidant effects of both total ethanol and polyphenolic extracts from the plant leaves on hydrogen peroxide and hypoxanthine-xanthine oxidase induced damage to human fibroblasts and keratinocytes were investigated. Cell viability was monitored by a colorimetric assay. The results showed that for fibroblasts, toxicity of hydrogen peroxide or hypoxanthine xanthine oxidase on cells was dose-dependent. Total ethanol extract (TEE) at 400 and 800 microg/ml showed maximum and consistent protective cellular effect on oxidant toxicity at low or high doses of oxidants. The 50 microg/ml concentration of TEE also had significant and slightly protective effects on fibroblasts against hydrogen peroxide and hypoxanthine-xanthine oxidase induced damage, respectively. For keratinocytes, a dose-dependent relationship of oxidant toxicity was only seen with hydrogen peroxide but the protective action of the extract correlated with oxidant dosage. TEE at 400 and 800 microg/ml showed dose-dependent effects with both low and high concentration of oxidants. TEE at 50 microg/ml had no

  19. Interactions at the Dimer Interface Influence the Relative Efficiencies for Purine Nucleotide Synthesis and Pyrophosphorolysis in a Phosphoribosyltransferase

    SciTech Connect

    Canyuk, Bhutorn; Medrano, Francisco J.; Wenck, MaryAnne; Focia, Pamela J.; Eakin, Ann E.; Craig III, Sydney P.

    2010-03-05

    Enzymes that salvage 6-oxopurines, including hypoxanthine phosphoribosyltransferases (HPRTs), are potential targets for drugs in the treatment of diseases caused by protozoan parasites. For this reason, a number of high-resolution X-ray crystal structures of the HPRTs from protozoa have been reported. Although these structures did not reveal why HPRTs need to form dimers for catalysis, they revealed the existence of potentially relevant interactions involving residues in a loop of amino acid residues adjacent to the dimer interface, but the contributions of these interactions to catalysis remained poorly understood. The loop, referred to as active-site loop I, contains an unusual non-proline cis-peptide and is composed of residues that are structurally analogous with Leu67, Lys68, and Gly69 in the human HPRT. Functional analyses of site-directed mutations (K68D, K68E, K68N, K68P, and K68R) in the HPRT from Trypanosoma cruzi, etiologic agent of Chagas disease, show that the side-chain at position 68 can differentially influence the K{sub m} values for all four substrates as well as the k{sub cat} values for both IMP formation and pyrophosphorolysis. Also, the results for the K68P mutant are inconsistent with a cis-trans peptide isomerization-assisted catalytic mechanism. These data, together with the results of structural studies of the K68R mutant, reveal that the side-chain of residue 68 does not participate directly in reaction chemistry, but it strongly influences the relative efficiencies for IMP formation and pyrophosphorolysis, and the prevalence of lysine at position 68 in the HPRT of the majority of eukaryotes is consistent with there being a biological role for nucleotide pyrophosphorolysis.

  20. In vivo footprint analysis and genomic sequencing of the human hypoxanthine-phosphoribosyl transferase (HPRT) 5 prime region on the active and inactive X chromosome

    SciTech Connect

    Hornstra, I.K.; Yang, T.P. )

    1991-03-11

    In female placental mammals, one of the two X chromosome in each somatic cell is randomly inactivated during female embryogenesis as a mechanism for dosage compensation. Once a given X chromosome is inactivated, all mitotic progeny maintain the same X chromosome in the inactive state. DNA-protein interactions and DNA methylation are hypothesized to maintain this allele-specific system of differential gene expression. Ligation-mediated polymerase chain reaction (LMPCR) in vivo footprinting and genomic sequencing were used to study DNA-protein interactions and DNA-methylation within the 5{prime} region of the X-linked human HPRT gene on the active and inactive X chromosomes. In vivo footprint analysis reveals at least one DNA-protein interaction specific to the active HPRT allele in human male fibroblast cells and hamster-human hybrid cells containing only the active human X chromosome. In the region examined, all CpG dinucleotides are methylated on the inactive HPRT allele and unmethylated on the active X allele in hamster-human hybrid cells carrying either the inactive or active human X chromosome, respectively. Thus, DNA-methylation may be mediating the differential binding of sequence-specific DNA-binding proteins to the active or inactive HPRT alleles.

  1. Cytotoxicity, differentiating activity and metabolism of tiazofurin in human neuroblastoma cells.

    PubMed

    Pillwein, K; Schuchter, K; Ressmańn, G; Gharehbaghi, K; Knoflach, A; Cermak, B; Jayaram, H N; Szalay, S M; Szekeres, T; Chiba, P

    1993-08-19

    The IMP dehydrogenase inhibitor, tiazofurin (TR)-2-beta-D-ribofuranosylthiazole-4-carboxamide, which exhibited oncolytic activity in patients with chronic myelogenous leukaemia (CML) in blast crisis was found to inhibit the growth of human neuroblastoma SK-N-SH cells with an IC50 of 4.2 microM. TR treatment of cells perturbed nucleic acid and catecholamine pathways. As biochemical markers of TR action decreased cellular GTP pools, increased inosine and hypoxanthine concentrations and depleted dopamine content were found. Incubation of tumour specimens obtained from paediatric patients with grade-IV neuroblastoma with TR resulted in the formation of the active metabolite, thiazole-4-carboxamide adenine dinucleotide, in concentrations sufficient to inhibit tumour growth. Cytotoxic and biochemical effects of TR were enhanced by combining it with allopurinol (an inhibitor of xanthine dehydrogenase), and hypoxanthine (an alternate substrate for hypoxanthine-guanine phosphoribosyltransferase). Induction of transdifferentiation of SK-N-SH cells from a neuroblast to an epitheloid, substrate-adherent phenotype was more pronounced with TR than with all-trans-retinoic acid. Transdifferentiating treatment with TR resulted in a 2-fold-enhanced sensitivity towards adriamycin. However, differentiation with all-trans-retinoic acid rendered the cells more resistant to adriamycin. Our results suggest that TR might be a promising agent for the treatment of children suffering from neuroblastoma. PMID:8344756

  2. IR spectra of guanine and hypoxanthine isolated molecules

    NASA Astrophysics Data System (ADS)

    Sheina, G. G.; Stepanian, S. G.; Radchenko, E. D.; Blagoi, Yu. P.

    1987-05-01

    High resolution spectra of guanine, hypoxanthine, isocytosine, 2-aminopyrimidine and their deutero- and methyl derivatives obtained in Ar matrices by the low temperature IR spectroscopy method are reported. Normal modes of enol tautomers of guanine, 9-CH 3-guanine, hypoxanthine and 2-aminopurine are calculated. Force fields are the same as for purine. Results calculated are used to interpret the experimental spectra. Keto—enol tautomerism is shown to exist in guanine and hypoxanthine, the proportions of enol tautomer being 50 and 5%, respectively. Possible biological applications of the results obtained are discussed.

  3. Extracellular nicotinamide phosphoribosyltransferase, a new cancer metabokine.

    PubMed

    Grolla, Ambra A; Travelli, Cristina; Genazzani, Armando A; Sethi, Jaswinder K

    2016-07-01

    In this review, we focus on the secreted form of nicotinamide phosphoribosyltransferase (NAMPT); extracellular NAMPT (eNAMPT), also known as pre-B cell colony-enhancing factor or visfatin. Although intracellular NAMPT is a key enzyme in controlling NAD metabolism, eNAMPT has been reported to function as a cytokine, with many roles in physiology and pathology. Circulating eNAMPT has been associated with several metabolic and inflammatory disorders, including cancer. Because cytokines produced in the tumour micro-environment play an important role in cancer pathogenesis, in part by reprogramming cellular metabolism, future improvements in cancer immunotherapy will require a better understanding of the crosstalk between cytokine action and tumour biology. In this review, the knowledge of eNAMPT in cancer will be discussed, focusing on its immunometabolic function as a metabokine, its secretion, its mechanism of action and possible roles in the cancer micro-environment. PMID:27128025

  4. Urinary hypoxanthine and xanthine levels in acute coronary syndromes.

    PubMed

    Turgan, N; Boydak, B; Habif, S; Gülter, C; Senol, B; Mutaf, I; Ozmen, D; Bayindir, O

    1999-01-01

    Ischemia leads to impaired ATP metabolism, with increased production of purine degradation products, such as hypoxanthine and xanthine, which are useful markers of tissue hypoxia. These extracellular markers of ischemia have been studied extensively in many clinical conditions of oxidative stress, including perinatal asphyxia, acute respiratory distress syndrome, cerebral ischemia, and preeclampsia. The aim of this study was to explore the usefulness of urinary hypoxanthine and xanthine as ischemia markers in acute coronary syndromes. Urinary excretion of hypoxanthine and xanthine was assessed by high-performance liquid chromatography in 30 patients with acute coronary syndromes and in 30 age- and sex-matched controls. Serum and urine uric acid, creatinine, and urea concentrations were also determined. Hypoxanthine excretion was significantly elevated in patients compared with healthy controls (84.37+/-8.63 and 42.70+/-3.97 nmol/mg creatinine, mean+/-SEM, P<0.0001). Urinary xanthine levels were also increased in patients with acute coronary syndromes (100.13+/-12.14 and 34.74+/-4.07 nmol/mg creatinine patients and controls, respectively; P<0.0001). Hypoxanthine and xanthine excretion showed a strong positive correlation in both groups. Significant negative correlations between urinary hypoxanthine and uric acid and xanthine and uric acid were observed in the patients, but not in controls. In conclusion, increased levels of ATP degradation products hypoxanthine and xanthine are observed in various hypoxic clinical conditions. This study suggests that these parameters may be useful markers of ischemia in patients with acute coronary syndromes. PMID:10784378

  5. V(D)J RECOMBINASE-MEDIATED DELETION OF THE HPRT GENE IN T-LYMPHOCYTES FROM ADULT HUMANS

    EPA Science Inventory

    The hprt T-cell cloning assay allows the detection of mutations occurring in vivo in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene of T-lymphocytes. e have shown previously that the illegitimate activity of V(D)J recombinase accounts for about 40% of the hprt mut...

  6. Cerebrospinal fluid concentrations of hypoxanthine, xanthine, uridine and inosine: high concentrations of the ATP metabolite, hypoxanthine, after hypoxia.

    PubMed Central

    Harkness, R A; Lund, R J

    1983-01-01

    CSF obtained for clinical purposes from newborn, children and adults has been analysed by high pressure liquid chromatography for hypoxanthine, xanthine, inosine, uridine and urate. Large rises in hypoxanthine and to a lesser extent xanthine occur for about 24 h after hypoxia. High concentrations were associated with later evidence of brain damage or subsequent death. Changes in CSF could be independent of those in plasma. Small or negligible rises were associated with localised and generalised infections including bacterial meningitis, fits, or both. Marked and rapid rises were found after death. These estimations may "predict" the extent of brain damage or brain death. PMID:6681617

  7. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines.

    PubMed

    López-Cruz, Roberto I; Crocker, Daniel E; Gaxiola-Robles, Ramón; Bernal, Jaime A; Real-Valle, Roberto A; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  8. Plasma Hypoxanthine-Guanine Phosphoribosyl Transferase Activity in Bottlenose Dolphins Contributes to Avoiding Accumulation of Non-recyclable Purines

    PubMed Central

    López-Cruz, Roberto I.; Crocker, Daniel E.; Gaxiola-Robles, Ramón; Bernal, Jaime A.; Real-Valle, Roberto A.; Lugo-Lugo, Orlando; Zenteno-Savín, Tania

    2016-01-01

    Marine mammals are exposed to ischemia/reperfusion and hypoxia/reoxygenation during diving. During oxygen deprivation, adenosine triphosphate (ATP) breakdown implies purine metabolite accumulation, which in humans is associated with pathological conditions. Purine recycling in seals increases in response to prolonged fasting and ischemia. Concentrations of metabolites and activities of key enzymes in purine metabolism were examined in plasma and red blood cells from bottlenose dolphins (Tursiops truncatus) and humans. Hypoxanthine and inosine monophosphate concentrations were higher in plasma from dolphins than humans. Plasma hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity in dolphins suggests an elevated purine recycling rate, and a mechanism for avoiding accumulation of non-recyclable purines (xanthine and uric acid). Red blood cell concentrations of hypoxanthine, adenosine diphosphate, ATP and guanosine triphosphate were lower in dolphins than in humans; adenosine monophosphate and nicotinamide adenine dinucleotide concentrations were higher in dolphins. HGPRT activity in red blood cells was higher in humans than in dolphins. The lower concentrations of purine catabolism and recycling by-products in plasma from dolphins could be beneficial in providing substrates for recovery of ATP depleted during diving or vigorous swimming. These results suggest that purine salvage in dolphins could be a mechanism for delivering nucleotide precursors to tissues with high ATP and guanosine triphosphate requirements. PMID:27375492

  9. Antimalarial activity of prodrugs of N-branched acyclic nucleoside phosphonate inhibitors of 6-oxopurine phosphoribosyltransferases.

    PubMed

    Hocková, Dana; Janeba, Zlatko; Naesens, Lieve; Edstein, Michael D; Chavchich, Marina; Keough, Dianne T; Guddat, Luke W

    2015-09-01

    Acyclic nucleoside phosphonates (ANPs) that contain a 6-oxopurine base are good inhibitors of the human and Plasmodium falciparum 6-oxopurine phosphoribosyltransferases (PRTs), key enzymes of the purine salvage pathway. Chemical modifications, based on the crystal structures of several inhibitors in complex with the human PRTase, led to the design of a new class of inhibitors--the aza-ANPs. Because of the negative charges of the phosphonic acid moiety, their ability to cross cell membranes is, however, limited. Thus, phosphoramidate prodrugs of the aza-ANPs were prepared to improve permeability. These prodrugs arrest parasitemia with IC50 values in the micromolar range against Plasmodium falciparum-infected erythrocyte cultures (both chloroquine-sensitive and chloroquine-resistant Pf strains). The prodrugs exhibit low cytotoxicity in several human cell lines. Thus, they fulfill two essential criteria to qualify them as promising antimalarial drug leads. PMID:26275679

  10. An amperometric hypoxanthine biosensor based on Au@FeNPs for determination of hypoxanthine in meat samples.

    PubMed

    Devi, Rooma; Yadav, Sujata; Nehra, Renuka; Pundir, C S

    2013-11-01

    A xanthine oxidase (XOD) from buttermilk was immobilized covalently onto boronic acid functionalized gold coated iron nanoparticles (Au@FeNPs) electrodeposited on pencil graphite (PG) electrode, via the boroester linkages, between free hydroxyl groups of boronic acid, α-COOH and -NH2 groups of enzyme. The surface functionalization of Fe/Au nanoparticles with boronic acid (Au@FeNPs) on pencil graphite (PG) electrode was characterized by Fourier transform infrared (FTIR), cyclic voltammetry (CV), scanning electron microscopy (SEM), atomic force microscopy (AFM) and Electrochemical impedance spectroscopy (EIS) before and after immobilization of XOD. The biosensor exhibited optimum response within 3s at pH 7.2 and 30 °C and linearity in the range, 0.05 μM to 150 μM for hypoxanthine with a detection limit of 0.05 μM (S/N=3). Apparent Michaelis Menten constant (Km(app)) for hypoxanthine was 40 μM and Imax 0.125 mA. The biosensor was employed to determine hypoxanthine in fish, chicken, pork, beef meat and lost 50% of its initial activity after its 200 uses over 100 days, when stored at 4 °C. PMID:24140402

  11. Regulatory elements in the introns of the human HPRT gene are necessary for its expression in embryonic stem cells.

    PubMed Central

    Reid, L H; Gregg, R G; Smithies, O; Koller, B H

    1990-01-01

    We have examined the expression of transfected human hypoxanthine phosphoribosyltransferase minigenes (HPRT) in mouse embryonic stem (ES) cells. cDNA constructs of this gene that have been successfully used in somatic cell lines failed to confer hypoxanthine/aminopterin/thymidine (HAT) resistance in ES cells. In contrast, constructs containing introns 1 and 2 from the HPRT gene produced a high frequency of HAT-resistant colonies. This observation allowed us to identify two sequences in these introns that influence expression of the HPRT gene in ES cells. One element, located in intron 2, is required for effective HPRT expression in these cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, we have constructed an HPRT minigene that can be used for either positive or negative selection in ES cell experiments. This dual capability allows the design of "in-out" procedures to create subtle changes in target genes by homologous recombination with the aid of this selectable minigene. PMID:2349238

  12. Dissociative electron attachment to the gas-phase nucleobase hypoxanthine

    SciTech Connect

    Dawley, M. Michele; Tanzer, Katrin; Denifl, Stephan E-mail: Sylwia.Ptasinska.1@nd.edu; Carmichael, Ian; Ptasińska, Sylwia E-mail: Sylwia.Ptasinska.1@nd.edu

    2015-06-07

    We present high-resolution measurements of the dissociative electron attachment (DEA) to isolated gas-phase hypoxanthine (C{sub 5}H{sub 4}N{sub 4}O, Hyp), a tRNA purine base. The anion mass spectra and individual ion efficiency curves from Hyp were measured as a function of electron energy below 9 eV. The mass spectra at 1 and 6 eV exhibit the highest anion yields, indicating possible common precursor ions that decay into the detectable anionic fragments. The (Hyp − H) anion (C{sub 5}H{sub 3}N{sub 4}O{sup −}) exhibits a sharp resonant peak at 1 eV, which we tentatively assign to a dipole-bound state of the keto-N1H,N9H tautomer in which dehydrogenation occurs at either the N1 or N9 position based upon our quantum chemical computations (B3LYP/6-311+G(d,p) and U(MP2-aug-cc-pVDZ+)) and prior studies with adenine. This closed-shell dehydrogenated anion is the dominant fragment formed upon electron attachment, as with other nucleobases. Seven other anions were also observed including (Hyp − NH){sup −}, C{sub 4}H{sub 3}N{sub 4}{sup −}/C{sub 4}HN{sub 3}O{sup −}, C{sub 4}H{sub 2}N{sub 3}{sup −}, C{sub 3}NO{sup −}/HC(HCN)CN{sup −}, OCN{sup −}, CN{sup −}, and O{sup −}. Most of these anions exhibit broad but weak resonances between 4 and 8 eV similar to many analogous anions from adenine. The DEA to Hyp involves significant fragmentation, which is relevant to understanding radiation damage of biomolecules.

  13. Biochemical Characterization of Uracil Phosphoribosyltransferase from Mycobacterium tuberculosis

    PubMed Central

    Villela, Anne Drumond; Ducati, Rodrigo Gay; Rosado, Leonardo Astolfi; Bloch, Carlos Junior; Prates, Maura Vianna; Gonçalves, Danieli Cristina; Ramos, Carlos Henrique Inacio; Basso, Luiz Augusto; Santos, Diogenes Santiago

    2013-01-01

    Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-α-1-pyrophosphate (PRPP) to uridine 5′-monophosphate (UMP) and pyrophosphate (PPi). UPRT plays an important role in the pyrimidine salvage pathway since UMP is a common precursor of all pyrimidine nucleotides. Here we describe cloning, expression and purification to homogeneity of upp-encoded UPRT from Mycobacterium tuberculosis (MtUPRT). Mass spectrometry and N-terminal amino acid sequencing unambiguously identified the homogeneous protein as MtUPRT. Analytical ultracentrifugation showed that native MtUPRT follows a monomer-tetramer association model. MtUPRT is specific for uracil. GTP is not a modulator of MtUPRT ativity. MtUPRT was not significantly activated or inhibited by ATP, UTP, and CTP. Initial velocity and isothermal titration calorimetry studies suggest that catalysis follows a sequential ordered mechanism, in which PRPP binding is followed by uracil, and PPi product is released first followed by UMP. The pH-rate profiles indicated that groups with pK values of 5.7 and 8.1 are important for catalysis, and a group with a pK value of 9.5 is involved in PRPP binding. The results here described provide a solid foundation on which to base upp gene knockout aiming at the development of strategies to prevent tuberculosis. PMID:23424660

  14. Nicotinamide phosphoribosyltransferase (NAMPT/PBEF/visfatin) is a tumoural cytokine released from melanoma.

    PubMed

    Grolla, Ambra A; Torretta, Simone; Gnemmi, Ilaria; Amoruso, Angela; Orsomando, Giuseppe; Gatti, Marco; Caldarelli, Antonio; Lim, Dmitry; Penengo, Lorenza; Brunelleschi, Sandra; Genazzani, Armando A; Travelli, Cristina

    2015-11-01

    High plasma levels of nicotinamide phosphoribosyltransferase (NAMPT), traditionally considered an intracellular enzyme with a key role in NAD synthesis, have been reported in several oncological, inflammatory and metabolic diseases. We now show that eNAMPT can be actively released by melanoma cells in vitro. We analysed the mechanisms of its release, and we found both classical and non-classical pathway involvement. eNAMPT released by melanoma cells, in our hands, has paracrine and autocrine effects: it activates MAPK, AKT and NF-κB pathways and increases colony formation in anchorage-independent conditions. eNAMPT also induces M1 polarization in human monocytes. Last, we demonstrate, for the first time in any cancer type, that eNAMPT levels in plasma of tumour-bearing mice increase and that this increase can be reconducted to the tumour itself. This provides an important cue on previous observations that eNAMPT is increased in patients with cancer. Moreover, silencing NAMPT in melanoma cells leads to a reduction in the tumour growth rate. Our findings extend the basis to consider eNAMPT as a cytokine involved in tumour progression. PMID:26358657

  15. Adenine phosphoribosyltransferase deficiency as a rare cause of renal allograft dysfunction.

    PubMed

    Kaartinen, Kati; Hemmilä, Ulla; Salmela, Kaija; Räisänen-Sokolowski, Anne; Kouri, Timo; Mäkelä, Satu

    2014-04-01

    Adenine phosphoribosyltransferase deficiency is a rare autosomal recessive disorder manifesting as urolithiasis or crystalline nephropathy. It leads to the generation of large amounts of poorly soluble 2,8-dihydroxyadenine excreted in urine, yielding kidney injury and in some patients, kidney failure. Early recognition of the disease, institution of xanthine analog therapy to block the formation of 2,8-dihydroxyadenine, high fluid intake, and low purine diet prevent CKD. Because of symptom variability and lack of awareness, however, the diagnosis is sometimes extremely deferred. We describe a patient with adenine phosphoribosyltransferase deficiency who was diagnosed during evaluation of a poorly functioning second kidney allograft. This report highlights the risk of renal allograft loss in patients with undiagnosed adenine phosphoribosyltransferase deficiency and the need for improved early detection of this disease. PMID:24459232

  16. Plasmodium falciparum: assessment of in vitro growth by (/sup 3/H)hypoxanthine incorporation

    SciTech Connect

    Chulay, J.D.; Haynes, J.D.; Diggs, C.L.

    1983-02-01

    To evaluate rapidly Plasmodium falciparum growth in Vitro, (/sup 3/H)hypoxanthine was added to parasite microcultures and radioisotope incorporation was measured. When culture parameters were carefully controlled, (/sup 3/H)hypoxanthine incorporation was proportional to the number of parasitized erythrocytes present. Factors affecting (/sup 3/H)hypoxanthine incorporation included initial parasitemia, duration of culture, duration of radioisotope pulse, parasite stage, concentration of uninfected erythrocytes, the use of serum or plasma to supplement growth, and the concentration of a variety of purines in the culture medium. The method described can be used to measure inhibition of P. falciparum growth by immune serum and has previously been used to study antimalarial drug activity in vitro.

  17. Phenotype and Genotype Characterization of Adenine Phosphoribosyltransferase Deficiency

    PubMed Central

    Bollée, Guillaume; Dollinger, Cécile; Boutaud, Lucile; Guillemot, Delphine; Bensman, Albert; Harambat, Jérôme; Deteix, Patrice; Daudon, Michel; Knebelmann, Bertrand

    2010-01-01

    Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder causing 2,8-dihydroxyadenine stones and renal failure secondary to intratubular crystalline precipitation. Little is known regarding the clinical presentation of APRT deficiency, especially in the white population. We retrospectively reviewed all 53 cases of APRT deficiency (from 43 families) identified at a single institution between 1978 and 2009. The median age at diagnosis was 36.3 years (range 0.5 to 78.0 years). In many patients, a several-year delay separated the onset of symptoms and diagnosis. Of the 40 patients from 33 families with full clinical data available, 14 (35%) had decreased renal function at diagnosis. Diagnosis occurred in six (15%) patients after reaching ESRD, with five diagnoses made at the time of disease recurrence in a renal allograft. Eight (20%) patients reached ESRD during a median follow-up of 74 months. Thirty-one families underwent APRT sequencing, which identified 54 (87%) mutant alleles on the 62 chromosomes analyzed. We identified 18 distinct mutations. A single T insertion in a splice donor site in intron 4 (IVS4 + 2insT), which produces a truncated protein, accounted for 40.3% of the mutations. We detected the IVS4 + 2insT mutation in two (0.98%) of 204 chromosomes of healthy newborns. This report, which is the largest published series of APRT deficiency to date, highlights the underdiagnosis and potential severity of this disease. Early diagnosis is crucial for initiation of effective treatment with allopurinol and for prevention of renal complications. PMID:20150536

  18. The Role of Nicotinamide Phosphoribosyltransferase in Cerebral Ischemia.

    PubMed

    Chen, Xinzhi; Zhao, Shangfeng; Song, Yang; Shi, Yejie; Leak, Rehana K; Cao, Guodong

    2015-01-01

    As recombinant tissue plasminogen activator is the only drug approved for the clinical treatment of acute ischemic stroke, there is an urgent unmet need for novel stroke treatments. Endogenous defense mechanisms against stroke may hold the key to new therapies for stroke. A large number of studies suggest that nicotinamide phosphoribosyl-transferase (NAMPT is an attractive candidate to improve post-stroke recovery. NAMPT is a multifunctional protein and plays important roles in immunity, metabolism, aging, inflammation, and stress responses. NAMPT exists in both the intracellular and extracellular space. As a rate-limiting enzyme, the intracellular form (iNAMPT catalyzes the first step in the biosynthesis of nicotinamide adenine dinucleotide (NAD from nicotinamide. iNAMPT closely regulates energy metabolism, enhancing the proliferation of endothelial cells, inhibiting apoptosis, regulating vascular tone, and stimulating autophagy in disease conditions such as stroke. Extracellular NAMPT (eNAMPT is also known as visfatin (visceral fat-derived adipokine and has pleotropic effects. It is widely believed that the diverse biological functions of eNAMPT are attributed to its NAMPT enzymatic activity. However, the effects of eNAMPT on ischemic injury are still controversial. Some authors have argued that eNAMPT exacerbates ischemic neuronal injury non-enzymatically by triggering the release of TNF-α from glial cells. In addition, NAMPT also participates in several pathophysiological processes such as hypertension, atherosclerosis, and ischemic heart disease. Thus, it remains unclear under what conditions NAMPT is beneficial or destructive. Recent work using in vitro and in vivo genetic/ pharmacologic manipulations, including our own studies, has greatly improved our understanding of NAMPT. This review focuses on the multifaceted and complex roles of NAMPT under both normal and ischemic conditions. PMID:26059356

  19. Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells

    SciTech Connect

    Mendez, M.J.; Abderrahim, H.; Noguchi, M.

    1995-03-20

    With the goal of creating a strain of mice capable of producing human antibodies, we are cloning and reconstructing the human immunoglobulin germline repertoire in yeast artificial chromosomes (YACs). We describe the identification of YACs containing variable and constant region sequences from the human heavy chain (IgH) and kappa light chain (IgK) loci and the characterization of their integrity in yeast and in mouse embryonic stem (ES) cells. The IgH locus-derived YAC contains five variable (V{sub H}) genes, the major diversity (D) gene cluster, the joining (J{sub H}) genes, the intronic enhancer (E{sub H}), and the constant region genes, mu (C{mu}) and delta (C{delta}). Two IgK locus-derived YACs each contain three variable (V{kappa}) genes, the joining (J{kappa}) region, the intronic enhancer (E{kappa}), the constant gene (C{kappa}), and the kappa deleting element (kde). The IgH YAC was unstable in yeast, generating a variety of deletion derivatives, whereas both IgK YACs were stable. YACs encoding heavy chain and kappa light chain, retrofitted with the mammalian selectable marker, hypoxanthine phosphoribosyltransferase (HPRT), were each introduced into HPRT-deficient mouse ES cells. Analysis of YAC integrity in ES cell lines revealed that the majority of DNA inserts were integrated in substantially intact form. 78 refs., 7 figs.

  20. Effects of Hypoxanthine Substitution in Peptide Nucleic Acids Targeting KRAS2 Oncogenic mRNA Molecules: Theory and Experiment

    PubMed Central

    Sanders, Jeffrey M.; Wampole, Matthew E.; Chen, Chang-Po; Sethi, Dalip; Singh, Amrita; Dupradeau, François-Yves; Wang, Fan; Gray, Brian D.; Thakur, Mathew L.; Wickstrom, Eric

    2013-01-01

    Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multi-mutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick basepairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA-PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA-PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition. PMID:23972113

  1. Human antiglioma monoclonal antibodies from patients with astrocytic tumors.

    PubMed

    Dan, M D; Schlachta, C M; Guy, J; McKenzie, R G; Dorscheid, D R; Sandor, V A; Villemure, J G; Price, G B

    1992-04-01

    The current management of malignant gliomas is unsatisfactory compared to that of other solid tumors; the expected median survival period is less than 1 year with the patient undergoing conventional surgery, radiotherapy, and chemotherapy treatment. Immunological reagents could be a useful adjunct. Human monoclonal antibodies derived from patients with astrocytic tumors might recognize subtle antigenic specificities that would differ from those recognized by xenogeneic (murine) systems. Five hybridomas, designated as BT27/1A2, BT27/2A3, BT32/A6, BT34/A5, and BT54/B8, were produced from the fusion of peripheral blood lymphocytes of four patients with astrocytic tumors to the human myeloma-like cell line TM-H2-SP2. This cell line has a 46, XX karyotype and is negative for hypoxanthine guanine phosphoribosyltransferase. All five human monoclonal antibodies produced 2.4 to 44 micrograms/ml of immunoglobulin M, had a similar but not identical pattern of reactivity against a panel of human tumor cell lines, and failed to react with normal human astrocytes. Labeling of four neuroectodermal tumor explant cultures by BT27/2A3 was demonstrated by flow cytometry. Karyotyping of three of the five hybridomas demonstrated that two were pseudodiploid (2-3n) and one hypodiploid (less than 2n). The monoclonality of the hybridomas was evaluated by Southern blot analysis of JH gene rearrangements, revealing two types of rearrangements for each hybridoma, both consistent with monoclonality. Preliminary antigen characterization indicated that at least four of the five human monoclonal antibodies were directed to cell-surface glycolipids. PMID:1545260

  2. The Small Molecule GMX1778 Is a Potent Inhibitor of NAD+ Biosynthesis: Strategy for Enhanced Therapy in Nicotinic Acid Phosphoribosyltransferase 1-Deficient Tumors▿

    PubMed Central

    Watson, Mark; Roulston, Anne; Bélec, Laurent; Billot, Xavier; Marcellus, Richard; Bédard, Dominique; Bernier, Cynthia; Branchaud, Stéphane; Chan, Helen; Dairi, Kenza; Gilbert, Karine; Goulet, Daniel; Gratton, Michel-Olivier; Isakau, Henady; Jang, Anne; Khadir, Abdelkrim; Koch, Elizabeth; Lavoie, Manon; Lawless, Michael; Nguyen, Mai; Paquette, Denis; Turcotte, Émilie; Berger, Alvin; Mitchell, Matthew; Shore, Gordon C.; Beauparlant, Pierre

    2009-01-01

    GMX1777 is a prodrug of the small molecule GMX1778, currently in phase I clinical trials for the treatment of cancer. We describe findings indicating that GMX1778 is a potent and specific inhibitor of the NAD+ biosynthesis enzyme nicotinamide phosphoribosyltransferase (NAMPT). Cancer cells have a very high rate of NAD+ turnover, which makes NAD+ modulation an attractive target for anticancer therapy. Selective inhibition by GMX1778 of NAMPT blocks the production of NAD+ and results in tumor cell death. Furthermore, GMX1778 is phosphoribosylated by NAMPT, which increases its cellular retention. The cytotoxicity of GMX1778 can be bypassed with exogenous nicotinic acid (NA), which permits NAD+ repletion via NA phosphoribosyltransferase 1 (NAPRT1). The cytotoxicity of GMX1778 in cells with NAPRT1 deficiency, however, cannot be rescued by NA. Analyses of NAPRT1 mRNA and protein levels in cell lines and primary tumor tissue indicate that high frequencies of glioblastomas, neuroblastomas, and sarcomas are deficient in NAPRT1 and not susceptible to rescue with NA. As a result, the therapeutic index of GMX1777 can be widended in the treatment animals bearing NAPRT1-deficient tumors by coadministration with NA. This provides the rationale for a novel therapeutic approach for the use of GMX1777 in the treatment of human cancers. PMID:19703994

  3. Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis, Leads to Altered Carbohydrate Metabolism in Cancer Cells*

    PubMed Central

    Tan, Bo; Dong, Sucai; Shepard, Robert L.; Kays, Lisa; Roth, Kenneth D.; Geeganage, Sandaruwan; Kuo, Ming-Shang; Zhao, Genshi

    2015-01-01

    Nicotinamide phosphoribosyltransferase (NAMPT) has been extensively studied due to its essential role in NAD+ biosynthesis in cancer cells and the prospect of developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the glyceraldehyde 3-phosphate dehydrogenase step (Tan, B., Young, D. A., Lu, Z. H., Wang, T., Meier, T. I., Shepard, R. L., Roth, K., Zhai, Y., Huss, K., Kuo, M. S., Gillig, J., Parthasarathy, S., Burkholder, T. P., Smith, M. C., Geeganage, S., and Zhao, G. (2013) Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD+ biosynthesis, in human cancer cells: metabolic basis and potential clinical implications. J. Biol. Chem. 288, 3500–3511). Due to technical limitations, we failed to separate isotopomers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and glyceraldehyde, and sedoheptulose 1-phosphate was derived from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased glyceraldehyde and

  4. Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT), an Enzyme Essential for NAD+ Biosynthesis, Leads to Altered Carbohydrate Metabolism in Cancer Cells.

    PubMed

    Tan, Bo; Dong, Sucai; Shepard, Robert L; Kays, Lisa; Roth, Kenneth D; Geeganage, Sandaruwan; Kuo, Ming-Shang; Zhao, Genshi

    2015-06-19

    Nicotinamide phosphoribosyltransferase (NAMPT) has been extensively studied due to its essential role in NAD(+) biosynthesis in cancer cells and the prospect of developing novel therapeutics. To understand how NAMPT regulates cellular metabolism, we have shown that the treatment with FK866, a specific NAMPT inhibitor, leads to attenuation of glycolysis by blocking the glyceraldehyde 3-phosphate dehydrogenase step (Tan, B., Young, D. A., Lu, Z. H., Wang, T., Meier, T. I., Shepard, R. L., Roth, K., Zhai, Y., Huss, K., Kuo, M. S., Gillig, J., Parthasarathy, S., Burkholder, T. P., Smith, M. C., Geeganage, S., and Zhao, G. (2013) Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD(+) biosynthesis, in human cancer cells: metabolic basis and potential clinical implications. J. Biol. Chem. 288, 3500-3511). Due to technical limitations, we failed to separate isotopomers of phosphorylated sugars. In this study, we developed an enabling LC-MS methodology. Using this, we confirmed the previous findings and also showed that NAMPT inhibition led to accumulation of fructose 1-phosphate and sedoheptulose 1-phosphate but not glucose 6-phosphate, fructose 6-phosphate, and sedoheptulose 7-phosphate as previously thought. To investigate the metabolic basis of the metabolite formation, we carried out biochemical and cellular studies and established the following. First, glucose-labeling studies indicated that fructose 1-phosphate was derived from dihydroxyacetone phosphate and glyceraldehyde, and sedoheptulose 1-phosphate was derived from dihydroxyacetone phosphate and erythrose via an aldolase reaction. Second, biochemical studies showed that aldolase indeed catalyzed these reactions. Third, glyceraldehyde- and erythrose-labeling studies showed increased incorporation of corresponding labels into fructose 1-phosphate and sedoheptulose 1-phosphate in FK866-treated cells. Fourth, NAMPT inhibition led to increased glyceraldehyde and

  5. Isolation of the human chromosomal band Xq28 within somatic cell hybrids by fragile X site breakage.

    PubMed Central

    Warren, S T; Knight, S J; Peters, J F; Stayton, C L; Consalez, G G; Zhang, F P

    1990-01-01

    The chromosomal fragile-site mapping to Xq27.3 is associated with a frequent form of mental retardation and is prone to breakage after induced deoxyribonucleotide pool perturbation. The human hypoxanthine phosphoribosyltransferase (HPRT) and glucose-6-phosphate dehydrogenase (G6PD) genes flank the fragile X chromosome site and can be used to monitor integrity of the site in human-hamster somatic cell hybrids deficient in the rodent forms of these activities. After induction of the fragile X site, negative selection for HPRT and positive enrichment for G6PD resulted in 31 independent colonies of HPRT-,G6PD+ phenotype. Southern blot analysis demonstrated the loss of all tested markers proximal to the fragile X site with retention of all tested human Xq28 loci in a majority of the hybrids. In situ hybridization with a human-specific probe demonstrated the translocation of a small amount of human DNA to rodent chromosomes in these hybrids, suggesting chromosome breakage at the fragile X site and the subsequent translocation of Xq28. Southern blot hybridization of hybrid-cell DNA, resolved by pulsed-field gel electrophoresis, for human-specific repetitive sequences revealed abundant CpG-islands within Xq28, consistent with its known gene density. The electrophoretic banding patterns of human DNA among the hybrids were remarkably consistent, suggesting that fragile X site breakage is limited to a relatively small region in Xq27-28. These somatic cell hybrids, containing Xq27.3-qter as the sole human DNA, will aid the search for DNA associated with the fragile X site and will augment the high resolution genomic analysis of Xq28, including the identification of candidate genes for genetic-disease loci mapping to this region. Images PMID:2339126

  6. Mutation in the Human HPRT1 Gene and the Lesch-Nyhan Syndrome.

    PubMed

    Nguyen, Khue Vu; Nyhan, William L

    2016-08-01

    Lesch-Nyhan syndrome (LNS) is a rare X-linked inherited neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt) is defective. The authors report a novel mutation which led to HGprt-related neurological dysfunction (HND) in two brothers from the same family with a missense mutation in exon 6 of the coding region of the HPRT1 gene: c.437T>C, p.L146S. Molecular diagnosis discloses the genetic heterogeneity of the HPRT1 gene responsible for HGprt deficiency. It allows fast, accurate carrier detection and genetic counseling. PMID:27379977

  7. Archaeoglobus Fulgidus DNA Polymerase D: A Zinc-Binding Protein Inhibited by Hypoxanthine and Uracil.

    PubMed

    Abellón-Ruiz, Javier; Waldron, Kevin J; Connolly, Bernard A

    2016-07-17

    Archaeal family-D DNA polymerases (Pol-D) comprise a small (DP1) proofreading subunit and a large (DP2) polymerase subunit. Pol-D is one of the least studied polymerase families, and this publication investigates the enzyme from Archaeoglobus fulgidus (Afu Pol-D). The C-terminal region of DP2 contains two conserved cysteine clusters, and their roles are investigated using site-directed mutagenesis. The cluster nearest the C terminus is essential for polymerase activity, and the cysteines are shown to serve as ligands for a single, critical Zn(2+) ion. The cysteines farthest from the C terminal were not required for activity, and a role for these amino acids has yet to be defined. Additionally, it is shown that Afu Pol-D activity is slowed by the template strand hypoxanthine, extending previous results that demonstrated inhibition by uracil. Hypoxanthine was a weaker inhibitor than uracil. Investigations with isolated DP2, which has a measurable polymerase activity, localised the deaminated base binding site to this subunit. Uracil and hypoxanthine slowed Afu Pol-D "in trans", that is, a copied DNA strand could be inhibited by a deaminated base in the alternate strand of a replication fork. The error rate of Afu Pol-D, measured in vitro, was 0.24×10(-5), typical for a polymerase that has been proposed to carry out genome replication in the Archaea. Deleting the 3'-5' proofreading exonuclease activity reduced fidelity twofold. The results presented in this publication considerably increase our knowledge of Pol-D. PMID:27320386

  8. Adenine phosphoribosyltransferase from Sulfolobus solfataricus is an enzyme with unusual kinetic properties and a crystal structure that suggests it evolved from a 6-oxopurine phosphoribosyltransferase.

    PubMed

    Jensen, Kaj Frank; Hansen, Michael Riis; Jensen, Kristine Steen; Christoffersen, Stig; Poulsen, Jens-Christian Navarro; Mølgaard, Anne; Kadziola, Anders

    2015-04-14

    The adenine phosphoribosyltransferase (APRTase) encoded by the open reading frame SSO2342 of Sulfolobus solfataricus P2 was subjected to crystallographic, kinetic, and ligand binding analyses. The enzyme forms dimers in solution and in the crystals, and binds one molecule of the reactants 5-phosphoribosyl-α-1-pyrophosphate (PRPP) and adenine or the product adenosine monophosphate (AMP) or the inhibitor adenosine diphosphate (ADP) in each active site. The individual subunit adopts an overall structure that resembles a 6-oxopurine phosphoribosyltransferase (PRTase) more than known APRTases implying that APRT functionality in Crenarchaeotae has its evolutionary origin in this family of PRTases. Only the N-terminal two-thirds of the polypeptide chain folds as a traditional type I PRTase with a five-stranded β-sheet surrounded by helices. The C-terminal third adopts an unusual three-helix bundle structure that together with the nucleobase-binding loop undergoes a conformational change upon binding of adenine and phosphate resulting in a slight contraction of the active site. The inhibitor ADP binds like the product AMP with both the α- and β-phosphates occupying the 5'-phosphoribosyl binding site. The enzyme shows activity over a wide pH range, and the kinetic and ligand binding properties depend on both pH and the presence/absence of phosphate in the buffers. A slow hydrolysis of PRPP to ribose 5-phosphate and pyrophosphate, catalyzed by the enzyme, may be facilitated by elements in the C-terminal three-helix bundle part of the protein. PMID:25790177

  9. Genetic Regulation of Charged Particle Mutagenesis in Human Cells

    NASA Technical Reports Server (NTRS)

    Kronenberg, Amy; Gauny, S.; Cherbonnel-Lasserre, C.; Liu, W.; Wiese, C.

    1999-01-01

    Our studies use a series of syngeneic, and where possible, isogenic human B-lymphoblastoid cell lines to assess the genetic factors that modulate susceptibility apoptosis and their impact on the mutagenic risks of low fluence exposures to 1 GeV Fe ions and 55 MeV protons. These ions are representative of the types of charged particle radiation that are of particular significance for human health in the space radiation environment. The model system employs cell lines derived from the male donor WIL-2. These cells have a single X chromosome and they are hemizygous for one mutation marker, hypoxanthine phosphoribosyltransferase (HPRT). TK6 and WTK1 cells were each derived from descendants of WIL-2 and were each selected as heterozygotes for a second mutation marker, the thymidine kinase (TK) gene located on chromosome 17q. The HPRT and TK loci can detect many different types of mutations, from single basepair substitutions up to large scale loss of heterozygosity (LOH). The single expressing copy of TK in the TK6 and WTKI cell lines is found on the same copy of chromosome 17, and this allele can be identified by a restriction fragment length polymorphism (RFLP) identified when high molecular weight DNA is digested by the SacI restriction endonuclease and hybridized against the cDNA probe for TK. A large series of polymorphic linked markers has been identified that span more than 60 cM of DNA (approx. 60 megabasepairs) and distinguish the copy of chromosome 17 bearing the initially active TK allele from the copy of chromosome 17 bearing the silent TK allele in both TK6 and WTKI cells. TK6 cells express normal p53 protein while WTKI cells express homozygous mutant p53. Expression of mutant p53 can increase susceptibility to x-ray-induced mutations. It's been suggested that the increased mutagenesis in p53 mutant cells might be due to reduced apoptosis.

  10. Mutagenesis in human cells with accelerated H and Fe ions

    NASA Technical Reports Server (NTRS)

    Kronenberg, Amy

    1994-01-01

    The overall goals of this research were to determine the risks of mutation induction and the spectra of mutations induced by energetic protons and iron ions at two loci in human lymphoid cells. During the three year grant period the research has focused in three major areas: (1) the acquisition of sufficient statistics for human TK6 cell mutation experiments using Fe ions (400 MeV/amu), Fe ions (600 MeV/amu) and protons (250 MeV/amu); (2) collection of thymidine kinase- deficient (tk) mutants or hypoxanthine phosphoribosyltransferase deficient (hprt) mutants induced by either Fe 400 MeV/amu, Fe 600 MeV/amu, or H 250 MeV/amu for subsequent molecular analysis; and (3) molecular characterization of mutants isolated after exposure to Fe ions (600 MeV/amu). As a result of the shutdown of the BEVALAC heavy ion accelerator in December 1992, efforts were rearranged somewhat in time to complete our dose-response studies and to complete mutant collections in particular for the Fe ion beams prior to the shutdown. These goals have been achieved. A major effort was placed on collection, re-screening, and archiving of 3 different series of mutants for the various particle beam exposures: tk-ng mutants, tk-sg mutants, and hprt-deficient mutants. Where possible, groups of mutants were isolated for several particle fluences. Comparative analysis of mutation spectra has occured with characterization of the mutation spectrum for hprt-deficient mutants obtained after exposure of TK6 cells to Fe ions (600 MeV/amu) and a series of spontaneous mutants.

  11. Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells.

    PubMed Central

    Margolskee, R F; Kavathas, P; Berg, P

    1988-01-01

    Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromycin B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBO-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8). Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(4) total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines. Images PMID:2841588

  12. Dynamic architecture of the purinosome involved in human de novo purine biosynthesis.

    PubMed

    Kyoung, Minjoung; Russell, Sarah J; Kohnhorst, Casey L; Esemoto, Nopondo N; An, Songon

    2015-01-27

    Enzymes in human de novo purine biosynthesis have been demonstrated to form a reversible, transient multienzyme complex, the purinosome, upon purine starvation. However, characterization of purinosomes has been limited to HeLa cells and has heavily relied on qualitative examination of their subcellular localization and reversibility under wide-field fluorescence microscopy. Quantitative approaches, which are particularly compatible with human disease-relevant cell lines, are necessary to explicitly understand the purinosome in live cells. In this work, human breast carcinoma Hs578T cells have been utilized to demonstrate the preferential utilization of the purinosome under purine-depleted conditions. In addition, we have employed a confocal microscopy-based biophysical technique, fluorescence recovery after photobleaching, to characterize kinetic properties of the purinosome in live Hs578T cells. Quantitative characterization of the diffusion coefficients of all de novo purine biosynthetic enzymes reveals the significant reduction of their mobile kinetics upon purinosome formation, the dynamic partitioning of each enzyme into the purinosome, and the existence of three intermediate species in purinosome assembly under purine starvation. We also demonstrate that the diffusion coefficient of the purine salvage enzyme, hypoxanthine phosphoribosyltransferase 1, is not sensitive to purine starvation, indicating exclusion of the salvage pathway from the purinosome. Furthermore, our biophysical characterization of nonmetabolic enzymes clarifies that purinosomes are spatiotemporally different cellular bodies from stress granules and cytoplasmic protein aggregates in both Hs578T and HeLa cells. Collectively, quantitative analyses of the purinosome in Hs578T cells led us to provide novel insights for the dynamic architecture of the purinosome assembly. PMID:25540829

  13. Structure of Plasmodium falciparum orotate phosphoribosyltransferase with autologous inhibitory protein–protein interactions

    SciTech Connect

    Kumar, Shiva; Krishnamoorthy, Kalyanaraman; Mudeppa, Devaraja G.; Rathod, Pradipsinh K.

    2015-04-21

    P. falciparum orotate phosphoribosyltransferase, a potential target for antimalarial drugs and a conduit for prodrugs, crystallized as a structure with eight molecules per asymmetric unit that included some unique parasite-specific auto-inhibitory interactions between catalytic dimers. The most severe form of malaria is caused by the obligate parasite Plasmodium falciparum. Orotate phosphoribosyltransferase (OPRTase) is the fifth enzyme in the de novo pyrimidine-synthesis pathway in the parasite, which lacks salvage pathways. Among all of the malaria de novo pyrimidine-biosynthesis enzymes, the structure of P. falciparum OPRTase (PfOPRTase) was the only one unavailable until now. PfOPRTase that could be crystallized was obtained after some low-complexity sequences were removed. Four catalytic dimers were seen in the asymmetic unit (a total of eight polypeptides). In addition to revealing unique amino acids in the PfOPRTase active sites, asymmetric dimers in the larger structure pointed to novel parasite-specific protein–protein interactions that occlude the catalytic active sites. The latter could potentially modulate PfOPRTase activity in parasites and possibly provide new insights for blocking PfOPRTase functions.

  14. Gold(I)-triphenylphosphine complexes with hypoxanthine-derived ligands: in vitro evaluations of anticancer and anti-inflammatory activities.

    PubMed

    Křikavová, Radka; Hošek, Jan; Vančo, Ján; Hutyra, Jakub; Dvořák, Zdeněk; Trávníček, Zdeněk

    2014-01-01

    A series of gold(I) complexes involving triphenylphosphine (PPh3) and one N-donor ligand derived from deprotonated mono- or disubstituted hypoxanthine (HLn) of the general composition [Au(Ln)(PPh3)] (1-9) is reported. The complexes were thoroughly characterized, including multinuclear high resolution NMR spectroscopy as well as single crystal X-ray analysis (for complexes 1 and 3). The complexes were screened for their in vitro cytotoxicity against human cancer cell lines MCF7 (breast carcinoma), HOS (osteosarcoma) and THP-1 (monocytic leukaemia), which identified the complexes 4-6 as the most promising representatives, who antiproliferative activity was further tested against A549 (lung adenocarcinoma), G-361 (melanoma), HeLa (cervical cancer), A2780 (ovarian carcinoma), A2780R (ovarian carcinoma resistant to cisplatin), 22Rv1 (prostate cancer) cell lines. Complexes 4-6 showed a significantly higher in vitro anticancer effect against the employed cancer cells, except for G-361, as compared with the commercially used anticancer drug cisplatin, with IC50 ≈ 1-30 µM. Anti-inflammatory activity was evaluated in vitro by the assessment of the ability of the complexes to modulate secretion of the pro-inflammatory cytokines, i.e. tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), in the lipopolysaccharide-activated macrophage-like THP-1 cell model. The results of this study identified the complexes as auspicious anti-inflammatory agents with similar or better activity as compared with the clinically applied gold-based antiarthritic drug Auranofin. In an effort to explore the possible mechanisms responsible for the biological effect, the products of interactions of selected complexes with sulfur-containing biomolecules (L-cysteine and reduced glutathione) were studied by means of the mass-spectrometry study. PMID:25226034

  15. Gold(I)-Triphenylphosphine Complexes with Hypoxanthine-Derived Ligands: In Vitro Evaluations of Anticancer and Anti-Inflammatory Activities

    PubMed Central

    Křikavová, Radka; Hošek, Jan; Vančo, Ján; Hutyra, Jakub; Dvořák, Zdeněk; Trávníček, Zdeněk

    2014-01-01

    A series of gold(I) complexes involving triphenylphosphine (PPh3) and one N-donor ligand derived from deprotonated mono- or disubstituted hypoxanthine (HLn) of the general composition [Au(Ln)(PPh3)] (1–9) is reported. The complexes were thoroughly characterized, including multinuclear high resolution NMR spectroscopy as well as single crystal X-ray analysis (for complexes 1 and 3). The complexes were screened for their in vitro cytotoxicity against human cancer cell lines MCF7 (breast carcinoma), HOS (osteosarcoma) and THP-1 (monocytic leukaemia), which identified the complexes 4–6 as the most promising representatives, who antiproliferative activity was further tested against A549 (lung adenocarcinoma), G-361 (melanoma), HeLa (cervical cancer), A2780 (ovarian carcinoma), A2780R (ovarian carcinoma resistant to cisplatin), 22Rv1 (prostate cancer) cell lines. Complexes 4–6 showed a significantly higher in vitro anticancer effect against the employed cancer cells, except for G-361, as compared with the commercially used anticancer drug cisplatin, with IC50 ≈ 1–30 µM. Anti-inflammatory activity was evaluated in vitro by the assessment of the ability of the complexes to modulate secretion of the pro-inflammatory cytokines, i.e. tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), in the lipopolysaccharide-activated macrophage-like THP-1 cell model. The results of this study identified the complexes as auspicious anti-inflammatory agents with similar or better activity as compared with the clinically applied gold-based antiarthritic drug Auranofin. In an effort to explore the possible mechanisms responsible for the biological effect, the products of interactions of selected complexes with sulfur-containing biomolecules (L-cysteine and reduced glutathione) were studied by means of the mass-spectrometry study. PMID:25226034

  16. In vitro mutational spectrum of cyclopenta[cd]pyrene in the human HPRT gene.

    PubMed

    Keohavong, P; Melacrinos, A; Shukla, R

    1995-04-01

    Cyclopenta[cd]pyrene (CPP) is a widely distributed polycyclic aromatic hydrocarbon with potent mutagenic and carcinogenic activity. In order to acquire an understanding of the mutagenic pathways of CPP, we studied mutations induced by this chemical in human cells. Four independent cultures of a human cell line expressing cytochrome P450 CYP1A1 (cell line MCL-5) were treated with CPP, and mutants at the hypoxanthine phosphoribosyltransferase (HPRT) locus were selected en masse by 6-thioguanine (6TG) resistance. The kinds and positions of the mutations were analyzed using the combination of high-fidelity polymerase chain reaction (hifi-PCR) and denaturing gradient gel electrophoresis (DGGE). The third exon of the HPRT gene was amplified from the 6TG-resistant cells using the hifi-PCR and the amplified fragment was subsequently analyzed by DGGE to separate mutant sequences from the wild-type sequence. Mutant bands were excised from the gel, amplified using PCR and sequenced. Sixteen different mutations were identified and consisted mostly of the G to T and A to T transversions. Other mutations identified included G to A and A to G transitions, a G to C transversion, and a single G deletion. Of these mutations, six occurred within a run of six guanines. The predominance of transversions involving a guanine or an adenine observed with CPP is similar to the data previously reported for the racemic mixtures of benzo[a]pyrene (B[a]P), suggesting that the mechanisms of mutation induced by CPP may be similar to those induced by B[a]P. PMID:7728967

  17. UV Radiation Induces Delayed Hyperrecombination Associated with Hypermutation in Human Cells†

    PubMed Central

    Durant, Stephen T.; Paffett, Kimberly S.; Shrivastav, Meena; Timmins, Graham S.; Morgan, William F.; Nickoloff, Jac A.

    2006-01-01

    Ionizing radiation induces delayed genomic instability in human cells, including chromosomal abnormalities and hyperrecombination. Here, we investigate delayed genome instability of cells exposed to UV radiation. We examined homologous recombination-mediated reactivation of a green fluorescent protein (GFP) gene in p53-proficient human cells. We observed an ∼5-fold enhancement of delayed hyperrecombination (DHR) among cells surviving a low dose of UV-C (5 J/m2), revealed as mixed GFP+/− colonies. UV-B did not induce DHR at an equitoxic (75 J/m2) dose or a higher dose (150 J/m2). UV is known to induce delayed hypermutation associated with increased oxidative stress. We found that hypoxanthine phosphoribosyltransferase (HPRT) mutation frequencies were ∼5-fold higher in strains derived from GFP+/− (DHR) colonies than in strains in which recombination was directly induced by UV (GFP+ colonies). To determine whether hypermutation was directly caused by hyperrecombination, we analyzed hprt mutation spectra. Large-scale alterations reflecting large deletions and insertions were observed in 25% of GFP+ strains, and most mutants had a single change in HPRT. In striking contrast, all mutations arising in the hypermutable GFP+/− strains were small (1- to 2-base) changes, including substitutions, deletions, and insertions (reminiscent of mutagenesis from oxidative damage), and the majority were compound, with an average of four hprt mutations per mutant. The absence of large hprt deletions in DHR strains indicates that DHR does not cause hypermutation. We propose that UV-induced DHR and hypermutation result from a common source, namely, increased oxidative stress. These two forms of delayed genome instability may collaborate in skin cancer initiation and progression. PMID:16880516

  18. A high-performance liquid chromatographic method for the determination of hypoxanthine, xanthine, uric acid and allantoin in serum.

    PubMed

    Kock, R; Delvoux, B; Greiling, H

    1993-05-01

    A method was developed for the simultaneous determination of hypoxanthine, xanthine, uric acid and allantoin based on isocratic reversed-phase chromatography. This HPLC-method additionally allows the direct determination with UV-detection of inosine-5'-phosphate, uridine, thymine, orotic acid, allopurinol and oxipurinol, besides hypoxanthine, xanthine and uric acid in the same chromatographic run. Allantoin elutes in this system near the void volume and a fraction is collected covering the retention time range for this substance. After hydrolysis allantoin is converted to glyoxylate-2,4-dinitrophenylhydrazone, rechromatographed and detected at 360 nm. The coefficient of variation for this method does not exceed 5.0% for a serum concentration of 0.3 mumol/l hypoxanthine and is not greater than 5.3% for a xanthine concentration of 0.3 mumol/l serum. Recoveries were 90-110% for both hypoxanthine and xanthine. The determination of uric acid had an imprecision and inaccuracy not exceeding 1.45% in the concentration range of 103-568 mumol/l. Due to the more complex procedure required for the determination of allantoin, the coefficient of variation between days was 13.6% for a sample containing 0.8 mumol/l allantoin and the recoveries for this analyte were in the range of 86-93%. Reference ranges (mean +/- SD) determined on 171 serum samples from healthy adults were 12.7 +/- 6.6 mumol/l for hypoxanthine, 3.3 +/- 1.4 mumol/l for xanthine, and 15.7 +/- 7.9 mumol/l for allantoin. No significant age or sex dependence was observed. Uric acid concentrations were 320 +/- 55 mumol/l serum for men and 206 +/- 55 mumol/l for women. PMID:8357939

  19. Structure of Plasmodium falciparum orotate phosphoribosyltransferase with autologous inhibitory protein–protein interactions

    PubMed Central

    Kumar, Shiva; Krishnamoorthy, Kalyanaraman; Mudeppa, Devaraja G.; Rathod, Pradipsinh K.

    2015-01-01

    The most severe form of malaria is caused by the obligate parasite Plasmodium falciparum. Orotate phosphoribosyltransferase (OPRTase) is the fifth enzyme in the de novo pyrimidine-synthesis pathway in the parasite, which lacks salvage pathways. Among all of the malaria de novo pyrimidine-biosynthesis enzymes, the structure of P. falciparum OPRTase (PfOPRTase) was the only one unavailable until now. PfOPRTase that could be crystallized was obtained after some low-complexity sequences were removed. Four catalytic dimers were seen in the asymmetic unit (a total of eight polypeptides). In addition to revealing unique amino acids in the PfOPRTase active sites, asymmetric dimers in the larger structure pointed to novel parasite-specific protein–protein interactions that occlude the catalytic active sites. The latter could potentially modulate PfOPRTase activity in parasites and possibly provide new insights for blocking PfOPRTase functions. PMID:25945715

  20. Discovery of potent and efficacious cyanoguanidine-containing nicotinamide phosphoribosyltransferase (Nampt) inhibitors.

    PubMed

    Zheng, Xiaozhang; Baumeister, Timm; Buckmelter, Alexandre J; Caligiuri, Maureen; Clodfelter, Karl H; Han, Bingsong; Ho, Yen-Ching; Kley, Nikolai; Lin, Jian; Reynolds, Dominic J; Sharma, Geeta; Smith, Chase C; Wang, Zhongguo; Dragovich, Peter S; Oh, Angela; Wang, Weiru; Zak, Mark; Wang, Yunli; Yuen, Po-Wai; Bair, Kenneth W

    2014-01-01

    A co-crystal structure of amide-containing compound (4) in complex with the nicotinamide phosphoribosyltransferase (Nampt) protein and molecular modeling were utilized to design and discover a potent novel cyanoguanidine-containing inhibitor bearing a sulfone moiety (5, Nampt Biochemical IC50=2.5nM, A2780 cell proliferation IC50=9.7nM). Further SAR exploration identified several additional cyanoguanidine-containing compounds with high potency and good microsomal stability. Among these, compound 15 was selected for in vivo profiling and demonstrated good oral exposure in mice. It also exhibited excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model. The co-crystal structure of this compound in complex with the NAMPT protein was also determined. PMID:24279990

  1. Nicotinamide Phosphoribosyltransferase Attenuates Methotrexate Response in Juvenile Idiopathic Arthritis and In Vitro.

    PubMed

    Funk, R S; Singh, R; Pramann, L; Gigliotti, N; Islam, S; Heruth, D P; Ye, S Q; Chan, M A; Leeder, J S; Becker, M L

    2016-06-01

    Variability in response to methotrexate (MTX) in the treatment of juvenile idiopathic arthritis (JIA) remains unpredictable and poorly understood. Based on previous studies implicating an interaction between nicotinamide phosphoribosyltransferase (NAMPT) expression and MTX therapy in inflammatory arthritis, we hypothesized that increased NAMPT expression would be associated with reduced therapeutic response to MTX in patients with JIA. A significant association was found between increased plasma concentrations of NAMPT and reduced therapeutic response in patients with JIA treated with MTX. Inhibition of NAMPT in cell culture by either siRNA-based gene silencing or pharmacological inhibition with FK-866 was found to result in a fourfold increase in the pharmacological activity of MTX. Collectively, these findings provide evidence that NAMPT inhibits the pharmacological activity of MTX and may represent a predictive biomarker of response, as well as a therapeutic target, in the treatment of JIA with MTX. PMID:27166432

  2. Radiation-quality dependent cellular response in mutation induction in normal human cells.

    PubMed

    Suzuki, Masao; Tsuruoka, Chizuru; Uchihori, Yukio; Kitamura, Hisashi; Liu, Cui Hua

    2009-09-01

    We studied cellular responses in normal human fibroblasts induced with low-dose (rate) or low-fluence irradiations of different radiation types, such as gamma rays, neutrons and high linear energy transfer (LET) heavy ions. The cells were pretreated with low-dose (rate) or low-fluence irradiations (approximately 1 mGy/7-8 h) of 137Cs gamma rays, 241Am-Be neutrons, helium, carbon and iron ions before irradiations with an X-ray challenging dose (1.5 Gy). Helium (LET = 2.3 keV/microm), carbon (LET = 13.3 keV/microm) and iron (LET = 200 keV/microm) ions were produced by the Heavy Ion Medical Accelerator in Chiba (HIMAC), Japan. No difference in cell-killing effect, measured by a colony forming assay, was observed among the pretreatment with different radiation types. In mutation induction, which was detected in the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus to measure 6-thioguanine resistant clones, there was no difference in mutation frequency induced by the X-ray challenging dose between unpretreated and gamma-ray pretreated cells. In the case of the pretreatment of heavy ions, X-ray-induced mutation was around 1.8 times higher in helium-ion pretreated and 4.0 times higher in carbon-ion pretreated cells than in unpretreated cells (X-ray challenging dose alone). However, the mutation frequency in cells pretreated with iron ions was the same level as either unpretreated or gamma-ray pretreated cells. In contrast, it was reduced at 0.15 times in cells pretreated with neutrons when compared to unpretreated cells. The results show that cellular responses caused by the influence of hprt mutation induced in cells pretreated with low-dose-rate or low-fluence irradiations of different radiation types were radiation-quality dependent manner. PMID:19680011

  3. A Mycobacterial Phosphoribosyltransferase Promotes Bacillary Survival by Inhibiting Oxidative Stress and Autophagy Pathways in Macrophages and Zebrafish*

    PubMed Central

    Mohanty, Soumitra; Jagannathan, Lakshmanan; Ganguli, Geetanjali; Padhi, Avinash; Roy, Debasish; Alaridah, Nader; Saha, Pratip; Nongthomba, Upendra; Godaly, Gabriela; Gopal, Ramesh Kumar; Banerjee, Sulagna; Sonawane, Avinash

    2015-01-01

    Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented ΔmimG:Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant MsmRv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-κB, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs. PMID:25825498

  4. A mycobacterial phosphoribosyltransferase promotes bacillary survival by inhibiting oxidative stress and autophagy pathways in macrophages and zebrafish.

    PubMed

    Mohanty, Soumitra; Jagannathan, Lakshmanan; Ganguli, Geetanjali; Padhi, Avinash; Roy, Debasish; Alaridah, Nader; Saha, Pratip; Nongthomba, Upendra; Godaly, Gabriela; Gopal, Ramesh Kumar; Banerjee, Sulagna; Sonawane, Avinash

    2015-05-22

    Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented ΔmimG:Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant MsmRv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-κB, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs. PMID:25825498

  5. Substrate Orientation and Catalytic Specificity in the Action of Xanthine Oxidase: The Sequential Hydroxylation of Hypoxanthine to Uric Acid

    SciTech Connect

    Cao, Hongnan; Pauff, James M.; Hille, Russ

    2010-11-29

    Xanthine oxidase is a molybdenum-containing enzyme catalyzing the hydroxylation of a sp{sup 2}-hybridized carbon in a broad range of aromatic heterocycles and aldehydes. Crystal structures of the bovine enzyme in complex with the physiological substrate hypoxanthine at 1.8 {angstrom} resolution and the chemotherapeutic agent 6-mercaptopurine at 2.6 {angstrom} resolution have been determined, showing in each case two alternate orientations of substrate in the two active sites of the crystallographic asymmetric unit. One orientation is such that it is expected to yield hydroxylation at C-2 of substrate, yielding xanthine. The other suggests hydroxylation at C-8 to give 6,8-dihydroxypurine, a putative product not previously thought to be generated by the enzyme. Kinetic experiments demonstrate that >98% of hypoxanthine is hydroxylated at C-2 rather than C-8, indicating that the second crystallographically observed orientation is significantly less catalytically effective than the former. Theoretical calculations suggest that enzyme selectivity for the C-2 over C-8 of hypoxanthine is largely due to differences in the intrinsic reactivity of the two sites. For the orientation of hypoxanthine with C-2 proximal to the molybdenum center, the disposition of substrate in the active site is such that Arg880 and Glu802, previous shown to be catalytically important for the conversion of xanthine to uric acid, play similar roles in hydroxylation at C-2 as at C-8. Contrary to the literature, we find that 6,8-dihydroxypurine is effectively converted to uric acid by xanthine oxidase.

  6. Estimation of time since death by vitreous humor hypoxanthine, potassium, and ambient temperature.

    PubMed

    Rognum, T O; Holmen, S; Musse, M A; Dahlberg, P S; Stray-Pedersen, A; Saugstad, O D; Opdal, S H

    2016-05-01

    Measurement of vitreous humor potassium (K(+)) has since the 1960s been recognized as an adjunct for estimation of time since death. In 1991 we introduced hypoxanthine (Hx) as a new marker. Furthermore we demonstrated that time since death estimation was more accurate when ambient temperature was included in the calculations, both for K(+) and for Hx. In this paper we present a refined method. The subjects consist of 132 cases with known time of death and ambient temperature. One sample from each subject was used in the calculations. Vitreous humor Hx levels were available in all subjects, while K(+) was measured in 106 of the subjects, due to insufficient volume of vitreous humor. Linear regression analysis was applied to model the correlation between vitreous humor Hx and K(+), taking the interactions with temperature into consideration. The diagrams published in 1991, which also included ambient temperature, estimated median time since death with range between the 10th and 90th percentile, whereas the linear regression analysis presented in this paper estimates mean time since death with a corresponding 95% interval of confidence. We conclude that time since death may be estimated with relatively high precision applying vitreous humor Hx and K(+) concentrations combined with ambient temperature. PMID:26994446

  7. Hepatic FoxOs Regulate Lipid Metabolism via Modulation of Expression of the Nicotinamide Phosphoribosyltransferase Gene*

    PubMed Central

    Tao, Rongya; Wei, Dan; Gao, Hanlin; Liu, Yunlong; DePinho, Ronald A.; Dong, X. Charlie

    2011-01-01

    FoxO transcription factors have been implicated in lipid metabolism; however, the underlying mechanisms are not well understood. Here, in an effort to elucidate such mechanisms, we examined the phenotypic consequences of liver-specific deletion of three members of the FoxO family: FoxO1, FoxO3, and FoxO4. These liver-specific triply null mice, designated LTKO, exhibited elevated triglycerides in the liver on regular chow diet. More remarkably, LTKO mice developed severe hepatic steatosis following placement on a high fat diet. Further analyses revealed that hepatic NAD+ levels and Sirt1 activity were decreased in the liver of the LTKO mice relative to controls. At the mechanistic level, expression profile analyses showed that LTKO livers had significantly down-regulated expression of the nicotinamide phosphoribosyltransferase (Nampt) gene encoding the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis. Luciferase reporter assays and chromatin immunoprecipitation analyses demonstrated that Nampt is a transcriptional target gene of FoxOs. Significantly, overexpression of Nampt gene reduced, whereas knockdown increased, hepatic triglyceride levels in vitro and in vivo. Thus, FoxOs control the Nampt gene expression and the NAD+ signaling in the regulation of hepatic triglyceride homeostasis. PMID:21388966

  8. Increasing NAD synthesis in muscle via nicotinamide phosphoribosyltransferase is not sufficient to promote oxidative metabolism.

    PubMed

    Frederick, David W; Davis, James G; Dávila, Antonio; Agarwal, Beamon; Michan, Shaday; Puchowicz, Michelle A; Nakamaru-Ogiso, Eiko; Baur, Joseph A

    2015-01-16

    The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle. PMID:25411251

  9. Increasing NAD Synthesis in Muscle via Nicotinamide Phosphoribosyltransferase Is Not Sufficient to Promote Oxidative Metabolism*

    PubMed Central

    Frederick, David W.; Davis, James G.; Dávila, Antonio; Agarwal, Beamon; Michan, Shaday; Puchowicz, Michelle A.; Nakamaru-Ogiso, Eiko; Baur, Joseph A.

    2015-01-01

    The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle. PMID:25411251

  10. Targeting Nicotinamide Phosphoribosyltransferase as a Potential Therapeutic Strategy to Restore Adult Neurogenesis.

    PubMed

    Wang, Shu-Na; Xu, Tian-Ying; Li, Wen-Lin; Miao, Chao-Yu

    2016-06-01

    Adult neurogenesis is the process of generating new neurons throughout life in the olfactory bulb and hippocampus of most mammalian species, which is closely related to aging and disease. Nicotinamide phosphoribosyltransferase (NAMPT), also an adipokine known as visfatin, is the rate-limiting enzyme for mammalian nicotinamide adenine dinucleotide (NAD) salvage synthesis by generating nicotinamide mononucleotide (NMN) from nicotinamide. Recent findings from our laboratory and other laboratories have provided much evidence that NAMPT might serve as a therapeutic target to restore adult neurogenesis. NAMPT-mediated NAD biosynthesis in neural stem/progenitor cells is important for their proliferation, self-renewal, and formation of oligodendrocytes in vivo and in vitro. Therapeutic interventions by the administration of NMN, NAD, or recombinant NAMPT are effective for restoring adult neurogenesis in several neurological diseases. We summarize adult neurogenesis in aging, ischemic stroke, traumatic brain injury, and neurodegenerative disease and review the advances of targeting NAMPT in restoring neurogenesis. Specifically, we provide emphasis on the P7C3 family, a class of proneurogenic compounds that are potential NAMPT activators, which might shed light on future drug development in neurogenesis restoration. PMID:27018006

  11. Kinetic analysis and chemical modification studies of nicotinate phosphoribosyltransferase from yeast

    SciTech Connect

    Hess, S.L.

    1988-01-01

    Nicotinate phosphoribosyltransferase (NaPRTase) from Baker's yeast catalyzes the formation of nicotinate mononucleotide (NaMN) and pyrophosphate from phosphoribosyl {alpha}-1-pyrophosphate and nicotinate, concomitant with ATP hydrolysis. Using purified NaPRTase, initial velocity measurements were performed varying one substrate concentration at different fixed levels of the second substrate and maintaining the third substrate constant. Subsequently, an exchange of label was observed between ATP and ({sup 14}C)-ADP. This rate of exchange was inhibited by PRibPP and pyrophosphate. Incubations of NaPRTase with pyridoxal 5{prime}-phosphate followed by sodium borohydride reduction led to inactivation of the enzyme. Pyridoxal was a less effective inhibitor than pyridoxal 5{prime}-phosphate. The inactivation of the enzyme by pyridoxal 5{prime}-phosphate was reversible upon flow dialysis, whereas reduction of the enzyme-pyridoxal complex with sodium borohydride rendered the inhibition irreversible. The presence of ATP or PRibPP, with or with Mg{sup 2+}, provided protection against this inactivation, while a kinetic analysis revealed the inhibition to be competitive, and noncompetitive, respectively. One mole of ({sup 3}H)-pyridoxal phosphate was required to completely inactivate the enzyme, which was reduced in the presence of MgATP and MgPRibPP to 0.2 and 0.6, respectively. No incorporation of pyridoxal 5{prime}-phosphate was observed in the combination of both of the two substrates.

  12. An ultrasensitive electrochemical sensor for simultaneous determination of xanthine, hypoxanthine and uric acid based on Co doped CeO2 nanoparticles.

    PubMed

    Lavanya, N; Sekar, C; Murugan, R; Ravi, G

    2016-08-01

    A novel electrochemical sensor has been fabricated using Co doped CeO2 nanoparticles for selective and simultaneous determination of xanthine (XA), hypoxanthine (HXA) and uric acid (UA) in a phosphate buffer solution (PBS, pH5.0) for the first time. The Co-CeO2 NPs have been prepared by microwave irradiation method and characterized by Powder XRD, Raman spectroscopy, HRTEM and VSM measurements. The electrochemical behaviours of XA, HXA and UA at the Co-CeO2 NPs modified glassy carbon electrode (GCE) were studied by cyclic voltammetry and square wave voltammetry methods. The modified electrode exhibited remarkably well-separated anodic peaks corresponding to the oxidation of XA, HXA and UA over the concentration range of 0.1-1000, 1-600 and 1-2200μM with detection limits of 0.096, 0.36, and 0.12μM (S/N=3), respectively. For simultaneous detection by synchronous change of the concentrations of XA, HXA and UA, the linear responses were in the range of 1-400μM each with the detection limits of 0.47, 0.26, and 0.43μM (S/N=3), respectively. The fabricated sensor was further applied to the detection of XA, HXA and UA in human urine samples with good selectivity and high reproducibility. PMID:27157753

  13. Orotate phosphoribosyltransferase is overexpressed in malignant pleural mesothelioma: Dramatically responds one case in high OPRT expression

    PubMed Central

    Hamamoto, Yoichiro; Takeoka, Shinjiro; Mouri, Atsuto; Fukusumi, Munehisa; Wakuda, Kazushige; Ibe, Tatsuya; Honma, Chie; Arimoto, Yoshihito; Yamada, Kazuaki; Wagatsuma, Miyuki; Tashiro, Akito; Kamoshida, Shingo; Kamimura, Mitsuhiro

    2016-01-01

    ABSTRACT Objective: Malignant pleural mesothelioma (MPM) is a rare and aggressive, treatment-resistant cancer. Pemetrexed, an inhibitor of thymidylate synthase (TS), is used worldwide for MPM as a first-line chemotherapy regimen. However, there is little consensus for a second-line chemotherapy. S-1, a highly effective dihydropyrimidine dehydrogenase (DPD)-inhibitory fluoropyrimidine, mainly acts via a TS inhibitory mechanism similar to pemetrexed. Orotate phosphoribosyltransferase (OPRT) is a key enzyme related to the first step activation of 5-fluorouracil (5-FU) for inhibiting RNA synthesis. We investigated 5-FU related-metabolism proteins, especially focusing on OPRT expression, in MPM Methods and Patients: Fifteen MPM patients who were diagnosed between July 2004 and December 2013 were enrolled. We examined the protein levels of 5-FU metabolism-related enzymes (TS, DPD, OPRT, and thymidine phosphorylase [TP]) in 14 cases Results: High TS, DPD, OPRT, and TP expressions were seen in 28.6%, 71.4%, 85.7%, and 35.7% of patients, respectively. We found that OPRT expression was extremely high in MPM tissue. We experienced one remarkable case of highly effective S-1 combined therapy for pemetrexed refractory MPM. This case also showed high OPRT protein expression Conclusion: The present study suggests that OPRT expression is high in MPM tumors. Although pemetrexed is mainly used for MPM chemotherapy as a TS inhibitor, S-1 has potential as an anticancer drug not only as a TS inhibitor but also inhibiting RNA synthesis through the OPRT pathway. This is the first report investigating OPRT protein expressions in MPM. PMID:27274438

  14. Perspectives on fast-neutron mutagenesis of human lymphoblastoid cells.

    PubMed

    Kronenberg, A

    1991-10-01

    The effects of low-fluence exposures to (Pu, Be) neutrons (En = 4.2 MeV) have been studied in a sensitive human B-lymphoblastoid cell line, TK6. Mutations were scored for two genetic loci, hypoxanthine phosphoribosyltransferase (hgprt) and thymidine kinase (tk), as a function of dose and dose rate. For exposures limited to less than one cell cycle, the mutation frequency for the hgprt locus was 1.92 X 10(-7)/cGy. When exposures were protracted over multiple cell generations, mutation yields were increased to 6.07 X 10(-7)/cGy. Similar yields were obtained for the induction of tk-deficient mutants with a normal cell generation time (tk-ng) when exposures were carried out at very low dose rates over multiple cell generations. In the series of data presented here, the results obtained for short-duration neutron exposures are compared with data obtained for monoenergetic heavy charged particles of defined linear energy transfer (LET) produced at the BEVALAC accelerator at Lawrence Berkeley Laboratory. TK6 cells have been exposed to beams ranging in atomic number from 20Ne to 40Ar over an energy range from 330 to 670 MeV/amu. Mutation induction was evaluated for both loci for a subset of these beams. The results obtained with 20Ne ions of 425 MeV/amu (LET = 32 keV/microns) and 28Si ions of 670 MeV/amu (LET = 50 keV/microns) closely resemble the mutation yields obtained for brief exposures to (Pu, Be) neutrons. The nature of alterations in DNA structure induced within the tk locus of tk-ng mutants is reviewed for a series of neutron-induced mutants and a series of mutants induced by exposure to 40Ar ions (470 MeV/amu, LET = 95 keV/microns). The mutational spectra for these two types of mutants were similar and were dominated by allele loss mutations. Multilocus deletions inclusive of the c-erbA1 locus were common among tk-deficient mutants induced by these densely ionizing radiations. For the mutants induced by 40Ar ions, it is likely that the mutations were produced by

  15. Coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts

    SciTech Connect

    Ipata, P.L.; Mura, U.; Camici, M.; Giovannitti, M.P.

    1987-08-01

    A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H/sub 2/O----adenosine + Pi (5'-nucleotidase); adenosine + H/sub 2/O----inosine + NH/sub 3/ (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5'-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into (/sup 14/C)inosine via the same combined reaction. Tissue extracts are incubated with excess (/sup 14/C)adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.

  16. Metabolic engineering of the purine biosynthetic pathway in Corynebacterium glutamicum results in increased intracellular pool sizes of IMP and hypoxanthine

    PubMed Central

    2012-01-01

    Background Purine nucleotides exhibit various functions in cellular metabolism. Besides serving as building blocks for nucleic acid synthesis, they participate in signaling pathways and energy metabolism. Further, IMP and GMP represent industrially relevant biotechnological products used as flavor enhancing additives in food industry. Therefore, this work aimed towards the accumulation of IMP applying targeted genetic engineering of Corynebacterium glutamicum. Results Blocking of the degrading reactions towards AMP and GMP lead to a 45-fold increased intracellular IMP pool of 22 μmol gCDW-1. Deletion of the pgi gene encoding glucose 6-phosphate isomerase in combination with the deactivated AMP and GMP generating reactions, however, resulted in significantly decreased IMP pools (13 μmol gCDW-1). Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 μmol gCDW-1) derived from IMP degradation. Conclusions The purine biosynthetic pathway is strongly interconnected with various parts of the central metabolism and therefore tightly controlled. However, deleting degrading reactions from IMP to AMP and GMP significantly increased intracellular IMP levels. Due to the complexity of this pathway further degradation from IMP to the corresponding nucleobase drastically increased suggesting additional targets for future strain optimization. PMID:23092390

  17. Radioprotector WR1065 reduces radiation-induced mutations at the hypoxanthine-guanine phosphoribosyl transferase locus in V79 cells

    SciTech Connect

    Grdina, D.J.; Hill, C.K.; Peraino, C. ); Biserka, N. ); Wells, R.L. . Dept. of Radiology and Radiation Biology)

    1985-06-01

    N-(2-mercaptoethyl)-1,3-diaminopropane (WR1065) protects against radiation-induced cell killing and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster lung fibroblast cells. WR1065 (4 mm) was found to be effective in protecting against radiation-induced cell lethality only if present during irradiation. No protective effect was observed if the protector was added within 5 min after irradiation or 3 h later. The effect of WR1065 on radiation-induced mutation, expressed as resistance to the cytotoxic purine analogue 6-thioguanine (HGPRT), was also investigated. This agent was effective in reducing radiation-induced mutations regardless of when it was administered. Following 10 Gy of /sup 60/Co ..gamma..-rays, the mutation frequencies observed per 10/sup 6/ survivors were 77 +- 8, 27 +- 6, 42 +- 7, and 42 +- 7 for radiation only, and WR1065 present during, immediately after, or 3 h after irradiation. These data suggest that although a segment of radiation-induced damage leading to reproductive death cannot be modulated through the postirradiation action of WR1065, processes leading to the fixation of gross genetic damage and mutation induction in surviving cells can be effectively altered and interfered with leading to a marked reduction in mutation frequency.

  18. Structure-Based Design of Potent Nicotinamide Phosphoribosyltransferase Inhibitors with Promising in Vitro and in Vivo Antitumor Activities.

    PubMed

    Bai, Jinhong; Liao, Chenzhong; Liu, Yanghan; Qin, Xiaochu; Chen, Jiaxuan; Qiu, Yatao; Qin, Dongguang; Li, Zheng; Tu, Zheng-Chao; Jiang, Sheng

    2016-06-23

    Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) has the potential to directly limit NAD production in cancer cells and is an effective strategy for cancer treatment. Using a structure-based strategy, we have designed a new class of potent small-molecule inhibitors of NAMPT. Several designed compounds showed promising antiproliferative activities in vitro. (E)-N-(5-((4-(((2-(1H-Indol-3-yl)ethyl)(isopropyl)amino)methyl)phenyl)amino)pentyl)-3-(pyridin-3-yl)acrylamide, 30, bearing an indole moiety, has an IC50 of 25.3 nM for binding to the NAMPT protein and demonstrated promising inhibitory activities in the nanomolar range against several cancer cell lines (MCF-7 GI50 = 0.13 nM; MDA-MB-231 GI50 = 0.15 nM). Triple-negative breast cancer is the most malignant subtype of breast cancer with no effective targeted treatments currently available. Significant antitumor efficacy of compound 30 was achieved (TGI was 73.8%) in an orthotopic MDA-MB-231 triple-negative breast cancer xenograft tumor model. This paper reports promising lead molecules for the inhibition of NAMPT which could serve as a basis for further investigation. PMID:27224875

  19. Down-Regulation of a Nicotinate Phosphoribosyltransferase Gene, OsNaPRT1, Leads to Withered Leaf Tips.

    PubMed

    Wu, Liwen; Ren, Deyong; Hu, Shikai; Li, Gengmi; Dong, Guojun; Jiang, Liang; Hu, Xingming; Ye, Weijun; Cui, Yongtao; Zhu, Li; Hu, Jiang; Zhang, Guangheng; Gao, Zhenyu; Zeng, Dali; Qian, Qian; Guo, Longbiao

    2016-06-01

    Premature leaf senescence affects plant growth and yield in rice. NAD plays critical roles in cellular redox reactions and remains at a sufficient level in the cell to prevent cell death. Although numerous factors affecting leaf senescence have been identified, few involving NAD biosynthetic pathways have been described for plants. Here, we report the cloning and characterization of Leaf Tip Senescence 1 (LTS1) in rice (Oryza sativa), a recessive mutation in the gene encoding O. sativa nicotinate phosphoribosyltransferase (OsNaPRT1) in the NAD salvage pathway. A point mutation in OsNaPRT1 leads to dwarfism and the withered leaf tip phenotype, and the lts1 mutant displays early leaf senescence compared to the wild type. Leaf nicotinate and nicotinamide contents are elevated in lts1, while NAD levels are reduced. Leaf tissue of lts1 exhibited significant DNA fragmentation and H2O2 accumulation, along with up-regulation of genes associated with senescence. The lts1 mutant also showed reduced expression of SIR2-like genes (OsSRT1 and OsSRT2) and increased acetylation of histone H3K9. Down-regulation of OsSRTs induced histone H3K9 acetylation of senescence-related genes. These results suggest that deficiency in the NAD salvage pathway can trigger premature leaf senescence due to transcriptional activation of senescence-related genes. PMID:27208230

  20. Campylobacter jejuni adenosine triphosphate phosphoribosyltransferase is an active hexamer that is allosterically controlled by the twisting of a regulatory tail.

    PubMed

    Mittelstädt, Gerd; Moggré, Gert-Jan; Panjikar, Santosh; Nazmi, Ali Reza; Parker, Emily J

    2016-08-01

    Adenosine triphosphate phosphoribosyltransferase (ATP-PRT) catalyzes the first committed step of the histidine biosynthesis in plants and microorganisms. Here, we present the functional and structural characterization of the ATP-PRT from the pathogenic ε-proteobacteria Campylobacter jejuni (CjeATP-PRT). This enzyme is a member of the long form (HisGL ) ATP-PRT and is allosterically inhibited by histidine, which binds to a remote regulatory domain, and competitively inhibited by AMP. In the crystalline form, CjeATP-PRT was found to adopt two distinctly different hexameric conformations, with an open homohexameric structure observed in the presence of substrate ATP, and a more compact closed form present when inhibitor histidine is bound. CjeATP-PRT was observed to adopt only a hexameric quaternary structure in solution, contradicting previous hypotheses favoring an allosteric mechanism driven by an oligomer equilibrium. Instead, this study supports the conclusion that the ATP-PRT long form hexamer is the active species; the tightening of this structure in response to remote histidine binding results in an inhibited enzyme. PMID:27191057

  1. Enhancement of succinate production by metabolically engineered Escherichia coli with co-expression of nicotinic acid phosphoribosyltransferase and pyruvate carboxylase.

    PubMed

    Ma, Jiangfeng; Gou, Dongmei; Liang, Liya; Liu, Rongming; Chen, Xu; Zhang, Changqing; Zhang, Jiuhua; Chen, Kequan; Jiang, Min

    2013-08-01

    Escherichia coli BA002, in which the ldhA and pflB genes are deleted, cannot utilize glucose anaerobically due to the inability to regenerate NAD(+). To restore glucose utilization, overexpression of nicotinic acid phosphoribosyltransferase (NAPRTase) encoded by the pncB gene, a rate-limiting enzyme of NAD(H) synthesis pathway, resulted in a significant increase in cell mass and succinate production under anaerobic conditions. However, a high concentration of pyruvate accumulated. Thus, co-expression of NAPRTase and the heterologous pyruvate carboxylase (PYC) of Lactococcus lactis subsp. cremoris NZ9000 in recombinant E. coli BA016 was investigated. The total concentration of NAD(H) was 9.8-fold higher in BA016 than in BA002, and the NADH/NAD(+) ratio decreased from 0.60 to 0.04. Under anaerobic conditions, BA016 consumed 17.50 g l(-1) glucose and produced 14.08 g l(-1) succinate with a small quantity of pyruvate. Furthermore, when the reducing agent dithiothreitol or reduced carbon source sorbitol was added, the cell growth and carbon source consumption rate of BA016 was reasonably enhanced and succinate productivity increased. PMID:23740313

  2. Down-Regulation of a Nicotinate Phosphoribosyltransferase Gene, OsNaPRT1, Leads to Withered Leaf Tips1[OPEN

    PubMed Central

    Ren, Deyong; Li, Gengmi; Jiang, Liang; Hu, Xingming; Ye, Weijun; Zhu, Li; Hu, Jiang; Zhang, Guangheng; Gao, Zhenyu; Guo, Longbiao

    2016-01-01

    Premature leaf senescence affects plant growth and yield in rice. NAD plays critical roles in cellular redox reactions and remains at a sufficient level in the cell to prevent cell death. Although numerous factors affecting leaf senescence have been identified, few involving NAD biosynthetic pathways have been described for plants. Here, we report the cloning and characterization of Leaf Tip Senescence 1 (LTS1) in rice (Oryza sativa), a recessive mutation in the gene encoding O. sativa nicotinate phosphoribosyltransferase (OsNaPRT1) in the NAD salvage pathway. A point mutation in OsNaPRT1 leads to dwarfism and the withered leaf tip phenotype, and the lts1 mutant displays early leaf senescence compared to the wild type. Leaf nicotinate and nicotinamide contents are elevated in lts1, while NAD levels are reduced. Leaf tissue of lts1 exhibited significant DNA fragmentation and H2O2 accumulation, along with up-regulation of genes associated with senescence. The lts1 mutant also showed reduced expression of SIR2-like genes (OsSRT1 and OsSRT2) and increased acetylation of histone H3K9. Down-regulation of OsSRTs induced histone H3K9 acetylation of senescence-related genes. These results suggest that deficiency in the NAD salvage pathway can trigger premature leaf senescence due to transcriptional activation of senescence-related genes. PMID:27208230

  3. Alternative substrates reveal catalytic cycle and key binding events in the reaction catalysed by anthranilate phosphoribosyltransferase from Mycobacterium tuberculosis.

    PubMed

    Cookson, Tammie V M; Castell, Alina; Bulloch, Esther M M; Evans, Genevieve L; Short, Francesca L; Baker, Edward N; Lott, J Shaun; Parker, Emily J

    2014-07-01

    AnPRT (anthranilate phosphoribosyltransferase), required for the biosynthesis of tryptophan, is essential for the virulence of Mycobacterium tuberculosis (Mtb). AnPRT catalyses the Mg2+-dependent transfer of a phosphoribosyl group from PRPP (5'-phosphoribosyl-1'-pyrophosphate) to anthranilate to form PRA (5'-phosphoribosyl anthranilate). Mtb-AnPRT was shown to catalyse a sequential reaction and significant substrate inhibition by anthranilate was observed. Antimycobacterial fluoroanthranilates and methyl-substituted analogues were shown to act as alternative substrates for Mtb-AnPRT, producing the corresponding substituted PRA products. Structures of the enzyme complexed with anthranilate analogues reveal two distinct binding sites for anthranilate. One site is located over 8 Å (1 Å=0.1 nm) from PRPP at the entrance to a tunnel leading to the active site, whereas in the second, inner, site anthranilate is adjacent to PRPP, in a catalytically relevant position. Soaking the analogues for variable periods of time provides evidence for anthranilate located at transient positions during transfer from the outer site to the inner catalytic site. PRPP and Mg2+ binding have been shown to be associated with the rearrangement of two flexible loops, which is required to complete the inner anthranilate-binding site. It is proposed that anthranilate first binds to the outer site, providing an unusual mechanism for substrate capture and efficient transfer to the catalytic site following the binding of PRPP. PMID:24712732

  4. A QM/QTAIM microstructural analysis of the tautomerisationviathe DPT of the hypoxanthine·adenine nucleobase pair

    NASA Astrophysics Data System (ADS)

    Brovarets', Ol'ha O.; Zhurakivsky, Roman O.; Hovorun, Dmytro M.

    2014-08-01

    We provide a pathway for the tautomerisation of the biologically important hypoxanthine.adenine (Hyp.Ade) nucleobase pair (Cs) formed by the keto tautomer of the Hyp and the amino tautomer of the Ade into the Hyp*.Ade* base pair (Cs) formed by the enol tautomer of the Hyp and the imino tautomer of the Ade by applying quantum-mechanical calculations and Bader's Quantum Theory of Atoms in Molecules analysis. It was found out that the dipole active Hyp.Ade↔Hyp*.Ade* tautomerisation occurs via the asynchronous concerted double proton transfer (DPT) through the TSHyp.Ade↔Hyp*.Ade* (Cs). Based on the sweeps of the energies of the intermolecular H-bonds along the intrinsic reaction coordinate, it was established that the N6H...O6 H-bond (5.40) is cooperative with the N1H...N1 H-bond (6.99) in the Hyp.Ade base pair, as well as the O6H...N6 H-bond (11.50) is cooperative with the N1H...N1 H-bond (7.28 kcal.mol-1) in the Hyp*.Ade* base pair, mutually strengthening each other. The Hyp*.Ade* base pair possesses an extremely short lifetime 2.68.10-14 s, which is predetermined by the negative value of the Gibbs free energy of the reverse barrier of its tautomerisation, and all of the six low-frequency intermolecular vibrations cannot develop during this period of time. Consequently, the Hyp.Ade→Hyp*.Ade* DPT tautomerisation cannot serve as a source of the rare tautomers of the bases.

  5. Biochemical characterization of quinolinic acid phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv and inhibition of its activity by pyrazinamide.

    PubMed

    Kim, Hyun; Shibayama, Keigo; Rimbara, Emiko; Mori, Shigetarou

    2014-01-01

    Quinolinic acid phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) is a key enzyme in the de novo pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis and a target for the development of new anti-tuberculosis drugs. QAPRTase catalyzes the synthesis of nicotinic acid mononucleotide from quinolinic acid (QA) and 5-phosphoribosyl-1-pyrophosphate (PRPP) through a phosphoribosyl transfer reaction followed by decarboxylation. The crystal structure of QAPRTase from Mycobacterium tuberculosis H37Rv (MtQAPRTase) has been determined; however, a detailed functional analysis of MtQAPRTase has not been published. Here, we analyzed the enzymatic activities of MtQAPRTase and determined the effect on catalysis of the anti-tuberculosis drug pyrazinamide (PZA). The optimum temperature and pH for MtQAPRTase activity were 60°C and pH 9.2. MtQAPRTase required bivalent metal ions and its activity was highest in the presence of Mg2+. Kinetic analyses revealed that the Km values for QA and PRPP were 0.08 and 0.39 mM, respectively, and the kcat values for QA and PRPP were 0.12 and 0.14 [s-1], respectively. When the amino acid residues of MtQAPRTase, which may interact with QA, were substituted with alanine residues, catalytic activity was undetectable. Further, PZA, which is an anti-tuberculosis drug and a structural analog of QA, markedly inhibited the catalytic activity of MtQAPRTase. The structure of PZA may provide the basis for the design of new inhibitors of MtQAPRTase. These findings provide new insights into the catalytic properties of MtQAPRTase. PMID:24949952

  6. Co-expression of phosphoenolpyruvate carboxykinase and nicotinic acid phosphoribosyltransferase for succinate production in engineered Escherichia coli.

    PubMed

    Jiang, Min; Chen, Xu; Liang, Liya; Liu, Rongming; Wan, Qing; Wu, Mingke; Zhang, Hanwen; Ma, Jiangfeng; Chen, Kequan; Ouyang, Pingkai

    2014-03-01

    Succinate is not the dominant fermentation product from xylose in wild-type Escherichia coli K12. E. coli BA 203 is a lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), and phosphoenolpyruvate (PEP)-carboxylase (ppc) deletion strain. To increase succinate accumulation and reduce byproduct formation, engineered E. coli BA204, in which ATP-forming PEP-carboxykinase (PEPCK) is overexpressed in BA203, was constructed and produced 2.17-fold higher succinate yield. To further improve the biomass and the consumption rate of xylose, nicotinic acid phosphoribosyltransferase (NAPRTase), a rate limiting enzyme in the synthesis of NAD(H), was also overexpressed. Thus, co-expression of PEPCK and NAPRTase in recombinant E. coli BA209 was investigated. In BA209, the pck gene and the pncB gene each have a trc promoter, hence, both genes are well expressed. During a 72-h anaerobic fermentation in sealed bottles, the total concentration of NAD(H) in BA209 was 1.25-fold higher than that in BA204, and the NADH/NAD+ ratio decreased from 0.28 to 0.11. During the exclusively anaerobic fermentation in a 3-L bioreactor, BA209 consumed 17.1 g L⁻¹ xylose and produced 15.5 g L⁻¹ succinate. Furthermore, anaerobic fermentation of corn stalk hydrolysate contained 30.1 g L⁻¹ xylose, 2.1 g L⁻¹ glucose and 1.5 g L⁻¹ arabinose, it produced a final succinate concentration of 17.2 g L⁻¹ with a yield of 0.94 g g⁻¹ total sugars. PMID:24564896

  7. Increased plasma nicotinamide phosphoribosyltransferase is associated with a hyperproliferative phenotype and restrains disease progression in MPN-associated myelofibrosis.

    PubMed

    Rosti, Vittorio; Campanelli, Rita; Massa, Margherita; Viarengo, Gianluca; Villani, Laura; Poletto, Valentina; Bonetti, Elisa; Catarsi, Paolo; Magrini, Umberto; Grolla, Ambra A; Travelli, Cristina; Genazzani, Armando A; Barosi, Giovanni

    2016-07-01

    Myeloproliferative neoplasm (MPN)-associated myelofibrosis is a clonal, neoplastic disorder of the hematopoietic stem cells, in which inflammation and immune dysregulation play an important role. Extracellular nicotinamide phosphoribosyltransferase (eNAMPT), also known as visfatin, is a cytokine implicated in a number of inflammatory and neoplastic diseases. Here plasma levels of eNAMPT in patients with MPN-associated myelofibrosis and their effects on disease phenotype and outcomes were examined. The concordance of eNAMPT levels with the marker of general inflammation high-sensitivity C-reactive protein (hs-CRP) was also studied. A total of 333 MPN-associated myelofibrosis patients (187 males and 146 females) and 31 age- and gender-matched normal-weight healthy subjects were enrolled in the study main body. Levels of eNAMPT and hs-CRP were simultaneously assayed in 209 MPN-associated myelofibrosis patients. Twenty-four polycythemia vera or essential thrombocythemia patients were used as controls. eNAMPT was over expressed in MPN-associated myelofibrosis, and eNAMPT expression was correlated with higher white blood cell count, higher hemoglobin, and higher platelet count, suggesting that eNAMPT is an indispensable permissive agent for myeloproliferation of MPN-associated myelofibrosis. The lack of correlation between eNAMPT and hs-CRP revealed that eNAMPT in MPN-associated myelofibrosis does not behave as a canonical inflammatory cytokine. In addition, higher levels of eNAMPT predicted longer time to blast transformation, and protected against progression toward thrombocytopenia and large splenomegaly. In conclusion, in MPN-associated myelofibrosis high levels of eNAMPT mark the myeloproliferative potential and, at variance with a high number of cancers, are protective against disease progression. Am. J. Hematol. 91:709-713, 2016. © 2016 Wiley Periodicals, Inc. PMID:27074203

  8. Quantifaction of mutagens at the Na/sup +/-K/sup +/-ATPase and hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene loci in Chinese hamster ovary cells

    SciTech Connect

    Li, A.P.

    1982-01-01

    The Chinese hamster ovary (CHO) cell/hypoxanthine guanine phosphoribosyl transferase (HGPRT) mutagen assay developed by Hsie et al., was simplified by culturing the cells as unattached cultures, and also modified to include mutation at the Na/sup +/-K/sup +/ ATPase (ouabain resistance) gene locus. The cost and time involved were decreased by culturing the CHO cells unattached on nontissue culture plates during the expression period. The inclusion of a second gene locus ensures that mutagenicities observed were not due to the peculiar properties of a specific gene locus. These procedures are now used in our laboratory for routine testing of environmental chemicals and complex mixtures.

  9. Bilirubin UDP-Glucuronosyltransferase 1A1 (UGT1A1) Gene Promoter Polymorphisms and HPRT, Glycophorin A, and Micronuclei Mutant Frequencies in Human Blood

    SciTech Connect

    Grant, D; Hall, I J; Eastmond, D; Jones, I M; Bell, D A

    2004-10-06

    A dinucleotide repeat polymorphism (5-, 6-, 7-, or 8-TA units) has been identified within the promoter region of UDP-glucuronosyltransferase 1A1 gene (UGT1A1). The 7-TA repeat allele has been associated with elevated serum bilirubin levels that cause a mild hyperbilirubinemia (Gilbert's syndrome). Studies suggest that promoter transcriptional activity of UGT1A1 is inversely related to the number of TA repeats and that unconjugated bilirubin concentration increases directly with the number of TA repeat elements. Because bilirubin is a known antioxidant, we hypothesized that UGT1A1 repeats associated with higher bilirubin may be protective against oxidative damage. We examined the effect of UGT1A1 genotype on somatic mutant frequency in the hypoxanthine-guanine phosphoribosyl-transferase (HPRT) gene in human lymphocytes and the glycophorin A (GPA) gene of red blood cells (both N0, NN mutants), and the frequency of lymphocyte micronuclei (both kinetochore (K) positive or micronuclei K negative) in 101 healthy smoking and nonsmoking individuals. As hypothesized, genotypes containing 7-TA and 8-TA displayed marginally lower GPA{_}NN mutant frequency relative to 5/5, 5/6, 6/6 genotypes (p<0.05). In contrast, our analysis showed that lower expressing UGT1A1 alleles (7-TA and 8-TA) were associated with modestly increased HPRT mutation frequency (p<0.05) while the same low expression genotypes were not significantly associated with micronuclei frequencies (K-positive or K-negative) when compared to high expression genotypes (5-TA and 6-TA). We found weak evidence that UGT1A1 genotypes containing 7-TA and 8-TA were associated with increased GPA{_}N0 mutant frequency relative to 5/5, 5/6, 6/6 genotypes (p<0.05). These data suggest that UGT1A1 genotype may modulate somatic mutation of some types, in some cell lineages, by a mechanism not involving bilirubin antioxidant activity. More detailed studies examining UGT1A1 promoter variation, oxidant/antioxidant balance and genetic

  10. The role of human cytochrome P4503A4 in biotransformation of tissue-specific derivatives of 7H-dibenzo[c,g]carbazole

    SciTech Connect

    Mesarosova, Monika; Valovicova, Zuzana; Srancikova, Annamaria; Krajcovicova, Zdenka; Milcova, Alena; Sokolova, Romana; Schmuczerova, Jana; Topinka, Jan; Gabelova, Alena

    2011-09-15

    The environmental pollutant 7H-dibenzo[c,g]carbazole (DBC) and its derivative, 5,9-dimethylDBC (DiMeDBC), produced significant and dose-dependent levels of micronuclei followed by a substantial increase in the frequency of apoptotic cells in the V79MZh3A4 cell line stably expressing the human cytochrome P450 (hCYP) 3A4. In contrast, neither micronuclei nor apoptosis were found in cells exposed to the sarcomagenic carcinogen, N-methylDBC (N-MeDBC). A slight but significant level of gene mutations and DNA adducts detected in V79MZh3A4 cells treated with N-MeDBC, only at the highest concentration (30 {mu}M), revealed that this sarcomagenic carcinogen was also metabolized by hCYP3A4. Surprisingly, DBC increased the frequency of 6-thioguanine resistant (6-TG{sup r}) mutations only at the highest concentration (30 {mu}M), while DiMeDBC failed to increase the frequency of these mutations. The resistance to 6-thioguanine is caused by the mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene. The molecular analysis of the coding region of Hprt gene showed a deletion of the entire exon 8 in DiMeDBC-induced 6-TG{sup r} mutants, while no changes in the nucleotide sequences were identified in 6-TG{sup r} mutants produced by DBC and N-MeDBC. Based on our results, we suggest that hCYP3A4 is involved in the metabolism of DBC and its tissue-specific derivatives. While hCYP3A4 probably plays an important role in biotransformation of the liver carcinogens, DBC and DiMeDBC, it might only have a marginal function in N-MeDBC metabolism. - Highlights: > DBC activation via CYP3A4 resulted in micronuclei, DNA adduct formation and mutations in V79MZh3A4 cells. > The CYP3A4-mediated DiMeDBC activation caused micronuclei followed by apoptosis in V79MZh3A4 cells. > The genotoxic effects produced by N-MeDBC in V79MZh3A4 cells were negligible. > The hCYP3A4 may play an important role in DBC and DiMeDBC metabolism. > The CYP3A4 might only have a marginal function in N

  11. Simultaneous assay of glucose, lactate, L-glutamate and hypoxanthine levels in a rat striatum using enzyme electrodes based on neutral red-doped silica nanoparticles.

    PubMed

    Zhang, Fen-Fen; Wan, Qiao; Li, Chen-Xin; Wang, Xiao-Li; Zhu, Zi-Qiang; Xian, Yue-Zhong; Jin, Li-Tong; Yamamoto, Katsunobu

    2004-10-01

    An electrochemical method suitable for the simultaneous measurement of cerebral glucose, lactate, L-glutamate and hypoxanthine concentrations from in vivo microdialysis sampling has been successfully performed for the first time using a neutral red-doped silica (NRDS) nanoparticle-derived enzyme sensor system. These uniform NRDS nanoparticles (about 50 +/- 3 nm) were prepared by a water-in-oil (W/O) microemulsion method, and characterized by a TEM technique. The neutral red-doped interior maintained its high electron-activity, while the exterior nano-silica surface prevented the mediator from leaching out into the aqueous solution, and showed high biocompability. These nanoparticles were then mixing with the glucose oxidase (GOD), lactate oxidase (LOD), L-glutamate oxidase (L-GLOD) or xanthine oxidase (XOD), and immobilized on four glassy carbon electrodes, respectively. A thin Nafion film was coated on the enzyme layer to prevent interference from molecules such as ascorbic acid and uric acid in the dialysate. The high sensitivity of the NRDS modified enzyme electrode system enables the simultaneous monitoring of trace levels of glucose, L-glutamate, lactate and hypoxanthine in diluted dialysate samples from a rat striatum. PMID:15517210

  12. A comparative study of the concentrations of hypoxanthine, xanthine, uric acid and allantoin in the peripheral blood of normals and patients with acute myocardial infarction and other ischaemic diseases.

    PubMed

    Kock, R; Delvoux, B; Sigmund, M; Greiling, H

    1994-11-01

    The aim of this study was the elucidation of the role of the xanthine oxidoreductase in the purine metabolism in ischaemic diseases of man. The serum concentrations of hypoxanthine, xanthine, uric acid and allantoin were determined in peripheral blood samples from patients with angina pectoris, cerebral insult and myocardial infarction with thrombolytic therapy and were compared with the concentrations obtained for healthy males and females. No significant differences were observed for the serum hypoxanthine concentrations, xanthine concentrations, the sum (hypoxanthine+xanthine) and the ratio (xanthine/hypoxanthine) between the healthy males, healthy females, the patients suffering from angina pectoris and the patients suffering from cerebral insult. An increase of the serum xanthine concentration in patients with myocardial infarction indicates a significant metabolic involvement of xanthine oxidoreductase in this disease and therefore a possible role in the development of tissue damage in the postischaemic phase due to oxygen radicals generated by the oxidase activity of this enzyme. The serum concentrations of uric acid and allantoin showed no differences between any of the studied groups. Study of the non-enzymatic oxidation of uric acid to allantoin by oxygen radicals, a relevant radical-scavenging mechanism in other diseases, provided no indication of an increased concentration of oxygen radicals due to the xanthine oxidoreductase reaction or other radical-producing mechanisms. PMID:7888480

  13. Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

    SciTech Connect

    Bethlenfalvay, N.C.; White, J.C.; Chadwick, E.; Lima, J.E. )

    1990-06-01

    High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of (G-{sup 3}H) hypoxanthine (Hy) into ({sup 3}H) labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate ({sup 3}H) ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation.

  14. Hypoxanthine uptake by skeletal muscle microvascular endothelial cells from equilibrative nucleoside transporter 1 (ENT1)-null mice: effect of oxidative stress.

    PubMed

    Bone, D B J; Antic, M; Quinonez, D; Hammond, J R

    2015-03-01

    Adenosine is an endogenous regulator of vascular tone. This activity of adenosine is terminated by its uptake and metabolism by microvascular endothelial cells (MVEC). The predominant transporter involved is ENT1 (equilibrative nucleoside transporter subtype 1). MVEC also express the nucleobase transporter (ENBT1) which is involved in the cellular flux of adenosine metabolites such as hypoxanthine. Changes in either of these transport systems would impact the bioactivity of adenosine and its metabolism, including the formation of oxygen free radicals. MVEC isolated from skeletal muscle of ENT1(+/+) and ENT1(-/-) mice were subjected to oxidative stress induced by simulated ischemia/reperfusion or menadione. The functional activities of ENT1 and ENBT1 were assessed based on zero-trans influx kinetics of radiolabeled substrates. There was a reduction in the rate of ENBT1-mediated hypoxanthine uptake by ENT1(+/+) MVEC treated with menadione or after exposure to conditions that simulate ischemia/reperfusion. In both cases, the superoxide dismutase mimetic MnTMPyP attenuated the loss of ENBT1 activity, implicating superoxide radicals in the response. In contrast, MVEC isolated from ENT1(-/-) mice showed no reduction in ENBT1 activity upon treatment with menadione or simulated ischemia/reperfusion, but they did have a significantly higher level of catalase activity relative to ENT1(+/+) MVEC. These data suggest that ENBT1 activity is decreased in MVEC in response to the increased superoxide radical that is associated with ischemia/reperfusion injury. MVEC isolated from ENT1(-/-) mice do not show this reduction in ENBT1, possibly due to increased catalase activity. PMID:25448155

  15. The role of the C-terminal region on the oligomeric state and enzymatic activity of Trypanosoma cruzi hypoxanthine phosphoribosyl transferase.

    PubMed

    Valsecchi, Wanda M; Cousido-Siah, Alexandra; Defelipe, Lucas A; Mitschler, André; Podjarny, Alberto; Santos, Javier; Delfino, José M

    2016-06-01

    Hypoxanthine phosphoribosyl transferase from Trypanosoma cruzi (TcHPRT) is a critical enzyme for the survival of the parasite. This work demonstrates that the full-length form in solution adopts a stable and enzymatically active tetrameric form, exhibiting large inter-subunit surfaces. Although this protein irreversibly aggregates during unfolding, oligomerization is reversible and can be modulated by low concentrations of urea. When the C-terminal region, which is predicted as a disordered stretch, is excised by proteolysis, TcHPRT adopts a dimeric state, suggesting that the C-terminal region acts as a main guide for the quaternary arrangement. These results are in agreement with X-ray crystallographic data presented in this work. On the other hand, the C-terminal region exhibits a modulatory role on the enzyme, as attested by the enhanced activity observed for the dimeric form. Bisphosphonates act as substrate-mimetics, uncovering long-range communications among the active sites. All in all, this work contributes to establish new ways applicable to the design of novel inhibitors that could eventually result in new drugs against parasitic diseases. PMID:26969784

  16. Photosensitized Oxidation of Hypoxanthine and Xanthine by Aluminum Phthalocyanine Tetrasulfonate. Role of the Alkylating Quinone 2,5-Dichloro-diaziridinyl-1,4-benzoquinone

    PubMed Central

    Alegria, Antonio E.; Inostroza, Yaritza; Kumar, Ajay

    2009-01-01

    Photoirradiation of nitrogen-saturated aqueous solutions containing aluminum phthalocyanine tetrasulfonate (AlPcS4) at 675 nm in the presence of 2,5-dichloro-diaziridinyl-1,4-benzoquinone (AZDClQ) and hypoxanthine (HX) produces the oxidized HX derivatives, xanthine (X) and uric acid (UA). Concentrations of the AZDClQ semiquinone, X and UA increase at the expense of HX with an increase in irradiation time. Almost negligible decomposition of HX, as well as very low amounts of X, are detected if photolysis occurs under identical conditions but in the absence of AZDClQ. Addition of calf-thymus DNA produces quinone-DNA covalent adducts after photolysis of anaerobic samples containing quinone, DNA and AlPcS4, in the presence or absence of HX and at pH 5.5. However, larger amounts of quinone-DNA adducts are detected if HX is present. The results presented here could have applications in the photodynamic treatment of hypoxic tissues such as solid tumors, under conditions of high HX concentration, where Type-I pathways could be more important than singlet oxygen generation. PMID:18627517

  17. Unravelling the potential of a new uracil phosphoribosyltransferase (UPRT) from Arabidopsis thaliana in sensitizing HeLa cells towards 5-fluorouracil.

    PubMed

    Narayanan, Sharmila; Sanpui, Pallab; Sahoo, Lingaraj; Ghosh, Siddhartha Sankar

    2016-10-01

    In silico studies with uracil phosphoribosyltransferase from Arabidopsis thaliana (AtUPRT) revealed its lower binding energies for uracil and 5-fluorouracil (5-FU) as compared to those of bacterial UPRT indicating the prospective of AtUPRT in gene therapy implications. Hence, AtUPRT was cloned and stably expressed in cervical cancer cells (HeLa) to investigate the effect of prodrug 5-FU on these transfected cancer cells. The treatment of AtUPRT-expressing HeLa (HeLa-UPP) cells with 5-FU for 72h resulted in significant decrease in cell viability. Moreover, 5-FU was observed to induce apoptosis and perturb mitochondrial membrane potential in HeLa-UPP cells. While cell cycle analysis revealed significant S-phase arrest as a result of 5-FU treatment in HeLa-UPP cells, quantitative gene expression analysis demonstrated simultaneous upregulation of important cell cycle related genes, cyclin D1 and p21. The survival fractions of non-transfected, vector-transfected and AtUPRT-transfected HeLa cells, following 5-FU treatment, were calculated to be 0.425, 0.366 and 0.227, respectively. PMID:27180296

  18. Orotate phosphoribosyltransferase localizes to the Golgi complex and its expression levels affect the sensitivity to anti-cancer drug 5-fluorouracil.

    PubMed

    Hozumi, Yasukazu; Tanaka, Toshiaki; Nakano, Tomoyuki; Matsui, Hirooki; Nasu, Takashi; Koike, Shuji; Kakehata, Seiji; Ito, Tsukasa; Goto, Kaoru

    2015-01-01

    Orotate phosphoribosyltransferase (OPRT) is engaged in de novo pyrimidine synthesis. It catalyzes oronitine to uridine monophosphate (UMP), which is used for RNA synthesis. De novo pyrimidine synthesis has long been known to play an important role in providing DNA/RNA precursors for rapid proliferative activity of cancer cells. Furthermore, chemotherapeutic drug 5-fluorouracil (5-FU) is taken up into cancer cells and is converted to 5-fluoro-UMP (FUMP) by OPRT or to 5-fluoro-dUMP (FdUMP) through intermediary molecules by thymidine phosphorylase. These 5-FU metabolites are misincorporated into DNA/RNA, thereby producing dysfunction of these information processing. However, it remains unclear how the subcellular localization of OPRT and how its variable expression levels affect the response to 5-FU at the cellular level. In this study, immunocytochemical analysis reveals that OPRT localizes to the Golgi complex. Results also show that not only overexpression but also downregulation of OPRT render cells susceptible to 5-FU exposure, but it has no effect on DNA damaging agent doxorubicin. This study provides clues to elucidate the cellular response to 5-FU chemotherapy in relation to the OPRT expression level. PMID:26700594

  19. Structures of Mycobacterium tuberculosis Anthranilate Phosphoribosyltransferase Variants Reveal the Conformational Changes That Facilitate Delivery of the Substrate to the Active Site.

    PubMed

    Cookson, Tammie V M; Evans, Genevieve L; Castell, Alina; Baker, Edward N; Lott, J Shaun; Parker, Emily J

    2015-10-01

    Anthranilate phosphoribosyltransferase (AnPRT) is essential for the biosynthesis of tryptophan in Mycobacterium tuberculosis (Mtb). This enzyme catalyzes the second committed step in tryptophan biosynthesis, the Mg²⁺-dependent reaction between 5'-phosphoribosyl-1'-pyrophosphate (PRPP) and anthranilate. The roles of residues predicted to be involved in anthranilate binding have been tested by the analysis of six Mtb-AnPRT variant proteins. Kinetic analysis showed that five of six variants were active and identified the conserved residue R193 as being crucial for both anthranilate binding and catalytic function. Crystal structures of these Mtb-AnPRT variants reveal the ability of anthranilate to bind in three sites along an extended anthranilate tunnel and expose the role of the mobile β2-α6 loop in facilitating the enzyme's sequential reaction mechanism. The β2-α6 loop moves sequentially between a "folded" conformation, partially occluding the anthranilate tunnel, via an "open" position to a "closed" conformation, which supports PRPP binding and allows anthranilate access via the tunnel to the active site. The return of the β2-α6 loop to the "folded" conformation completes the catalytic cycle, concordantly allowing the active site to eject the product PRA and rebind anthranilate at the opening of the anthranilate tunnel for subsequent reactions. Multiple anthranilate molecules blocking the anthranilate tunnel prevent the β2-α6 loop from undergoing the conformational changes required for catalysis, thus accounting for the unusual substrate inhibition of this enzyme. PMID:26356348

  20. Biomarker analysis in patients with advanced gastric cancer treated with S-1 plus cisplatin chemotherapy: orotate phosphoribosyltransferase expression is associated with treatment outcomes.

    PubMed

    Choi, In Sil; Lee, Hye Seung; Lee, Keun-Wook; Kim, Haeryoung; Kim, Ki Hwan; Kim, Yu Jung; Kim, Jee Hyun; Kim, Woo Ho; Lee, Jong Seok

    2011-12-01

    This study was performed to analyze the impact of protein expression related to fluoropyrimidine and cisplatin metabolism (thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, orotate phosphoribosyltransferase [OPRT], excision repair cross-complementation 1, Fanconi anemia complementation group D2, glutathione S-transferase P1, and X-ray repair cross-complementing group 1) on treatment outcomes in patients with metastatic or relapsed gastric cancer (MRGC) receiving S-1/cisplatin chemotherapy. Protein expression was measured by immunohistochemistry (IHC). Of the 43 patients who had received S-1 (80 mg/m2/day; days 1-14) and cisplatin (60 mg/m2; day 1) every 3 weeks and had available tissue blocks, IHC was successfully performed in 41 patients. Patients with high OPRT levels in tumor tissues (IHC score≥6) had superior progression-free survival (PFS) (23.3 vs. 14.1 weeks [median]) and overall survival (OS) (72.4 vs. 55.4 weeks [median]) to those with low OPRT levels (IHC score≤5; P-values<.05). Expression levels of other proteins were not predictive of treatment outcomes. In multivariate analysis, both a good performance status and a high OPRT level were independently associated with prolonged PFS and OS. The OPRT expression level may be a good predictive marker in S-1/cisplatin-treated patients with MRGC. PMID:20533001

  1. Molecular analyses of in vivo hprt mutations in human T-lymphocytes: IV. Studies in newborns

    SciTech Connect

    McGinniss, M.J.; Nicklas, J.A.; Albertini, R.J. )

    1989-01-01

    In order to characterize in vivo gene mutations that occur during fetal development, molecular analyses were undertaken of mutant 6-thioguanine resistant T-lymphocytes isolated from placental cord blood samples of 13 normal male newborns. These mutant T-cells were studied to define hypoxanthine-guanine phosphoribosyltransferase (hprt) gene structural alterations and to determine T-cell receptor (TCR) gene rearrangement patterns. Structural hprt alterations, as shown by Southern blot analyses, occurred in 85% of these mutant clones. These alterations consisted mostly of deletion of exons 2 and 3. These findings contrast with the 10-20% of gross structural alterations occurring randomly across the entire gene previously reported for T-cell mutants isolated from normal young adults. Iterative analyses of hprt structural alterations and TCR gene rearrangement patterns show that approximately one-third of the newborn derived mutants may have originated as pre- or intrathymic hprt mutations. This too contrasts with previous findings in adults where the background in vivo hprt mutations appeared to originate in postthymic T-lymphocytes.

  2. Human somatic mutation assays as biomarkers of carcinogenesis

    SciTech Connect

    Compton, P.J.E.; Smith, M.T. ); Hooper, K. )

    1991-08-01

    This paper describes four assays that detect somatic gene mutations in humans: the hypoxanthine-guanine phosphoribosyl transferase assay, the glycophorin A assay, the HLA-A assay, and the sickle cell hemoglobin assay. Somatic gene mutations can be considered a biomarker of carcinogenesis, and assays for somatic mutation may assist epidemiologists in studies that attempt to identify factors associated with increased risks of cancer. Practical aspects of the use of these assays are discussed.

  3. Molecular analysis of mutations affecting hprt mRNA splicing in human T-lymphocytes in vivo

    SciTech Connect

    Rossi, A.M. Pisa Univ. ); Tates, A.D.; van Zeeland, A.A.; Vrieling, H. )

    1992-01-01

    Molecular analysis of hypoxanthine-guanine phosphoribosyltransferase (hprt) cDNA from 6-thioguanine-resistant T-lymphocytes cloned from smoking and non-smoking adult donors showed that 35% of these mutants were defective in splicing of hprt mRNA. Among a set of 42 hprt splice mutants, the authors observed (1) complete loss of one or more exons, (2) partial loss of one exon, or (3) inclusion of part of an intron sequence between adjacent exons. Loss of exon 4 was significantly more frequent than of the other exons, suggesting that the sequences that regulate splicing of this exon are either larger than those of the other exons or especially prone to mutation. In order to identify the molecular nature of DNA alterations causing aberrant splicing of hprt mRNA, 17 splice mutants were analyzed in more detail by sequencing the genomic regions flanking the mis-spliced exon. Base pair substitutions or small deletions causing defective splicing were either detected in exon sequences or in splice site consensus sequences of introns.

  4. The effect of phorbols on metabolic cooperation between human fibroblasts

    SciTech Connect

    Mosser, D.D.; Bols, N.C.

    1982-01-01

    Autoradiography has been used to study the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), 4-O-methyl TPA, and phorbol on metabolic cooperation between human diploid fibroblasts. When the donors, hypoxanthine-guanine phosphoribosyl transferase+ (HGPRT+) cells, and recipients, HGPRT- cells, were plated together in the presence of (/sup 3/H)hypoxanthine and either 4-O-methyl TPA or phorbol, nearly all interactions that developed in 4 h were positive for metabolic cooperation whereas when high concentrations of TPA were used, the number of positive interactions was significantly less than the control. If the phorbol analogs were added after the donors and recipients had made contact, the number of positive interactions was the same as the control in all cases. However, although primary recipients in the cultures that had been treated with phorbol had the same number of grains as those in the control, primary recipients in cultures that had been treated with TPA or high concentrations of 4-O-methyl TPA had significantly fewer grains than those in the control. TPA treatment for 4 h had no effect on total (/sup 3/H)hypoxanthine incorporation or incorporation into acid-soluble and acid-insoluble fractions. Thus, the effect of TPA on metabolic cooperation is interpreted as a reduction in the transfer of (/sup 3/H)nucleotides and is an indication of an interference with intercellular communication.

  5. Quantitative assay for mutation in diploid human lymphoblasts using microtiter plates

    SciTech Connect

    Furth, E.A.; Thilly, W.G.; Penman, B.W.; Liber, H.L.; Rand, W.M.

    1981-01-01

    A microtiter plating technique which eliminates the need for soft agar and fibroblast feeder layers to determine the colony-forming ability of diploid human lymphoblast lines was described. The calculation of cloning efficiency is based on the Poisson distribution, and a statistical method for calculating confidence intervals is presented. This technique has been applied to the comcomitant examination of induced mutation at the putative loci for hypoxanthine guanine phosphoribosyl transferase, thymidine, kinase, and Na/sup +//K/sup +/ adenosine triphosphatase.

  6. Nature of 6-methylpurine inhibition and characterization of two 6-methylpurine-resistant mutants of Neurospora crassa.

    PubMed Central

    Pendyala, L; Smyth, J; Wellman, A M

    1979-01-01

    6-Methylpurine, an analog of adenine, inhibits the growth of Neurospora crassa. From kinetic studies it was found that 6-methylpurine is converted to its nucleotide form by adenine phosphoribosyltransferase (EC 2.4.2.7), and inhibits the de novo purine biosynthesis. Adenine relieves the growth inhibition caused by 6-methylpurine, whereas hypoxanthine is not very effective. Studies dealing with hypoxanthine utilization in the presence of 6-methylpurine indicated a severely reduced uptake of hypoxanthine and a general slowdown in its further metabolism. Two mutants (Mepr-3 and Mepr-10) which are resistant to 6-methylpurine were characterized. Studies of purine base uptake and the in vivo and in vitro conversion to nucleotides indicated that Mepr-10 may be an adenine phosphoribosyltransferase-defective mutant, whereas Mepr-3 may be a mutant with altered feedback response to 6-methylpurine. Both mutants showed a severely lowered hypoxanthine phosphoribosyltransferase activity, but because 6-methylpurine did not have any effect on the conversion of hypoxanthine to IMP in the wild type, it was concluded that 6-methylpurine resistance in these mutants cannot be due to lowered hypoxanthine phosphoribosyltransferase activity, but rather that the lowering of enzyme activity may be a secondary effect. PMID:153898

  7. High-performance liquid chromatographic assay for N2-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-arginine (ST 789) in plasma by cyclization with benzoin and fluorimetric detection.

    PubMed

    Bruno, G; Curti, S; Longo, A; Marzo, A; Arrigoni Martelli, E

    1993-09-01

    This paper describes a new highly sensitive assay for N2-[5-(hypoxanthin-9-yl)pentyloxycarbonyl]-L-arginine, an immunomodulatory agent, required for clinical pharmacokinetic investigation. A pre-column derivatization by cyclization with benzoin in aqueous medium produces the fluorescent 2-substituted amino-4,5-diphenylimidazole fluorescing at 450 nm (excitation wavelength 310 nm). L-Arginine-acetyl-L-carnitinamide chloride (ST 857, II), another arginine derivative, was used as an internal standard. A C18 DB column (5 microns, 250 mm x 4.6 mm I.D.) and a 45:55 (v/v) mixture of 0.05 M ammonium phosphate at pH 7.2 and methanol as mobile phase were used. Linearity was ascertained in the range 5-100 ng. Extraction recovery from plasma proved to be higher than 90% in the range 5-50 ng/ml. Intra-day precision, expressed as coefficient of variation, was in the range 4.7-6.0%. The limit of quantification proved to be 5 ng/ml and the limit of detection 2 ng/ml at a signal-to-noise ratio of 5. The method is specific. PMID:8245158

  8. Orotate phosphoribosyltransferase from baker's yeast: I. Kinetic analysis, chemical modification, and proton NMR spectroscopy of the enzyme substrate complex. II. Amino acid analysis and NMR spectroscopy of the protein

    SciTech Connect

    Strauss, R.S.

    1986-01-01

    Kinetic analysis of the effect of pH on the reversible reaction catalyzed by orotate phosphoribosyltransferase (OPRTase) from Baker's yeast revealed that different amino acid residues may enable the enzyme-catalyzed reactions to proceed in the forward and reverse directions, respectively. For the forward reaction, there appear to be at least two critical amino acid residues (pK's 4.6 and 7.1) which must be in a deprotonated state to reach a maximum activity near pH 8 which is maintained through pH 9.5. For the reverse reaction, maximum activity is reached near pH 7 (pK's at 5.4) and then the activity decreases at higher pH (pK's at 7.9 and possibly above 9). A theoretical proton NMR spectrum was generated for OPRTase, based on its amino acid composition. The spectrum thus produced has a similar number of major peaks to that of the actual spectrum taken at 300 MHz. Spectra collected at various pH values between 8 and 5, were consistent with the maintenance of the gross conformational structure of the enzyme over that pH range.

  9. Slow base excision by human alkyladenine DNA glycosylase limits the rate of formation of AP sites and AP endonuclease 1 does not stimulate base excision.

    PubMed

    Maher, Robyn L; Vallur, Aarthy C; Feller, Joyce A; Bloom, Linda B

    2007-01-01

    The base excision repair pathway removes damaged DNA bases and resynthesizes DNA to replace the damage. Human alkyladenine DNA glycosylase (AAG) is one of several damage-specific DNA glycosylases that recognizes and excises damaged DNA bases. AAG removes primarily damaged adenine residues. Human AP endonuclease 1 (APE1) recognizes AP sites produced by DNA glycosylases and incises the phophodiester bond 5' to the damaged site. The repair process is completed by a DNA polymerase and DNA ligase. If not tightly coordinated, base excision repair could generate intermediates that are more deleterious to the cell than the initial DNA damage. The kinetics of AAG-catalyzed excision of two damaged bases, hypoxanthine and 1,N6-ethenoadenine, were measured in the presence and absence of APE1 to investigate the mechanism by which the base excision activity of AAG is coordinated with the AP incision activity of APE1. 1,N6-ethenoadenine is excised significantly slower than hypoxanthine and the rate of excision is not affected by APE1. The excision of hypoxanthine is inhibited to a small degree by accumulated product, and APE1 stimulates multiple turnovers by alleviating product inhibition. These results show that APE1 does not significantly affect the kinetics of base excision by AAG. It is likely that slow excision by AAG limits the rate of AP site formation in vivo such that AP sites are not created faster than can be processed by APE1. PMID:17018265

  10. Morin, a dietary bioflavonol suppresses monosodium urate crystal-induced inflammation in an animal model of acute gouty arthritis with reference to NLRP3 inflammasome, hypo-xanthine phospho-ribosyl transferase, and inflammatory mediators.

    PubMed

    Dhanasekar, Chitra; Rasool, Mahaboobkhan

    2016-09-01

    The anti-inflammatory effect of morin, a dietary bioflavanol was explored on monosodium urate (MSU) crystal-induced inflammation in rats, an experimental model for acute gouty arthritis. Morin treatment (30mg/kg b.wt) significantly attenuated the ankle swelling and the levels of lipid peroxidation, nitric oxide, serum pro-inflammatory cytokines (tumor necrosis factor (TNF) -α, interleukin (IL)-1β, and IL-6), monocyte chemoattractant protein (MCP)-1, vascular endothelial growth factor (VEGF), prostaglandin E2 (PGE2), and articular elastase along with an increased anti-oxidant status (catalase (CAT) and superoxide dismutase (SOD)) in the joint homogenate of MSU crystal-induced rats. Histological assessment revealed that morin limited the diffusion of joint space, synovial hyperplasia, and inflammatory cell infiltrations. The mRNA expression of NLRP3 (nucleotide oligomerization domain (NOD)-like receptor family, pyrin domain containing 3) inflammasome, caspase-1, pro-inflammatory cytokines, MCP-1, inflammatory enzymes (inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2)), and nuclear factor-kappa B (NF-κB) p65 was found downregulated and HPRT (hypo-xanthine phospho-ribosyl transferase) mRNA expression was upregulated in morin treated MSU crystal-induced rats. In addition, morin treatment reduced the protein expression of NF-κB p65, p-NF-κB p65, iNOS, COX-2, and TNF-α. The results clearly demonstrated that morin exert a potent anti-inflammatory effect on MSU crystal-induced inflammation in rats. PMID:27268719

  11. Assay of SF/sub 6/ and spark-decomposed SF/sub 6/ for mutagenic activity in the CHO/HGPRT (Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase) mammalian cell system

    SciTech Connect

    Kurka, K.; Griffin, G.D.

    1987-01-01

    The potential mutagenic (and cytotoxic) activity of SF/sub 6/ and spark-decomposed SF/sub 6/ was investigated in an in vitro mammalian cell culture system using Chinese Hamster Ovary (CHO) cells. The CHO cells were exposed to the gases in vacutainer tubes which were constantly rotated. After a 4 h exposure the mutagenic and cytotoxic activity was assayed with the CHO/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system. Results indicated that SF/sub 6/ was neither cytotoxic nor mutagenic to CHO cells. Spark-decomposed SF/sub 6/ was found to be strongly cytotoxic (-80% cell death following 4 h exposure to 2 kJ spark discharge in 60 cm/sup 3/ at 1000 torr of SF/sub 6/) but not mutagenic. Increasing spark energy increased cytotoxicity but the spark samples remained nonmutagenic. The CHO/HGPRT system was coupled with a metabolic activation (S9 fraction) system used for detecting promutagens. When exposures were carried out in the presence of S9 fraction, SF/sub 6/ was still neither cytotoxic nor mutagenic; spark-decomposed SF/sub 6/ was again strongly cytotoxic but not mutagenic. It appears that SF/sub 6/ and sparked SF/sub 6/ are neither promutagens nor direct acting mutagens in the CHO/HGPRT system. Studies have begun using a more mutagenically sensitive subclone of the CHO cells known as CHO-AS/sub 52/. The results of initial experiments using the CHO-AS/sub 52/ cells remain unchanged. 9 refs., 1 tab.

  12. Effect of expression of adenine phosphoribosyltransferase on the in vivo anti-tumor activity of prodrugs activated by E. coli purine nucleoside phosphorylase.

    PubMed

    Parker, W B; Allan, P W; Waud, W R; Hong, J S; Sorscher, E J

    2011-06-01

    The use of E. coli purine nucleoside phosphorylase (PNP) to activate prodrugs has demonstrated excellent activity in the treatment of various human tumor xenografts in mice. E. coli PNP cleaves purine nucleoside analogs to generate toxic adenine analogs, which are activated by adenine phosphoribosyl transferase (APRT) to metabolites that inhibit RNA and protein synthesis. We created tumor cell lines that encode both E. coli PNP and excess levels of human APRT, and have used these new cell models to test the hypothesis that treatment of otherwise refractory human tumors could be enhanced by overexpression of APRT. In vivo studies with 6-methylpurine-2'-deoxyriboside (MeP-dR), 2-F-2'-deoxyadenosine (F-dAdo) or 9-β-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate (F-araAMP) indicated that increased APRT in human tumor cells coexpressing E. coli PNP did not enhance either the activation or the anti-tumor activity of any of the three prodrugs. Interestingly, expression of excess APRT in bystander cells improved the activity of MeP-dR, but diminished the activity of F-araAMP. In vitro studies indicated that increasing the expression of APRT in the cells did not significantly increase the activation of MeP. These results provide insight into the mechanism of bystander killing of the E. coli PNP strategy, and suggest ways to enhance the approach that are independent of APRT. PMID:21394111

  13. PRTFDC1 Is a Genetic Modifier of HPRT-Deficiency in the Mouse

    PubMed Central

    Gaval-Cruz, Meriem; Freeman, Kimberly G.; Edwards, Gaylen L.; Weinshenker, David; Thomas, James W.

    2011-01-01

    Lesch-Nyhan disease (LND) is a severe X-linked neurological disorder caused by a deficiency of hypoxanthine phosphoribosyltransferase (HPRT). In contrast, HPRT-deficiency in the mouse does not result in the profound phenotypes such as self-injurious behavior observed in humans, and the genetic basis for this phenotypic disparity between HPRT-deficient humans and mice is unknown. To test the hypothesis that HPRT deficiency is modified by the presence/absence of phosphoribosyltransferase domain containing 1 (PRTFDC1), a paralog of HPRT that is a functional gene in humans but an inactivated pseudogene in mice, we created transgenic mice that express human PRTFDC1 in wild-type and HPRT-deficient backgrounds. Male mice expressing PRTFDC1 on either genetic background were viable and fertile. However, the presence of PRTFDC1 in the HPRT-deficient, but not wild-type mice, increased aggression as well as sensitivity to a specific amphetamine-induced stereotypy, both of which are reminiscent of the increased aggressive and self-injurious behavior exhibited by patients with LND. These results demonstrate that PRTFDC1 is a genetic modifier of HPRT-deficiency in the mouse and could therefore have important implications for unraveling the molecular etiology of LND. PMID:21818316

  14. MULTIPLE-ENDPOINT MUTAGENESIS WITH CHINESE HAMSTER OVARY (CHO) CELLS: EVALUATION WITH EIGHT CARCINOGENIC AND NON-CARCINOGENIC COMPOUNDS

    EPA Science Inventory

    Using Chinese hamster ovary (CHO) cells in culture, the authors have defined an assay, CHO/HGPRT, to quantify mutagen-induced cytotoxicity and mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus. This assay permits elucidation of the structure-activity r...

  15. COMPARISON OF MUTAGENICITY RESULTS FOR NINE COMPOUNDS EVALUATED AT THE HGPRT LOCUS IN THE STANDARD AND SUSPENSION CHO ASSAYS

    EPA Science Inventory

    The Chinese hamster ovary (CHO) assay which measures newly induced mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus has been widely used for mutagenesis testing. he insensitivity of the standard assay to some genotoxic agents has been speculated to be...

  16. Attenuated Variants of Lesch-Nyhan Disease

    ERIC Educational Resources Information Center

    Jinnah, H. A.; Ceballos-Picot, Irene; Torres, Rosa J.; Visser, Jasper E.; Schretlen, David J.; Verdu, Alfonso; Larovere, Laura E.; Chen, Chung-Jen; Cossu, Antonello; Wu, Chien-Hui; Sampat, Radhika; Chang, Shun-Jen; de Kremer, Raquel Dodelson; Nyhan, William; Harris, James C.; Reich, Stephen G.; Puig, Juan G.

    2010-01-01

    Lesch-Nyhan disease is a neurogenetic disorder caused by deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase. The classic form of the disease is described by a characteristic syndrome that includes overproduction of uric acid, severe generalized dystonia, cognitive disability and self-injurious behaviour. In addition to the…

  17. Rapid and High-Throughput Detection and Quantitation of Radiation Biomarkers in Human and Nonhuman Primates by Differential Mobility Spectrometry-Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Chen, Zhidan; Coy, Stephen L.; Pannkuk, Evan L.; Laiakis, Evagelia C.; Hall, Adam B.; Fornace, Albert J.; Vouros, Paul

    2016-07-01

    Radiation exposure is an important public health issue due to a range of accidental and intentional threats. Prompt and effective large-scale screening and appropriate use of medical countermeasures (MCM) to mitigate radiation injury requires rapid methods for determining the radiation dose. In a number of studies, metabolomics has identified small-molecule biomarkers responding to the radiation dose. Differential mobility spectrometry-mass spectrometry (DMS-MS) has been used for similar compounds for high-throughput small-molecule detection and quantitation. In this study, we show that DMS-MS can detect and quantify two radiation biomarkers, trimethyl-L-lysine (TML) and hypoxanthine. Hypoxanthine is a human and nonhuman primate (NHP) radiation biomarker and metabolic intermediate, whereas TML is a radiation biomarker in humans but not in NHP, which is involved in carnitine synthesis. They have been analyzed by DMS-MS from urine samples after a simple strong cation exchange-solid phase extraction (SCX-SPE). The dramatic suppression of background and chemical noise provided by DMS-MS results in an approximately 10-fold reduction in time, including sample pretreatment time, compared with liquid chromatography-mass spectrometry (LC-MS). DMS-MS quantitation accuracy has been verified by validation testing for each biomarker. Human samples are not yet available, but for hypoxanthine, selected NHP urine samples (pre- and 7-d-post 10 Gy exposure) were analyzed, resulting in a mean change in concentration essentially identical to that obtained by LC-MS (fold-change 2.76 versus 2.59). These results confirm the potential of DMS-MS for field or clinical first-level rapid screening for radiation exposure.

  18. Nicotinamide phosphoribosyltransferase leukocyte overexpression in Graves' opthalmopathy.

    PubMed

    Sawicka-Gutaj, Nadia; Budny, Bartłomiej; Zybek-Kocik, Ariadna; Sowiński, Jerzy; Ziemnicka, Katarzyna; Waligórska-Stachura, Joanna; Ruchała, Marek

    2016-08-01

    To investigate the role of NAMPT/visfatin in euthyroid patients with Graves' disease without (GD) and with Graves' ophthalmopathy (GO), we analyzed NAMPT leukocyte expression and its serum concentration. This was a single-center, cross-sectional study with consecutive enrollment. In total, 149 patients diagnosed with Graves' disease were enrolled in the study. We excluded subjects with hyper- or hypothyroidism, diabetes mellitus, other autoimmune disorders, active neoplastic disease, and infection. The control group was recruited among healthy volunteers adjusted for age, sex, and BMI with normal thyroid function and negative thyroid antibodies. Serum levels of visfatin, TSH, FT4, FT3, antibodies against TSH receptor (TRAb), antithyroperoxidase antibodies, antithyroglobulin antibodies, fasting glucose, and insulin were measured. NAMPT mRNA leukocyte expression was assessed using RT-qPCR. NAMPT/visfatin serum concentration was higher in GD (n = 44) and GO (n = 49) patients than in the control group (n = 40) (p = 0.0275). NAMPT leukocyte expression was higher in patients with GO (n = 30) than in GD patients (n = 27) and the control group (n = 29) (p < 0.0001). Simple linear regression analysis revealed that NAMPT/visfatin serum concentration was significantly associated with GD (β = 1.5723; p = 0.021). When NAMPT leukocyte expression was used as a dependent variable, simple regression analysis found association with TRAb, fasting insulin level, HOMA-IR, GD, and GO. In the stepwise multiple regression analysis, we confirmed the association between higher serum NAMPT/visfatin level and GD (coefficient = 1.5723; p = 0.0212), and between NAMPT leukocyte expression and GO (coefficient = 2.4619; p = 0.0001) and TRAb (coefficient = 0.08742; p = 0.006). Increased NAMPT leukocyte expression in patients with GO might suggest a presently undefined role in the pathogenesis of GO. PMID:26767650

  19. Multiple dispersed spontaneous mutations: A novel pathway of mutation in a malignant human cell line

    SciTech Connect

    Harwood, J.; Tachibana, Akira; Meuth, M. )

    1991-06-01

    The authors analyzed the nature of spontaneous mutations at the autosomal locus coding for adenine phosphoribosyltransferase in the human colorectal carcinoma cell line SW620 to establish whether distinctive mutational pathways exist that might underlie the more complex genome rearrangements arising in tumor cells. Point mutations occur at a low rate in part hemizygotes derived from SW620, largely as a result of base substitutions at G {center dot} C base pairs to yield transversions and transitions. However, a novel pathway is evident in the form of multiple dispersed mutations in which two errors, separated by as much as 1,800 bp, fall in the same mutant gene. Such mutations could be the result of error-prone DNA synthesis occurring during normal replication or during long-patch excision-repair of spontaneously arising DNA lesions. This process could also contribute to the chromosomal instability evident in these tumor cells.

  20. Inhibition of an NAD+ Salvage Pathway Provides Efficient and Selective Toxicity to Human Pluripotent Stem Cells

    PubMed Central

    Kropp, Erin M.; Oleson, Bryndon J.; Broniowska, Katarzyna A.; Bhattacharya, Subarna; Chadwick, Alexandra C.; Diers, Anne R.; Hu, Qinghui; Sahoo, Daisy; Hogg, Neil; Boheler, Kenneth R.; Corbett, John A.

    2015-01-01

    The tumorigenic potential of human pluripotent stem cells (hPSCs) is a major limitation to the widespread use of hPSC derivatives in the clinic. Here, we demonstrate that the small molecule STF-31 is effective at eliminating undifferentiated hPSCs across a broad range of cell culture conditions with important advantages over previously described methods that target metabolic processes. Although STF-31 was originally described as an inhibitor of glucose transporter 1, these data support the reclassification of STF-31 as a specific NAD+ salvage pathway inhibitor through the inhibition of nicotinamide phosphoribosyltransferase (NAMPT). These findings demonstrate the importance of an NAD+ salvage pathway in hPSC biology and describe how inhibition of NAMPT can effectively eliminate hPSCs from culture. These results will advance and accelerate the development of safe, clinically relevant hPSC-derived cell-based therapies. PMID:25834119

  1. Characterization of a TK6-Bcl-xL gly-159-ala Human Lymphoblast Clone

    SciTech Connect

    Chyall, L.: Gauny, S.; Kronenberg, A.

    2006-01-01

    TK6 cells are a well-characterized human B-lymphoblast cell line derived from WIL-2 cells. A derivative of the TK6 cell line that was stably transfected to express a mutated form of the anti-apoptotic protein Bcl-xL (TK6-Bcl-xL gly-159- ala clone #38) is compared with the parent cell line. Four parameters were evaluated for each cell line: growth under normal conditions, plating efficiency, and frequency of spontaneous mutation to 6‑thioguanine resistance (hypoxanthine phosphoribosyl transferase locus) or trifluorothymidine resistance (thymidine kinase locus). We conclude that the mutated Bcl-xL protein did not affect growth under normal conditions, plating efficiency or spontaneous mutation frequencies at the thymidine kinase (TK) locus. Results at the hypoxanthine phosphoribosyl transferase (HPRT) locus were inconclusive. A mutant fraction for TK6‑Bcl-xL gly-159-ala clone #38 cells exposed to 150cGy of 160kVp x-rays was also calculated. Exposure to x-irradiation increased the mutant fraction of TK6‑Bcl-xL gly-159-ala clone #38 cells.

  2. Production of monoclonal and polyclonal antibodies against human alphafetoprotein, a hepatocellular tumor marker.

    PubMed

    Chou, Shu-Fen; Hsu, Wen-Lin; Hwang, Jing-Min; Chen, Chien-Yuan

    2002-08-01

    The objective of this study is to produce and purify monoclonal antibodies and polyclonal antibodies (PAbs) against human alphafetoprotein (AFP). Hyperimmune ICR mice produced PAbs after injection with 0.5 mL pristane, and were injected with NS-1 myeloma cells 2 weeks later. Hyperimmune Balb/c mice were used for the production of MAbs. Mice were immunized four times, given a final boost, and their spleen cells were collected and fused with NS-1 myeloma cells under the presence of PEG 1500. The fused cells were then selected in the hypoxanthine, aminopterine, and thymidine (HAT)-RPMIX medium. Anti-AFP antibody-secreting hybridoma cell lines with high titer were cloned by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution in 15% fetal bovine serum (FBS), hypoxanthine, thymidine (HT)-RPMIX medium. Twelve murine hybridoma producing anti-AFP MAbs were obtained and designated as A73F3, A73E8, B73C5, A73G3, A73F8, 67B3, B73C2, B73E1, A73G2, B73G7, B73D7, and B73F4. Isotypes of these MAbs were identified as IgG(1) heavy chain and kappa light chain. The MAbs with high purity were obtained by affinity chromatography. The purity analysis of AFP and the MAbs was performed by capillary electrophoresis. PMID:12193284

  3. Genotoxicity of alpha particles in human embryonic skin fibroblasts

    SciTech Connect

    Chen, D.J.; Strniste, G.F.; Tokita, N.

    1984-11-01

    Cell inactivation and induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus have been measured in cultured human fibroblasts (GM10) exposed to ..cap alpha.. particles from /sup 238/ Pu and 250 kVp X rays. The survival curves resulting from exposure to ..cap alpha.. particles are exponential. The mean lethal dose, D/sub 0/, is approximately 1.3 Gy for X rays and 0.25 Gy for ..cap alpha.. particles. As a function of radiation dose, mutation induction at the HGPRT locus was linear for ..cap alpha.. particles whereas the X-ray-induced mutation data were better fitted by a quadratic function. When mutation frequencies were plotted against the log of survival, mutation frequency at a given survival level was greater in cells exposed to ..cap alpha.. particles than to X rays.

  4. GMP synthase is essential for viability and infectivity of Trypanosoma brucei despite a redundant purine salvage pathway.

    PubMed

    Li, Qiong; Leija, Christopher; Rijo-Ferreira, Filipa; Chen, Jun; Cestari, Igor; Stuart, Kenneth; Tu, Benjamin P; Phillips, Margaret A

    2015-09-01

    The causative agent of human African trypanosomiasis, Trypanosoma brucei, lacks de novo purine biosynthesis and depends on purine salvage from the host. The purine salvage pathway is redundant and contains two routes to guanosine-5'-monophosphate (GMP) formation: conversion from xanthosine-5'-monophosphate (XMP) by GMP synthase (GMPS) or direct salvage of guanine by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). We show recombinant T. brucei GMPS efficiently catalyzes GMP formation. Genetic knockout of GMPS in bloodstream parasites led to depletion of guanine nucleotide pools and was lethal. Growth of gmps null cells was only rescued by supraphysiological guanine concentrations (100 μM) or by expression of an extrachromosomal copy of GMPS. Hypoxanthine was a competitive inhibitor of guanine rescue, consistent with a common uptake/metabolic conversion mechanism. In mice, gmps null parasites were unable to establish an infection demonstrating that GMPS is essential for virulence and that plasma guanine is insufficient to support parasite purine requirements. These data validate GMPS as a potential therapeutic target for treatment of human African trypanosomiasis. The ability to strategically inhibit key metabolic enzymes in the purine pathway unexpectedly bypasses its functional redundancy by exploiting both the nature of pathway flux and the limited nutrient environment of the parasite's extracellular niche. PMID:26043892

  5. Monochromosomal hybrids for the analysis of the human genome

    SciTech Connect

    Athwal, R.S.

    1990-01-01

    In this research project the authors proposed to develop rodent/human hybrid cell lines each containing a single different human chromosome. The human chromosomes will be marked with Ecogpt and stably maintained by selection in the hybrid cells. The experimental approach to produce the proposed cell lines involve the following: they will first transfer a cloned selectable marker, Ecogpt (an E. coli gene for xanthine-guanine phosphoribosyltransferase: XGPRT) to normal diploid human cells using a retroviral vector. The transferred gene will integrate at random into multiple sites in the recipient cell genome. Clonal cell lines from independent transgenotes will each carry the selectable marker integrated into a different site and perhaps a different chromosome. The chromosome carrying the selectable marker will then be transferred further to mouse cells by microcell fusion. In addition they also use directed integration of Ecogpt into the chromosome present in rodent cells, otherwise not marked with a selectable marker. This allows them to complete the bank of proposed cell line. The human chromosome, since it will be marked with a selectable marker, can be transferred to any other cell line of interest for complementation analysis. Clones of each cell line, containing varying size segments of the same chromosome produced by selection for the retention or loss of the selectable marker following x-irradiation or by metaphase chromosome transfer method will facilitate physical mapping and determination of gene order on a chromosome. 1 fig.

  6. GMP synthase is essential for viability and infectivity of Trypanosoma brucei despite a redundant purine salvage pathway

    PubMed Central

    Li, Qiong; Leija, Christopher; Rijo-Ferreira, Filipa; Chen, Jun; Cestari, Igor; Stuart, Kenneth; Tu, Benjamin P.; Phillips, Margaret A.

    2015-01-01

    Summary The causative agent of human African trypanosomiasis, Trypanosoma brucei, lacks de novo purine biosynthesis and depends on purine salvage from the host. The purine salvage pathway is redundant and contains two routes to guanosine-5′-monophosphate (GMP) formation: conversion from xanthosine-5′-monophosphate (XMP) by GMP synthase (GMPS) or direct salvage of guanine by hypoxanthine-guanine phosphoribosyltransferase (HGPRT). We show recombinant T. brucei GMPS efficiently catalyzes GMP formation. Genetic knockout of GMPS in bloodstream parasites led to depletion of guanine nucleotide pools and was lethal. Growth of gmps null cells was only rescued by supraphysiological guanine concentrations (100 μM) or by expression of an extrachromosomal copy of GMPS. Hypoxanthine was a competitive inhibitor of guanine rescue, consistent with a common uptake/metabolic conversion mechanism. In mice, gmps null parasites were unable to establish an infection demonstrating that GMPS is essential for virulence and that plasma guanine is insufficient to support parasite purine requirements. These data validate GMPS as a potential therapeutic target for treatment of HAT. The ability to strategically inhibit key metabolic enzymes in the purine pathway unexpectedly bypasses its functional redundancy by exploiting both the nature of pathway flux and the limited nutrient environment of the parasite's extracellular niche. PMID:26043892

  7. Genetic and physiological characterization of Bacillus subtilis mutants resistant to purine analogs.

    PubMed Central

    Saxild, H H; Nygaard, P

    1987-01-01

    Bacillus subtilis mutants defective in purine metabolism have been isolated by selecting for resistance to purine analogs. Mutants resistant to 2-fluoroadenine were found to be defective in adenine phosphoribosyltransferase (apt) activity and slightly impaired in adenine uptake. By making use of apt mutants and mutants defective in adenosine phosphorylase activity, it was shown that adenine deamination is an essential step in the conversion of both adenine and adenosine to guanine nucleotides. Mutants resistant to 8-azaguanine, pbuG mutants, appeared to be defective in hypoxanthine and guanine transport and normal in hypoxanthine-guanine phosphoribosyltransferase activity. Purine auxotrophic pbuG mutants grew in a concentration-dependent way on hypoxanthine, while normal growth was observed on inosine as the purine source. Inosine was taken up by a different transport system and utilized after conversion to hypoxanthine. Two mutants resistant to 8-azaxanthine were isolated: one was defective in xanthine phosphoribosyltransferase (xpt) activity and xanthine transport, and another had reduced GMP synthetase activity. The results obtained with the various mutants provide evidence for the existence of specific purine base transport systems. The genetic lesions causing the mutant phenotypes, apt, pbuG, and xpt, have been located on the B. subtilis linkage map at 243, 55, and 198 degrees, respectively. PMID:3110131

  8. Purine salvage pathways of Bacillus subtilis and effect of guanine on growth of GMP reductase mutants.

    PubMed

    Endo, T; Uratani, B; Freese, E

    1983-07-01

    We have isolated numerous mutants containing mutations in the salvage pathways of purine synthesis. The mutations cause deficiencies in adenine phosphoribosyltransferase (adeF), in hypoxanthine-guanine phosphoribosyltransferase (guaF), in adenine deaminase (adeC), in inosine-guanosine phosphorylase, (guaP), and in GMP reductase (guaC). The physiological properties of mutants containing one or more of these mutations and corresponding enzyme measurements have been used to derive a metabolic chart of the purine salvage pathway of Bacillus subtilis. PMID:6408059

  9. Purine salvage pathways of Bacillus subtilis and effect of guanine on growth of GMP reductase mutants.

    PubMed Central

    Endo, T; Uratani, B; Freese, E

    1983-01-01

    We have isolated numerous mutants containing mutations in the salvage pathways of purine synthesis. The mutations cause deficiencies in adenine phosphoribosyltransferase (adeF), in hypoxanthine-guanine phosphoribosyltransferase (guaF), in adenine deaminase (adeC), in inosine-guanosine phosphorylase, (guaP), and in GMP reductase (guaC). The physiological properties of mutants containing one or more of these mutations and corresponding enzyme measurements have been used to derive a metabolic chart of the purine salvage pathway of Bacillus subtilis. PMID:6408059

  10. Insights into the pyrimidine biosynthetic pathway of human malaria parasite Plasmodium falciparum as chemotherapeutic target.

    PubMed

    Krungkrai, Sudaratana R; Krungkrai, Jerapan

    2016-06-01

    Malaria is a major cause of morbidity and mortality in humans. Artemisinins remain as the first-line treatment for Plasmodium falciparum (P. falciparum) malaria although drug resistance has already emerged and spread in Southeast Asia. Thus, to fight this disease, there is an urgent need to develop new antimalarial drugs for malaria chemotherapy. Unlike human host cells, P. falciparum cannot salvage preformed pyrimidine bases or nucleosides from the extracellular environment and relies solely on nucleotides synthesized through the de novo biosynthetic pathway. This review presents significant progress on understanding the de novo pyrimidine pathway and the functional enzymes in the human parasite P. falciparum. Current knowledge in genomics and metabolomics are described, particularly focusing on the parasite purine and pyrimidine nucleotide metabolism. These include gene annotation, characterization and molecular mechanism of the enzymes that are different from the human host pathway. Recent elucidation of the three-dimensional crystal structures and the catalytic reactions of three enzymes: dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase, as well as their inhibitors are reviewed in the context of their therapeutic potential against malaria. PMID:27262062

  11. Analysis of purine metabolic enzymes in human CD4 Leu 8- and CD4 Leu 8+ lymphocyte subpopulations.

    PubMed

    Fernandez-Mejia, C; Polmar, S H; Peralta-Zaragoza, O; Madrid-Marina, V

    1993-02-01

    1. Specific activities of adenosine deaminase, purine nucleoside phosphorylase, adenosine kinase, 5'-nucleotidase, S-adenosyl-L-homocysteine hydrolase, AMP deaminase, adenine phosphoribosyl transferase, and hypoxanthine phosphoribosyl transferase were analyzed in human CD4 T-lymphocyte subsets. 2. CD4 Leu 8- (helper/inducer) and CD4 Leu 8+ (suppressor/inducer) subpopulations were obtained by panning or fluorescence activated cell sorting techniques using specific monoclonal antibodies. 3. A 45% decrease of 5'-NT AMP activity in the CD4 Leu 8- cells (suppressor/inducer) compared with CD4 total cell population. 4. No statistical significant differences in enzyme activity were found between the subsets analyzed in other purine enzymes. 5. These results suggest that the distribution of purine metabolic enzymes is homogeneous in CD4 Leu 8- and CD4 Leu 8+ T-lymphocyte subpopulations. PMID:8444317

  12. Use of PCR amplification of cDNA to study mechanisms of human cell mutagenesis and malignant transformation

    SciTech Connect

    Maher, V.M.; Yang, Jialing; Chen, Rueyhwa; McGregor, W.G.; Lukash, L.; Scheid, J.M.; Reinhold, D.S.; McCormick, J.J. )

    1991-01-01

    PCR is widely employed to amplify short segments of genomic DNA to determine if a specific change has occurred. But some investigators need to sequence the entire coding region of mammalian genes to determine what specific changes have occurred. In 1989, the authors described a method to copy mRNA of the hypoxanthine (guanine) phosphoribosyl transferase (HPRT) gene directly from the lysate of a clone of 6-thioguanine-resistant mutant diploid human fibroblasts without the need for RNA extraction or DNA template purification. The consensus sequence of the cDNA is determined by direct nucleotide sequencing. Using this method, they have investigated the kinds of mutations induced by carcinogens in the coding region of the HPRT gene and their location in the gene and examined the role of DNA repair were exposed to mutagens in exponential growth or synchronized and exposed at the beginning of S phase or in G{sub 1} phase several hr prior to DNA replication.

  13. Alzheimer's disease shares gene expression aberrations with purinergic dysregulation of HPRT deficiency (Lesch-Nyhan disease).

    PubMed

    Kang, Tae Hyuk; Friedmann, Theodore

    2015-03-17

    Transcriptomic studies of murine D3 embryonic stem (ES) cells deficient in the purinergic biosynthetic function hypoxanthine guanine phosphoribosyltransferase (HPRT) and undergoing dopaminergic neuronal differentiation has demonstrated a marked shift from neuronal to glial gene expression and aberrant expression of multiple genes also known to be aberrantly expressed in Alzheimer's and other CNS disorders. Such genetic dysregulations may indicate some shared pathogenic metabolic mechanisms in diverse CNS diseases. PMID:25636690

  14. Purine oversecretion in cultured murine lymphoma cells deficient in adenylosuccinate synthetase: genetic model for inherited hyperuricemia and gout.

    PubMed Central

    Ullman, B; Wormsted, M A; Cohen, M B; Martin, D W

    1982-01-01

    Alterations in several specific enzymes have been associated with increased rates of purine synthesis de novo in human and other mammalian cells. However, these recognized abnormalities in humans account for only a few percent of the clinical cases of hyperuricemia and gout. We have examined in detail the rates of purine production de novo and purine excretion by normal and by mutant (AU-100) murine lymphoma T cells (S49) 80% deficient in adenylosuccinate synthetase [IMP:L-aspartate ligase (GDP-forming), EC 6.3.4.4]. The intracellular ATP concentration of the mutant cells is slightly diminished, but their GTP is increased 50% and their IMP, four-fold. Compared to wild-type cells, the AU-100 cells excrete into the culture medium 30- to 50-fold greater amounts of purine metabolites consisting mainly of inosine. Moreover, the AU-100 cell line overproduces total purines. In an AU-100-derived cell line, AU-TG50B, deficient in adenylosuccinate synthetase and hypoxanthine/guanine phosphoribosyltransferase (IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), purine nucleoside excretion is increased 50- to 100-fold, and de novo synthesis is even greater than that for AU-100 cells. The overexcretion of purine metabolites by the AU-100 cells seems to be due to the primary genetic deficiency of adenylosuccinate synthetase, a deficiency that requires the cell to increase intracellular IMP in an attempt to maintain ATP levels. As a consequence of elevated IMP pools, large amounts of inosine are secreted into the culture medium. We propose that a similar primary genetic defect may account for the excessive purine excretion in some patients with dominantly inherited hyperuricemia and gout. Images PMID:6957854

  15. Visfatin expression analysis in association with recruitment and activation of human and rodent brown and brite adipocytes.

    PubMed

    Pisani, Didier F; Dumortier, Olivier; Beranger, Guillaume E; Casamento, Virginie; Ghandour, Rayane A; Giroud, Maude; Gautier, Nadine; Balaguer, Thierry; Chambard, Jean-Claude; Virtanen, Kirsi A; Nuutila, Pirjo; Niemi, Tarja; Taittonen, Markku; Van Obberghen, Emmanuel; Hinault, Charlotte; Amri, Ez-Zoubir

    2016-01-01

    Human brown adipocytes are able to burn fat and glucose and are now considered as a potential strategy to treat obesity, type 2 diabetes and metabolic disorders. Besides their thermogenic function, brown adipocytes are able to secrete adipokines. One of these is visfatin, a nicotinamide phosphoribosyltransferase involved in nicotinamide dinucleotide synthesis, which is known to participate in the synthesis of insulin by pancreatic β cells. In a therapeutic context, it is of interest to establish whether a potential correlation exists between brown adipocyte activation and/or brite adipocyte recruitment, and adipokine expression. We analyzed visfatin expression, as a pre-requisite to its secretion, in rodent and human biopsies and cell models of brown/brite adipocytes. We found that visfatin was preferentially expressed in mature adipocytes and that this expression was higher in brown adipose tissue of rodents compared to other fat depots. However, using various rodent models we were unable to find any correlation between visfatin expression and brown or brite adipocyte activation or recruitment. Interestingly, the situation is different in humans where visfatin expression was found to be equivalent between white and brown or brite adipocytes in vivo and in vitro. In conclusion, visfatin can be considered only as a rodent brown adipocyte biomarker, independently of tissue activation. PMID:27386154

  16. Adipose Tissue–derived Mesenchymal Stem Cells Expressing Prodrug-converting Enzyme Inhibit Human Prostate Tumor Growth

    PubMed Central

    Cavarretta, Ilaria T; Altanerova, Veronika; Matuskova, Miroslava; Kucerova, Lucia; Culig, Zoran; Altaner, Cestmir

    2009-01-01

    The ability of human adipose tissue–derived mesenchymal stem cells (AT-MSCs), engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase (CD::UPRT), to convert the relatively nontoxic 5-fluorocytosine (5-FC) into the highly toxic antitumor 5-fluorouracil (5-FU) together with their ability to track and engraft into tumors and micrometastases makes these cells an attractive tool to activate prodrugs directly within the tumor mass. In this study, we tested the feasibility and efficacy of these therapeutic cells to function as cellular vehicles of prodrug-activating enzymes in prostate cancer (PC) therapy. In in vitro migration experiments we have shown that therapeutic AT-MSCs migrated to all the prostate cell lines tested. In a pilot preclinical study, we observed that coinjections of human bone metastatic PC cells along with the transduced AT-MSCs into nude mice treated with 5-FC induced a complete tumor regression in a dose dependent manner or did not even allow the establishment of the tumor. More importantly, we also demonstrated that the therapeutic cells were effective in significantly inhibiting PC tumor growth after intravenous administration that is a key requisite for any clinical application of gene-directed enzyme prodrug therapies. PMID:19844197

  17. Mutagenic effects of alpha particles in normal human skin fibroblasts

    SciTech Connect

    Chen, D.J.; Carpenter, S.; Hanks, T.

    1992-12-31

    Alpha-irradiation to the bronchial airways from inhaled radon progeny increases the risk of developing lung cancer. The molecular mechanism of radon-induced lung cancer is not clear, but one of the most important genetic effects of ionizing radiation is the induction of gene mutation. Mutations, especially those associated with visible chromosome abnormalities in humans, have been associated with cancer. Therefore, our objective is to use a well-defined model system to determine the mutagenic potential of alpha particles in normal human skin cells and to define this action at the molecular level. Normal human skin fibroblasts were irradiated with alpha particles (3.59 MeV, LET 115 keV {mu}m{sup {minus}1}) emitted from the decay of {sup 238}Pu. Mutagenicity was determined at the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) locus. Results from this study indicate that beta particles were more efficient in mutation induction than gamma rays. Based on the initial slopes of the dose-response curves, the RBE for mutation is about 8 for alpha particles. HPRT-deficient mutants which are resistant to 6-thioguanine have been isolated and analyzed by the Southern blot technique. To date, we have characterized 69 gamma-ray-induced and 195 alpha-particle-induced HPRT-deficient mutants. Our data indicate that more than 50% of all gamma-ray-induced mutants have band patterns identical to that observed for the normal structural HPRT gene, whereas the remaining mutants (45%) contain either a rearrangement, partial deletion, or total deletion of the HPRT gene. In contrast, only 30% of alpha-particle-induced human HPRT mutants contain a normal Southern blot pattern, and about 50% indicate total deletion of the HPRT gene. Our results support the notion that high-LET radiation produces more unrepaired or misrepaired DNA damage than do gamma rays.

  18. The clastogenicity of 4NQO is cell-type dependent and linked to cytotoxicity, length of exposure and p53 proficiency.

    PubMed

    Brüsehafer, Katja; Manshian, Bella B; Doherty, Ann T; Zaïr, Zoulikha M; Johnson, George E; Doak, Shareen H; Jenkins, Gareth J S

    2016-03-01

    4-Nitroquinoline 1-oxide (4NQO) is used as a positive control in various genotoxicity assays because of its known mutagenic and carcinogenic properties. The chemical is converted into 4-hydroxyaminoquinoline 1-oxide and gives rise to three main DNA adducts, N-(deoxyguanosin-8-yl)-4AQO, 3-(desoxyguanosin-N (2)-yl)-4AQO and 3-(deoxyadenosin-N (6)-yl)-4AQO. This study was designed to assess the shape of the dose-response curve at low concentrations of 4NQO in three human lymphoblastoid cell lines, MCL-5, AHH-1 and TK6 as well as the mouse lymphoma L5178Y cell line in vitro. Chromosomal damage was investigated using the in vitro micronucleus assay, while further gene mutation and DNA damage studies were carried out using the hypoxanthine-guanine phosphoribosyltransferase forward mutation and comet assays. 4NQO showed little to no significant increases in micronucleus induction in the human lymphoblastoid cell lines, even up to 55±5% toxicity. A dose-response relationship could only be observed in the mouse lymphoma cell line L5178Y after 4NQO treatment, even at concentrations with no reduction in cell viability. Further significant increases in gene mutation and DNA damage induction were observed. Hence, 4NQO is a more effective point mutagen than clastogen, and its suitability as a positive control for genotoxicity testing has to be evaluated for every individual assay. PMID:26362870

  19. Molecular epidemiology studies on occupational and environmental exposure to mutagens and carcinogens, 1997-1999.

    PubMed Central

    Srám, R J; Binková, B

    2000-01-01

    Molecular epidemiology is a new and evolving area of research, combining laboratory measurement of internal dose, biologically effective dose, biologic effects, and influence of individual susceptibility with epidemiologic methodologies. Biomarkers evaluated were selected according to basic scheme: biomarkers of exposure--metabolites in urine, DNA adducts, protein adducts, and Comet assay parameters; biomarkers of effect--chromosomal aberrations, sister chromatid exchanges, micronuclei, mutations in the hypoxanthine-guanine phosphoribosyltransferase gene, and the activation of oncogenes coding for p53 or p21 proteins as measured on protein levels; biomarkers of susceptibility--genetic polymorphisms of genes CYP1A1, GSTM1, GSTT1, NAT2. DNA adducts measured by 32P-postlabeling are the biomarker of choice for the evaluation of exposure to polycyclic aromatic hydrocarbons. Protein adducts are useful as a biomarker for exposure to tobacco smoke (4-aminobiphenyl) or to smaller molecules such as acrylonitrile or 1,3-butadiene. Of the biomarkers of effect, the most common are cytogenetic end points. Epidemiologic studies support the use of chromosomal breakage as a relevant biomarker of cancer risk. The use of the Comet assay and methods analyzing oxidative DNA damage needs reliable validation for human biomonitoring. Until now there have not been sufficient data to interpret the relationship between genotypes, biomarkers of exposure, and biomarkers of effect for assessing the risk of human exposure to mutagens and carcinogens. PMID:10698723

  20. In vitro evaluation of genotoxic effects under magnetic resonant coupling wireless power transfer.

    PubMed

    Mizuno, Kohei; Shinohara, Naoki; Miyakoshi, Junji

    2015-04-01

    Wireless power transfer (WPT) technology using the resonant coupling phenomenon has been widely studied, but there are very few studies concerning the possible relationship between WPT exposure and human health. In this study, we investigated whether exposure to magnetic resonant coupling WPT has genotoxic effects on WI38VA13 subcloned 2RA human fibroblast cells. WPT exposure was performed using a helical coil-based exposure system designed to transfer power with 85.4% efficiency at a 12.5-MHz resonant frequency. The magnetic field at the positions of the cell culture dishes is approximately twice the reference level for occupational exposure as stated in the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. The specific absorption rate at the positions of the cell culture dishes matches the respective reference levels stated in the ICNIRP guidelines. For assessment of genotoxicity, we studied cell growth, cell cycle distribution, DNA strand breaks using the comet assay, micronucleus formation, and hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation, and did not detect any significant effects between the WPT-exposed cells and control cells. Our results suggest that WPT exposure under the conditions of the ICNIRP guidelines does not cause detectable cellular genotoxicity. PMID:25853218

  1. Data supporting the design and evaluation of a universal primer pair for pseudogene-free amplification of HPRT1 in real-time PCR

    PubMed Central

    Valadan, Reza; Hedayatizadeh-Omran, Akbar; Alhosseini-Abyazani, Mahdyieh Naghavi; Amjadi, Omolbanin; Rafiei, Alireza; Tehrani, Mohsen; Alizadeh-Navaei, Reza

    2015-01-01

    Hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) is a common housekeeping gene for sample normalization in the quantitative reverse transcriptase polymerase chain (qRT-PCR). However, co-amplification of HPRT1 pseudogenes may affect accurate results obtained in qRT-PCR. We designed a primer pair (HPSF) for pseudogene-free amplification of HPRT1 in qRT-PCR [1]. We showed specific amplification of HPRT1 mRNA in some common laboratory cell lines, including HeLa, NIH/3T3, CHO, BHK, COS-7 and VERO. This article provides data supporting the presence and location of HPRT1 pseudogenes within human and mouse genome, and the strategies used for designing primers that avoid the co-amplification of contaminating pseudogenes in qRT-PCR. In silico analysis of human genome showed three homologous sequences for HPRT1 on chromosomes 4, 5 and 11. The mRNA sequence of HPRT1 was aligned with the pseudogenes, and the primers were designed toward 5′ end of HPRT1 mRNA that was only specific to HPRT1 mRNA not to the pseudogenes. The standard curve plot generated by HPSF primers showed the correlation coefficient of 0.999 and the reaction efficiency of 99.5%. Our findings suggest that HPSF primers can be recommended as a candidate primer pair for accurate and reproducible qRT-PCR assays. PMID:26217821

  2. A Nampt inhibitor FK866 mimics vitamin B3 deficiency by causing senescence of human fibroblastic Hs68 cells via attenuation of NAD(+)-SIRT1 signaling.

    PubMed

    Song, Tuzz-Ying; Yeh, Shu-Lan; Hu, Miao-Lin; Chen, Mei-Yau; Yang, Nae-Cherng

    2015-12-01

    Vitamin B3 (niacin) deficiency can cause pellagra with symptoms of dermatitis, diarrhea and dementia. However, it is unclear whether the vitamin B3 deficiency causes human aging. FK866 (a Nampt inhibitor) can reduce intracellular NAD(+) level and induce senescence of human Hs68 cells. However, the mechanisms underlying FK866-induced senescence of Hs68 cells are unclear. In this study, we used FK866 to mimic the effects of vitamin B3 deficiency to reduce the NAD(+) level and investigated the mechanisms of FK866-induced senescence of Hs68 cells. We hypothesized that FK866 induced the senescence of Hs68 cells via an attenuation of NAD(+)-silent information regulator T1 (SIRT1) signaling. We found that FK866 induced cell senescence and diminished cellular NAD(+) levels and SIRT1 activity (detected by acetylation of p53), and these effects were dramatically antagonized by co-treatment with nicotinic acid, nicotinamide, or NAD(+). In contrast, the protein expression of SIRT1, AMP-activated protein kinase, mammalian target of rapamycin, and nicotinamide phosphoribosyltransferase (Nampt) was not affected by FK866. In addition, the role of GSH in the FK866-induced cells senescence may be limited, as N-acetylcysteine did not antagonize FK866-induced cell senescence. These results suggest that FK866 induces cell senescence via attenuation of NAD(+)-SIRT1 signaling. The effects of vitamin B3 deficiency on human aging warrant further investigation. PMID:26330291

  3. Purine Salvage Pathways among Borrelia Species▿

    PubMed Central

    Pettersson, Jonas; Schrumpf, Merry E.; Raffel, Sandra J.; Porcella, Stephen F.; Guyard, Cyril; Lawrence, Kevin; Gherardini, Frank C.; Schwan, Tom G.

    2007-01-01

    Genome sequencing projects on two relapsing fever spirochetes, Borrelia hermsii and Borrelia turicatae, revealed differences in genes involved in purine metabolism and salvage compared to those in the Lyme disease spirochete Borrelia burgdorferi. The relapsing fever spirochetes contained six open reading frames that are absent from the B. burgdorferi genome. These genes included those for hypoxanthine-guanine phosphoribosyltransferase (hpt), adenylosuccinate synthase (purA), adenylosuccinate lyase (purB), auxiliary protein (nrdI), the ribonucleotide-diphosphate reductase alpha subunit (nrdE), and the ribonucleotide-diphosphate reductase beta subunit (nrdF). Southern blot assays with multiple Borrelia species and isolates confirmed the presence of these genes in the relapsing fever group of spirochetes but not in B. burgdorferi and related species. TaqMan real-time reverse transcription-PCR demonstrated that the chromosomal genes (hpt, purA, and purB) were transcribed in vitro and in mice. Phosphoribosyltransferase assays revealed that, in general, B. hermsii exhibited significantly higher activity than did the B. burgdorferi cell lysate, and enzymatic activity was observed with adenine, hypoxanthine, and guanine as substrates. B. burgdorferi showed low but detectable phosphoribosyltransferase activity with hypoxanthine even though the genome lacks a discernible ortholog to the hpt gene in the relapsing fever spirochetes. B. hermsii incorporated radiolabeled hypoxanthine into RNA and DNA to a much greater extent than did B. burgdorferi. This complete pathway for purine salvage in the relapsing fever spirochetes may contribute, in part, to these spirochetes achieving high cell densities in blood. PMID:17502392

  4. Differential effects of immunosuppressants and antibiotics on human monoclonal antibody production in SCID mouse ascites by five heterohybridomas.

    PubMed

    Yoshinari, K; Arai, K

    1998-02-01

    SCID mice were inoculated with five human-mouse heterohybridomas derived by fusion of human lymph node lymphocytes from lung cancer patients with murine myeloma cells or human-mouse heteromyeloma cells, and the production of their human monoclonal antibodies (MAb) in the mouse ascites was investigated. In a comparison of the effects of pretreatment by i.p. (intraperitoneal) injection of pristane and anti-asialo GM1 serum on the antibody production of three of the hybridomas, pristane pretreatment resulted in substantial antibody production by all three, and pretreatment with anti-asialo GM1 serum resulted in similar or slightly lower levels of antibody production by two of the hybridomas but none by the third. In a second series of experiments using four of the hybridomas with pristane pretreatment, the co-injection of either penicillin G and streptomycin or kanamycin together with the hybridoma at the time of i.p. inoculation resulted in enhanced MAb production by the two heterohybridomas that had been propagated in medium containing hypoxanthine-aminopterin-thymidine (HAT) but not by the two that had been propagated in HAT-free medium. PMID:9523236

  5. Mutations Associated with Functional Disorder of Xanthine Oxidoreductase and Hereditary Xanthinuria in Humans

    PubMed Central

    Ichida, Kimiyoshi; Amaya, Yoshihiro; Okamoto, Ken; Nishino, Takeshi

    2012-01-01

    Xanthine oxidoreductase (XOR) catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid with concomitant reduction of either NAD+ or O2. The enzyme is a target of drugs to treat hyperuricemia, gout and reactive oxygen-related diseases. Human diseases associated with genetically determined dysfunction of XOR are termed xanthinuria, because of the excretion of xanthine in urine. Xanthinuria is classified into two subtypes, type I and type II. Type I xanthinuria involves XOR deficiency due to genetic defect of XOR, whereas type II xanthinuria involves dual deficiency of XOR and aldehyde oxidase (AO, a molybdoflavo enzyme similar to XOR) due to genetic defect in the molybdenum cofactor sulfurase. Molybdenum cofactor deficiency is associated with triple deficiency of XOR, AO and sulfite oxidase, due to defective synthesis of molybdopterin, which is a precursor of molybdenum cofactor for all three enzymes. The present review focuses on mutation or chemical modification studies of mammalian XOR, as well as on XOR mutations identified in humans, aimed at understanding the reaction mechanism of XOR and the relevance of mutated XORs as models to estimate the possible side effects of clinical application of XOR inhibitors. PMID:23203137

  6. Selection of suitable reference genes for expression analysis in human glioma using RT-qPCR.

    PubMed

    Grube, Susanne; Göttig, Tatjana; Freitag, Diana; Ewald, Christian; Kalff, Rolf; Walter, Jan

    2015-05-01

    In human glioma research, quantitative real-time reverse-transcription PCR is a frequently used tool. Considering the broad variation in the expression of candidate reference genes among tumor stages and normal brain, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. This study aimed at testing a panel of nine reference genes [beta-2-microglobulin, cytochrome c-1 (CYC1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hydroxymethylbilane synthase, hypoxanthine guanine phosphoribosyl transferase 1, ribosomal protein L13a (RPL13A), succinate dehydrogenase, TATA-box binding protein and 14-3-3 protein zeta] to identify and validate the most suitable reference genes for expression studies in human glioma of different grades (World Health Organization grades II-IV). After analysis of the stability values calculated using geNorm, NormFinder, and BestKeeper algorithms, GAPDH, RPL13A, and CYC1 can be indicated as reference genes applicable for accurate normalization of gene expression in glioma compared with normal brain and anaplastic astrocytoma or glioblastoma alone within this experimental setting. Generally, there are no differences in expression levels and variability of candidate genes in glioma tissue compared to normal brain. But stability analyses revealed just a small number of genes suitable for normalization in each of the tumor subgroups and across these groups. Nevertheless, our data show the importance of validation of adequate reference genes prior to every study. PMID:25862007

  7. The human T-cell cloning assay: identifying genotypes susceptible to drug toxicity and somatic mutation.

    PubMed

    Hou, Sai-Mei

    2014-01-01

    Humans exhibit marked genetic polymorphisms in drug metabolism that contribute to high incidence of adverse effects in susceptible individuals due to altered balance between metabolic activation and detoxification. The T-cell cloning assay, which detects mutations in the gene for hypoxanthine-guanine phosphoribosyl transferase (HPRT), is the most well-developed reporter system for studying specific locus mutation in human somatic cells. The assay is based on a mitogen- and growth factor-dependent clonal expansion of peripheral T-lymphocytes in which the 6-thioguanine-resistant HPRT mutants can be selected, enumerated, and collected for molecular analysis of the mutational nature. The assay provides a unique tool for studying in vivo and in vitro mutagenesis, for investigating the functional impact of common polymorphism in metabolism and repair genes, and for identifying risk genotypes for drug-induced toxicity and mutagenicity. This chapter presents a simple and reliable method for the enumeration of HPRT mutant frequency induced in vitro without using any source of recombinant interleukin-2. The other main feature is that only truly induced and unique mutants are collected for further analysis. PMID:24623236

  8. Crucial involvement of xanthine oxidase in the intracellular signalling networks associated with human myeloid cell function

    PubMed Central

    Abooali, Maryam; Lall, Gurprit S.; Coughlan, Karen; Lall, Harjinder S.; Gibbs, Bernhard F.; Sumbayev, Vadim V.

    2014-01-01

    Xanthine oxidase (XOD) is an enzyme which plays a central role in purine catabolism by converting hypoxanthine into xanthine and then further into uric acid. Here we report that XOD is activated in THP-1 human myeloid cells in response to pro-inflammatory and growth factor stimulation. This effect occurred following stimulation of THP-1 cells with ligands of plasma membrane associated TLRs 2 and 4, endosomal TLRs 7 and 8 as well as stem cell growth factor (SCF). Hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) transcription complexes were found to be responsible for XOD upregulation. Importantly, the mammalian target of rapamycin (mTOR), a major myeloid cell translation regulator, was also found to be essential for XOD activation. Specific inhibition of XOD by allopurinol and sodium tungstate led to an increase in intracellular AMP levels triggering downregulation of mTOR activation by phosphorylation of its T2446 residue. Taken together, our results demonstrate for the first time that XOD is not only activated by pro-inflammatory stimuli or SCF but also plays an important role in maintaining mTOR-dependent translational control during the biological responses of human myeloid cells. PMID:25200751

  9. Crucial involvement of xanthine oxidase in the intracellular signalling networks associated with human myeloid cell function.

    PubMed

    Abooali, Maryam; Lall, Gurprit S; Coughlan, Karen; Lall, Harjinder S; Gibbs, Bernhard F; Sumbayev, Vadim V

    2014-01-01

    Xanthine oxidase (XOD) is an enzyme which plays a central role in purine catabolism by converting hypoxanthine into xanthine and then further into uric acid. Here we report that XOD is activated in THP-1 human myeloid cells in response to pro-inflammatory and growth factor stimulation. This effect occurred following stimulation of THP-1 cells with ligands of plasma membrane associated TLRs 2 and 4, endosomal TLRs 7 and 8 as well as stem cell growth factor (SCF). Hypoxia-inducible factor 1 (HIF-1) and activator protein 1 (AP-1) transcription complexes were found to be responsible for XOD upregulation. Importantly, the mammalian target of rapamycin (mTOR), a major myeloid cell translation regulator, was also found to be essential for XOD activation. Specific inhibition of XOD by allopurinol and sodium tungstate led to an increase in intracellular AMP levels triggering downregulation of mTOR activation by phosphorylation of its T2446 residue. Taken together, our results demonstrate for the first time that XOD is not only activated by pro-inflammatory stimuli or SCF but also plays an important role in maintaining mTOR-dependent translational control during the biological responses of human myeloid cells. PMID:25200751

  10. Mutator activity induced by microRNA-155 (miR-155) links inflammation and cancer.

    PubMed

    Tili, Esmerina; Michaille, Jean-Jacques; Wernicke, Dorothee; Alder, Hansjuerg; Costinean, Stefan; Volinia, Stefano; Croce, Carlo M

    2011-03-22

    Infection-driven inflammation has been implicated in the pathogenesis of ~15-20% of human tumors. Expression of microRNA-155 (miR-155) is elevated during innate immune response and autoimmune disorders as well as in various malignancies. However, the molecular mechanisms providing miR-155 with its oncogenic properties remain unclear. We examined the effects of miR-155 overexpression and proinflammatory environment on the frequency of spontaneous hypoxanthine phosphoribosyltransferase (HPRT) mutations that can be detected based on the resistance to 6-thioguanine. Both miR-155 overexpression and inflammatory environment increased the frequency of HPRT mutations and down-regulated WEE1 (WEE1 homolog-S. pombe), a kinase that blocks cell-cycle progression. The increased frequency of HPRT mutation was only modestly attributable to defects in mismatch repair machinery. This result suggests that miR-155 enhances the mutation rate by simultaneously targeting different genes that suppress mutations and decreasing the efficiency of DNA safeguard mechanisms by targeting of cell-cycle regulators such as WEE1. By simultaneously targeting tumor suppressor genes and inducing a mutator phenotype, miR-155 may allow the selection of gene alterations required for tumor development and progression. Hence, we anticipate that the development of drugs reducing endogenous miR-155 levels might be key in the treatment of inflammation-related cancers. PMID:21383199

  11. Reduced levels of dopamine and altered metabolism in brains of HPRT knock-out rats: a new rodent model of Lesch-Nyhan Disease.

    PubMed

    Meek, Stephen; Thomson, Alison J; Sutherland, Linda; Sharp, Matthew G F; Thomson, Julie; Bishop, Valerie; Meddle, Simone L; Gloaguen, Yoann; Weidt, Stefan; Singh-Dolt, Karamjit; Buehr, Mia; Brown, Helen K; Gill, Andrew C; Burdon, Tom

    2016-01-01

    Lesch-Nyhan disease (LND) is a severe neurological disorder caused by loss-of-function mutations in the gene encoding hypoxanthine phosphoribosyltransferase (HPRT), an enzyme required for efficient recycling of purine nucleotides. Although this biochemical defect reconfigures purine metabolism and leads to elevated levels of the breakdown product urea, it remains unclear exactly how loss of HPRT activity disrupts brain function. As the rat is the preferred rodent experimental model for studying neurobiology and diseases of the brain, we used genetically-modified embryonic stem cells to generate an HPRT knock-out rat. Male HPRT-deficient rats were viable, fertile and displayed normal caged behaviour. However, metabolomic analysis revealed changes in brain biochemistry consistent with disruption of purine recycling and nucleotide metabolism. Broader changes in brain biochemistry were also indicated by increased levels of the core metabolite citrate and reduced levels of lipids and fatty acids. Targeted MS/MS analysis identified reduced levels of dopamine in the brains of HPRT-deficient animals, consistent with deficits noted previously in human LND patients and HPRT knock-out mice. The HPRT-deficient rat therefore provides a new experimental platform for future investigation of how HPRT activity and disruption of purine metabolism affects neural function and behaviour. PMID:27185277

  12. Reduced levels of dopamine and altered metabolism in brains of HPRT knock-out rats: a new rodent model of Lesch-Nyhan Disease

    PubMed Central

    Meek, Stephen; Thomson, Alison J.; Sutherland, Linda; Sharp, Matthew G. F.; Thomson, Julie; Bishop, Valerie; Meddle, Simone L.; Gloaguen, Yoann; Weidt, Stefan; Singh-Dolt, Karamjit; Buehr, Mia; Brown, Helen K.; Gill, Andrew C.; Burdon, Tom

    2016-01-01

    Lesch-Nyhan disease (LND) is a severe neurological disorder caused by loss-of-function mutations in the gene encoding hypoxanthine phosphoribosyltransferase (HPRT), an enzyme required for efficient recycling of purine nucleotides. Although this biochemical defect reconfigures purine metabolism and leads to elevated levels of the breakdown product urea, it remains unclear exactly how loss of HPRT activity disrupts brain function. As the rat is the preferred rodent experimental model for studying neurobiology and diseases of the brain, we used genetically-modified embryonic stem cells to generate an HPRT knock-out rat. Male HPRT-deficient rats were viable, fertile and displayed normal caged behaviour. However, metabolomic analysis revealed changes in brain biochemistry consistent with disruption of purine recycling and nucleotide metabolism. Broader changes in brain biochemistry were also indicated by increased levels of the core metabolite citrate and reduced levels of lipids and fatty acids. Targeted MS/MS analysis identified reduced levels of dopamine in the brains of HPRT-deficient animals, consistent with deficits noted previously in human LND patients and HPRT knock-out mice. The HPRT-deficient rat therefore provides a new experimental platform for future investigation of how HPRT activity and disruption of purine metabolism affects neural function and behaviour. PMID:27185277

  13. Escape from Het-6 Incompatibility in Neurospora Crassa Partial Diploids Involves Preferential Deletion within the Ectopic Segment

    PubMed Central

    Smith, M. L.; Yang, C. J.; Metzenberg, R. L.; Glass, N. L.

    1996-01-01

    Self-incompatible het-6(OR)/het-6(PA) partial diploids of Neurospora crassa were selected from a cross involving the translocation strain, T(IIL -> IIIR)AR18, and a normal sequence strain. About 25% of the partial diploids exhibited a marked increase in growth rate after 2 weeks, indicating that ``escape'' from het-6 incompatibility had occurred. Near isogenic tester strains with different alleles (het-6(OR) and het-6(PA)) were constructed and used to determine that 80 of 96 escape strains tested were het-6(PA), retaining the het-6 allele found in the normal-sequence LGII position; 16 were het-6(OR), retaining the allele in the translocated position. Restriction fragment length polymorphisms in 45 escape strains were examined with probes made from cosmids that spanned the translocated region. Along with electrophoretic analysis of chromosomes from three escape strains, RFLPs showed that escape is associated with deletion of part of one or the other of the duplicated DNA segments. Deletions ranged in size from ~70 kbp up to putatively the entire 270-kbp translocated region but always included a 35-kbp region wherein we hypothesize het-6 is located. The deletion spectrum at het-6 thus resembles other cases where mitotic deletions occur such as of tumor suppressor genes and of the hprt gene (coding for hypoxanthine-guanine phosphoribosyl-transferase) in humans. PMID:8889517

  14. Expression of the Kynurenine Pathway in Human Peripheral Blood Mononuclear Cells: Implications for Inflammatory and Neurodegenerative Disease.

    PubMed

    Jones, Simon P; Franco, Nunzio F; Varney, Bianca; Sundaram, Gayathri; Brown, David A; de Bie, Josien; Lim, Chai K; Guillemin, Gilles J; Brew, Bruce J

    2015-01-01

    The kynurenine pathway is a fundamental mechanism of immunosuppression and peripheral tolerance. It is increasingly recognized as playing a major role in the pathogenesis of a wide variety of inflammatory, neurodegenerative and malignant disorders. However, the temporal dynamics of kynurenine pathway activation and metabolite production in human immune cells is currently unknown. Here we report the novel use of flow cytometry, combined with ultra high-performance liquid chromatography and gas chromatography-mass spectrometry, to sensitively quantify the intracellular expression of three key kynurenine pathway enzymes and the main kynurenine pathway metabolites in a time-course study. This is the first study to show that up-regulation of indoleamine 2,3-dioxygenase (IDO-1), kynurenine 3-monoxygenase (KMO) and quinolinate phosphoribosyltransferase (QPRT) is lacking in lymphocytes treated with interferon gamma. In contrast, peripheral monocytes showed a significant elevation of kynurenine pathway enzymes and metabolites when treated with interferon gamma. Expression of IDO-1, KMO and QPRT correlated significantly with activation of the kynurenine pathway (kynurenine:tryptophan ratio), quinolinic acid concentration and production of the monocyte derived, pro-inflammatory immune response marker: neopterin. Our results also describe an original and sensitive methodological approach to quantify kynurenine pathway enzyme expression in cells. This has revealed further insights into the potential role of these enzymes in disease processes. PMID:26114426

  15. Effects of 8-aminoguanosine and 2'-deoxyguanosine on the human mixed lymphocyte reaction (MLR). I. Description of the inhibition

    SciTech Connect

    Scott, M.E.; Gilbertsen, R.B.

    1986-03-05

    Patients deficient in purine nucleoside phosphorylase (PNP) generally have a pronounced deficiency in T cell function with no deleterious effect on B cell function. It has been demonstrated that high concentrations of 8-amino-guanosine (8-AG), an inhibitor of PNP, in combination with the PNP substrate 2'-deoxyguanosine (dGuo) can inhibit mitogen and antigen responsiveness of human lymphocytes in culture. The studies described here evaluated the effects of 8-AG and dGuo on the human MLR. Normal human lymphocytes were isolated from venous blood using Ficoll-Hypaque gradients. Stimulator cells, pretreated with mitomycin C (50 ..mu..g/ml), were added with an equal number of responder cells (2 x 10/sup 5/) to the wells of flat-bottomed microtiter plates. Cell proliferation was quantitated by the uptake of /sup 3/H-thymidine (TdR) during the final 4 hrs of six-day cultures. dGuo at 10-50 ..mu..M had no effect or stimulated proliferation, while dGuo at > 75 ..mu..M was markedly inhibitory. 8-AG alone at 100 ..mu..M had essentially no effect on TdR uptake. 8-AG (10-100 ..mu..M) plus dGuo (50 ..mu..M) produced a concentration-dependent inhibition of the MLR resulting in 8-AG IC/sub 50/s generally < 60 ..mu..M. Coaddition of 50 ..mu..M 2'-deoxycytidine, hypoxanthine or adenine partially prevented inhibition of the MLR.

  16. Development and application of human cell lines engineered to metabolically activate structurally diverse environmental mutagens

    NASA Astrophysics Data System (ADS)

    Crespi, C. I.; Langenbach, Robert; Gonzalez, Frank J.; Gelboin, Harry V.; Penman, B. W.

    1993-03-01

    Cytochromes P450 are responsible for the mutagenic/carcinogenic activation of many environmental promutagens/procarcinogens. These enzymes are present at highest concentrations in liver in vivo but are markedly absent in tester organisms for most in vitro mutagenicity test systems. Two approaches have been used to supply needed metabolic activation, incorporation of an extracellular activating system, usually derived from a rodent liver and introduction of activating enzymes into the target cell. The latter approach appears to result in a more sensitive testing system because of the close proximity of the activating enzymes and the target DNA. Human cell lines have been developed which stably express human cytochromes P450 and other cDNAs which have been introduced individually or in combination. The resulting cell lines are exquisitely sensitive to exposure to promutagens and procarcinogens. Mutagenicity is measured at the hypoxanthine phosphoribosyl transferase (hprt) and thymidine kinase (tk) gene loci. The most versatile cell line, designated MCL-5, stably express five cDNAs encoding all of the human hepatic P450s known to be principally responsible for known human procarcinogen activation. The induction of mutation is observed in MCL-5 cells upon exposure to ng/ml levels of model compounds such as nitrosamines, aflatoxin B1 and benzo(a)pyrene. A lower volume mutagenicity assay has been developed for use with samples available in limited amounts. Human lymphoblast mutation assays have been used to screen for mutagenic activity sediment samples from a polluted watershed. Two sediment samples were found to have mutagenic activity to human lymphoblasts.

  17. Membrane permeation characteristics of abacavir in human erythrocytes and human T-lymphoblastoid CD4+ CEM cells: comparison with (-)-carbovir.

    PubMed

    Mahony, William B; Domin, Barbara A; Daluge, Susan M; Zimmerman, Thomas P

    2004-11-01

    Abacavir, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol, is a novel purine carbocyclic nucleoside analogue that has been approved by the FDA for the treatment of HIV (as Ziagen trade mark [abacavir sulfate]). Chemically, abacavir and (-)-carbovir (CBV) differ only at the 6-position of the purine ring; abacavir contains a cyclopropylamino moiety in place of the 6-lactam functionality of CBV. Intracellularly both are ultimately metabolized to CBV triphosphate. We compared the membrane permeation characteristics of these two compounds at 20 degrees C in human erythrocytes and in human T-lymphoblastoid CD4+ CEM cells, using a "papaverine-stop" assay. In erythrocytes, abacavir influx was rapid, nonsaturable (rate constant=200 pmol/s/mM/microl cell water), and unaffected by inhibitors of nucleoside or nucleobase transport. CBV influx was slow, saturable, strongly inhibited by adenine or hypoxanthine, and occurred via both the nucleobase carrier (Vmax=0.67 pmol/s/microl cell water; Km=50 microM) and the nucleoside carrier (Vmax=0.47 pmol/s/microl cell water; Km=440 microM). Similar qualitative results were obtained with CD4+ CEM cells, although CBV influx rates were somewhat higher and abacavir influx rates lower, compared to the corresponding rates in erythrocytes. Equilibrium studies further revealed that both compounds are concentrated intracellularly, but nonmetabolically, in both cell types, apparently due to cytosolic protein binding (absent in erythrocyte ghosts). We conclude that, in both cell types, while CBV influx is slow and carrier-dependent, abacavir influx occurs rapidly by nonfacilitated diffusion. The membrane permeation characteristics of abacavir are consistent with its superior oral bioavailability and its impressive ability to penetrate the central nervous system. PMID:15450945

  18. Clock Gene Bmal1 Modulates Human Cartilage Gene Expression by Crosstalk With Sirt1.

    PubMed

    Yang, Wei; Kang, Xiaomin; Liu, Jiali; Li, Huixia; Ma, Zhengmin; Jin, Xinxin; Qian, Zhuang; Xie, Tianping; Qin, Na; Feng, Dongxu; Pan, Wenjie; Chen, Qian; Sun, Hongzhi; Wu, Shufang

    2016-08-01

    The critical regulation of the peripheral circadian gene implicated in osteoarthritis (OA) has been recently recognized; however, the causative role and clinical potential of the peripheral circadian rhythm attributable to such effects remain elusive. The purpose of this study was to elucidate the role of a circadian gene Bmal1 in human cartilage and pathophysiology of osteoarthritis. In our present study, the mRNA and protein levels of circadian rhythm genes, including nicotinamide adenine dinucleotide oxidase (NAD(+)) and sirtuin 1 (Sirt1), in human knee articular cartilage were determined. In OA cartilage, the levels of both Bmal1 and NAD(+) decreased significantly, which resulted in the inhibition of nicotinamide phosphoribosyltransferase activity and Sirt1 expression. Furthermore, the knockdown of Bmal1 was sufficient to decrease the level of NAD(+) and aggravate OA-like gene expression changes under the stimulation of IL-1β. The overexpression of Bmal1 relieved the alteration induced by IL-1β, which was consistent with the effect of the inhibition of Rev-Erbα (known as NR1D1, nuclear receptor subfamily 1, group D). On the other hand, the transfection of Sirt1 small interfering RNA not only resulted in a reduction of the protein expression of Bmal1 and a moderate increase of period 2 (per2) and Rev-Erbα but also further exacerbated the survival of cells and the expression of cartilage matrix-degrading enzymes induced by IL-1β. Overexpression of Sirt1 restored the metabolic imbalance of chondrocytes caused by IL-1β. These observations suggest that Bmal1 is a key clock gene to involve in cartilage homeostasis mediated through sirt1 and that manipulating circadian rhythm gene expression implicates an innovative strategy to develop novel therapeutic agents against cartilage diseases. PMID:27253997

  19. Capillary bioreactors based on human purine nucleoside phosphorylase: a new approach for ligands identification and characterization.

    PubMed

    de Moraes, Marcela Cristina; Ducati, Rodrigo Gay; Donato, Augusto José; Basso, Luiz Augusto; Santos, Diógenes Santiago; Cardoso, Carmen Lucia; Cass, Quezia Bezerra

    2012-04-01

    The enzyme purine nucleoside phosphorylase (PNP) is a target for the discovery of new lead compounds employed on the treatment severe T-cell mediated disorders. Within this context, the development of new, direct, and reliable methods for ligands screening is an important task. This paper describes the preparation of fused silica capillaries human PNP (HsPNP) immobilized enzyme reactor (IMER). The activity of the obtained IMER is monitored on line in a multidimensional liquid chromatography system, by the quantification of the product formed throughout the enzymatic reaction. The K(M) value for the immobilized enzyme was about twofold higher than that measured for the enzyme in solution (255 ± 29.2 μM and 133 ± 14.9 μM, respectively). A new fourth-generation immucillin derivative (DI4G; IC(50)=40.6 ± 0.36 nM), previously identified and characterized in HsPNP free enzyme assays, was used to validate the IMER as a screening method for HsPNP ligands. The validated method was also used for mechanistic studies with this inhibitor. This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay. PMID:22099222

  20. A system for assaying homologous recombination at the endogenous human thymidine kinase gene

    SciTech Connect

    Benjamin, M.B.; Little, J.B. ); Potter, H. ); Yandell, D.W. Massachusetts Eye and Ear Infirmary, Boston Harvard Medical School, Boston, MA )

    1991-08-01

    A system for assaying human interchromosomal recombination in vitro was developed, using a cell line containing two different mutant thymidine kinase genes (TK) on chromosomes 17. Heteroalleles were generated in the TK{sup +/+} parent B-lymphoblast cell line WIL-2 by repeated exposure to the alkylating nitrogen mustard ICR-191, which preferentially causes +1 or {minus}1 frameshifts. Resulting TK{sup {minus}/{minus}} mutants were selected in medium containing the toxic thymidine analog trifluorothymidine. In two lines, heterozygous frameshifts were located in exons 4 and 7 of the TK gene separated by {approx}8 kilobases. These lines undergo spontaneous reversion to TK{sup +} at a frequency of < 10{sup {minus}7}, and revertants can be selected in cytidine/hypoxanthine/aminopterin/thymidine medium. The nature and location of these heteroallelic mutations make large deletions, rearrangements, nondisjunction, and reduplication unlikely mechanisms for reversion to TK{sup +}. The mode of reversion to TK{sup +} was specifically assessed by DNA sequencing, use of single-strand conformation polymorphisms, and analysis of various restriction fragment length polymorphisms (RFLPs) linked to the TK gene on chromosome 17. The data suggest that a proportion of revertants has undergone recombination and gene conversion at the TK locus, with concomitant loss of frameshifts and allele loss at linked RFLPs. Models are presented for the origin of two recombinants.

  1. Induction of sister chromatid exchanges by direct and indirect chemical agents in a human teratoma cell line

    SciTech Connect

    Murison, G. . Dept. of Biological Sciences)

    1989-01-01

    In the present work, we have extended the characterization of the P3 cell line, derived from a human epithelial teratocarcinoma, by studying the induction of sister chromatid exchanges (SCEs) by direct and indirect carcinogens. Several direct-acting carcinogens produce a dose-dependent increase in SCEs. Most notably, N-methyl-N{prime}-nitro-N-nitrosoguanidine and 7{beta}, 8{alpha}-dihydroxy-9 {alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene produce increases in SCEs at dosages comparable to those used to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase locus. The indirect carcinogens elicit SCEs only when the P3 cells are cocultured with cells capable of metabolizing the indirect carcinogens to the active form. Human breast carcinoma (BJ-015) and rat hepatoma (RL12) cells are equally efficient in activating polycyclic aromatic hydrocarbons to the active form. This cell-mediated induction of SCEs is obtained when P3 cells are incubated with live, x-irradiated, or UV-irradiated BJ or RL cells. This P3 cell line is thus equally suitable to study the induction of mutations or the induction of SCEs with direct and indirect carcinogens. 35 refs., 3 tabs.

  2. Differential Proteomic Analysis of Human Placenta-Derived Mesenchymal Stem Cells Cultured on Normal Tissue Culture Surface and Hyaluronan-Coated Surface

    PubMed Central

    Wong, Tzyy Yue; Chen, Ying-Hui; Liu, Szu-Heng; Solis, Mairim Alexandra; Yu, Chen-Hsiang; Chang, Chiung-Hsin; Huang, Lynn L. H.

    2016-01-01

    Our previous results showed that hyaluronan (HA) preserved human placenta-derived mesenchymal stem cells (PDMSC) in a slow cell cycling mode similar to quiescence, the pristine state of stem cells in vivo, and HA was found to prevent murine adipose-derived mesenchymal stem cells from senescence. Here, stable isotope labeling by amino acid in cell culture (SILAC) proteomic profiling was used to evaluate the effects of HA on aging phenomenon in stem cells, comparing (1) old and young passage PDMSC cultured on normal tissue culture surface (TCS); (2) old passage on HA-coated surface (CHA) compared to TCS; (3) old and young passage on CHA. The results indicated that senescence-associated protein transgelin (TAGLN) was upregulated in old TCS. Protein CYR61, reportedly senescence-related, was downregulated in old CHA compared to old TCS. The SIRT1-interacting Nicotinamide phosphoribosyltransferase (NAMPT) increased by 2.23-fold in old CHA compared to old TCS, and is 0.48-fold lower in old TCS compared to young TCS. Results also indicated that components of endoplasmic reticulum associated degradation (ERAD) pathway were upregulated in old CHA compared to old TCS cells, potentially for overcoming stress to maintain cell function and suppress senescence. Our data points to pathways that may be targeted by HA to maintain stem cells youth. PMID:27057169

  3. Gonococci causing disseminated gonococcal infection are resistant to the bactericidal action of normal human sera.

    PubMed Central

    Schoolnik, G K; Buchanan, T M; Holmes, K K

    1976-01-01

    The susceptibility of strains of Neisseria gonorrhoeae to the bactericidal action of normal human sera was determined for isolates from patients with disseminated gonococcal infection and uncomplicated gonorrhea. Serum susceptibility was correlated with penicillin susceptibility and auxotype. 38 of 39 strains (97%) of N. gonorrhoeae from Seattle patients with disseminated gonococcal infection were resistant to the complement-dependent bactericidal action of normal human sera. 36 of these were inhibited by less than or equal to mug/ml of penicillin G and required arginine, hypoxanthine, and uracil for growth on chemically defined medium (Arg-Hyx-Ura- auxotype). 12 of 43 isolates from patients with uncomplicated gonorrhea were also of the Arg-Hyx-Ura-auxotype, inhibited by less than or equal to 0.030 mug/ml of penicillin G, and serum resistant. Of the 31 remaining strains of other auxotypes isolated from patients with uncomplicated gonorrhea, 18 (58.1%) were sensitive to normal human sera in titers ranging from 2 to 2,048. The bactericidal action of normal human sera may prevent the dissemination of serum-sensitive gonococci. However, since only a small proportion of individuals infected by serum-resistant strains develop disseminated gonococcal infection, serum resistance appears to be a necessary but not a sufficient virulence factor for dissemination. Host factors such as menstruation and pharyngeal gonococcal infection may favor the dissemination of serum-resistant strains. Since serum-resistant Arg-Hyx-Ura strains are far more frequently isolated from patients with disseminated gonococcal infection than serum-resistant strains of other auxotypes, Arg-Hyx-Ura-strains may possess other virulence factors in addition to serum resistance. PMID:825532

  4. Human See, Human Do.

    ERIC Educational Resources Information Center

    Tomasello, Michael

    1997-01-01

    A human demonstrator showed human children and captive chimpanzees how to drag food or toys closer using a rakelike tool. One side of the rake was less efficient than the other for dragging. Chimps tried to reproduce results rather than methods while children imitated and used the more efficient rake side. Concludes that imitation leads to…

  5. Lesch-Nyhan Syndrome in a Family with a Deletion Followed by an Insertion within the HPRT1 Gene.

    PubMed

    Nguyen, Khue Vu; Nyhan, William L

    2015-01-01

    Lesch-Nyhan syndrome (LNS) is a rare X-linked inherited neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine-guanine phosphoribosyltransferase(HGprt) is defective. The authors report a novel mutation which led to LNS in a family with a deletion followed by an insertion (INDELS) via the serial replication slippage mechanism: c.428_432delTGCAGinsAGCAAA, p.Met143Lysfs*12 in exon 6 of HPRT1 gene. Molecular diagnosis discloses the genetic heterogeneity of HPRT1 gene responsible for HGprt deficiency. It allows fast, accurate carrier detection and genetic counseling. PMID:25965333

  6. Use of HeLa cell guanine nucleotides by Chlamydia psittaci.

    PubMed Central

    Ceballos, M M; Hatch, T P

    1979-01-01

    Exogenous guanine was found to be incorporated into the nucleic acids of Chlamydia psittaci when the parasite was grown in HeLa cells containing hypoxanthine guanine phosphoribosyltransferase (EC 2.4.2.8) activity but not when the parasite was grown in transferase-deficient HeLa cells. No evidence for a chlamydia-specific transferase activity was found in either transferase-containing or transferase-deficient infected HeLa cells. It is concluded that C. psittaci is incapable of metabolizing guanine, but that the parasite can use host-generated guanine nucleotides as precursors for nucleic acid synthesis. Images PMID:478649

  7. Assay method for monitoring the inhibitory effects of antimetabolites on the activity of inosinate dehydrogenase in intact human CEM lymphocytes.

    PubMed Central

    Balzarini, J; De Clercq, E

    1992-01-01

    A rapid and convenient method has been developed to monitor the inhibition of inosinate (IMP) dehydrogenase by antimetabolites in intact human CEM lymphocytes. This method is based on the determination of 3H release from [2,8-3H]hypoxanthine ([2,8-3H]Hx) or [2,8-3H]inosine ([2,8-3H]Ino). The validity of this procedure was assessed by evaluating IMP dehydrogenase inhibition in intact CEM cells by the well-known IMP dehydrogenase inhibitors ribavirin, mycophenolic acid and tiazofurin. As reference materials, several compounds that are targeted at other enzymes in de novo purine nucleotide anabolism (i.e. hadacidine, acivicin) or catabolism (i.e. 8-aminoguanosine, allopurinol) were evaluated. There was a strong correlation between the inhibitory effects of the IMP dehydrogenase inhibitors (ribavirin, mycophenolic acid, tiazofurin) on 3H release from [2,8-3H]Hx and [2,8-3H]Ino in intact CEM cells and their ability to decrease intracellular GTP pool levels. The other compounds (hadacidine, acivicin, 8-aminoguanosine, allopurinol) had no marked effect on 3H release from [2,8-3H]Hx. Using this method, we demonstrated that the novel ribavirin analogue, 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide, is a potent inhibitor of IMP dehydrogenase in intact cells. PMID:1359876

  8. Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts.

    PubMed

    Bae, Seunghee; An, In-Sook; An, Sungkwan

    2015-09-01

    Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fibroblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identification of putative materials regulating DNA repair in skin cells. First, we established a modified lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fibroblast WS-1 cells were infected with the modified lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The first step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity after pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain effective reagents identified in the first step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fibroblasts. PMID:26431110

  9. Synthesis and antiviral properties of (+/-)-5'-noraristeromycin and related purine carbocyclic nucleosides. A new lead for anti-human cytomegalovirus agent design.

    PubMed

    Patil, S D; Schneller, S W; Hosoya, M; Snoeck, R; Andrei, G; Balzarini, J; De Clercq, E

    1992-09-01

    (+/-)-5'-Noraristeromycin (3) has been prepared in three steps beginning with the 2,3-O-isopropylidene derivative of (+/-)-(1 alpha, 2 beta, 3 beta, 4 alpha)-4-amino-1,2,3-cyclopentanetriol (7). Also prepared from the same starting material were the related hypoxanthine (4), guanine (5), and 2,6-diaminopurine (6) analogues. Compounds 3-6 were evaluated for antiviral activity against a large number of viruses with marked activity being observed for 3 towards vaccinia virus, human cytomegalovirus, vesicular stomatitis virus, parainfluenza (type 3) virus, measles virus, respiratory syncytial virus, reovirus (type 1), and the arenaviruses Junin and Tacaribe. None of the compounds showed cytotoxicity to the host cell monolayers used in the antiviral studies. Both 3 and 6 have been found to be inhibitors of S-adenosyl-L-homocysteine hydrolase (AdoHcy hydrolase), which likely accounts for their antiviral activity. Inhibition of AdoHcy hydrolase represents a new approach to human cytomegalovirus drug design that should be pursued. Also, the activity of 3 should be further scrutinized for the treatment of pox-, rhabdo-, paramyxo-, reo-, and arenavirus infections. PMID:1326633

  10. Toxoplasma gondii: mechanism of the parasitostatic action of 6-thioxanthine.

    PubMed

    Pfefferkorn, E R; Bzik, D J; Honsinger, C P

    2001-12-01

    In contrast to the cytocidal effect of 6-thiopurines on mammalian cells, the action of 6-thioxanthine on Toxoplasma gondii was only parasitostatic. 6-Thioxanthine was a substrate of the parasite's hypoxanthine-guanine phosphoribosyltransferase. That enzyme converted 6-thioxanthine to 6-thioxanthosine 5'-phosphate which accumulated to near millimolar concentrations within parasites incubated intracellularly in medium containing the drug. 6-Thioxanthosine 5'-phosphate was the only detectable metabolite of 6-thioxanthine. The absence of 6-thioguanine nucleotides explains the lack of a parasitocidal effect because the incorporation of 6-thiodeoxyguanosine triphosphate into DNA is the mechanism of the lethal effect of 6-thiopurines on mammalian cells. Extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate incorporated more labeled hypoxanthine or xanthine into their nucleotide pools than did control parasites. The basis for this increased nucleobase salvage remains unexplained. It was not due to up-regulation of hypoxanthine-guanine phosphoribosyltransferase and could not be explained by reduced use of labeled nucleotides for nucleic acid synthesis. Extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate used labeled hypoxanthine almost entirely to make adenine nucleotides while control parasites made both adenine and guanine nucleotides. Both extracellular parasites that had accumulated a high concentration of 6-thioxanthosine 5'-phosphate and control parasites efficiently used labeled xanthine to make guanine nucleotides. These observations suggested that inosine 5'-phosphate-dehydrogenase was inhibited while guanosine 5'-phosphate synthase was not. Assay of inosine 5'-phosphate dehydrogenase in soluble extracts of T. gondii confirmed that 6-thioxanthosine 5'-phosphate was an inhibitor. We conclude that 6-thioxanthine blocks the growth of T. gondii by a depletion a guanine

  11. Genotoxicity of 2,6- and 3,5-Dimethylaniline in Cultured Mammalian Cells: The Role of Reactive Oxygen Species

    PubMed Central

    Chao, Ming-Wei; Kim, Min Young; Wogan, Gerald N.

    2012-01-01

    Several alkylanilines with structures more complex than toluidines have been associated epidemiologically with human cancer. Their mechanism of action remains largely undetermined, and there is no reported evidence that it replicates that of multicyclic aromatic amines even though the principal metabolic pathways of P450-mediated hydroxylation and phase II conjugation are very similar. As a means to elucidate their mechanisms of action, lethality and mutagenicity in the adenine phosphoribosyltransferase (aprt +/−) gene induced in several Chinese hamster ovary cell types by 2,6- and 3,5-dimethylaniline (2,6-DMA, 3,5-DMA) and their N- and ring-hydroxyl derivatives (N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, 3,5-DMAP) were assessed. Dose-response relationships were determined in the parental AA8 cell line, its repair-deficient UV5 subclone and other repair-deficient 5P3NAT2 or -proficient 5P3NAT2R9 subclones engineered to express mouse cytochrome P4501A2 (CYP1A2) and human N-acetyltransferase (NAT2), and also in AS52 cells harboring the bacterial guanine-hypoxanthine phosphoribosyltransferase (gpt) gene. Mutations in the gpt gene of AS52 cells were characterized and found to be dominated by G:C to A:T and A:T to G:C transitions. Separately, treatment of AS52 cells with N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, 3,5-DMAP, and 3,5-DMAP led to intracellular production of reactive oxygen species (ROS) for at least 24h after removal of the mutagens in every case. Using the comet assay, DNA strand breaks were observed in a dose-dependent manner in AS52 cells when treated with each of the four N-OH-2,6-DMA, N-OH-3,5-DMA, 2,6-DMAP, and 3,5-DMAP derivatives. Comparative evaluation of the results indicates that the principal mechanism of mutagenic action is likely to be through redox cycling of intracellularly bound aminophenol/quinone imine structures to generate ROS rather than through formation of covalent DNA adducts. PMID:22831970

  12. Human Development, Human Evolution.

    ERIC Educational Resources Information Center

    Smillie, David

    One of the truly remarkable events in human evolution is the unprecedented increase in the size of the brain of "Homo" over a brief span of 2 million years. It would appear that some significant selective pressure or opportunity presented itself to this branch of the hominid line and caused a rapid increase in the brain, introducing a wholly new…

  13. Ex vivo Expansion of Human Adult Pancreatic Cells with Properties of Distributed Stem Cells by Suppression of Asymmetric Cell Kinetics

    PubMed Central

    Paré, JF; Sherley, JL

    2013-01-01

    Transplantation therapy for type I diabetes (T1D) might be improved if pancreatic stem cells were readily available for investigation. Unlike macroscopic islets, pancreatic tissue stem cells could more easily access the retroperitoneal pancreatic environment and thereby might achieve more effective pancreatic regeneration. Unfortunately, whether the adult pancreas actually contains renewing stem cells continues as a controversial issue in diabetes research. We evaluated a new method developed in our lab for expanding renewing distributed stem cells (DSCs) from adult tissues as a means to provide more evidence for adult pancreatic stem cells, and potentially advance their availability for future clinical investigation. The new method was designed to switch DSCs from asymmetric self-renewal to symmetric self-renewal, which promotes their exponential expansion in culture with reduced production of differentiated cells. Called suppression of asymmetric cell kinetics (SACK), the method uses natural purine metabolites to accomplish the self-renewal pattern shift. The SACK purine metabolites xanthine, xanthosine, and hypoxanthine were evaluated for promoting expansion of DSCs from the pancreas of adult human postmortem donors. Xanthine and xanthosine were effective for deriving both pooled and clonal populations of cells with properties indicative of human pancreatic DSCs. The expanded human cell strains had signature SACK agent-suppressible asymmetric cell kinetics, produced Ngn3+ bipotent precursors for α-cells and β-cells, and were non-tumorigenic in immunodeficient mice. Our findings support the existence of pancreatic DSCs in the adult human pancreas and indicate a potential path to increasing their availability for future clinical evaluation. PMID:25197614

  14. Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

    NASA Technical Reports Server (NTRS)

    Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

    2002-01-01

    Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

  15. Cleavage of Human Transferrin by Porphyromonas gingivalis Gingipains Promotes Growth and Formation of Hydroxyl Radicals

    PubMed Central

    Goulet, Véronique; Britigan, Bradley; Nakayama, Koji; Grenier, Daniel

    2004-01-01

    Porphyromonas gingivalis, a gram-negative anaerobic bacterium associated with active lesions of chronic periodontitis, produces several proteinases which are presumably involved in host colonization, perturbation of the immune system, and tissue destruction. The aims of this study were to investigate the degradation of human transferrin by gingipain cysteine proteinases of P. gingivalis and to demonstrate the production of toxic hydroxyl radicals (HO·) catalyzed by the iron-containing transferrin fragments generated or by release of iron itself. Analysis by polyacrylamide gel electrophoresis and Western immunoblotting showed that preparations of Arg- and Lys-gingipains of P. gingivalis cleave transferrin (iron-free and iron-saturated forms) into fragments of various sizes. Interestingly, gingival crevicular fluid samples from diseased periodontal sites but not samples from healthy periodontal sites contained fragments of transferrin. By using 55Fe-transferrin, it was found that degradation by P. gingivalis gingipains resulted in the production of free iron, as well as iron bound to lower-molecular-mass fragments. Subsequent to the degradation of transferrin, bacterial cells assimilated intracellularly the radiolabeled iron. Growth of P. gingivalis ATCC 33277, but not growth of an Arg-gingipain- and Lys-gingipain-deficient mutant, was possible in a chemically defined medium containing 30% iron-saturated transferrin as the only source of iron and peptides, suggesting that gingipains play a critical role in the acquisition of essential growth nutrients. Finally, the transferrin degradation products generated by Arg-gingipains A and B were capable of catalyzing the formation of HO·, as determined by a hypoxanthine/xanthine oxidase system and spin trapping-electron paramagnetic resonance spectrometry. Our study indicates that P. gingivalis gingipains degrade human transferrin, providing sources of iron and peptides. The iron-containing transferrin fragments or the

  16. Human Health

    MedlinePlus

    ... effects of climate change Video not supported Human Health Climate change threatens human health and well-being ... Copy link to clipboard Key Message: Wide-ranging Health Impacts Climate change threatens human health and well- ...

  17. Pseudogenes as Weaknesses of ACTB (Actb) and GAPDH (Gapdh) Used as Reference Genes in Reverse Transcription and Polymerase Chain Reactions

    PubMed Central

    Sun, Yuan; Li, Yan; Luo, Dianzhong; Liao, D. Joshua

    2012-01-01

    The genes encoding β-actin (ACTB in human or Actb in mouse) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH in human or Gapdh in mouse) are the two most commonly used references for sample normalization in determination of the mRNA level of interested genes by reverse transcription (RT) and ensuing polymerase chain reactions (PCR). In this study, bioinformatic analyses revealed that the ACTB, Actb, GAPDH and Gapdh had 64, 69, 67 and 197 pseudogenes (PGs), respectively, in the corresponding genome. Most of these PGs are intronless and similar in size to the authentic mRNA. Alignment of several PGs of these genes with the corresponding mRNA reveals that they are highly homologous. In contrast, the hypoxanthine phosphoribosyltransferase-1 gene (HPRT1 in human or Hprt in mouse) only had 3 or 1 PG, respectively, and the mRNA has unique regions for primer design. PCR with cDNA or genomic DNA (gDNA) as templates revealed that our HPRT1, Hprt and GAPDH primers were specific, whereas our ACTB and Actb primers were not specific enough both vertically (within the cDNA) and horizontally (compared cDNA with gDNA). No primers could be designed for the Gapdh that would not mis-prime PGs. Since most of the genome is transcribed, we suggest to peers to forgo ACTB (Actb) and GAPDH (Dapdh) as references in RT-PCR and, if there is no surrogate, to use our primers with extra caution. We also propose a standard operation procedure in which design of primers for RT-PCR starts from avoiding mis-priming PGs and all primers need be tested for specificity with both cDNA and gDNA. PMID:22927912

  18. Biomarker triplet NAMPT/VEGF/HER2 as a de novo detection panel for the diagnosis and prognosis of human breast cancer.

    PubMed

    Zhu, Yanyan; Guo, Meiyan; Zhang, Lingyun; Xu, Tao; Wang, Li; Xu, Guoxiong

    2016-01-01

    The early detection of breast cancer, the most common malignant tumor disease in women worldwide, relies on mammography and self breast examination. Here we evaluated the concentration of nicotinamide phosphoribosyltransferase (NAMPT), vascular endothelial growth factor (VEGF) and human epidermal growth factor receptor-2 (HER2) in serum and their expression in breast tissues associated with the clinicopathological features of patients with benign and malignant breast tumors. The immunohistochemical analysis showed that NAMPT, VEGF and HER2 proteins were overexpressed in breast tumors. The highest expression was observed in malignant tumors, low in benign tumors and negative in the adjacent normal tissue, indicating that the triplets may be progression markers and correlated with each other. The detection rate of NAMPT, VEGF and HER2 alone in tissue was 54.17, 64.58 and 60.42%, respectively, and was increased to about 79% in double combination and to 90% in triple combination. The basal levels of serum NAMPT, VEGF and HER2 in healthy controls were 94.90±4.24 pg/ml, 87.02±2.41 pg/ml and 1.12±0.04 ng/ml, respectively, measured by ELISA and found to be increased by 6.64-, 1.76- and 2.52-fold, respectively, in patients with malignant breast tumor. These elevated serum levels of NAMPT, VEGF and HER2 in patients were decreased after tumor removal, suggesting that these molecules are the indicators of treatment efficacy. The combined measurement of these triplets together may improve the sensitivity of breast cancer diagnosis and may potentially be used as a testing panel for the detection of malignant tumors, the assessment of treatment effectiveness and the monitoring of the disease progression in patients with breast cancer. Thus, we propose that the biomarker triplet NAMPT/VEGF/HER2 can be used as a de novo detection panel for the diagnosis and prognosis of human breast cancer. PMID:26531769

  19. Steroid sulfatase gene in XX males.

    PubMed Central

    Mohandas, T K; Stern, H J; Meeker, C A; Passage, M B; Müller, U; Page, D C; Yen, P H; Shapiro, L J

    1990-01-01

    The human X and Y chromosomes pair and recombine at their distal short arms during male meiosis. Recent studies indicate that the majority of XX males arise as a result of an aberrant exchange between X and Y chromosomes such that the testis-determining factor gene (TDF) is transferred from a Y chromatid to an X chromatid. It has been shown that X-specific loci such as that coding for the red cell surface antigen, Xg, are sometimes lost from the X chromosome in this aberrant exchange. The steroid sulfatase functional gene (STS) maps to the distal short arm of the X chromosome proximal to XG. We have asked whether STS is affected in the aberrant X-Y interchange leading to XX males. DNA extracted from fibroblasts of seven XX males known to contain Y-specific sequences in their genomic DNA was tested for dosage of the STS gene by using a specific genomic probe. Densitometry of the autoradiograms showed that these XX males have two copies of the STS gene, suggesting that the breakpoint on the X chromosome in the aberrant X-Y interchange is distal to STS. To obtain more definitive evidence, cell hybrids were derived from the fusion of mouse cells, deficient in hypoxanthine phosphoribosyltransferase, and fibroblasts of the seven XX males. The X chromosomes in these patients could be distinguished from each other when one of three X-linked restriction-fragment-length polymorphisms was used. Hybrid clones retaining a human X chromosome containing Y-specific sequences in the absence of the normal X chromosome could be identified in six of the seven cases of XX males.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 PMID:2301402

  20. ALDH16A1 is a novel non-catalytic enzyme that may be involved in the etiology of gout via protein–protein interactions with HPRT1

    PubMed Central

    Vasiliou, Vasilis; Sandoval, Monica; Backos, Donald S.; Jackson, Brian C.; Chen, Ying; Reigan, Philip; Lanaspa, Miguel A.; Johnson, Richard J.; Koppaka, Vindhya; Thompson, David C.

    2013-01-01

    Gout, a common form of inflammatory arthritis, is strongly associated with elevated uric acid concentrations in the blood (hyperuricemia). A recent study in Icelanders identified a rare missense single nucleotide polymorphism (SNP) in the ALDH16A1 gene, ALDH16A1*2, to be associated with gout and serum uric acid levels. ALDH16A1 is a novel and rather unique member of the ALDH superfamily in relation to its gene and protein structures. ALDH16 genes are present in fish, amphibians, protista, bacteria but absent from archaea, fungi and plants. In most mammalian species, two ALDH16A1 spliced variants (ALDH16A1, long form and ALDH16A1_v2, short form) have been identified and both are expressed in HepG-2, HK-2 and HK-293 human cell lines. The ALDH16 proteins contain two ALDH domains (as opposed to one in the other members of the superfamily), four transmembrane and one coiled-coil domains. The active site of ALDH16 proteins from bacterial, frog and lower animals contain the catalytically important cysteine residue (Cys-302); this residue is absent from the mammalian and fish orthologs. Molecular modeling predicts that both the short and long forms of human ALDH16A1 protein would lack catalytic activity but may interact with the hypoxanthine-guanine phosphoribosyltransferase (HPRT1) protein, a key enzyme involved in uric acid metabolism and gout. Interestingly, such protein-protein interactions with HPRT1 are predicted to be impaired for the long or short forms of ALDH16A1*2. These results lead to the intriguing possibility that association between ALDH16A1 and HPRT1 may be required for optimal HPRT activity with disruption of this interaction possibly contributing to the hyperuricemia seen in ALDH16A1*2 carriers. PMID:23348497

  1. Effects of cell cycle position on ionizing radiation mutagenesis. I. Quantitative assays of two genetic loci in a human lymphoblastoid cell line

    SciTech Connect

    Chuang, Yao-Yu; Liber, H.L.

    1996-11-01

    Relatively little work has been done on the influence of the position of the cell in the cell cycle on ionizing radiation-induced mutagenesis. We synchronized WTK1 human lymphoblastoid cells with 200 {mu}M lovastatin for 48 h; under these conditions more than 80% of the cells were arrested in G{sub 1} phase. Upon release, there was a 12-15-h lag followed by movement of a large fraction into S phase. We irradiated cells with either 1.5 Gy X rays at 1, 15, 18, 21 or 24 h or 1.5 Gy {gamma} rays at 1, 5, 10, 15 or 24 h after release from lovastatin. We showed that WTK1 cells were most sensitive to ionizing radiation-induced toxicity in G{sub 1} and into S phase, and more resistant in mid to late S and G{sub 2}/M phase. Somewhat surprisingly, we found that the two different gene loci had different sensitivities to radiation-induced mutation through the cell cycle. Cells in late G{sub 1} through mid-S phase were most sensitive to radiation-induced mutations at the autosomal thymidine kinase (TK) locus, whereas G{sub 1} phase was the most sensitive phase at the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) locus. 29 refs., 6 figs., 1 tab.

  2. Release of nucleosides from canine and human hearts as an index of prior ischemia.

    PubMed

    Fox, A C; Reed, G E; Meilman, H; Silk, B B

    1979-01-01

    During ischemia, myocardial adenosine triphosphate is degraded to adenosine, inosine and hypoxanthine. These nucleosides are released into coronary venous blood and may provide an index of ischemia; adenosine may also participate in the autoregulation of coronary flow. In dogs, the temporal relations between reactive hyperemic flow and nucleoside concentrations in regional venous blood were correlated after brief occlusions of a segmental coronary artery. Reactive hyperemia and adenosine release peaked together in 10 seconds, persisted for 10 to 30 seconds and then decreased in a pattern consistent with the hypothesis that they are related. During initial reflow after 45 seconds of ischemia, mean concentrations of adenosine, inosine and hypoxanthine increased, respectively, to 52, 67 and 114 nmol/100 ml plasma; after 5 minutes of ischemia, the respective levels increased to 58, 1,570 and 1,134 nmol and fell quickly. In nine patients there was a similar release of nucleosides into coronary sinus blood during reperfusion after 59 to 80 minutes of ischemic arrest during cardiac surgery. With initial reflow, adenosine, inosine and hypoxanthine levels reached 65, 655 and 917 nmol/100 ml of blood, respectively. Inosine and hypoxanthine concentrations remained high for 5 to 10 minutes after cardiac beating resumed, often when production of lactate had decreased. The results indicate that postischemic release of nucleosides reaches significant levels in man as well as animals, is parallel with the duration of ischemia, is temporary and may be a useful supplement to measurement of lactate as an index of prior myocardial ischemia. PMID:758770

  3. Mutagenicity and cytotoxicity of five antitumor ellipticines in mammalian cells and their structure-activity relationships in Salmonella

    SciTech Connect

    DeMarini, D.M.; Cros, S.; Paoletti, C.; Lecointe, P.; Hsie, A.W.

    1983-08-01

    The mutagenicity and cytotoxicity of five antitumor compounds (ellipticines) were investigated in the Chinese hamster ovary cell hypoxanthine-guanine phosphoribosyltransferase assay and in six strains of Salmonella. All five compounds (ellipticine, 9-methoxyellipticine, 9-hydroxyellipticine, 9-aminoellipticine, and 2-methyl-9-hydroxyellipticinium) were cytotoxic and mutagenic in the Chinese hamster ovary cell hypoxanthine-guanine phosphoribosyltransferase assay in the presence or absence of Aroclor 1254-induced rat liver S9, and all except the last compound were mutagenic in Salmonella. Based on the reversion pattern obtained in various frame-shift and DNA repair-proficient (uvrB/sup +/) or -deficient (uvrB) strains of Salmonella in the presence or absence of S9, the first three compounds appear to cause frameshift mutations by both intercalation and covalent bonding with DNA; thus, these are classified as reactive intercalators. However, 9-aminoellipticine intercalates only weakly and may instead exert its mutagenic activity primarily (or exclusively) by forming a covalent adduct with DNA. Compared to the published antitumor data obtained in mice, the results in Salmonella and Chinese hamster ovary cells suggest that the ability of ellipticine, 9-methoxyellipticine, and 9-hydroxyellipticine to intercalate with DNA, induce frame-shift mutations, and cause cell killing is associated with and may be the basis for their antitumor activity. The observation that the ellipticines are mutagenic in mammalian cells suggests that these antitumor agents may be carcinogenic.

  4. Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts

    NASA Technical Reports Server (NTRS)

    Chen, D. J.; Tsuboi, K.; Nguyen, T.; Yang, T. C.

    1994-01-01

    The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 micrometer2 and 0.09 to 5.56 x 10(-3) micrometer2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(-5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

  5. Charged-particle mutagenesis II. Mutagenic effects of high energy charged particles in normal human fibroblasts

    NASA Astrophysics Data System (ADS)

    Chen, D. J.; Tsuboi, K.; Nguyen, T.; Yang, T. C.

    1994-10-01

    The biological effects of high LET charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/μm to 975 KeV/gmm with particle energy (on the cells) between 94 - 603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/μm. The inactivation cross-section (αi) and the action-section for mutant induction (αm) ranged from 2.2 to 92.0 μm2 and 0.09 to 5.56 × 10-3 μm2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/μm. The mutagenicity (αm/αi) ranged from 2.05 to 7.99 × 10-5 with the maximum value at 150 keV/μm. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

  6. Charged-particle mutagenesis 2. Mutagenic effects of high energy charged particles in normal human fibroblasts

    NASA Technical Reports Server (NTRS)

    Chen, D. J.; Tsuboi, K.; Nguyen, T.; Yang, T. C.

    1994-01-01

    The biological effects of high Linear Energy Transfer (LET) charged particles are a subject of great concern with regard to the prediction of radiation risk in space. In this report, mutagenic effects of high LET charged particles are quantitatively measured using primary cultures of human skin fibroblasts, and the spectrum of induced mutations are analyzed. The LET of the charged particles ranged from 25 KeV/micrometer to 975 KeV/micrometer with particle energy (on the cells) between 94-603 MeV/u. The X-chromosome linked hypoxanthine guanine phosphoribosyl transferase (hprt) locus was used as the target gene. Exposure to these high LET charged particles resulted in exponential survival curves; whereas, mutation induction was fitted by a linear model. The Relative Biological Effect (RBE) for cell-killing ranged from 3.73 to 1.25, while that for mutant induction ranged from 5.74 to 0.48. Maximum RBE values were obtained at the LET of 150 keV/micrometer. The inactivation cross-section (alpha i) and the action cross-section for mutant induction (alpha m) ranged from 2.2 to 92.0 sq micrometer and 0.09 to 5.56 x 10(exp -3) sq micrometer respectively. The maximum values were obtained by Fe-56 with an LET of 200 keV/micrometer. The mutagenicity (alpha m/alpha i) ranged from 2.05 to 7.99 x 10(exp -5) with the maximum value at 150 keV/micrometer. Furthermore, molecular analysis of mutants induced by charged particles indicates that higher LET beams are more likely to cause larger deletions in the hprt locus.

  7. Effect of zinc supplementation on resistance of cultured human skin fibroblasts toward oxidant stress.

    PubMed

    Richard, M J; Guiraud, P; Leccia, M T; Beani, J C; Favier, A

    1993-01-01

    In purified system zinc has been shown to have an antioxidant role. Its effects on the resistance of cultured cells towards oxidative stress in vitro were examined. Diploid human skin fibroblasts were grown for 21 d in culture media (RPMI 1640 containing 15% fetal calf serum) added with different zinc (Zn) concentrations (100, 125, and 150 microM as Zinc chlorur ZnCl2). In comparison, cell controls were grown in standard culture media (6.5 microM Zn). The intracellular zinc levels of treated fibroblasts increased from 3- to 7-fold (2330 +/- 120 ng/mg protein in 150-microM Zn-treated cells versus 331 +/- 21 ng/mg protein in control cells). The intracellular copper increased 3- fold whereas the iron content slightly but not significantly decreased. The index of basal lipid peroxidation measured as thiobarbituric acid reactants (TBARs) of zinc-supplemented cells was lower than that of non zinc supplemented controls (0.89 mumol/g protein in 150 microM Zn-treated cells versus 1.59 mumol/g protein in controls). At these high doses of zinc, fibroblasts expressed lower antioxidant metalloenzymes activities. Diminished TBARs in Zn treated cells tends to support that Zn acts protectively against free radical mediated damage. However when the cells were challenged with extracellular oxidant stresses mediated by hypoxanthine/xanthine oxidase or hydrogen peroxide (H2O2), an increased toxicity in Zn-supplemented cells was observed. When we applied an intracellular oxidative stress as UV-B or UV-A radiation, Zn-treated fibroblasts were more resistant than cells grown in normal medium. If Zn has shown antioxidant effect in some in vitro or in vivo systems our observations clearly demonstrate that this role is not mediated by antioxidant metalloenzymes. PMID:7688532

  8. Human Trafficking

    MedlinePlus

    ... to debt bondage or peonage in which traffickers demand labor as a means repayment for a real ... human smuggling are two separate crimes under federal law. There are several important differences between them. Human ...

  9. Human Trafficking

    MedlinePlus

    ... TRAFFICKING (English) Listen < Back to Search FACT SHEET: HUMAN TRAFFICKING (English) Published: August 2, 2012 Topics: Public Awareness , ... organizations that protect and serve trafficking victims. National Human Trafficking Resource Center at 1.888.373.7888 Last ...

  10. A Phosphoenzyme Mimic, Overlapping Catalytic Sites and Reaction Coordinate Motion for Human NAMPT

    SciTech Connect

    Burgos, E.; Ho, M; Almo, S; Schramm, V

    2009-01-01

    Nicotinamide phosphoribosyltransferase (NAMPT) is highly evolved to capture nicotinamide (NAM) and replenish the nicotinamide adenine dinucleotide (NAD+) pool during ADP-ribosylation and transferase reactions. ATP-phosphorylation of an active-site histidine causes catalytic activation, increasing NAM affinity by 160,000. Crystal structures of NAMPT with catalytic site ligands identify the phosphorylation site, establish its role in catalysis, demonstrate unique overlapping ATP and phosphoribosyltransferase sites, and establish reaction coordinate motion. NAMPT structures with beryllium fluoride indicate a covalent H247-BeF3- as the phosphohistidine mimic. Activation of NAMPT by H247-phosphorylation causes stabilization of the enzyme-phosphoribosylpyrophosphate complex, permitting efficient capture of NAM. Reactant and product structures establish reaction coordinate motion for NAMPT to be migration of the ribosyl anomeric carbon from the pyrophosphate leaving group to the nicotinamide-N1 while the 5-phosphoryl group, the pyrophosphate moiety, and the nicotinamide ring remain fixed in the catalytic site.

  11. Different mechanisms of radiation-induced loss of heterozygosity in two human lymphoid cell lines from a single donor

    NASA Technical Reports Server (NTRS)

    Wiese, C.; Gauny, S. S.; Liu, W. C.; Cherbonnel-Lasserre, C. L.; Kronenberg, A.

    2001-01-01

    Allelic loss is an important mutational mechanism in human carcinogenesis. Loss of heterozygosity (LOH) at an autosomal locus is one outcome of the repair of DNA double-strand breaks (DSBs) and can occur by deletion or by mitotic recombination. We report that mitotic recombination between homologous chromosomes occurred in human lymphoid cells exposed to densely ionizing radiation. We used cells derived from the same donor that express either normal TP53 (TK6 cells) or homozygous mutant TP53 (WTK1 cells) to assess the influence of TP53 on radiation-induced mutagenesis. Expression of mutant TP53 (Met 237 Ile) was associated with a small increase in mutation frequencies at the hemizygous HPRT (hypoxanthine phosphoribosyl transferase) locus, but the mutation spectra were unaffected at this locus. In contrast, WTK1 cells (mutant TP53) were 30-fold more susceptible than TK6 cells (wild-type TP53) to radiation-induced mutagenesis at the TK1 (thymidine kinase) locus. Gene dosage analysis combined with microsatellite marker analysis showed that the increase in TK1 mutagenesis in WTK1 cells could be attributed, in part, to mitotic recombination. The microsatellite marker analysis over a 64-cM region on chromosome 17q indicated that the recombinational events could initiate at different positions between the TK1 locus and the centromere. Virtually all of the recombinational LOH events extended beyond the TK1 locus to the most telomeric marker. In general, longer LOH tracts were observed in mutants from WTK1 cells than in mutants from TK6 cells. Taken together, the results demonstrate that the incidence of radi-ation-induced mutations is dependent on the genetic background of the cell at risk, on the locus examined, and on the mechanisms for mutation available at the locus of interest.

  12. A study of allelic polymorphism of four short tandem repeats in the population of northwestern Russia

    SciTech Connect

    Aseev, M.V.; Skakun, V.N.; Baranov, V.S.

    1995-06-01

    Characteristics of the allelic polymorphisms of the trimeric AGC repeat of the androgen receptor gene (Xq11-12), exon 1 (AR); the tetrameric ATCT repeat of the von Willebrand factor gene (12p12), intron 40 (vWF); the AGAT repeat of the hypoxanthine phosphoribosyltransferase gene (Xq26) (HPRT); and the AGAT repeat of anonymous DNA sequences of the short arm of chromosome X (STRX1) were studied in 160 DNA samples from unrelated inhabitants of northwestern Russia using the method of polymerase chain reaction. Seventeen, ten, eight, and nine alleles were revealed electrophoretically for short tandem repeats of AR, vWF, HPRT, and STRX1, respectively. The heterozygosity indices for these repeats were 0.80, 0.70, 0.54, and 0.58, respectively. The values for AR and vWF correlated with those expected according to the Hardy-Weinberg equilibrium, whereas the values for HPRT and STRX1 differed significantly from those theoretically expected. The individualization potentials were 0.045, 0.135, 0.095, and 0.061 for the short tandem repeats of AR, vWF, HPRT, and STRX1, respectively. The distribution of genotypes for the set of these four loci in the population studied was determined. The possibilities of using the studied polymorphic marker systems in molecular diagnosis of the corresponding monogenic diseases - spinal and bulbar muscle atrophy (AR), Lesch-Nyhan disease (HPRT), and von Willebrand disease (vWF) - as well as in population human genetics, testing of personal identity, and molecular approaches to the estimation of mutagenic activity are discussed. 17 refs., 2 figs., 6 tabs.

  13. Carboxylated Short Single-Walled Carbon Nanotubes But Not Plain and Multi-walled Short Carbon Nanotubes Show in vitro Genotoxicity

    PubMed Central

    Mrakovcic, Maria; Meindl, Claudia; Leitinger, Gerd; Roblegg, Eva; Fröhlich, Eleonore

    2015-01-01

    Long carbon nanotubes (CNTs) resemble asbestos fibers due to their high length to diameter ratio and they thus have genotoxic effects. Another parameter that might explain their genotoxic effects is contamination with heavy metal ions. On the other hand, short (1–2 μm) CNTs do not resemble asbestos fibers, and, once purified from contaminations, they might be suitable for medical applications. To identify the role of fiber thickness and surface properties on genotoxicity, well-characterized short pristine and carboxylated single-walled (SCNTs) and multi-walled (MCNTs) CNTs of different diameters were studied for cytotoxicity, the cell’s response to oxidative stress (immunoreactivity against hemoxygenase 1 and glutathione levels), and in a hypoxanthine guanine phosphoribosyltransferase (HPRT) assay using V79 chinese hamster fibroblasts and human lung adenocarcinoma A549 cells. DNA repair was demonstrated by measuring immunoreactivity against activated histone H2AX protein. The number of micronuclei as well as the number of multinucleated cells was determined. CNTs acted more cytotoxic in V79 than in A549 cells. Plain and carboxylated thin (<8 nm) SCNTs and MCNTs showed greater cytotoxic potential and carboxylated CNTs showed indication for generating oxidative stress. Multi-walled CNTs did not cause HPRT mutation, micronucleus formation, DNA damage, interference with cell division, and oxidative stress. Carboxylated, but not plain, SCNTs showed indication for in vitro DNA damage according to increase of H2AX-immunoreactive cells and HPRT mutation. Although short CNTs presented a low in vitro genotoxicity, functionalization of short SCNTs can render these particles genotoxic. PMID:25505129

  14. Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

    PubMed Central

    Rommereim, Leah M.; Hortua Triana, Miryam A.; Falla, Alejandra; Sanders, Kiah L.; Guevara, Rebekah B.; Bzik, David J.; Fox, Barbara A.

    2013-01-01

    Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination. Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The Δku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The Δku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4. Here, we report methods for using type I and type II Δku80Δhxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human

  15. Genetic manipulation in Δku80 strains for functional genomic analysis of Toxoplasma gondii.

    PubMed

    Rommereim, Leah M; Hortua Triana, Miryam A; Falla, Alejandra; Sanders, Kiah L; Guevara, Rebekah B; Bzik, David J; Fox, Barbara A

    2013-01-01

    Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination. Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein(1,2). The Δku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The Δku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale(1-4). Here, we report methods for using type I and type II Δku80Δhxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human

  16. Clonal Expansions of 6-Thioguanine Resistant T Lymphocytes in the Blood and Tumor of Melanoma Patients1

    PubMed Central

    Albertini, Mark R.; Macklin, Michael D.; Zuleger, Cindy L.; Newton, Michael A.; Judice, Stephen A.; Albertini, Richard J.

    2011-01-01

    The identification of specific lymphocyte populations that mediate tumor immune responses is required for elucidating the mechanisms underlying these responses and facilitating therapeutic interventions in humans with cancer. To this end, mutant hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficient (HPRT-) T-cells were employed as probes to detect T-cell clonal amplifications and trafficking in vivo in patients with advanced melanoma. Mutant T-cells from peripheral blood were obtained as clonal isolates or in mass cultures in the presence of 6-thioguanine (TG) selection, and from tumor-bearing lymph nodes or metastatic melanoma tissues by TG-selected mass cultures. Non-mutant (wild-type) cells were obtained from all sites by analogous means, but without TG selection. cDNA sequences of the T-cell receptor (TCR) beta chains (TCR-β), determined directly (clonal isolates) or following insertion into plasmids (mass cultures), were used as unambiguous biomarkers of in vivo clonality of mature T-cell clones. Clonal amplifications, identified as repetitive TCR-β V-region, complementarity determining region 3 (CDR3), and J-region gene sequences, were demonstrated at all sites studied, i.e., peripheral blood, lymph nodes, and metastatic tumors. Amplifications were significantly enriched among the mutant compared with the wild-type T-cell fractions. Importantly, T-cell trafficking was manifest by identical TCR-β cDNA sequences, including the hyper-variable CDR3 motifs, being found in both blood and tissues in individual patients. The findings described herein indicate that the mutant T-cell fractions from melanoma patients are enriched for proliferating T-cells that infiltrate the tumor, making them candidates for investigations of potentially protective immunological responses. PMID:18712786

  17. Relative photomutagenicity of furocoumarins and limettin in the hypoxanthine phosphoribosyl transferase assay in V79 cells.

    PubMed

    Raquet, Nicole; Schrenk, Dieter

    2009-09-01

    Furocoumarins are phototoxic and photomutagenic natural plant constituents found in many medicinal plants and food items. Because plants contain mixtures of several furocoumarins, there is a need for a comparative risk assessment of a large number of furocoumarins. Little is known about the photomutagenicity of the structurally related family of coumarins, which are also abundant in many plant species. In this study, we analyzed the photomutagenic potency of the linear furocoumarins 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MOP), the angular furocoumarin angelicin, and the coumarin limettin. Above certain concentrations, all test compounds were more or less phototoxic in the presence of UVA doses between 50 and 200 mJ/cm(2), 5-MOP being the most phototoxic compound. At nonphototoxic concentrations, linear correlations were found between concentration and mutagenicity at a UVA dose of 125 mJ/cm(2) for all test compounds including limettin. For 5-MOP, strictly linear correlations were also found for the relationships of mutagenicity vs concentration at various UVA doses or vs UVA dose at given concentrations, respectively. These data indicate that the photomutagenicity of 5-MOP is proportional to the UVA dose x concentration product for noncytotoxic combinations of both factors. They also suggest that the slope of the concentration-photomutagenicity correlation at a given UVA dose may provide a basis for comparison between individual compounds. Applying this concept, in vitro photomutagenicity equivalency factors at 125 mJ/cm(2) were as follows: 1.0 (5-MOP, reference compound), 0.25 (8-MOP), and 0.02 (angelicin and limettin, respectively). These findings provide a new concept for the description of the relative photomutagenic potency of coumarins and furocoumarins and indicate that, in V79 cells, 8-MOP is less photomutagenic and limettin and angelicin are much less photomutagenic than 5-MOP. PMID:19725558

  18. Human Issues in Human Rights

    ERIC Educational Resources Information Center

    Kates, Robert W.

    1978-01-01

    Presents the report of the National Academy of Sciences' Committee in Human Rights which seeks to ease the plight of individual scientists, engineers, and medical personnel suffering severe repression. Case studies of instances of negligence of human rights are provided. (CP)

  19. Biochemistry and metabolism of Giardia.

    PubMed

    Jarroll, E L; Manning, P; Berrada, A; Hare, D; Lindmark, D G

    1989-01-01

    Giardia lamblia, an aerotolerant anaerobe, respires in the presence of oxygen by a flavin, iron-sulfur protein-mediated electron transport system. Glucose appears to be the only sugar catabolized by the Embden-Meyerhof-Parnas and hexose monophosphate pathways, and energy is produced by substrate level phosphorylation. Substrates are incompletely oxidized to CO2, ethanol and acetate by nonsedimentable enzymes. The lack of incorporation of inosine, hypoxanthine, xanthine, formate or glycine into nucleotides indicates an absence of de novo purine synthesis. Only adenine, adenosine, guanine and guanosine are salvaged, and no interconversion of these purines was detected. Salvage of these purines and their nucleosides is accomplished by adenine phosphoribosyltransferase, adenosine hydrolase, guanosine phosphoribosyltransferase and guanine hydrolase. The absence of de novo pyrimidine synthesis was confirmed by the lack of incorporation of bicarbonate, orotate and aspartate into nucleotides, and by the lack of detectable levels of the enzymes of de novo pyrimidine synthesis. Salvage appears to be accomplished by the action of uracil phosphoribosyltransferase, uridine hydrolase, uridine phosphotransferase, cytidine deaminase, cytidine hydrolase, cytosine phosphoribosyltransferase and thymidine phosphotransferase. Nucleotides of uracil may be converted to nucleotides of cytosine by cytidine triphosphate synthetase, but thymidylate synthetase and dihydrofolate reductase activities were not detected. Uptake of pyrmidine nucleosides, and perhaps pyrimidines, appears to be accomplished by carrier-mediated transport, and the common site for uptake of uridine and cytidine is distinct from the site for thymidine. Thymine does not appear to be incorporated into nucleotide pools. Giardia trophozoites appear to rely on preformed lipids rather than synthesizing them de novo.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2657035

  20. Action, human.

    PubMed

    Russo, M T

    2010-01-01

    The term "human action" designates the intentional and deliberate movement that is proper and exclusive to mankind. Human action is a unified structure: knowledge, intention or volition, deliberation, decision or choice of means and execution. The integration between these dimensions appears as a task that demands strength of will to achieve the synthesis of self-possession and self-control that enables full personal realisation. Recently, the debate about the dynamism of human action has been enriched by the contribution of neurosciences. Thanks to techniques of neuroimaging, neurosciences have expanded the field of investigation to the nature of volition, to the role of the brain in decision-making processes and to the notion of freedom and responsibility. PMID:20393686

  1. Classical Humanities

    ERIC Educational Resources Information Center

    Goodwin, Donn; And Others

    1975-01-01

    This article reports on a pilot course in humanities team-taught by three teachers, two from a senior high-school and one from a junior high-school, in Brookfield, Wisconsin. The specific subject matter is Greek and Roman culture. The curriculum is outlined and the basic reading list is included. (CLK)

  2. [Human monkeypox].

    PubMed

    Chastel, C

    2009-03-01

    Unlike other recent viral emergences, which were in majority caused by RNA viruses, the monkeypox results from infection by a DNA virus, an orthopoxvirus closely related to both vaccine and smallpox viruses and whose two genomic variants are known. Unexpectedly isolated from captive Asiatic monkeys and first considered as an laboratory curiosity, this virus was recognised in 1970 as an human pathogen in tropical Africa. Here it was responsible for sporadic cases following intrusions (for hunting) into tropical rain forests or rare outbreak with human-to-human transmission as observed in 1996 in Democratic Republic of Congo. As monkeypox in humans is not distinguishable from smallpox (a disease globally eradicated in 1977) it was only subjected to vigilant epidemiological surveillance and not considered as a potential threat outside Africa. This point of view radically changed in 2003 when monkeypox was introduced in the USA by African wild rodents and spread to 11 different states of this country. Responsible for 82 infections in American children and adults, this outbreak led to realize the sanitary hazards resulting from international trade of exotic animals and scientific investigations increasing extensively our knowledge of this zoonosis. PMID:18394820

  3. Humanizing Calculus

    ERIC Educational Resources Information Center

    Cirillo, Michelle

    2007-01-01

    In this article, the author explores the history and the mathematics used by Newton and Leibniz in their invention of calculus. The exploration of this topic is intended to show students that mathematics is a human invention. Suggestions are made to help teachers incorporate the mathematics and the history into their own lessons. (Contains 3…

  4. Human Trafficking

    ERIC Educational Resources Information Center

    Wilson, David McKay

    2011-01-01

    The shadowy, criminal nature of human trafficking makes evaluating its nature and scope difficult. The U.S. State Department and anti-trafficking groups estimate that worldwide some 27 million people are caught in a form of forced servitude today. Public awareness of modern-day slavery is gaining momentum thanks to new abolitionist efforts. Among…

  5. Nothing Human

    ERIC Educational Resources Information Center

    Wharram, C. C.

    2014-01-01

    In this essay C. C. Wharram argues that Terence's concept of translation as a form of "contamination" anticipates recent developments in philosophy, ecology, and translation studies. Placing these divergent fields of inquiry into dialogue enables us read Terence's well-known statement "I am a human being--I deem nothing…

  6. Human Rights in the Humanities

    ERIC Educational Resources Information Center

    Harpham, Geoffrey

    2012-01-01

    Human rights are rapidly entering the academic curriculum, with programs appearing all over the country--including at Duke, Harvard, Northeastern, and Stanford Universities; the Massachusetts Institute of Technology; the Universities of Chicago, of Connecticut, of California at Berkeley, and of Minnesota; and Trinity College. Most of these…

  7. Human Protothecosis

    PubMed Central

    Lass-Flörl, Cornelia; Mayr, Astrid

    2007-01-01

    Human protothecosis is a rare infection caused by members of the genus Prototheca. Prototheca species are generally considered to be achlorophyllic algae and are ubiquitous in nature. The occurrence of protothecosis can be local or disseminated and acute or chronic, with the latter being more common. Diseases have been classified as (i) cutaneous lesions, (ii) olecranon bursitis, or (iii) disseminated or systemic manifestations. Infections can occur in both immunocompetent and immunosuppressed patients, although more severe and disseminated infections tend to occur in immunocompromised individuals. Prototheca wickerhamii and Prototheca zopfii have been associated with human disease. Usually, treatment involves medical and surgical approaches; treatment failure is not uncommon. Antifungals such as ketoconazole, itraconazole, fluconazole, and amphotericin B are the most commonly used drugs to date. Among them, amphotericin B displays the best activity against Prototheca spp. Diagnosis is largely made upon detection of characteristic structures observed on histopathologic examination of tissue. PMID:17428884

  8. Human genetics

    SciTech Connect

    Carlson, E.A.

    1984-01-01

    This text provides full and balanced coverage of the concepts requisite for a thorough understanding of human genetics. Applications to both the individual and society are integrated throughout the lively and personal narrative, and the essential principles of heredity are clearly presented to prepare students for informed participation in public controversies. High-interest, controversial topics, including recombinant DNA technology, oncogenes, embryo transfer, environmental mutagens and carcinogens, IQ testing, and eugenics encourage understanding of important social issues.

  9. Transient induction of a nuclear antigen unrelated to Epstein-Barr nuclear antigen in cells of two human B-lymphoma lines converted by Epstein-Barr virus.

    PubMed

    Fresen, K O; zur Hausen, H

    1977-01-01

    Infection of cells of the Epstein-Barr virus (EBV)-negative human B-lymphoma lines BJAB and Ramos with EBV preparations from P3HR-1 or B 95-8 cells converted these cells to EBV genome carriers expressing Epstein-Barr nuclear antigen (EBNA) in almost 100% of these cells. Induction of these cells as well as of clones from P3HR-1 EBV-converted BJAB cells with iododeoxyuridine, aminopterin, and hypoxanthine resulted in the appearance of a nuclear antigen in about 1-6% of the cells 1-4 days after induction. The antigen is different from known EBV-induced antigens like EBNA, viral capsid antigen (VCA) or the D- and R-subspecificities of the early antigen (EA) complex. It is demonstrated by indirect immunofluorescence and inactivated after acetone fixation. The antigen was not detectable after induction of uninfected BJAB and Ramos cells nor has it been found in noninduced or induced P3HR-1 and Raji cells. Thus, it appears that EBV-infection mediates the expression of this antigen, for which the name TINA (transiently induced nuclear antigen) is suggested. Sera reacting against TINA generally contained high antibody titers against EBV-induced EA. Only a limited number of highly EA-reactive sera, however, were also positive for TINA. Among 200 sera tested thus far, TINA reactivity was most frequently observed in sera of patients with nasopharyngeal carcinoma (7 out of 28), in sera of the only two patients with immunoblastoma tested and occasionally in sera from patients with Hodgkin's disease and chronic lymphatic leukemia. Among 70 sera from nontumor patients, TINA reactivity was observed three times: two patients suffered from "chronic" infectious mononucleosis, the other revealed persistent splenomegaly. PMID:189313

  10. Human evolution.

    PubMed

    Wood, B

    1996-12-01

    The common ancestor of modern humans and the great apes is estimated to have lived between 5 and 8 Myrs ago, but the earliest evidence in the human, or hominid, fossil record is Ardipithecus ramidus, from a 4.5 Myr Ethiopian site. This genus was succeeded by Australopithecus, within which four species are presently recognised. All combine a relatively primitive postcranial skeleton, a dentition with expanded chewing teeth and a small brain. The most primitive species in our own genus, Homo habilis and Homo rudolfensis, are little advanced over the australopithecines and with hindsight their inclusion in Homo may not be appropriate. The first species to share a substantial number of features with later Homo is Homo ergaster, or 'early African Homo erectus', which appears in the fossil record around 2.0 Myr. Outside Africa, fossil hominids appear as Homo erectus-like hominids, in mainland Asia and in Indonesia close to 2 Myr ago; the earliest good evidence of 'archaic Homo' in Europe is dated at between 600-700 Kyr before the present. Anatomically modern human, or Homo sapiens, fossils are seen first in the fossil record in Africa around 150 Kyr ago. Taken together with molecular evidence on the extent of DNA variation, this suggests that the transition from 'archaic' to 'modern' Homo may have taken place in Africa. PMID:8976151