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Partial purification and characterization of the mRNA for human thymidine kinase and hypoxanthine/guanine phosphoribosyltransferase.  

PubMed Central

We used direct microinjection of poly(A)+RNA into individual hypoxanthine/guanine phosphoribosyltransferase-deficient or thymidine kinase-deficient cells and detected the specific in vivo translation products as an assay for human hypoxanthine/guanine phosphoribosyltransferase or thymidine kinase mRNAs. The incorporation of [3H]hypoxanthine or [3H]thymidine into cells in response to injected mRNA was assayed in situ by autoradiography. Methylmercuric hydroxide/agarose gel analysis showed that human hypoxanthine/guanine phosphoribosyltransferase mRNA contains approximately 1,530 nucleotides, which is twice the number required for its protein coding capacity. The mRNA for human cytoplasmic thymidine kinase is estimated to be approximately the same length; thus, the size of the cytosol thymidine kinase subunit can be predicted to be approximately 47,000 daltons, if the full coding capacity of its mRNA is utilized. Images

Lin, P F; Yamaizumi, M; Murphy, P D; Egg, A; Ruddle, F H



Purine Requirement of Cells Cultured from Humans Affected with Lesch-Nyhan Syndrome (Hypoxanthine-Guanine Phosphoribosyltransferase Deficiency)  

Microsoft Academic Search

Humans with the Lesch-Nyhan syndrome have an X-chromosomal mutant gene that causes severe neurological and developmental abnormalities. The patients are deficient in hypoxanthine-guanine phosphoribosyltransferase, which converts hypoxanthine to inosinic acid, a major precursor of adenine and guanine nucleotides. Paradoxically, the enzyme defect causes hypernormal de novo synthesis of inosinic acid, which manifests itself as excesses of hypoxanthine, xanthine, and uric

Jeanette S. Felix; Robert Demars



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

SciTech Connect

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.

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



Purinergic signaling in human pluripotent stem cells is regulated by the housekeeping gene encoding hypoxanthine guanine phosphoribosyltransferase  

PubMed Central

Lesch–Nyhan disease (LND) is an X-linked genetic disorder caused by mutations of the hypoxanthine guanine phosphoribosyltransferase (HPRT) purine biosynthesis gene and characterized by aberrant purine metabolism, deficient basal ganglia dopamine levels, dystonia, and severe neurobehavioral manifestations, including compulsive self-injurious behavior. Although available evidence has identified important roles for purinergic signaling in brain development, the mechanisms linking HPRT deficiency, purinergic pathways, and neural dysfunction of LND are poorly understood. In these studies aimed at characterizing purinergic signaling in HPRT deficiency, we used a lentivirus vector stably expressing an shRNA targeted to the HPRT gene to produce HPRT-deficient human CVB induced pluripotent stem cells and human HUES11 embryonic stem cells. Both CVB and HUES11 cells show >99% HPRT knockdown and demonstrate markedly decreased expression of the purinergic P2Y1 receptor mRNA. In CVB cells, P2Y1 mRNA and protein down-regulation by HPRT knockdown is refractory to activation by the P2Y1 receptor agonist ATP and shows aberrant purinergic signaling, as reflected by marked deficiency of the transcription factor pCREB and constitutive activation of the MAP kinases phospho-ERK1/2. Moreover, HPRT-knockdown CVB cells also demonstrate marked reduction of phosphorylated ?-catenin. These results indicate that the housekeeping gene HPRT regulates purinergic signaling in pluripotent human stem cells, and that this regulation occurs at least partly through aberrant P2Y1-mediated expression and signaling. We propose that such mechanisms may play a role in the neuropathology of HPRT-deficiency LND and may point to potential molecular targets for modulation of this intractable neurological phenotype.

Mastrangelo, Lina; Kim, Ji-Eun; Miyanohara, Atsushi; Kang, Tae Hyuk; Friedmann, Theodore



Absence of hypoxanthine:guanine phosphoribosyltransferase activity in murine Dunn osteosarcoma  

SciTech Connect

The transplantable murine Dunn osteosarcoma has no detectable hypoxanthine:guanine phosphoribosyltransferase (EC 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.

Abelson, H.T.; Gorka, C.



Mutations which alter splicing in the human hypoxanthine-guanine phosphoribosyltransferase gene.  

PubMed Central

A large proportion of mutations at the human hprt locus result in aberrant splicing of the hprt mRNA. We have been able to relate the mutation to the splicing abnormality in 30 of these mutants. Mutations at the splice acceptor sites of introns 4, 6 and 7 result in splicing out of the whole of the downstream exons, whereas in introns 1, 7 or 8 a cryptic site in the downstream exon can be used. Mutations in the donor site of introns 1 and 5 result in the utilisation of cryptic sites further downstream, whereas in the other introns, the upstream exons are spliced out. Our most unexpected findings were mutations in the middle of exons 3 and 8 which resulted in splicing out of these exons in part of the mRNA populations. Our results have enabled us to assess current models of mRNA splicing. They emphasize the importance of the polypyrimidine tract in splice acceptor sites, they support the role of the exon as the unit of assembly for splicing, and they are consistent with a model proposing a stem-loop structure for exon 8 in the hprt mRNA. Images

Steingrimsdottir, H; Rowley, G; Dorado, G; Cole, J; Lehmann, A R



The effect of novel [3-fluoro-(2-phosphonoethoxy)propyl]purines on the inhibition of Plasmodium falciparum, Plasmodium vivax and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases.  


Protozoan parasites from the Plasmodiidae family are the causative agents of malaria. Inhibition of hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT) has been suggested as a target for development of new anti-malarial therapeutics. Acyclic nucleoside phosphonates (ANPs) are potent and selective inhibitors of plasmodial HG(X)PRTs. A new series of ANPs, based on the chemical structure and inhibitory activity of three ANPs, 2-(phosphonoethoxy)ethyl with either guanine or hypoxanthine as the base (PEEG and PEEHx) and 3-hydroxy-2-(phosphonomethoxy)propyl with guanine as the base (HPMPG), were prepared. These compounds are stereoisomers of 3-fluoro-(2-phosphonoethoxy)propyl (FPEPs) and 3-fluoro-(2-phosphonomethoxy)propyl (FPMPs) analogues. Both the (R)- and (S)-isomers of these fluorinated derivatives have higher Ki values (by 10- to 1000-fold) for human HGPRT and Plasmodium falciparum HGXPRT than the non-fluorinated ANPs. Possible explanations for these changes in affinity are proposed based on docking studies using the known crystal structures of human HGPRT in complex with PEEG. PMID:23850568

Baszczy?ski, Ond?ej; Hocková, Dana; Janeba, Zlatko; Holý, Antonín; Jansa, Petr; Dra?ínský, Martin; Keough, Dianne T; Guddat, Luke W



Characterization of guanine and hypoxanthine phosphoribosyltransferases in Methanococcus voltae.  

PubMed Central

Phosphoribosyltransferase (PRTase) and nucleoside phosphorylase (NPase) activities were detected by radiometric methods in extracts of Methanococcus voltae. Guanine PRTase activity was present at 2.7 nmol min(-1) mg of protein(-1) and had an apparent Km for guanine of 0.2 mM and a pH optimum of 9. The activity was inhibited 50% by 0.3 mM GMP. IMP and AMP were not inhibitory at concentrations up to 0.6 mM. Hypoxanthine inhibited by 50% at 0.16 mM, and adenine and xanthine were not inhibitory at concentrations up to 0.5 mM. Guanosine NPase activity was present at 0.01 nmol min(-1) mg of protein(-1). Hypoxanthine PRTase activity was present at 0.85 nmol min(-1) mg of protein(-1) with an apparent Km for hypoxanthine of 0.015 mM and a pH optimum of 9. Activity was stimulated at least twofold by 0.05 mM GMP and 0.2 mM IMP but was unaffected by AMP. Guanine inhibited by 50% at 0.06 mM, but adenine and xanthine were not inhibitory. Inosine NPase activity was present at 0.04 nmol min(-1) mg of protein(-1). PRTase activities were not sensitive to any base analogs examined, with the exception of 8-azaguanine, 8-azahypoxanthine, and 2-thioxanthine. Fractionation of cell extracts by ion-exchange chromatography resolved three peaks of activity, each of which contained both guanine and hypoxanthine PRTase activities. The specific activities of the PRTases were not affected by growth in medium containing the nucleobases. Mutants of M. voltae resistant to base analogs lacked PRTase activity. Two mutants resistant to both 8-azaguanine and 8-azahypoxanthine lacked activity for both guanine and hypoxanthine PRTase. These results suggest that analog resistance was acquired by the loss of PRTase activity.

Bowen, T L; Lin, W C; Whitman, W B



Hypoxanthine phosphoribosyltransferase: radiochemical assay procedures for the forward and reverse reactions  

SciTech Connect

Simple and rapid radiochemical assay procedures for the forward (IMP synthesis) and reverse (IMP pyrophosphorolysis) reactions catalyzed by hypoxanthine phosphoribosyltransferase have been developed. Enzyme activity in the forward direction was assessed by measuring the amount of (8-/sup 14/C)IMP formed from (8-/sup 14/C)hypoxanthine following their separation by polyethyleneimine-cellulose TLC in methanol:water (1:1, v/v). (8-/sup 14/C)IMP has been synthesized from (8-/sup 14/C)hypoxanthine, using hypoxanthine phosphoribosyltransferase derived from human brain, with subsequent purification by elution from phenyl boronate-agarose. Enzyme activity in the reverse direction was assessed by measuring the amount of (8-/sup 14/C)uric acid formed from the labeled IMP following their separation by polyethyleneimine-cellulose TLC in 0.2 M LiCl saturated with boric acid (pH 4.5):95% ethanol (1:1, v/v), the transferase reaction being coupled with excess xanthine oxidase and catalase to overcome the unfavorable equilibrium.

Smithers, G.W.; O'Sullivan, W.J.



Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma brucei.  

PubMed Central

The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme of Trypanosoma brucei and related parasites provides a rational target for the treatment of African sleeping sickness and several other parasitic diseases. To characterize the T. brucei HGPRT enzyme in detail, the T. brucei hgprt was isolated within a 4.2 kb SalI-KpnI genomic insert and sequenced. Nucleotide sequence analysis revealed an open reading frame of 630 bp that encoded a protein of 210 amino acids with a M(r) = 23.4 kd. After gap alignment, the T. brucei HGPRT exhibited 21-23% amino acid sequence identity, mostly in three clustered regions, with the HGPRTs from human, S. mansoni, and P falciparum, indicating that the trypanosome enzyme was the most divergent of the group. Surprisingly, the T. brucei HGPRT was more homologous to the hypoxanthine phosphoribosyltransferase (HPRT) from the prokaryote V. harveyi than to the eukaryotic HGPRTs. Northern blot analysis revealed two trypanosome transcripts of 1.4 and 1.9 kb, each expressed to equivalent degrees in insect vector and mammalian forms of the parasite. The T. brucei hgprt was inserted into an expression plasmid and transformed into S phi 606 E. coli that are deficient in both HPRT and xanthine-guanine phosphoribosyltransferase activities. Soluble, enzymatically active recombinant T. brucei HGPRT was expressed to high levels and purified to homogeneity by GTP-agarose affinity chromatography. The purified recombinant enzyme recognized hypoxanthine, guanine, and allopurinol, but not xanthine or adenine, as substrates and was inhibited by a variety of nucleotide effectors. The availability of a molecular clone encoding the T. brucei hgprt and large quantities of homogeneous recombinant HGPRT enzyme provides an experimentally manipulable molecular and biochemical system for the rational design of novel therapeutic agents for the treatment of African sleeping sickness and other diseases of parasitic origin. Images

Allen, T E; Ullman, B



Hypoxanthine-guanine phosphoribosyltransferase deficiency: analysis of HPRT mutations by direct sequencing and allele-specific amplification  

Microsoft Academic Search

The Lesch-Nyhan syndrome is a severe X chromosome-linked human disease caused by a virtual absence of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity. A partial deficiency in the activity of this enzyme can result in gouty arthritis. To determine the genetic basis for reduction or loss of enzyme activity, we have amplified and sequenced the coding region of HPRT cDNA from four patients:

Donna G. Sculley; Paul A. Dawson; Ifor R. Beacham; Bryan T. Emmerson; Ross B. Gordon



Acyclic Immucillin Phosphonates: Second generation inhibitors of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase  

PubMed Central

Summary Plasmodium falciparum, the primary cause of deaths from malaria, 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. Here we present a new 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.

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



Cloning, characterization and preliminary crystallographic analysis of Leishmania hypoxanthine–guanine phosphoribosyltransferase  

Microsoft Academic Search

Hypoxanthine–guanine phosphoribosyltransferase (HGPRT) (EC is an important enzyme involved in the recycling of purine nucleotides in all cells. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of nucleotides; therefore, this pathway is an attractive target for antiparasitic drug design. The hgprt gene was cloned from a

Paulo S. Monzani; Juan D. Alfonzo; Larry Simpson; Glaucius Oliva; Otavio H. Thiemann



Short sequence-paper Cloning, characterization and preliminary crystallographic analysis of Leishmania hypoxanthine-guanine phosphoribosyltransferase  

Microsoft Academic Search

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC is an important enzyme involved in the recycling of purine nucleotides in all cells. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of nucleotides; therefore, this pathway is an attractive target for antiparasitic drug design. The hgprt gene was cloned from a

Paulo S. Monzani; Juan D. Alfonzo; Larry Simpson; Glaucius Oliva; Otavio H. Thiemann


Nicotinamide Phosphoribosyltransferase in Human Diseases  

PubMed Central

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.

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



Analysis of cDNA encoding the hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) of Schistosoma mansoni; a putative target for chemotherapy.  

PubMed Central

Because of the lack of de novo purine biosynthesis, hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) is a critical enzyme in the purine metabolic pathway of the human parasite, Schistosoma mansoni. Using a cDNA clone encoding mouse HGPRTase and subsequently a synthetic oligonucleotide derived from sequencing a clone of genomic DNA, two clones were isolated from an adult schistosome cDNA library. One clone is 1.374 Kilobases (Kb) long and has an open reading frame of 693 bases. The deduced 231 amino acid sequence has 47.9% identity in a 217 amino acid overlap with human HGPRTase. Northern blot analysis indicates that the full length of mRNA for the S. mansoni HGPRTase is 1.45-1.6 Kb. Analysis of the primary structures of the putative active site for human and parasite enzymes reveal specific differences which may eventually be exploitable in the design of drugs for the treatment of schistosomiasis. Images

Craig, S P; McKerrow, J H; Newport, G R; Wang, C C



A new point mutation in a hypoxanthine phosphoribosyltransferase-deficient patient.  


A 12-year-old boy was referred because of abdominal pain, gross hematuria, and passage of stones. Further evaluation showed growth delay, low average range of intellectual functioning, and a speech articulation disorder. No signs of self-mutilation or self-injurious behavior were present. He had hyperuricemia, hyperuricosuria, uric acid crystalluria, uric acid calculi, macrocytosis, megaloblastic bone marrow changes, and mild anemia. Hypoxanthine phosphoribosyltransferase (HPRT) enzyme activity was reduced to approximately 26% of normal. Polymerase chain reaction-single strand conformational polymorphism analysis of the HPRT gene in DNA isolated from the patient's blood lymphocytes revealed a single nucleotide substitution at codon 200 in exon 8. The base change was a guanine to cytosine transversion, resulting in the conservative amino acid substitution of threonine in place of arginine. To our knowledge, this mutation has not previously been reported. PMID:9323299

Hidalgo-Laos, R I; Kedar, A; Williams, C A; Neiberger, R E



Chromium(III) tris(picolinate) is mutagenic at the hypoxanthine (guanine) phosphoribosyltransferase locus in Chinese hamster ovary cells  

Microsoft Academic Search

Chromium trispicolinate (CrPic) is a popular dietary supplement that is not regulated by the Food and Drug Administration. We are using this compound as a bio-available model to explore the role of Cr(III) in Cr(VI)-induced cancers. The ability of CrPic to cause mutations at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus of CHO AA8 cells has been measured after a 48h

Diane M. Stearns; Stacey M. Silveira; Kristina K. Wolf; April M. Luke



Identification of a single nucleotide change in a mutant gene for hypoxanthine-guanine phosphoribosyltransferase (HPRT Ann Arbor)  

Microsoft Academic Search

HPRTAnn Arbor is a variant of hypoxanthine (guanine) phosphoribosyl-transferase (HPRT: EC, which was identified in two brothers with hyperuricemia and nephrolithiasis. In previous studies, this mutant enzyme was characterized by an increased Km for both substrates, a normal Vmax, a decreased intracellular concentration of enzyme protein, a normal subunit molecular weight and an acidic isoelectric point under native isoelectric

Shin Fujimori; Yuji Hidaka; Beverly L. Davidson; Thomas D. Palella; William N. Kelley



Double-stranded RNA specific to adenosine kinase and hypoxanthine–xanthine–guanine-phosphoribosyltransferase retards growth of Toxoplasma gondii  

Microsoft Academic Search

Adenosine kinase (AK) and hypoxanthine–xanthine–guanine phosphoribosyltransferase (HXGPRT) are the two key enzymes involved\\u000a in the purine salvage pathway in Toxoplasma gondii. In this study, we targeted the genes encoding AK and HXGPRT in T. gondii for inhibition by exposing the parasites to the corresponding double stranded RNAs (dsRNAs). We report here that dsRNAs targeting\\u000a both AK and HXGPRT were effective

Li Yu; Yu-Feng Gao; Xia Li; Zeng-Pei Qiao; Ji-Long Shen



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


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

Kang, Tae Hyuk; Park, Yongjin; Bader, Joel S; Friedmann, Theodore



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

PubMed Central

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.

Bader, Joel S.; Friedmann, Theodore



Mutational spectra at the hypoxanthine–guanine phosphoribosyltransferase ( HPRT) locus in T-lymphocytes of nonsmoking and smoking lung cancer patients  

Microsoft Academic Search

Molecular analysis of mutations at the hypoxanthine–guanine phosphoribosyltransferase (HPRT) locus in peripheral blood T-lymphocytes can provide information on mechanisms of somatic in vivo mutation in populations exposed to exogenous carcinogens and in individuals with inherent susceptibility to cancer and other diseases. To study possible mutational changes associated with smoking as a risk factor for lung cancer, we analyzed HPRT mutations

Peter Hackman; Sai-Mei Hou; Fredrik Nyberg; Göran Pershagen; Bo Lambert



Substrate inhibition of adenosine phosphorylation in adenosine deaminase deficiency and adenosine-mediated inhibition of PP-ribose-P dependent nucleotide synthesis in hypoxanthine phosphoribosyltransferase deficient erythrocytes  

Microsoft Academic Search

Summary The metabolism of adenosine and its effects on phosphoribosylpyrophosphate, PP-ribose-P, dependent nucleotide synthesis were studied using erythrocytes from patients with adenosine deaminase and hypoxanthine phosphoribosyltransferase deficiency as models. The phosphorylation of adenosine was progressively inhibited by concentrations of adenosine greater than 1 µmol L-1 for control and ADA deficient erythrocytes. There was essentially no initial rate of phosphorylation at

F. F. Snyder; C. Dyer; J. E. Seegmiller; R. M. Goldblum; G. C. Mills; F. C. Schmalstieg



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

SciTech Connect

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.

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



Phenotypic variation among seven members of one family with deficiency of hypoxanthine-guanine phosphoribosyltransferase.  


We describe a family of seven boys affected by Lesch-Nyhan disease with various phenotypes. Further investigations revealed a mutation c.203T>C in the gene encoding HGprt of all members, with substitution of leucine to proline at residue 68 (p.Leu68Pro). Thus patients from this family display a wide variety of symptoms although sharing the same mutation. Mutant HGprt enzyme was prepared by site-directed mutagenesis and the kinetics of the enzyme revealed that the catalytic activity of the mutant was reduced, in association with marked reductions in the affinity towards phosphoribosylpyrophosphate (PRPP). Its Km for PRPP was increased 215-fold with hypoxanthine as substrate and 40-fold with guanine as substrate with associated reduced catalytic potential. Molecular modeling confirmed that the most prominent defect was the dramatically reduced affinity towards PRPP. Our studies suggest that the p.Leu68Pro mutation has a strong impact on PRPP binding and on stability of the active conformation. This suggests that factors other than HGprt activity per se may influence the phenotype of Lesch-Nyhan patients. PMID:24075303

Ceballos-Picot, Irène; Augé, Franck; Fu, Rong; Olivier-Bandini, Anne; Cahu, Julie; Chabrol, Brigitte; Aral, Bernard; de Martinville, Bérengère; Lecain, Jean-Paul; Jinnah, H A



Genetic modification of mouse bone marrow by lentiviral vector-mediated delivery of hypoxanthine-Guanine phosphoribosyltransferase short hairpin RNA confers chemoprotection against 6-thioguanine cytotoxicity.  


We have recently developed a novel and highly efficient strategy that exclusively uses the purine analog 6-thioguanine (6TG) for both pretransplantation conditioning and post-transplantation chemoselection of hypoxanthine-guanine phosphoribosyltransferase (HPRT)-deficient bone marrow (BM). In a mouse BM transplantation model, combined 6TG preconditioning and in vivo chemoselection consistently achieved >95% engraftment of HPRT-deficient donor BM and long-term reconstitution of histologically and immunophenotypically normal hematopoiesis in both primary and secondary recipients, without significant toxicity and in the absence of any other cytotoxic conditioning regimen. To translate this strategy for combined 6TG conditioning and chemoselection into a clinically feasible approach, it is necessary to develop methods for genetic modification of normal hematopoietic stem cells (HSC) to render them HPRT-deficient and thus 6TG-resistant. Here we investigated a strategy to reduce HPRT expression and thereby confer protection against 6TG myelotoxicity to primary murine BM cells by RNA interference (RNAi). Accordingly, we constructed and validated a lentiviral gene transfer vector expressing short-hairpin RNA (shRNA) that targets the murine HPRT gene. Our results showed that lentiviral vector-mediated delivery of HPRT-targeted shRNA could achieve effective and long-term reduction of HPRT expression. Furthermore, in both an established murine cell line as well as in primary murine BM cells, lentiviral transduction with HPRT-targeted shRNA was associated with enhanced resistance to 6TG cytotoxicity in vitro. Hence this represents a translationally feasible method to genetically engineer HSC for implementation of 6TG-mediated preconditioning and in vivo chemoselection. PMID:23769104

Hacke, K; Treger, J A; Bogan, B T; Schiestl, R H; Kasahara, N



Nitric oxide and ethylnitrosourea: relative mutagenicity in the p53 tumor suppressor and hypoxanthine-phosphoribosyltransferase genes.  


Nitric oxide (NO) is a cellular messenger which is mutagenic in bacteria and human TK6 cells and induces deamination of 5-methylcytosine (5meC) residues in vitro. The aims of this study were: (i) to investigate whether NO induces 5meC deamination in codon 248 of the p53 gene in cultured human bronchial epithelial cells (BEAS-2B); and (ii) to compare NO mutagenicity to that of ethylnitrosourea (ENU), a strong mutagen. Two approaches were used: (i) a novel genotypic assay, using RFLP/PCR technology on purified exon VII sequence of the p53 gene; and (ii) a phenotypic (HPRT) mutation assay using 6-thioguanine selection. BEAS-2B cells were either exposed to 4 mM DEA/NO (Et2N[N2O2]Na, an agent that spontaneously releases NO into the medium) or transfected with the inducible nitric oxide synthase (iNOS) gene. The genotypic mutation assay, which has a sensitivity of 1 x 10(-6), showed that 4 mM ENU induces detectable numbers of G --> A transitions in codon 248 of p53 while 5-methylcytosine deamination was not detected in either iNOS-transfected cells or cells exposed to 4 mM DEA/NO. Moreover, ENU was dose-responsively mutagenic in the phenotypic HPRT assay, reaching mutation frequencies of 24 and 96 times that of untreated control cells at ENU concentrations of 4 and 8 mM respectively; by contrast, 4 mM DEA/NO induced no detectable mutations in this assay, nor were any observed in cells transfected with murine iNOS. We conclude that if NO is at all promutagenic in these cells, it is significantly less so than the ethylating mutagen, ENU. PMID:7554056

Felley-Bosco, E; Mirkovitch, J; Ambs, S; Macé, K; Pfeifer, A; Keefer, L K; Harris, C C



Transition States of Plasmodium falciparum and Human Orotate Phosphoribosyltransferases†  

PubMed Central

Orotate phosphoribosyltransferases (OPRT) catalyze the formation of orotidine 5?-monophosphate (OMP) from ?-D-phosphoribosylpyrophosphate (PRPP) and orotate, an essential step in the de novo biosynthesis of pyrimidines. Pyrimidine de novo biosynthesis is required in Plasmodium falciparum, thus OPRT of the parasite (PfOPRT) is a target for anti-malarial drugs. De novo biosynthesis of pyrimidines is also a feature of rapidly-proliferating cancer cells. Human OPRT (HsOPRT) is therefore a target for neoplastic and autoimmune diseases. One approach to the inhibition of OPRTs is through analogues that mimic the transition states of PfOPRT and HsOPRT. The transition state structures of these OPRTs were analyzed by kinetic isotope effects (KIEs), substrate specificity and computational chemistry. With phosphonoacetic acid (PA), an analogue of pyrophosphate, the intrinsic KIEs of [1?-14C], [1, 3-15N2], [3-15N], [1?-3H], [2?-3H], [4?-3H] and [5?-3H2] are 1.034, 1.028, 0.997, 1.261, 1.116, 0.974 and 1.013 for PfOPRT and 1.035, 1.025, 0.993, 1.199, 1.129, 0.962 and 1.019 for HsOPRT, respectively. Transition state structures of PfOPRT and HsOPRT were determined computationally by matching the calculated and intrinsic KIEs. The enzymes form late associative DN*AN‡ transition states with complete orotate loss and partially-associative nucleophile. The C1?-OPA distances are approximately 2.1 Å at these transition states. The modest [1?-14C] KIEs and large [1?-3H] KIEs are characteristic of DN*AN‡ transition states. The large [2?-3H] KIEs indicate a ribosyl 2?-C-endo conformation at the transition states. p-Nitrophenyl ?-Dribose 5?-phosphate is a poor substrate of PfOPRT and HsOPRT but is a nanomolar inhibitor, supporting a reaction coordinate with strong leaving group activation.

Zhang, Yong; Luo, Minkui; Schramm, Vern L.



Multiplex PCR Analysis of In vivo-Arising Deletion Mutations in the 'hprt' Gene of Human T-Lymphocytes.  

National Technical Information Service (NTIS)

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. The hprt clonal assay was used to isolate in-v...

J. C. Fuscoe L. J. Zimmerman K. Harrington-Brock M. M. Moore



Regulation of purine utilization in bacteria. VI. Characterization of hypoxanthine and guanine uptake into isolated membrane vesicles from Salmonella typhimurium.  

PubMed Central

Uptake of hypoxanthine and guanine into isolated membrane vesicles of Salmonella typhimurium TR119 was stimulated by 5'-phosphoribosyl-1'-pyrophosphate (PRPP). For strain proAB47, a mutant that lacks guanine phosphoribosyltransferase, PRPP stimulated uptake of hypoxanthine into membrane vesicles. No PRPP-stimulated uptake of guanine was observed. For strain TR119, guanosine 5'-monophosphate and inosine 5'-monophosphate accumulated intravesicularly when guanine and hypoxanthine, respectively, were used with PRPP as transport substrates. For strain proAB47, IMP accumulated intravesicularly with hypoxanthine and PRPP as transport substrates. For strain TR119, hypoxanthine also accumulated when PRPP was absent. This free hypoxanthine uptake was completely inhibited by N-ethylmaleimide, but the PRPP-stimulated uptake of hypoxanthine was inhibited only 20% by N-ethylmaleimide. Hypoxanthine and guanine phosphoribosyltransferase activity paralleled uptake activity in both strains. But, when proAB47 vesicles were sonically treated to release the enzymes, a three- to sixfold activation of phosphoribosyltransferase molecules occurred. Since proAB47 vessicles lack the guanine phsophoribosyltransferase gene product and since hypoxanthine effectively competes out the phosphoribosylation of guanine by proAB47 vesicles, it was postulated that the hypoxanthine phosphoribosyltransferase gains specificity for both guanine and hypoxanthine when released from the membrane. A group translocation as the major mechanism for the uptake of guanine and hypoxanthine was proposed.

Jackman, L E; Hochstadt, J



Characterization of ouabain resistant, hypoxanthine phosphoribosyl transferase deficient human cells and their usefulness as a general method for the production of human cell hybrids  

Microsoft Academic Search

Ouabain resistant mutants of hypoxanthine phosphoribosyl transferase deficient HeLa cells and euploid human fibroblasts were isolated and extensively characterized. These double mutants were used to test the prediction that they would be “universal hybridizers” for intraspecific human cell hybrid production. Hybrids were selected from fusions with human cells of diverse somatic origin. In addition it was shown that multi-parental hybrids

B. Weissman; E. J. Stanbridge



Impairment of adenylyl cyclase 2 function and expression in hypoxanthine phosphoribosyltransferase-deficient rat B103 neuroblastoma cells as model for Lesch-Nyhan disease: BODIPY-forskolin as pharmacological tool.  


Hypoxanthine phosphoribosyl transferase (HPRT) deficiency results in Lesch-Nyhan disease (LND). The link between the HPRT defect and the self-injurious behavior in LND is still unknown. HPRT-deficient rat B103 neuroblastoma cells serve as a model system for LND. In B103 cell membranes, HPRT deficiency is associated with a decrease of basal and guanosine triphosphate-stimulated adenylyl cyclase (AC) activity (Pinto and Seifert, J Neurochem 96:454-459, 2006). Since recombinant AC2 possesses a high basal activity, we tested the hypothesis that AC2 function and expression is impaired in HPRT deficiency. We examined AC regulation in B103 cell membranes, cAMP accumulation in intact B103 cells, AC isoform expression, and performed morphological studies. As most important pharmacological tool, we used 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene forskolin (BODIPY-FS) that inhibits recombinant AC2 but activates ACs 1 and 5 (Erdorf et al., Biochem Pharmacol 82:1673-1681, 2011). In B103 control membranes, BODIPY-FS reduced catalysis, but in HPRT(-) membranes, BODIPY-FS was rather stimulatory. 2'(3')-O-(N-methylanthraniloyl) (MANT)-nucleoside 5'-[?-thio]triphosphates inhibit recombinant ACs 1 and 5 more potently than AC2. In B103 control membranes, MANT-guanosine 5'-[?-thio]triphosphate inhibited catalysis in control membranes less potently than in HPRT(-) membranes. Quantitative real-time PCR revealed that in HPRT deficiency, AC2 was virtually absent. In contrast, AC5 was up-regulated. Forskolin (FS) and BODIPY-FS induced cell clustering and rounding and neurite extension in B103 cells. The effects of FS and BODIPY-FS were much more prominent in control than in HPRT(-) cells, indicative for a differentiation defect in HPRT deficiency. Neither FS nor BODIPY-FS significantly changed cAMP concentrations in intact B103 cells. Collectively, our data show that HPRT deficiency in B103 cells is associated with impaired AC2 function and expression and reduced sensitivity to differentiation induced by FS and BODIPY-FS. We discuss the pathophysiological implications of our data for LND. PMID:22552731

Kinast, Liz; von der Ohe, Juliane; Burhenne, Heike; Seifert, Roland



Uptake and salvage of hypoxanthine mediates developmental arrest in preimplantation mouse embryos.  


Preimplantation mouse embryos become arrested after first or second cleavage when cultured in hypoxanthine-supplemented Whitten's medium. We present evidence that the hypoxanthine-induced arrest is dependent on uptake and salvage of hypoxanthine and depletion of phosphoribosylpyrophosphate (PRPP) levels. Hypoxanthine uptake increased during the 2-cell stage and was augmented by glucose. HPLC analysis of [14C]hypoxanthine metabolism revealed that hypoxanthine was salvaged and converted to ATP and guanosine triphosphate (GTP), with a shift to more guanyl nucleotide production at the 3- to 4-cell stage. In embryos from mice with a null mutation for the salvage enzyme hypoxanthine-guanine phosphoribosyltransferase, hypoxanthine did not block development nor was it taken up by the embryos. Glucose, which is required for the hypoxanthine-induced arrest, produced a 5.3-fold increase in PRPP levels at the 2-cell stage, which was eliminated by hypoxanthine. We conclude that metabolism of hypoxanthine to nucleotides mediates its inhibitory action on preimplantation mouse embryos via negative feedback on PRPP synthetase, ultimately resulting in decreased PRPP availability and arrest of other PRPP-dependent pathways. Finally, reversal of the block by EDTA and cAMP-elevating agents may be mediated by alterations in hypoxanthine or glucose uptake, or by changes in the relative metabolism of hypoxanthine. PMID:9002627

Dienhart, M K; O'Brien, M J; Downs, S M



Adenoviral-mediated transfer of Escherichia coli uracil phosphoribosyltransferase ( UPRT) gene to modulate the sensitivity of the human colon cancer cells to 5-fluorouracil  

Microsoft Academic Search

5-Fluorouracil (5-FU) has been used as a chemotherapeutic drug for colorectal cancer. Escherichia coli uracil phosphoribosyltransferase (UPRT), a pyrimidine salvage enzyme, converts 5-FU into 5-fluorouridine monophosphate (5-FUMP) at the initial step of 5-FU activation. We investigated the effects of adenoviral-mediated transfer of the E. coliUPRT gene into human colon cancer cells on 5-FU metabolism and 5-FU chemosensitivity. Three cell lines

F Koyama; H Sawada; H Fujii; H Hamada; T Hirao; M Ueno; H Nakano



Hypoxanthine-guanine phosophoribosyltransferase (HPRT) deficiency: Lesch-Nyhan syndrome  

Microsoft Academic Search

Deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity is an inborn error of purine metabolism associated with uric acid overproduction and a continuum spectrum of neurological manifestations depending on the degree of the enzymatic deficiency. The prevalence is estimated at 1\\/380,000 live births in Canada, and 1\\/235,000 live births in Spain. Uric acid overproduction is present inall HPRT-deficient patients and is associated

Rosa J Torres; Juan G Puig



Deletion Mutations in the hprt Gene of T-Lymphocytes as a Biomarker for Genomic Rearrangements Important in Human Cancers.  

National Technical Information Service (NTIS)

The DNA sequence of 11 in vivo-arising intragenic deletion junctions occurring in the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene of human T-lymphocytes was determined. These delections ranged in size from 16 bp to 4057 bp. Extensive homolo...

J. C. Fuscoe L. J. Zimmerman K. Harrington-Brock M. M. Moore



V(D)J Recombinase-like Activity Mediates hprt Gene Deletion in Human Fetal T-Lymphocytes1  

Microsoft Academic Search

Studies from several laboratories worldwide have developed a large database for in vivo hypoxanthine-guanine phosphoribosyltransferase gene mutations in human ï-lymphocytes. Sufficient differences have been found thus far between the spectrum for spontaneous mutations in adults and that observed in the fetus to suggest fundamental differences in in vivo mutagenic mechanisms at these two life stages. In adults, only -15% of

James C. Fuscoe; Lisa J. Zimmerman; Malcolm J. Lippert; Janice A. Nicklas; J. Patrick O'Neill; Richard J. Albertini


Complex hprt deletion events are recovered after exposure of human lymphoblastoid cells to high-LET carbon and neon ion beams  

Microsoft Academic Search

Hypoxanthine phosphoribosyltransferase gene (hprt) mutations were induced in human TK-6 lymphoblastoid cells by irradiation at a linear energy transfer (LET) of 250 or 310 keV\\/mm for carbon and neon ions, respectively. At such a high level of LET, ions will lose most of their total energy and stop shortly after passing through the cell. The hprt mutations were analyzed by

Yasuhiro Kagawa; Tsuneo Shimazu; Alasdair J. E. Gordon; Nobunao Fukunishi; Naohito Inabe; Masao Suzuki; Masahiko Hirano; Takesi Kato; Masami Watanabe; Fumio Hanaoka; Fumio Yatagai



Human T cell recognition of the blood stage antigen Plasmodium hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT) in acute malaria  

PubMed Central

Background The Plasmodium purine salvage enzyme, hypoxanthine guanine xanthine phosphoribosyl transferase (HGXPRT) can protect mice against Plasmodium yoelii pRBC challenge in a T cell-dependent manner and has, therefore, been proposed as a novel vaccine candidate. It is not known whether natural exposure to Plasmodium falciparum stimulates HGXPRT T cell reactivity in humans. Methods PBMC and plasma collected from malaria-exposed Indonesians during infection and 7–28 days after anti-malarial therapy, were assessed for HGXPRT recognition using CFSE proliferation, IFN? ELISPOT assay and ELISA. Results HGXPRT-specific T cell proliferation was found in 44% of patients during acute infection; in 80% of responders both CD4+ and CD8+ T cell subsets proliferated. Antigen-specific T cell proliferation was largely lost within 28 days of parasite clearance. HGXPRT-specific IFN-? production was more frequent 28 days after treatment than during acute infection. HGXPRT-specific plasma IgG was undetectable even in individuals exposed to malaria for at least two years. Conclusion The prevalence of acute proliferative and convalescent IFN? responses to HGXPRT demonstrates cellular immunogenicity in humans. Further studies to determine minimal HGXPRT epitopes, the specificity of responses for Plasmodia and associations with protection are required. Frequent and robust T cell proliferation, high sequence conservation among Plasmodium species and absent IgG responses distinguish HGXPRT from other malaria antigens.

Woodberry, Tonia; Pinzon-Charry, Alberto; Piera, Kim A; Panpisutchai, Yawalak; Engwerda, Christian R; Doolan, Denise L; Salwati, Ervi; Kenangalem, Enny; Tjitra, Emiliana; Price, Ric N; Good, Michael F; Anstey, Nicholas M



Chromosome-mediated transfer of murine alleles for hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and ouabain resistance into human cell lines  

Microsoft Academic Search

Genetic drug-resistance markers were transferred via purified metaphase chromosomes from mouse L cells into the human fibrosarcoma line HT1080 and HeLa S3 cells. Interspecific chromosome-mediated transfer of hypoxanthine-guanine phosphoribosyl transferase (HGPRT; EC from mouse L cells into HGPRT- HT1080 cells occurred at a frequency of approximately 1×10-7. The presence of the mouse allele for HGPRT in transferent isolates was

Tracy Gross Lugo; Raymond M. Baker



The Mutational Spectrum of the HPRT Gene from Human T Cells in Vivo Shares a Significant Concordant Set of Hot Spots with MNNG-treated Human Cells1  

Microsoft Academic Search

The preponderance of G:C to A:T transitions in inherited and somatic human mutations has led to the hypothesis that some of these mutations arise as a result of formation of O6-methylguanine in DNA. To test this hypothesis, the fine structure map of N-methyl-N'-nitro-N-nitrosoguani- dine (MNNG)-induced mutations was determined in human lymphoblas- toid cells in the human hypoxanthine-guanine-phosphoribosyltransferase (HPRT) gene and

Aoy Tomita-Mitchell; Losee Lucy Ling; Curtis L. Glover; Jacklene Goodluck-Griffith; William G. Thilly



High rate of mutation reporter gene inactivation during human T cell proliferation  

Microsoft Academic Search

Caspase activation and degradation of deoxyribonucleic acid (DNA) damage response factors occur during in vitro T-cell proliferation,\\u000a and an increased frequency of hypoxanthine-guanine phosphoribosyltransferase (HPRT)-negative variants have been reported in\\u000a conditions associated with in vivo T-cell proliferation. We have applied two human somatic cell mutation reporter assays,\\u000a for the HPRT and phosphatidylinositol glycan class A (PIG-A) genes, to human T

Aida Gabdoulkhakova; Gunnel Henriksson; Nadezhda Avkhacheva; Alexander Sofin; Anders Bredberg



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

Microsoft Academic Search

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

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



Isolation of the Human Chromosomal Band Xq28 Within Somatic Cell Hybrids by Fragile X Site Breakage  

Microsoft Academic Search

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

Stephen T. Warren; Samantha J. L. Knight; Jeanne F. Peters; Carol L. Stayton; G. Giacomo Consalez; Fuping Zhang



Identification of amides derived from 1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid as potent inhibitors of human nicotinamide phosphoribosyltransferase (NAMPT).  


Potent, 1H-pyrazolo[3,4-b]pyridine-containing inhibitors of the human nicotinamide phosphoribosyltransferase (NAMPT) enzyme were identified using structure-based design techniques. Many of these compounds exhibited nanomolar antiproliferation activities against human tumor lines in in vitro cell culture experiments, and a representative example (compound 26) demonstrated encouraging in vivo efficacy in a mouse xenograft tumor model derived from the A2780 cell line. This molecule also exhibited reduced rat retinal exposures relative to a previously studied imidazo-pyridine-containing NAMPT inhibitor. Somewhat surprisingly, compound 26 was only weakly active in vitro against mouse and monkey tumor cell lines even though it was a potent inhibitor of NAMPT enzymes derived from these species. The compound also exhibited only minimal effects on in vivo NAD levels in mice, and these changes were considerably less profound than those produced by an imidazo-pyridine-containing NAMPT inhibitor. The crystal structures of compound 26 and the corresponding PRPP-derived ribose adduct in complex with NAMPT were also obtained. PMID:24021463

Zheng, Xiaozhang; Bair, Kenneth W; Bauer, Paul; Baumeister, Timm; Bowman, Krista K; Buckmelter, Alexandre J; Caligiuri, Maureen; Clodfelter, Karl H; Feng, Yezhen; Han, Bingsong; Ho, Yen-Ching; Kley, Nikolai; Li, Hong; Liang, Xiaorong; Liederer, Bianca M; Lin, Jian; Ly, Justin; O'Brien, Thomas; Oeh, Jason; Oh, Angela; Reynolds, Dominic J; Sampath, Deepak; Sharma, Geeta; Skelton, Nicholas; Smith, Chase C; Tremayne, Jarrod; Wang, Leslie; Wang, Weiru; Wang, Zhongguo; Wu, Hongxing; Wu, Jiansheng; Xiao, Yang; Yang, Guangxing; Yuen, Po-Wai; Zak, Mark; Dragovich, Peter S



Adenine phosphoribosyltransferase deficiency.  


Complete adenine phosphoribosyltransferase (APRT) deficiency is a rare inherited metabolic disorder that leads to the formation and hyperexcretion of 2,8-dihydroxyadenine (DHA) into urine. The low solubility of DHA results in precipitation of this compound and the formation of urinary crystals and stones. The disease can present as recurrent urolithiasis or nephropathy secondary to crystal precipitation into renal parenchyma (DHA nephropathy). The diagnostic tools available-including stone analysis, crystalluria, and APRT activity measurement-make the diagnosis easy to confirm when APRT deficiency is suspected. However, the disease can present at any age, and the variability of symptoms can present a diagnostic challenge to many physicians. The early recognition and treatment of APRT deficiency are of crucial importance for preventing irreversible loss of renal function, which still occurs in a non-negligible proportion of cases. This review summarizes the genetic and metabolic mechanisms underlying stone formation and renal disease, along with the diagnosis and management of APRT deficiency. PMID:22700886

Bollée, Guillaume; Harambat, Jérôme; Bensman, Albert; Knebelmann, Bertrand; Daudon, Michel; Ceballos-Picot, Irène



Yeast artificial chromosomes spanning 8 megabases and 10-15 centimorgans of human cytogenetic band Xq26  

Microsoft Academic Search

A successful test is reported to generate long range contiguous coverage of DNA from a human cytogenetic band in overlapping yeast artificial chromosomes (YACs). Seed YACs in band Xq26 were recovered from a targeted library of clones from Xq24-q28 with 14 probes, including probes for the hypoxanthine guanine phosphoribosyltransferase- and coagulation factor IX-encoding genes and nine probes used in linkage

R. D. Little; G. Pilia; S. Johnson; D. Schlessinger; M. DUrso



Fibroblast growth factor-dependent metabolism of hypoxanthine via the salvage pathway for purine synthesis in porcine aortic endothelial cells.  


In this study we examined the metabolism of hypoxanthine in fibroblast growth factor (FGF)-stimulated porcine aortic endothelial cells (PAEC). Our previous report indicated that hypoxanthine in fetal bovine serum (FBS) was an essential component for both basal and FGF-dependent growth of PAEC (Hayashi et al., Exp Cell Res 185: 217-228, 1989). Besides hypoxanthine, the addition of various purine bases and purine nucleosides, but not xanthine, xanthosine or any pyrimidine metabolites, restored the limited growth of PAEC cultured in medium containing 10% dialyzed FBS in the presence or absence of FGF. The metabolism of [14C]hypoxanthine was compared in PAEC treated with and without FGF. Treatment of PAEC with FGF for 24 hr enhanced the radioactivity incorporation from [14C]hypoxanthine into both the acid-soluble and -insoluble fractions approximately 2-fold. Upon chromatographic analyses of hypoxanthine metabolites in the acid-soluble nucleotide fraction, it was found that in control PAEC hypoxanthine was largely metabolized to IMP, adenine nucleotides and uric acid, whereas in FGF-treated cells it was converted to ATP, ADP, GTP, xanthine and uric acid. The radioactivity of IMP was lowered in FGF-stimulated cells. The addition of FGF to PAEC increased phosphoribosyl pyrophosphate (PRPP) synthetase activity by approximately 8-fold and the PRPP content by approximately 2-fold, but it did not increase hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity or hypoxanthine transport. On the other hand, methotrexate, an inhibitor of de novo synthesis of purine, did not affect the growth of PAEC. Analyses of the rate of [14C]formate incorporation into total purine compounds showed that PAEC had a low capacity to synthesize purines de novo, which was not stimulated by FGF. These data indicate that FGF stimulates the synthesis of PRPP necessary for the salvage synthesis of purine nucleotides in conjunction with purine bases, e.g. hypoxanthine. PMID:7683470

Hirai, S; Hayashi, Y; Koizumi, T; Nakanishi, N; Fukui, T; Ichikawa, A



Hypoxanthine-guanine phosophoribosyltransferase (HPRT) deficiency: Lesch-Nyhan syndrome.  


Deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity is an inborn error of purine metabolism associated with uric acid overproduction and a continuum spectrum of neurological manifestations depending on the degree of the enzymatic deficiency. The prevalence is estimated at 1/380,000 live births in Canada, and 1/235,000 live births in Spain. Uric acid overproduction is present inall HPRT-deficient patients and is associated with lithiasis and gout. Neurological manifestations include severe action dystonia, choreoathetosis, ballismus, cognitive and attention deficit, and self-injurious behaviour. The most severe forms are known as Lesch-Nyhan syndrome (patients are normal at birth and diagnosis can be accomplished when psychomotor delay becomes apparent). Partial HPRT-deficient patients present these symptoms with a different intensity, and in the least severe forms symptoms may be unapparent. Megaloblastic anaemia is also associated with the disease. Inheritance of HPRT deficiency is X-linked recessive, thus males are generally affected and heterozygous female are carriers (usually asymptomatic). Human HPRT is encoded by a single structural gene on the long arm of the X chromosome at Xq26. To date, more than 300 disease-associated mutations in the HPRT1 gene have been identified. The diagnosis is based on clinical and biochemical findings (hyperuricemia and hyperuricosuria associated with psychomotor delay), and enzymatic (HPRT activity determination in haemolysate, intact erythrocytes or fibroblasts) and molecular tests. Molecular diagnosis allows faster and more accurate carrier and prenatal diagnosis. Prenatal diagnosis can be performed with amniotic cells obtained by amniocentesis at about 15-18 weeks' gestation, or chorionic villus cells obtained at about 10-12 weeks' gestation. Uric acid overproduction can be managed by allopurinol treatment. Doses must be carefully adjusted to avoid xanthine lithiasis. The lack of precise understanding of the neurological dysfunction has precluded development of useful therapies. Spasticity, when present, and dystonia can be managed with benzodiazepines and gamma-aminobutyric acid inhibitors such as baclofen. Physical rehabilitation, including management of dysarthria and dysphagia, special devices to enable hand control, appropriate walking aids, and a programme of posture management to prevent deformities are recommended. Self-injurious behaviour must be managed by a combination of physical restraints, behavioural and pharmaceutical treatments. PMID:18067674

Torres, Rosa J; Puig, Juan G



Hypoxanthine-guanine phosophoribosyltransferase (HPRT) deficiency: Lesch-Nyhan syndrome  

PubMed Central

Deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity is an inborn error of purine metabolism associated with uric acid overproduction and a continuum spectrum of neurological manifestations depending on the degree of the enzymatic deficiency. The prevalence is estimated at 1/380,000 live births in Canada, and 1/235,000 live births in Spain. Uric acid overproduction is present inall HPRT-deficient patients and is associated with lithiasis and gout. Neurological manifestations include severe action dystonia, choreoathetosis, ballismus, cognitive and attention deficit, and self-injurious behaviour. The most severe forms are known as Lesch-Nyhan syndrome (patients are normal at birth and diagnosis can be accomplished when psychomotor delay becomes apparent). Partial HPRT-deficient patients present these symptoms with a different intensity, and in the least severe forms symptoms may be unapparent. Megaloblastic anaemia is also associated with the disease. Inheritance of HPRT deficiency is X-linked recessive, thus males are generally affected and heterozygous female are carriers (usually asymptomatic). Human HPRT is encoded by a single structural gene on the long arm of the X chromosome at Xq26. To date, more than 300 disease-associated mutations in the HPRT1 gene have been identified. The diagnosis is based on clinical and biochemical findings (hyperuricemia and hyperuricosuria associated with psychomotor delay), and enzymatic (HPRT activity determination in haemolysate, intact erythrocytes or fibroblasts) and molecular tests. Molecular diagnosis allows faster and more accurate carrier and prenatal diagnosis. Prenatal diagnosis can be performed with amniotic cells obtained by amniocentesis at about 15–18 weeks' gestation, or chorionic villus cells obtained at about 10–12 weeks' gestation. Uric acid overproduction can be managed by allopurinol treatment. Doses must be carefully adjusted to avoid xanthine lithiasis. The lack of precise understanding of the neurological dysfunction has precluded development of useful therapies. Spasticity, when present, and dystonia can be managed with benzodiazepines and gamma-aminobutyric acid inhibitors such as baclofen. Physical rehabilitation, including management of dysarthria and dysphagia, special devices to enable hand control, appropriate walking aids, and a programme of posture management to prevent deformities are recommended. Self-injurious behaviour must be managed by a combination of physical restraints, behavioural and pharmaceutical treatments.

Torres, Rosa J; Puig, Juan G



Extracellular nicotinamide phosphoribosyltransferase (NAMPT/visfatin) inhibits insulin-like growth factor-1 signaling and proteoglycan synthesis in human articular chondrocytes  

PubMed Central

Introduction Obesity is one of the major risk factors for the development of osteoarthritis (OA). Although the mechanical factors appear to be critical, recent studies have suggested a role for adipokines in cartilage degradation. Chondrocytes from osteoarthritic cartilage respond poorly to insulin-like growth factor-1 (IGF-1) and the molecular mechanism(s) involved is not clearly understood. The purpose of the present study was to determine the role of extracellular nicotinamide phosphoribosyltransferase (eNAMPT/visfatin), a newly described adipokine, in regulating IGF-1 function in chondrocytes. Methods Human articular chondrocytes isolated from normal ankle cartilage were pretreated with eNAMPT (0.1 to 5.0 ?g/ml) overnight followed by stimulation with IGF-1 (50 ng/ml) for 24 hours, and proteoglycan synthesis was measured by [35S]sulfate incorporation. Chondrocytes were pretreated with eNAMPT overnight followed by IGF-1 for 10 minutes, and the cell lysates were immunoblotted for various signaling proteins that are activated by IGF-1 using phosphospecific antibodies. In addition, chondrocytes were pretreated with mitogen-activated protein kinase kinase inhibitor (U0126) prior to stimulation with eNAMPT and IGF-1. Results Pretreatment of chondrocytes with eNAMPT inhibited IGF-1-stimulated proteoglycan synthesis in a dose-dependent manner. Treatment of chondrocytes with eNAMPT inhibited IGF-1-induced phosphorylation of signaling molecules, including insulin receptor substrate-1 and AKT. Interestingly, pretreatment of chondrocytes with eNAMPT did not inhibit IGF-1-mediated phosphorylation of the IGF-1 receptor; however, it stimulated a sustained phosphorylation of the extracellular signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) signaling pathway. Inhibition of the ERK/MAPK signaling pathway restored IGF-1-mediated insulin receptor substrate-1 and AKT phosphorylation. Conclusions Our study demonstrates that eNAMPT/visfatin inhibits IGF-1 function in articular chondrocytes by activating the ERK/MAPK pathway independent of the IGF-1 receptor. Since eNAMPT levels are elevated in the synovial fluid of OA patients, the signaling pathway activated by eNAMPT could contribute to IGF-1 resistance in OA.



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

SciTech Connect

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.

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




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...


Stable association of the human transgenome and host murine chromosomes demonstrated with trispecific microcell hybrids.  

PubMed Central

Trispecific microcell hybrids were prepared by transferring limited numbers of chromosomes from a human/mouse gene-transfer cell line to a Chinese hamster recipient line. The donor cells employed were murine L-cells that stably expressed the human form of the enzyme hypoxanthine phosphoribosyltransferase. Karyotypic, zymographic, and back-selection tests of the resulting human/mouse/Chinese hamster microcell hybrids provided strong genetic evidence for a stable association of the human transgenome with host murine chromosomes in stable gene-transfer cell lines. This association, which may represent physical integration of the transgenome into the host cell genome, occurred at multiple chromosomal sites. Images

Fournier, R E; Ruddle, F H



Antigen-specific human T-cell hybridomas with helper activity.  

PubMed Central

Human T cell hybridomas were produced by fusing the hypoxanthine phosphoribosyltransferase-deficient line of the human T cell lymphoma Jurkat with a continuous line of normal human T cells specific for tetanus toxoid (TeT). The hybridomas were selected for their ability to produce interleukin 2 after exposure to TeT on semiautologous monocytes and for their ability to bind to TeT-pulsed semiautologous monocytes. These antigen-specific T hybridomas demonstrated potent helper activity for semiautologous B cells as determined by the production of high levels of anti-TeT antibody in vitro.

DeFreitas, E C; Vella, S; Linnenbach, A; Zmijewski, C; Koprowski, H; Croce, C M



The depletion of cellular ATP by AG2034 mediates cell death or cytostasis in a hypoxanthine-dependent manner in human prostate cancer cells  

Microsoft Academic Search

Purpose  4-[2-(2-Amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4] thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-l-glutamic acid (AG2034), is a classical antifolate, an analog of folic acid that has been shown to be an excellent inhibitor\\u000a of glycinamide ribonucleotide formyltransferase (GARFT), ultimately inhibiting the de novo synthesis of purines. We examined\\u000a the effect of this drug on cell proliferation, steady-state ATP levels, de novo and hypoxanthine salvage ATP synthesis, and\\u000a on the phosphorylation of

Oluwakemi Obajimi; Peter W. Melera



Pyrophosphate Activation in Hypoxanthine-Guanine Phosphoribosyltransferase with Transition State Analogue †  

PubMed Central

Isotope-edited difference Raman and FTIR studies complemented by ab initio calculations have been applied to the transition state analogue complex of HGPRT?ImmHP?MgPPi to determine the ionic states of the 5’-phosphate moiety of ImmHP and of PPi. These measurements characterize electrostatic interactions within the enzyme active site as deduced from frequency shifts of the phosphate groups. The bound 5’-phosphate moiety of ImmHP is di-anionic, and this phosphate group exists in two different conformations within the protein complex. In one conformation, a hydrogen bond between the 5’-phosphate of ImmHP and the OH group of Tyr104 in the catalytic loop appears to be stronger. With the stronger H-bond, the OH of Tyr104 approaches one of the P••O bonds from the bridging oxygen side to cause distortion of the PO3 moiety, as indicated by a lowered symmetric P••O stretch frequency. The asymmetric stretch frequencies are similar in both phosphate conformations. Bound PPi in this complex is fully ionized to P2O74?. Bond frequency changes for bound PPi indicate coordination to Mg2+ ions but show no indication of significant P••O bond polarization. Extrapolation of these results to reaction coordinate motion for HGPRT suggests that bond formation between C1’ of the nucleotide ribose and the oxygen of PPi is accomplished by migration of the ribocation toward immobilized pyrophosphate.

Deng, Hua; Callender, Robert; Schramm, Vern L.; Grubmeyer, Charles



Pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), an enzyme essential for NAD+ biosynthesis, in human cancer cells: metabolic basis and potential clinical implications.  


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

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



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

SciTech Connect

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.

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



Complementation of a DNA repair defect in xeroderma pigmentosum cells by transfer of human chromosome 9  

SciTech Connect

Complementation of the repair defect in xeroderma pigmentosum cells of complementation group A was achieved by the transfer of human chromosome 9. A set of mouse-human hybrid cell lines, each containing a single Ecogpt-marked human chromosome, was used as a source of donor chromosomes. Chromosome transfer to XPTG-1 cells, a hypoxanthine/guanine phosphoribosyltransferase-deficient mutant of simian virus 40-transformed complementation group A cells, was achieved by microcell fusion and selection for Ecogpt. Chromosome-transfer clones of XPTG-1 cells, each containing a different human donor chromosome, were analyzed for complementation of sensitivity to UV irradiation. Among all the clones, increased levels of resistance to UV was observed only in clones containing chromosome 9. Since our recipient cell line XPTG-1 is hypoxanthine/guanine phosphoribosyltransferase deficient, cultivation of Ecogpt+ clones in medium containing 6-thioguanine permits selection of cells for loss of the marker and, by inference, transferred chromosome 9. Clones isolated for growth in 6-thioguanine, which have lost the Ecogpt-marked chromosome, exhibited a UV-sensitive phenotype, confirming the presence of the repair gene(s) for complementation group A on chromosome 9.

Kaur, G.P.; Athwal, R.S. (Univ. of Medicine and Dentistry of New Jersey, Newark (USA))



Crystal structures of adenine phosphoribosyltransferase from Leishmania donovani.  


The enzyme adenine phosphoribosyltransferase (APRT) functions to salvage adenine by converting it to adenosine-5-monophosphate (AMP). APRT deficiency in humans is a well characterized inborn error of metabolism, and APRT may contribute to the indispensable nutritional role of purine salvage in protozoan parasites, all of which lack de novo purine biosynthesis. We determined crystal structures for APRT from Leishmania donovani in complex with the substrate adenine, the product AMP, and sulfate and citrate ions that appear to mimic the binding of phosphate moieties. Overall, these structures are very similar to each other, although the adenine and AMP complexes show different patterns of hydrogen-bonding to the base, and the active site pocket opens slightly to accommodate the larger AMP ligand. Whereas AMP adopts a single conformation, adenine binds in two mutually exclusive orientations: one orientation providing adenine-specific hydrogen bonds and the other apparently positioning adenine for the enzymatic reaction. The core of APRT is similar to that of other phosphoribosyltransferases, although the adenine-binding domain is quite different. A C-terminal extension, unique to Leishmania APRTs, extends an extensive dimer interface by wrapping around the partner molecule. The active site involves residues from both subunits of the dimer, indicating that dimerization is essential for catalysis. PMID:10393170

Phillips, C L; Ullman, B; Brennan, R G; Hill, C P



Effect of hypoxanthine, antioxidants and allopurinol on cholinesterase activities in rats.  


In the present study, we investigate the in vitro effect of hypoxanthine on acetylcholinesterase and butyrylcholinesterase activities in the hippocampus, striatum, cerebral cortex and serum of 15-, 30- and 60-day-old rats. Furthermore, we also evaluated the influence of antioxidants, namely ?-tocopherol (trolox) and ascorbic acid, and allopurinol to investigate the possible participation of free radicals and uric acid in the effects elicited by hypoxanthine on these parameters. Acetylcholinesterase and butyrylcholinesterase activities were determined according to Ellman et al. (Biochem Pharmacol 7:88-95, 1961), with some modifications. Hypoxanthine (10.0 ?M), when added to the incubation medium, enhanced acetylcholinesterase activity in the hippocampus and striatum of 15- and 30-day-old rats and reduced butyrylcholinesterase activity in the serum of 60-day-old rats. The administration of allopurinol and/or antioxidants partially prevented the alterations caused by hypoxanthine in acetylcholinesterase and butyrylcholinesterase activities in the cerebrum and serum of rats. Data indicate that hypoxanthine alters cholinesterase activities, probably through free radicals and uric acid production since the alterations were prevented by the administration of allopurinol and antioxidants. It is presumed that the cholinesterase system may be associated, at least in part, with the neuronal dysfunction observed in patients affected by Lesch-Nyhan disease. In addition, although extrapolation of findings from animal experiments to humans is difficult, it is conceivable that these vitamins and allopurinol might serve as an adjuvant therapy to avoid progression of brain damage in patients affected by this disease. PMID:23400363

Wamser, Morgahna Nathalie; Leite, Eduardo Fernandes; Ferreira, Vinícius Vialle; Delwing-de Lima, Daniela; da Cruz, José Geraldo Pereira; Wyse, Angela T S; Delwing-Dal Magro, Débora



High level glucose increases mutagenesis in human lymphoblastoid cells  

Microsoft Academic Search

Epidemiological data have suggested an increased cancer rates in diabetic patients, for which the underlying mechanism is poorly understood. We studied whether high level of glucose (HG) treatment that mimic the hyperglycemic condition in diabetes mellitus is mutagenic. Mutagenesis studies were carried out at both hypoxanthine phosphoribosyltransferase (hprt) and thymidine kinase (tk) loci. Role of p53 in HG-induced mutagenesis was

Ying Zhang; Junqing Zhou; Tieli Wang; Lu Cai


Yeast artificial chromosomes spanning 8 megabases and 10-15 centimorgans of human cytogenetic band Xq26  

SciTech Connect

A successful test is reported to generate long range contiguous coverage of DNA from a human cytogenetic band in overlapping yeast artificial chromosomes (YACs). Seed YACs in band Xq26 were recovered from a targeted library of clones from Xq24-q28 with 14 probes, including probes for the hypoxanthine guanine phosphoribosyltransferase- and coagulation factor IX-encoding genes and nine probes used in linkage mapping. Neighboring YACs were then identified by 25 walking' steps with end-clones, and the content of 71 probes in cognate YACs was verified by further hybridization analyses. The resultant contig extends across 8 million base pairs, including most of band Xq26, with an order of markers consistent with linkage data. YAC-based mapping, thus, permits steps toward a fully integrated physical and genetic map and is probably adequate to sustain most of the human genome project.

Little, R.D.; Pilia, G.; Johnson, S.; Schlessinger, D. (Washington Univ., St. Louis, MO (United States)); D'Urso, M. (International Inst. of Genetics and Biophysics, Naples (Italy))



Targeted disruption of the mouse adenine phosphoribosyltransferase (aprt) gene and the production of APRT-deficient mice  

Microsoft Academic Search

Adenine phosphoribosyltransferase (APRT: EC, a ubiquitously expressed purine salvage enzyme, catalyzes the synthesis of AMP and inorganic pyrophosphate from existing adenine and 5-phosphoribosyl-1-pyrophosphate. Deficiency of this enzyme in humans results in the accumulation of 2,8-dihydroxyadenine leading to crystalluria and nephrolithiasis. In order to facilitate our study of this rare, autosomal recessive disorder, we applied the advances in gene targeting

S. J. Engle; J. Chen; J. A. Tischfield



Purine metabolism of human glioblastoma in vivo.  


The aim of this study was to identify targets for rational chemotherapy of glioblastoma. In order to elucidate differences in the biochemistry of tumor and normal human brain, in vivo pool sizes of purine nucleotides, nucleosides, and nucleobases and of purine metabolizing enzymes in biopsy material from 14 grade IV astrocytomas and 4 normal temporal lobe samples were analyzed. Specimens were collected during surgery using the freeze-clamp sampling technique and analyzed by high pressure liquid chromatography. Total purine nucleotides, adenylates, and guanylates in the tumors were 2186, 1865, and 310 nmol/g (wet weight), respectively, which corresponds to 61, 60, and 71% of normal brain tissue concentrations. Relative to normal brain the tumors had significantly lower ATP and GTP levels, essentially normal pool sizes of purine nucleosides and bases, unchanged activities of the salvage enzymes hypoxanthine-guanine phosphoribosyltransferase, adenine phosphoribosyltransferase, and adenosine kinase (659, 456, and 98 nmol/h/mg protein, respectively) and 4-fold higher activities of IMP dehydrogenase (11.6 nmol/h/mg protein); the latter is the rate limiting enzyme for guanylate de novo synthesis. IMP pools in the tumors were 64% of values in normal brain. Modulation of the guanylate pathway in glioblastoma by inhibition of IMP dehydrogenase with tumor specific agents such as tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) appears to be a rational therapeutic approach. Preliminary in vitro experiments with normal and malignant tissue specimens from 2 additional patients revealed that significant amounts of the active metabolite thiazole-4-carboxamide adenine dinucleotide are formed from tiazofurin. At a concentration of 200 microM this drug was able to deplete guanylate pools in the tumors to a median of 54% of phosphate buffered saline treated controls. Flux studies with [14C]formate showed that tiazofurin strongly inhibited de novo synthesis of guanylates in glioblastoma to an average of 10% of controls. This effect was more pronounced in the tumors as compared to normal brain. No inhibition of salvage of [14C]guanine by tiazofurin could be observed in normal and malignant tissues. Supportive measures have to be considered to inhibit the highly active salvage enzyme hypoxanthine-guanine phosphoribosyltransferase that can partly antagonize a tiazofurin induced decrease in guanine nucleotides. PMID:2154328

Pillwein, K; Chiba, P; Knoflach, A; Czermak, B; Schuchter, K; Gersdorf, E; Ausserer, B; Murr, C; Goebl, R; Stockhammer, G



Purification of IMP:Pyrophosphate Phosphoribosyltransferases, Catalytically Incompetent Enzymes in Lesch-Nyhan Disease  

PubMed Central

IMP:pyrophosphate phosphoribosyltransferase (IPPase) (EC has been purified over 7000-fold from human erythrocytes. The purified enzyme moved as a single band on disc electrophoresis. Antisera prepared in rabbits and rats against the purified enzyme precipitated and neutralized the enzyme, but had no effect on AMP-pyrophosphate phosphoribosyltransferase (EC activity. Evidence was found for isozymes (enzyme variants) of IPPase in normal erythrocytes. Erythrocyte lysates of five patients with Lesch-Nyhan disease reacted with antisera against normal IPPase. Lysates from LN erythrocytes blocked the inactivation of normal enzyme by the antibody. LN erythrocytes had about the same concentration of enzyme protein as normal erythrocytes. The genetic defect in LN results in the production of essentially normal amounts of an immunologically identifiable but catalytically incompetent enzyme. Thus LN is apparently the result of a mutation in a structural gene and is not due to deletion of a structural gene or defect in a regulatory gene.

Rubin, Charles S.; Dancis, Joseph; Yip, Lily C.; Nowinski, Robert C.; Balis, M. Earl



Fibre-optic biosensor for hypoxanthine and xanthine based on a chemiluminescence reaction.  


Fibre-optic biosensors were constructed for determination of hypoxanthine and xanthine. Xanthine oxidase and peroxidase were immobilized on different preactivated membranes which were subsequently mounted onto the tip of a fibre-optic bundle. The H2O2 generated by the reaction of hypoxanthine and xanthine oxidase was measured by chemiluminescence (CL) detection using luminol and peroxidase. A linear calibration curve of the sensors in the range of 1-316 microM hypoxanthine and 3.1-316 microM xanthine, respectively, with a detection limit of 0.55 microM hypoxanthine was obtained. Recovery of hypoxanthine ranged between 91 and 102%. PMID:8060588

Hlavay, J; Haemmerli, S D; Guilbault, G G



Active Site Contacts in the Purine Nucleoside Phosphorylase?Hypoxanthine Complex by NMR and ab Initio Calculations †  

Microsoft Academic Search

Hypoxanthine (Hx) with specific 15N labels has been used to probe hydrogen-bonding interactions with purine nucleoside phosphorylase (PNP) by NMR spectroscopy. Hx binds to human PNP as the N-7H tautomer, and the N-7H 1H and 15N chemical shifts are located at 13.9 and 156.5 ppm, respectively, similar to the solution values. In contrast, the 1H and 15N chemical shifts of

Hua Deng; Sean M. Cahill; Jose ´-Luis Abad; Andrzej Lewandowicz; Robert H. Callender; Vern L. Schramm; Roger A. Jones



Hypoxanthine-guanine phosphoribosylotransferase deficiency--the spectrum of Polish mutations.  


Hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC deficiency (OMIM 308000) is an inborn error of purine metabolism. The defect causes three overlapping clinical syndromes: Lesch-Nyhan disease (LND; OMIM 300322), HPRT-related hyperuricaemia with neurologic dysfunction (HRND) and hyperuricaemia alone (HRH; OMIM 300322). During the period 1977-2007, 18 patients belonging to 12 Polish families and one Latvian family with HPRT deficiency have been identified. The majority of patients had a typical LND phenotype, three patients were classified as HRH and one patient as an intermediate phenotype (HRND). Genetic analysis revealed 12 different HPRT1 mutations, five of them being unique. In two typical Lesch-Nyhan families a novel single-base substitution, c.220T>G (p.Phe74Val), and a deletion of seven nucleotides, c.395_401del7 (p.Ile132LysfsX3), were found. Another novel single-base substitution, c.295T>G (p.Phe99Val), was identified in a patient with severe partial deficiency of HPRT with neurological dysfunction. In patients belonging to the HRH group, two transitions were detected: c.481G>A (p.Ala161Thr) and c.526C>T (p.Pro176Ser). Other mutations identified in Polish patients, c.131A>G (p.Asp44Gly), c.222C>A (p.Phe74Leu), c.385-1G>A (p.Asn129_Glu134del), c.482C>A (p.Ala161Glu), c.508C>T (p.Arg170Ter) and c.569G>A (p.Gly190Glu), have been reported previously in unrelated patients and are located within one of the clusters of hot spots of the HPRT1 gene (exons 3, 7 and 8). Patients with partial phenotypes presented mutations predicted to permit some degree of residual enzyme function (single-base substitutions). All mutations, except c.508C>T (p.Arg170Ter), were found in single families only, indicating the lack of any common mutation causing HPRT deficiency in Poland. PMID:19016344

Jurecka, A; Popowska, E; Tylki-Szymanska, A; Kubalska, J; Ciara, E; Krumina, Z; Sykut-Cegielska, J; Pronicka, E



Low-potential amperometric enzyme biosensor for xanthine and hypoxanthine.  


The bacterial xanthine dehydrogenase (XDH) from Rhodobacter capsulatus was immobilized on an edge-plane pyrolytic graphite (EPG) electrode to construct a hypoxanthine/xanthine biosensor that functions at physiological pH. Phenazine methosulfate (PMS) was used as a mediator which acts as an artificial electron-transfer partner for XDH. The enzyme catalyzes the oxidation of hypoxanthine to xanthine and also xanthine to uric acid by an oxidative hydroxylation mechanism. The present electrochemical biosensor was optimized in terms of applied potential and pH. The electrocatalytic oxidation response showed a linear dependence on the xanthine concentration ranging from 1.0 × 10(-5) to 1.8 × 10(-3) M with a correlation coefficient of 0.994. The modified electrode shows a very low detection limit for xanthine of 0.25 nM (signal-to-noise ratio = 3) using controlled potential amperometry. PMID:23134312

Kalimuthu, Palraj; Leimkühler, Silke; Bernhardt, Paul V



The kinetics of hypoxanthine efflux from the rat brain.  


The brain efflux of radiolabelled hypoxanthine in the rat was rapid in the first minute after injection [K(eff)(i)=0.21+/-0.06 min(-1)], which was saturable with a V(max)=13.08+/-0.81 nM min(-1) g(-1), and a high K(m,app) (67.2+/-13.4 microM); the K(i,app) for inosine was 31.5+/-7.6 microM. Capillary depletion analysis indicated that hypoxanthine accumulates in neurons and glia with the time. From cross-inhibition studies with different purines and pyrimidines, it suggests that these molecules could also be important substrates for this carrier. PMID:11311886

Redzic, Z B; Isakovic, A; Segal, M B; Thomas, S A; Rakic, L M



A semiempirical study of the prototropic tautomerism of hypoxanthine  

Microsoft Academic Search

The total and relative energies, bond order matrices and localized MOs for the eight possible tautomers of hypoxanthine (HYP) have been calculated, with full geometry optimization, using both AM1 and MNDO methods. The AM1 relative energies show that HYP(9,1), HYP(7,1) and HYP (9,10) are the predominant species at room temperature, the two former being in larger concentration that the latter.

J. Guillermo Contreras; Joel B. Alderete



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

SciTech Connect

The authors 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 them 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 thee cells; the other element, located in intron 1, acts as an enhancer of HPRT expression. Using this information, they 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.

Reid, L.H.; Smithies, O.; Koller, B.H. (Univ. of North Carolina, Chapel Hill (USA)); Gregg, R.G. (Univ. of Wisconsin, Madison (USA))



Isolation and properties of crystalline quinolinate phosphoribosyltransferase from hog kidney.  


Crystalline quinolinate phosphoribosyltransferase (nicotinatenucleotide: pyrophosphate phosphoribosyltransferase (carboxylating), EC was isolated from hog kidney and compared with the same enzyme prepared from hog liver. The enzyme preparation was homogeneous as shown by polyacrylamide gel electrophoresis and ultracentrifugation analysis. The enzyme had a molecular weight of 220 000 and the subunit 35 000. The physicochemical properties of the enzyme were: sedimentation coefficient (S200,W), 7.75 . 10(-13) s; difussion coefficient (D200,W), 5.04 . 10(-7) cm2/s; Stokes radius, 62.05 A, frictional ratio (f/f0), 1.62 and isoelectric point (pI), 4.5. The enzyme was stable at 37 degrees C for 30 min between pH 4.5 and 9.5. Enzyme activity was inhibited by various carboxylic acids; however, this inhibition was reversed by raising the Mg2+ concentration. Optimum pH was 5.5, and no detectable amounts of Mg2+, Mn2+, Fe2+, Cu2+, Zn2+ and Ca2+ were found by atomic absorption spectrophotometry. The enzyme was found to contain sugar. Mg2+ was completely replaceable by Mn2+. The reaction mechanism of this enzyme was suggested to be of the 'ping-pong' type. Km values of quinolinic acid and 5-phosphoribosyl 1-pyrophosphate were 4 . 10(-5) and 1.4 . 10(-4) M, respectively. PMID:7357010

Shibata, K; Iwai, K



Human Gene Targeting Favors Insertions Over Deletions  

PubMed Central

Abstract Gene targeting is a powerful technique for manipulating the human genome, but few studies have directly compared the targeting frequencies of various types of vector constructs. Here we show that similar targeting constructs are able to insert nucleotides at the homologous chromosomal target locus more efficiently than they can delete nucleotides, and combination insertion/deletion vectors appear to target at intermediate frequencies. This holds true for deletions ranging from 1 to 334 bp and insertions ranging from 1 to 1332 bp. In addition, vectors designed to inactivate the human hypoxanthine phosphoribosyltransferase gene (HPRT) by deleting nucleotides often produced rearrangements at the target locus that in many cases were due to insertions of multimerized vector constructs, effectively converting a deletion vector into an insertion vector. These findings were obtained when adeno-associated virus vectors were used to efficiently deliver single-stranded DNA targeting constructs, but the same phenomenon was also observed when transfecting linearized double-stranded plasmids. Thus human cells distinguish between deletion and insertion vectors and process their recombination intermediates differently, presumably at the heteroduplex stage, with implications for the design of gene-targeting vectors and the evolution of human genomes.

Hirata, Roli K.



Properties of Pea Seedling Uracil Phosphoribosyltransferase and Its Distribution in Other Plants 1  

PubMed Central

A uracil phosphoribosyltransferase (UMP-pyrophosphorylase) was found in several angiosperms and was partially purified from epicotyls of pea (Pisum sativum L. cv. Alaska) seedlings. Its pH optimum was about 8.5; its required approximately 0.3 mm MgCl2 for maximum activity but was inhibited by MnCl2; its molecular weight determined by chromatography on Sephadex G-150 columns was approximately 100,000; its Km values for uracil and 5-phosphorylribose 1-pyrophosphate were 0.7 ?m and 11 ?m; and it was partially resolved from a similar phosphoribosyltransferase converting orotic acid to orotodine 5?-phosphate. Enzyme fractions containing both uracil phosphoribosyl transferase and orotate phosphoribosyltransferase converted 6-azauracil and 5-fluorouracil to products with chromatographic properties of 6-azauradine 5?-phosphate and 5-fluorouridine 5?-phosphate. Uracil phosphoribosyltransferase probably functions in salvage of uracil for synthesis of pyrimidine nucleotides.

Bressan, Ray A.; Murray, Michael G.; Gale, James M.; Ross, Cleon W.



Expression of thymidylate synthase and orotate phosphoribosyltransferase in thymic carcinoma.  


Thymic carcinoma is a rare thymic epithelial tumor in which chemotherapy for advanced disease has not yet been established. Thymidylate synthase (TS) and orotate phosphoribosyltransferase (OPRT) protein expression levels in thymic carcinoma were evaluated as possible indicators of the anticancer activity of 5-fluorouracil (5-FU) drugs using immunohistochemistry (IHC). A total of 24 samples of thymic carcinoma were used in the present study. The tumor sections were immunohistochemically stained for TS and OPRT. As a comparison with thymic carcinoma, we also assessed the TS and OPRT protein expression levels in 55 lung cancer samples. The TS expression was positive in 12 of 24 thymic carcinoma samples (50%) and OPRT expression was positive in 10 (42%). The association between TS and OPRT expression and Masaoka stages of thymic carcinoma was analyzed. The TS and OPRT expressions in stage IV were significantly higher compared to that in stages I, II or III. We also compared the TS and OPRT expression levels between thymic carcinoma and lung cancer (33 adenocarcinomas and 22 squamous cell carcinomas). TS expression in thymic carcinoma was significantly lower compared with lung squamous cell carcinoma. OPRT expression in thymic carcinoma was significantly higher compared to lung adenocarcinoma. The combination of a relatively low expression of TS and high expression of OPRT suggests an improved antitumor effect of 5-FU drugs in thymic carcinoma compared to in lung carcinoma. PMID:23170110

Yokota, Keisuke; Sasaki, Hidefumi; Okuda, Katsuhiro; Shitara, Masayuki; Hikosaka, Yu; Moriyama, Satoru; Yano, Motoki; Fujii, Yoshitaka



Expression of thymidylate synthase and orotate phosphoribosyltransferase in thymic carcinoma  

PubMed Central

Thymic carcinoma is a rare thymic epithelial tumor in which chemotherapy for advanced disease has not yet been established. Thymidylate synthase (TS) and orotate phosphoribosyltransferase (OPRT) protein expression levels in thymic carcinoma were evaluated as possible indicators of the anticancer activity of 5-fluorouracil (5-FU) drugs using immunohistochemistry (IHC). A total of 24 samples of thymic carcinoma were used in the present study. The tumor sections were immunohistochemically stained for TS and OPRT. As a comparison with thymic carcinoma, we also assessed the TS and OPRT protein expression levels in 55 lung cancer samples. The TS expression was positive in 12 of 24 thymic carcinoma samples (50%) and OPRT expression was positive in 10 (42%). The association between TS and OPRT expression and Masaoka stages of thymic carcinoma was analyzed. The TS and OPRT expressions in stage IV were significantly higher compared to that in stages I, II or III. We also compared the TS and OPRT expression levels between thymic carcinoma and lung cancer (33 adenocarcinomas and 22 squamous cell carcinomas). TS expression in thymic carcinoma was significantly lower compared with lung squamous cell carcinoma. OPRT expression in thymic carcinoma was significantly higher compared to lung adenocarcinoma. The combination of a relatively low expression of TS and high expression of OPRT suggests an improved antitumor effect of 5-FU drugs in thymic carcinoma compared to in lung carcinoma.




Biochemical characterization of uracil phosphoribosyltransferase from Mycobacterium tuberculosis.  


Uracil phosphoribosyltransferase (UPRT) catalyzes the conversion of uracil and 5-phosphoribosyl-?-1-pyrophosphate (PRPP) to uridine 5'-monophosphate (UMP) and pyrophosphate (PP(i)). 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 PP(i) 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

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



Purine and pyrimidine metabolism in human gliomas: relation to chromosomal aberrations.  

PubMed Central

Chromosomal aberrations in human gliomas are principally numerical. In tumours of low malignancy, karyotypes are frequently normal, but occasionally an excess of chromosome 7 and a loss of sex chromosome are observed. In highly malignant tumours, the most frequent aberrations are gain of chromosome 7, loss of chromosome 10 and less frequently losses or deletions of chromosomes 9, 22, 6, 13 and 14 or gains of chromosomes 19 and 20. To understand the meaning of these chromosome imbalances, the relationships between chromosome abnormalities and metabolic disturbances were studied. The losses or deletions observed affected principally chromosomes carrying genes encoding enzymes involved in purine metabolism. The activities of ten enzymes were measured: adenosine kinase, adenine phosphoribosyltransferase, adenylate kinase, methylthioadenosine phosphorylase, hypoxanthine phosphoribosyltransferase, adenylosuccinate lyase, inosine monophosphate dehydrogenase, adenosine deaminase, nucleoside phosphorylase and adenosine monophosphate deaminase. In parallel, two enzymes involved in pyrimidine metabolism, thymidine kinase and thymidylate synthase (TS), were studied. The activities of all these enzymes were measured on samples from 30 human primary glial tumours with low or high malignancy, six xenografted tumours at different passages, four portions of normal brain tissue and four non-glial brain neoplasms. As suggested by cytogenetic data, the enzymatic results showed a relatively low activity of purine metabolism in glial tumours when compared with normal brain and non-glial brain neoplasms. Considering the two enzymes involved in pyrimidine metabolism, only TS had higher activity in glial tumours of high malignancy than in normal brain. In comparison with normal brain, the balance between salvage and de novo pathways changes in gliomas, and even more in grafted tumours, in favour of de novo synthesis. The relation between chromosomes and metabolic imbalances does not correspond to a simple gene dosage effect in these tumours. These data suggest that the decrease of adenosine metabolism occurs before chromosomal aberrations appear, since it is observed in tumours of low malignancy when most karyotypes are still normal, and that the de novo pathway increases with tumour progression. Images Figure 2

Bardot, V.; Dutrillaux, A. M.; Delattre, J. Y.; Vega, F.; Poisson, M.; Dutrillaux, B.; Luccioni, C.



Orotate Phosphoribosyltransferase from Corynebacterium ammoniagenes Lacking a Conserved Lysine? †  

PubMed Central

The pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase), was cloned by nested PCR and colony blotting from Corynebacterium ammoniagenes ATCC 6872, which is widely used in nucleotide production. Sequence analysis shows that there is a lack of an important conserved lysine (Lys 73 in Salmonella enterica serovar Typhimurium OPRTase) in the C. ammoniagenes OPRTase. This lysine has been considered to contribute to the initiation of catalysis. The enzyme was overexpressed and purified from a recombinant Escherichia coli strain. The molecular mass of the purified OPRTase was determined to be 45.4 ± 1.5 kDa by gel filtration. Since the molecular mass for the subunit of the enzyme was 21.3 ± 0.6 kDa, the native enzyme exists as a dimer. Divalent magnesium was necessary for the activity of the enzyme and can be substituted for by Mn2+ and Co2+. The optimal pH for the forward (phosphoribosyl transfer) reaction is 10.5 to 11.5, which is higher than that of other reported OPRTases, and the optimal pH for the reverse (pyrophosphorolysis) reaction is 5.5 to 6.5. The Km values for the four substrates were determined to be 33 ?M for orotate, 64 ?M for 5-phosphoribosyl-1-pyrophosphate (PRPP), 45 ?M for orotidine-5-phosphate (OMP), and 36 ?M for pyrophosphate. The Km value for OMP is much larger than those of other organisms. These differences may be due to the absence of Lys 73, which is present in the active sites of other OPRTases and is known to interact with OMP and PRPP.

Wang, Xing; Ma, Cuiqing; Wang, Xiuwen; Xu, Ping



Mutation induction in human lymphoid cells by energetic heavy ions  

NASA Astrophysics Data System (ADS)

One of the concerns for extended space flight outside the magnetosphere is exposure to galactic cosmic radiation. In the series of studies presented herein, the mutagenic effectiveness of high energy heavy ions is examined using human B-lymphoblastoid cells across an LET range from 32keV/?m to 190 keV/?m. Mutations were scored for an autosomal locus, thymidine kinase (tk), and for an X-linked locus, hypoxanthine phosphoribosyltransferase (hprt). For each of the radiations studied, the autosomal locus is more sensitive to mutation induction than is the X-linked locus. When mutational yields are expressed in terms of particle fluence, the two loci respond quite differently across the range of LET. The action cross section for mutation induction peaks at 61 keV/?m for the tk locus and then declines for particles of higher LET, including Fe ions. For the hprt locus, the action cross section for mutation is maximal at 95 keV/?m but is relatively constant across the range from 61 keV/?m to 190 keV/gmm. The yields of hprt-deficient mutants obtained after HZE exposure to TK6 lymphoblasts may be compared directly with published data on the induction of hprt-deficient mutants in human neonatal fibroblasts exposed to similar ions. The action cross section for induction of hprt-deficient mutants by energetic Fe ions is more than 10-fold lower for lymphoblastoid cells than for fibroblasts.

Kronenberg, A.



Intrastriatal injection of hypoxanthine reduces striatal serotonin content and impairs spatial memory performance in rats  

Microsoft Academic Search

The aim of this study was to investigate the effects of intrastriatal injection of hypoxanthine, a metabolite accumulated\\u000a in Lesch-Nyhan disease, on rats’ performance in the Morris water maze tasks, along with the monoamine content in striatum\\u000a of rats. Male adult Wistar rats were divided in two groups: (1) saline-injected and (2) hypoxanthine-injected group. Seven\\u000a days after solutions infusion, animals

Caren Serra Bavaresco; Fabria Chiarani; Eduardo Duringon; Marcelo Machado Ferro; Cláudio Da Cunha; Carlos Alexandre Netto; Angela Terezinha de Souza Wyse



On the accessibility to conical intersections in purines: hypoxanthine and its singly protonated and deprotonated forms.  


The dynamics following electronic excitation of hypoxanthine and its nucleoside inosine were studied by femtosecond fluorescence up-conversion. Our objective was to explore variants of the purinic DNA bases in order to determine the molecular parameters that increase or reduce the accessibility to ground state conical intersections. From experiments in water and methanol solution we conclude that both dominant neutral tautomers of hypoxanthine exhibit ultrashort excited state lifetimes (? < 0.2 ps), which are significantly shorter than in the related nucleobase guanine. This points to a more accessible conical intersection for the fluorescent state upon removal of the amino group, present in guanine but absent in hypoxanthine. The excited state dynamics of singly protonated hypoxanthine were also studied, showing biexponential decays with a 1.1 ps component (5%) besides a sub-0.2 ps ultrafast component. On the other hand, the S(1) lifetimes of the singly deprotonated forms of hypoxanthine and inosine show drastic differences, where the latter remains ultrafast but the singly deprotonated hypoxanthine shows a much longer lifetime of 19 ps. This significant variation is related to the different deprotonation sites in hypoxanthine versus inosine, which gives rise to significantly different resonance structures. In our study we also include multireference perturbation theory (MRMP2) excited state calculations in order to determine the nature of the initial electronic excitation in our experiments and clarify the ordering of the states in the singlet manifold at the ground state geometry. In addition, we performed multireference configuration interaction calculations (MR-CIS) that identify the presence of low-lying conical intersections for both prominent neutral tautomers of hypoxanthine. In both cases, the surface crossings occur at geometries reached by out of plane opposite motions of C2 and N3. The study of this simpler purine gives several insights into how small structural modifications, including amino substitution and protonation site and state, determine the accessibility to conical intersections in this kind of heterocycles. PMID:22486543

Villabona-Monsalve, Juan P; Noria, Raquel; Matsika, Spiridoula; Peón, Jorge



Nicotinamide Phosphoribosyltransferase\\/Visfatin Does Not Catalyze Nicotinamide Mononucleotide Formation in Blood Plasma  

Microsoft Academic Search

Nicotinamide (Nam) phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in mammalian NAD synthesis, catalyzing nicotinamide mononucleotide (NMN) formation from Nam and 5-phosphoribosyl 1-pyrophosphate (PRPP). NAMPT has also been described as an adipocytokine visfatin with a variety of actions, although physiological significance of this protein remains unclear. It has been proposed that possible actions of visfatin are mediated through the extracellular formation

Nobumasa Hara; Kazuo Yamada; Tomoko Shibata; Harumi Osago; Mikako Tsuchiya



A new function for a common fold: the crystal structure of quinolinic acid phosphoribosyltransferase  

Microsoft Academic Search

Background: Quinolinic acid (QA) is a neurotoxin and has been shown to be present at high levels in the central nervous system of patients with certain diseases, such as AIDS and meningitis. The enzyme quinolinic acid phosphoribosyltransferase (QAPRTase) provides the only route for QA metabolism and is also an essential step in de novo NAD biosynthesis. QAPRTase catalyzes the synthesis

Janina C Eads; Derya Ozturk; Tom B Wexler; Charles Grubmeyer; James C Sacchettini



Nicotinamide Phosphoribosyltransferase/Visfatin Does Not Catalyze Nicotinamide Mononucleotide Formation in Blood Plasma  

PubMed Central

Nicotinamide (Nam) phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in mammalian NAD synthesis, catalyzing nicotinamide mononucleotide (NMN) formation from Nam and 5-phosphoribosyl 1-pyrophosphate (PRPP). NAMPT has also been described as an adipocytokine visfatin with a variety of actions, although physiological significance of this protein remains unclear. It has been proposed that possible actions of visfatin are mediated through the extracellular formation of NMN. However, we did not detect NMN in mouse blood plasma, even with a highly specific and sensitive liquid chromatography/tandem mass spectrometry. Furthermore, there is no or little ATP, the activator of NAMPT, in extracellular spaces. We thus questioned whether visfatin catalyzes the in situ formation of NMN under such extracellular milieus. To address this question, we here determined Km values for the substrates Nam and PRPP in the NAMPT reaction without or with ATP using a recombinant human enzyme and found that 1 mM ATP dramatically decreases Km values for the substrates, in particular PRPP to its intracellular concentration. Consistent with the kinetic data, only when ATP is present at millimolar levels, NAMPT efficiently catalyzed the NMN formation at the intracellular concentrations of the substrates. Much lower concentrations of Nam and almost the absence of PRPP and ATP in the blood plasma suggest that NAMPT should not efficiently catalyze its reaction under the extracellular milieu. Indeed, NAMPT did not form NMN in the blood plasma. From these kinetic analyses of the enzyme and quantitative determination of its substrates, activator, and product, we conclude that visfatin does not participate in NMN formation under the extracellular milieus. Together with the absence of NMN in the blood plasma, our conclusion does not support the concept of “NAMPT-mediated systemic NAD biosynthesis.” Our study would advance current understanding of visfatin physiology.

Hara, Nobumasa; Yamada, Kazuo; Shibata, Tomoko; Osago, Harumi; Tsuchiya, Mikako



The kinetics of hypoxanthine transport across the perfused choroid plexus of the sheep.  


The uptake of principal salvageable nucleobase hypoxanthine was investigated across the basolateral membrane of the sheep choroid plexus (CP) perfused in situ. The results suggest that hypoxanthine uptake was Na+-independent, which means that transport system on the basolateral membrane can mediate the transport in both directions. Although the unlabelled nucleosides adenosine and inosine markedly reduce the transport it seems that this inhibition was due to nucleoside degradation into nucleobases in the cells, since non-metabolised nucleoside analogue NBTI did not inhibit the transport. The presence of adenine also inhibits hypoxanthine uptake while the addition of the pyrimidines does not show any effect, so it seems that the transport of purine nucleobases through basolateral membrane is mediated via a common transporter which is different from the nucleoside transporters. The inclusion of allopurinol in the perfusion fluid did not change the value and general shape of the curve for the uptake which suggest that degradation of hypoxanthine into xanthine and uric acid does not occur in the CP. The capacity of the CP basolateral membrane to transport hypoxanthine is high (90.63+/-3.79 nM/min/g) and close to the values obtained for some essential amino acids by the CP and blood-brain barrier, while the free diffusion is negligible. The derived value of Km (20.72+/-2.42 microM) is higher than the concentration of hypoxanthine in the sheep plasma (15.61+/-2.28 microM) but less than a half of the concentration in the CSF, which indicates that the transport system at basolateral membrane mostly mediates the efflux of hypoxanthine from the cerebrospinal fluid in vivo. PMID:11792365

Redzic, Zoran B; Gasic, Jovana M; Segal, Malcolm B; Markovic, Ivanka D; Isakovic, Aleksandra J; Rakic, Miodrag Lj; Thomas, Sarah A; Rakic, Ljubisa M



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

SciTech Connect

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.

Mendez, M.J.; Abderrahim, H.; Noguchi, M. [Cell Genesys, Inc., Foster City, CA (United States)] [and others



Quantification and molecular characterization of hprt mutants of human T-lymphocytes.  

PubMed Central

Somatic mutations have been implicated as critical early events in carcinogenesis. Point mutations, deletions, and translocation events have been shown to activate oncogenes or inactivate suppressor oncogenes. In human population monitoring, quantitative analysis of mutation events that affect gene function is limited to those genes whose cellular phenotypes can be identified by selection procedures and to those tissues (like blood) that are accessible for analysis. In an effort to determine the frequency and types of mutations that can be detected at the hypoxanthine guanine phosphoribosyltransferase (hprt) gene, we have used the T-cell cloning assay and have developed a strategy to propagate mutants and screen for point mutations and breakage events. Early in the clonal expansion of mutants, 1-2 x 10(4) cells are prepared as a crude cell lysate, and a sample is analyzed using the multiplex polymerase chain reaction (PCR). Those mutants that yield altered DNA fragments are then expanded for Southern blot hybridization, PCR, flanking probe isolation, and DNA sequencing. To date we have found presumed point mutations, intragenic deletions, and deletions that extend outside of the hprt gene. By analyzing mutations in selectable, nonessential gene markers, it should be possible to understand mechanisms of both spontaneous and induced genetic damage. An association of these specific genetic events with human diseases and the evaluation of the ability of environmental chemicals to induce these specific types of mutations will lead to a rational basis for evaluating risks from various chemical exposures. Images FIGURE 3.

Moore, M M; Harrington-Brock, K; Zimmerman, L J; Burnette, L P; Smith, T W; Everson, R B; O'Neill, J P; Fuscoe, J C



Kinetic analysis and chemical modification studies of nicotinate phosphoribosyltransferase from yeast  

Microsoft Academic Search

Nicotinate phosphoribosyltransferase (NaPRTase) from Baker's yeast catalyzes the formation of nicotinate mononucleotide (NaMN) and pyrophosphate from phosphoribosyl α-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




The H2O2 induced effects on purine metabolism in human endothelial cells.  


The effects of hydrogen peroxide (H2O2) on the purine metabolism of human endothelial cells were investigated. An incubation with 0.01 mM H2O2 over 60 min led to an increase in the intracellular adenosine-5-triphosphate (ATP) and creatine phosphate (CP) levels by 51.3% and 18.2%, respectively. A 60 min incubation with 0.1 mM H2O2 showed no effect. The uptake and salvage of 14C-adenine (14C-AD) and 14C-adenosine (14C-ADO) was significantly (p < 0.005) increased using 0.01 mM H2O2. Only an increase of 14C-ADO incorporation was observed using 0.1 mM H2O2. A concentration of 0.01 mM H2O2 reduced 5-phosphoribosyl-1-pyrophosphate synthetase (PRPP-S) activity by 60% and at the same time increased the activity of purine nucleoside phosphorylase, which converts inosine to hypoxanthine (PNP I), by 24%. Adenosine kinase (AK) activity was reduced by H2O2, whereas adenine phosphoribosyltransferase (APRT) activity was found to be elevated. In conclusion, the observed elevation of cellular ATP and CP levels could be partially caused by an increased purine salvage resulting from changes in purine enzyme activities. PMID:8138186

Griesmacher, A; Weigel, G; Schimke, I; Windischbauer, A; Mueller, M M



Enhanced cytotoxicity with methotrexate in conjunction with hypoxanthine in L1210 cells in culture  

Microsoft Academic Search

By inhibiting dihydrofolate reductase, methotrexate (MTX) depletes cellular stores of reduced folates, resulting in the inhibition of DNA and RNA synthesis. Inhibition of RNA synthesis arrests cells in the G1 phase of the cell cycle, preventing these cells from entering S phase and rendering them insensitive to MTX. Because MTX cytotoxicity can be enhanced by concurrent administration of hypoxanthine (HX),

Craig R. Fairchild; Jonathan Maybaum; James A. Straw



Targeted disruption of the mouse adenine phosphoribosyltransferase (aprt) gene and the production of APRT-deficient mice  

SciTech Connect

Adenine phosphoribosyltransferase (APRT: EC, a ubiquitously expressed purine salvage enzyme, catalyzes the synthesis of AMP and inorganic pyrophosphate from existing adenine and 5-phosphoribosyl-1-pyrophosphate. Deficiency of this enzyme in humans results in the accumulation of 2,8-dihydroxyadenine leading to crystalluria and nephrolithiasis. In order to facilitate our study of this rare, autosomal recessive disorder, we applied the advances in gene targeting technology and mouse embryonic stem (ES) cell culture to the production of APRT-deficient mice. A positive-negative targeting strategy was used. The tageting vector contain 5.6 kb of the mouse APRT gene, a neomycin resistance gene in exon 3 as a positive selection marker, and a HSV thymidine kinase gene at the 3{prime} end of the homology as a negative selection marker. The vector was introduced into D3 ES cells by electroporation and the cells were selected for G418 and ganciclovir (GANC) resistance. G418-GANC resistant clones were screened by Southern blot. One of several correctly targeted clones was expanded and used for blastocyst microinjection to produce chimeric mice. Chimeric animals were bred and agouti progeny heterozygous for the targeted allele were obtained. Heterozygous animals have been bred to produce APRT-deficient animals. Matings are currently underway to determine the phenotype of APRT/HPRT-deficient animals.

Engle, S.J.; Chen, J.; Tischfield, J.A. [Indiana Univ., School of Medicine, Indianapolis, IN (United States)] [and others



Mutations induced in the hypoxanthine phosphoribosyl transferase gene by three urban air pollutants: acetaldehyde, benzo[a]pyrene diolepoxide, and ethylene oxide.  

PubMed Central

Provisional mutational spectra at the hypoxanthine phosphoribosyl transferase (HPRT) locus in vitro have been worked out for acetaldehyde (AA) and benzo[a]pyrene diolepoxide (BPDE) in human (T)-lymphocytes and for ethylene oxide (EtO) in human diploid fibroblasts using Southern blotting and polymerase chain reaction (PCR)-based DNA sequencing techniques. The results indicate that large genomic deletions are the predominating hprt mutations caused by AA and EO, whereas BPDE induces point mutations that are mainly GC > TA transversions. The mutational spectra induced by the three agents are clearly different from the background spectrum in human T-cells. Thus, the hprt locus is a useful target for the study of chemical-specific mutational events that may help identify causes of background mutation in human cells in vivo.

Lambert, B; Andersson, B; Bastlova, T; Hou, S M; Hellgren, D; Kolman, A



Guanine, Adenine, and Hypoxanthine Production from UV-Irradiated Formamide: Relaxation of the Requirements for Prebiotic Purine Nucleobase Formation  

NASA Astrophysics Data System (ADS)

We observe the production of adenine, hypoxanthine, and guanine in heated and UV irradiated formamide solutions. These "one pot" reactions occur due to the synergy of thermal and UV photon-induced processes.

Orlando, T. M.; Barks, H.; Buckley, R.; Grieves, G.; Dimauro, E.; Hud, N. V.



Mixed-ligand iridium(IV) complexes with amino acids, adenine and hypoxanthine  

Microsoft Academic Search

The complexation in iridium(IV)-purine base (adenine, hypoxanthine)-amino acid (?-alanine, aspartic acid, lysine) systems\\u000a was studied by pH titration. The stability constants of 1: 1: 1 complexes were determined. The stability of 1: 1: 1 mixed-ligand\\u000a complexes with hypoxanthine and adenine increases in the series Ala < Lys < Asp. Reactions between aqueous solutions gave\\u000a the following coordination compounds: [Ir(C5H4N4O)(C3H6NO2)Cl]Cl2, [Ir(C5H4N4O)(C4H5NO4)]Cl2,

A. K. Molodkin; N. Ya. Esina; M. N. Kurasova



Ab initio Study on Ionization Energies of 1-Methyl-hypoxanthine  

NASA Astrophysics Data System (ADS)

Six low-lying tautomers of 1-methyl-hypoxanthine have been studied at the B3LYP/aug-cc-pVDZ level. Two tautomers N7H and N9H with the comparable energies are far more stable than the others. The vertical ionization energies of the tautomers calculated with ab initio electron propagator theory in the P3/aug-cc-pVDZ approximation are in agreement with the experimental data from photoelectron spectroscopy. According to the calculated relative energies and the comparison between the simulated and the experimental photoelectron spectra, it demonstrates that there are at least two tautomers of 1-methyl-hypoxanthine in the gas-phase experiments.

Wang, Ke-dong; Yang, Da-peng; Liu, Yu-fang



Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine.  


Riboswitches are genetic regulatory elements found in the 5' untranslated region of messenger RNA that act in the absence of protein cofactors. They are broadly distributed across bacteria and account for the regulation of more than 2% of all genes in Bacillus subtilis, underscoring their importance in the control of cellular metabolism. The 5' untranslated region of many mRNAs of genes involved in purine metabolism and transport contain a guanine-responsive riboswitch that directly binds guanine, hypoxanthine or xanthine to terminate transcription. Here we report the crystal structure at 1.95 A resolution of the purine-binding domain of the guanine riboswitch from the xpt-pbuX operon of B. subtilis bound to hypoxanthine, a prevalent metabolite in the bacterial purine salvage pathway. This structure reveals a complex RNA fold involving several phylogenetically conserved nucleotides that create a binding pocket that almost completely envelops the ligand. Hypoxanthine functions to stabilize this structure and to promote the formation of a downstream transcriptional terminator element, thereby providing a mechanism for directly repressing gene expression in response to an increase in intracellular concentrations of metabolite. PMID:15549109

Batey, Robert T; Gilbert, Sunny D; Montange, Rebecca K



Combined radiation and gene therapy for brain tumors with adenovirus-mediated transfer of cytosine deaminase and uracil phosphoribosyltransferase genes  

Microsoft Academic Search

Radiation therapy is an established modality for the treatment of malignant gliomas. Several reports have shown the advantage of additional radiation in combination with gene therapy. In this study, we investigated the ability of radiation therapy to enhance 5-fluorocytosine (5-FC)\\/cytosine deaminase (CD) plus uracil phosphoribosyltransferase (UPRT) gene therapy in malignant gliomas. In vitro study suggested evidence of a significant cytotoxic

Hirokazu Kambara; Takashi Tamiya; Yasuhiro Ono; Shinji Ohtsuka; Kinya Terada; Yoshiaki Adachi; Tomotsugu Ichikawa; Hirofumi Hamada; Takashi Ohmoto



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

PubMed Central

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

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



Structure-based identification of ureas as novel nicotinamide phosphoribosyltransferase (Nampt) inhibitors.  


Nicotinamide phosphoribosyltransferase (Nampt) is a promising anticancer target. Virtual screening identified a thiourea analogue, compound 5, as a novel highly potent Nampt inhibitor. Guided by the cocrystal structure of 5, SAR exploration revealed that the corresponding urea compound 7 exhibited similar potency with an improved solubility profile. These studies also indicated that a 3-pyridyl group was the preferred substituent at one inhibitor terminus and also identified a urea moiety as the optimal linker to the remainder of the inhibitor structure. Further SAR optimization of the other inhibitor terminus ultimately yielded compound 50 as a urea-containing Nampt inhibitor which exhibited excellent biochemical and cellular potency (enzyme IC50 = 0.007 ?M; A2780 IC50 = 0.032 ?M). Compound 50 also showed excellent in vivo antitumor efficacy when dosed orally in an A2780 ovarian tumor xenograft model (TGI of 97% was observed on day 17). PMID:23617784

Zheng, Xiaozhang; Bauer, Paul; 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; Gunzner-Toste, Janet; Zhao, Guiling; Yuen, Po-wai; Bair, Kenneth W



[The comparison of contents of uracil, xanthine and hypoxanthine in five species of leech].  


Three constituents(uracil, xanthine, hypoxanthine) were isolated from five species of leech and determined by reversed phase HPLC. The column employed was Shim-pack CLC-ODS C18(150 mm x 6 mm). The mobile phase was 0.05 mol/l ammonium phophate dibasic solution(pH = 8.4). The flow rate was 0.8 ml/min and detection was effected at 254 nm. This method is accurate, rapid and reproducible. Analytical data for five species and Whitmania pigra samples from different places were given. PMID:12575054

Liu, L; Jin, R; Xu, G



Photoion mass spectroscopy and valence photoionization of hypoxanthine, xanthine and caffeine  

NASA Astrophysics Data System (ADS)

Photoionization mass spectra of hypoxanthine, xanthine and caffeine were measured using the photoelectron-photoion coincidence technique and noble gas resonance radiation at energies from 8.4 to 21.2 eV for ionization. The fragmentation patterns for these compounds show that hydrogen cyanide is the main neutral loss species at higher photon energies, while photoionization below 16.67 eV led predominantly to the parent ion. The valence photoelectron spectra of this family of molecules were measured over an extended energy range, including the inner C, N and O 2s valence orbitals. The observed ion fragments were related to ionization of the valence orbitals.

Feyer, Vitaliy; Plekan, Oksana; Richter, Robert; Coreno, Marcello; Prince, Kevin C.



The gene for theta-globin is transcribed in human fetal erythroid tissues.  


A new gene like the alpha-globin gene has been identified in higher primates at the 3' end of the alpha-globin gene cluster. There is some controversy as to whether this gene, theta, is a functional globin gene or a non-functional pseudogene. The high degree of sequence conservation displayed by theta between primates and various mammals, such as horse and rabbit, suggests that this gene is functional in some species. Furthermore, theta encodes a 141-amino-acid polypeptide in sequence similar to alpha-globin and appears to possess functional RNA-processing signals. But the promoter region of theta is unlike the other globin genes because its CCAAT and ATA box sequences are displaced from the coding sequence by the insertion of a 200-base-pair GC-rich sequence. We demonstrate here the presence of theta-globin messenger RNA in human fetal erythroid tissue, but not in adult erythroid or other non-erythroid tissues. Furthermore, theta-globin mRNA is detectable in significant amounts in a human erythroleukaemic cell line. These results predict that theta-globin protein will be found in the early stages of human fetal development. Surprisingly, the promoter sequence of theta-globin does not correspond to the CCAAT and ATA box sequences of the gene but rather lies within the adjacent GC-rich sequence, resulting in a heterogeneous series of mRNA 5' ends 50-10 base pairs to 5' of the initiation codon. This type of promoter is reminiscent of that found in housekeeping genes such as adenine deaminase and hypoxanthine-guanine phosphoribosyl-transferase. PMID:3657976

Leung, S; Proudfoot, N J; Whitelaw, E


Expression of Escherichia coli uracil phosphoribosyltransferase gene in murine colon carcinoma cells augments the antitumoral effect of 5-fluorouracil and induces protective immunity  

Microsoft Academic Search

Uracil phosphoribosyltransferase (UPRT) of Escherichia coli origin can convert 5-fluorouracil (5-FU), a chemotherapeutic agent widely used for solid tumors, to an active intermediate, 5-fluorouridine-5?-monophosphate, as mammalian orotate phosphoribosyltransferase does. To examine whether the E. coli UPRT gene expressed in tumor cells can confer increased sensitivity to 5-FU, we retrovirally transduced Colon 26 cells, a murine colon carcinoma cell line, with

Kiyoko Kawamura; Kentaro Tasaki; Hirofumi Hamada; Keizo Takenaga; Shigeru Sakiyama; Masastoshi Tagawa



Estimation of postmortem interval by hypoxanthine and potassium evaluation in vitreous humor with a sequential injection system.  


The estimation of the time since death known as postmortem interval (PMI) is a main issue in the field of forensic science and legal medicine. In this work it is proposed a sequential injection system for the determination of hypoxanthine and potassium in the same sample of vitreous humor since the concentrations of both parameters change with PMI and the vitreous humor has been regarded as the ideal extracellular fluid for these kinds of determinations. By measuring both parameters the accuracy of estimation of PMI can be increased, and the effects of factors which influence the values in postmortem chemistry minimized. Hypoxanthine determination is based on its oxidation to uric acid (290 nm), catalyzed by immobilized xanthine oxidase, and the quantification of potassium levels in vitreous humor was performed using a tubular potassium ion-selective electrode. With a unique analytical cycle both analytes were evaluated being potassium levels determined during the degradation of hypoxanthine in the enzymatic reactor. Working concentration ranges between 6.04-40.00 micromol L(-1) and 7.00 x 10(-5) to 1.00 x 10(-1)mmol L(-1) were obtained, for hypoxanthine and potassium, respectively. The method proved to be reproducible with R.S.D. <5% for hypoxanthine and <3% for potassium. Sampling rate was approximately 30 per hour for the sequential determination of both parameters being 15 and 60 determinations per hour if hypoxanthine or potassium, where evaluated independently. Statistical evaluation at the 95% confidence level showed good agreement between the results obtained, for the vitreous humor samples, with both the SIA system and the comparison batch procedures. Moreover the methodology has low environmental impact in agreement with the demands of green analytical chemistry as only 2.7 mL of chemical waste is produced during both determinations. PMID:19615515

Passos, Marieta L C; Santos, Ana M; Pereira, Ana I; Santos, J Rodrigo; Santos, Agostinho J C; Saraiva, M Lúcia M F S; Lima, José L F C



Validation of adequate endogenous reference genes for the normalisation of qPCR gene expression data in human post mortem tissue  

Microsoft Academic Search

Gene expression analyses based on messenger RNA (mRNA) profiling require accurate data normalisation. When using endogenous\\u000a reference genes, these have to be validated carefully. Therefore, we examined the transcript stability of 10 potential reference\\u000a genes using quantitative real-time polymerase chain reaction: beta actin, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase,\\u000a TATA box-binding protein, hypoxanthine phosphoribosyl-transferase I, beta-2-microglobulin, hydroxymethylbilane synthase, succinate\\u000a dehydrogenase complex, subunit

Antje Koppelkamm; Benedikt Vennemann; Tony Fracasso; Sabine Lutz-Bonengel; Ulrike Schmidt; Marielle Heinrich



Determinants of Sensitivity of Human T Cell Leukemia CCRF-CEM cells to Immucillin-H  

PubMed Central

Immucillin-H (BCX-1777, forodesine) is a transition state analogue and potent inhibitor of PNP that shows promise as a specific agent against activated human T-cells and T-cell leukemias. The immunosuppressive or antileukemic effects of Immucillin-H (ImmH) require co-administration with deoxyguanosine (dGuo) to attain therapeutic levels of intracellular dGTP. In this study we investigated the requirements for sensitivity and resistance to ImmH and dGuo. 3H-ImmH transport assays demonstrated that the equilibrative nucleoside transporters (ENT1 and ENT2) facilitated the uptake of ImmH in human leukemia CCRF-CEM cells whereas 3H-dGuo uptake was primarily dependent upon concentrative nucleoside transporters (CNTs). Analysis of lysates from an ImmH-resistant CCRF-CEM-AraC-8D cells demonstrated undetectable deoxycytidine kinase (dCK) activity, suggesting that dCK and not deoxyguanosine kinase (dGK) was the rate-limiting enzyme for phosphorylation of dGuo in these cells. Examination of ImmH cytotoxicity in a hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient cell line CCRF-CEM-AraC-8C, demonstrated enhanced sensitivity to low concentrations of ImmH and dGuo. RT-PCR and sequencing of HGPRT from the HGPRT-deficient CCRF-CEM-AraC-8C cells identified an Exon 8 deletion mutation in this enzyme. Thus these studies show that specific nucleoside transporters are required for ImmH cytotoxicity and predict that ImmH may be more cytotoxic to 6-thioguanine (6-TG) or 6-thiopurine-resistant leukemia cells caused by HGPRT deficiency.

Huang, Min; Wang, Yanhong; Gu, Jingjin; Yang, Jing; Noel, Karen; Mitchell, Beverly S.; Schramm, Vern L.; Graves, Lee M.



Adenovirus-Mediated Transduction of Escherichia coli Uracil Phosphoribosyltransferase Gene Increases the Sensitivity of Esophageal Cancer Cells to 5Fluorouracil  

Microsoft Academic Search

Esophageal cancer is associated with the poorest prognosis among the digestive tract cancers, and chemotherapy is the treatment\\u000a of choice for many patients. In this study, we experimentally introduced an Escherichia coli-derived uracil phosphoribosyltransferase (UPRT) gene to cultured esophageal cancer cell lines to potentiate the antitumor\\u000a effects of a representative anticancer drug, 5-fluorouacil (5-FU). UPRT is a pyrimidine salvage enzyme

Hajime Nakamura; Hiroyuki Sekiguchi; Seiji Akiyama; Hirofumi Hamada; Michitaka Fujiwara; Yasushi Kasai; Katsuki Ito; Akimasa Nakao



Functional characterization of a gene encoding a dual domain for uridine kinase and uracil phosphoribosyltransferase in Arabidopsis thaliana  

Microsoft Academic Search

Uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) are enzymes catalyzing the formation of uridine 5?-monophosphate\\u000a (UMP) from uridine and adenine 5?-triphosphate (ATP) and from uracil and phosphoribosyl-?-1-pyrophosphate (PRPP), respectively, in the pyrimidine salvage pathway. Here, we report the characterization and functional\\u000a analysis of a gene AtUK\\/UPRT1 from Arabidopsis thaliana. Sequencing of an expressed sequence tag clone of this gene revealed

M. Rafiqul Islam; Hoyeun Kim; Shin-Wook Kang; Jung-Sup Kim; Young-Min Jeong; Hyun-Ju Hwang; So-Young Lee; Je-Chang Woo; Sang-Gu Kim



Preparing a new biosensor for hypoxanthine determination by immobilization of xanthine oxidase and uricase in polypyrrole-polyvinyl sulphonate film.  


Abstract In this study, a new amperometric biosensor for the determination of hypoxanthine was developed. To this aim, polypyrrole-polyvinyl sulphonate films were prepared on the platinum electrode by the electropolymerization of pyrrole in the presence of polyvinyl sulphonate. Xanthine oxidase and uricase enzymes were immobilized in polypyrrole-polyvinyl sulphonate via the entrapment method. Optimum conditions of enzyme electrode were determined. Hypoxanthine detection is based on the oxidation of hydrogen peroxide at +400 mV produced by the enzymatic reaction on the enzyme electrode surface. The linear working range of biosensor for hypoxanthine was determined. The effects of pH and temperature on the response of the hypoxanthine biosensor were investigated. Optimum pH and temperature were measured as 8 and 30°C, respectively. Operational and storage stability of the biosensor were determined. After 20 assays, the biosensor sustained 74.5% of its initial performance. After 33 days, the biosensor lost 36% of its initial performance. The performance of the biosensor was tested in real samples. PMID:23305069

Görgülü, Mustafa; Cete, Servet; Arslan, Halit; Ya?ar, Ahmet



Mediated xanthine oxidase potentiometric biosensors for hypoxanthine based on ferrocene carboxylic acid modified electrode.  


A potentiometric enzyme electrode for detection of hypoxanthine (Hx) in fish meat is described. The sensor was developed by entrapment of xanthine oxidase (XOD) and ferrocene carboxylic acid (Fc) into polypyrrole (PPy) film during galvanostatic polymerisation film formation. The responses for Hx were obtained in 0-05 M phosphate buffer (pH 7.1) at 0.0 mV vs Ag/AgCl (3M KCl). The optimum condition for the formation of PPy-XOD-Fc film include 0.4M PPy, 6.2U/mL XOD, 40 mM Fc, polymerisation time of 200 s and applied current density of 0-5 mA cm(-2). The sensor provides a linear response to Hx in concentration range of 5-20 ?M, (r=0.998) and was successfully used for determination of Hx in fish. PMID:22980900

Lawal, Abdulazeez T; Adeloju, Samuel B



Mutagenesis and cytotoxicity in human epithelial cells by far- and near-ultraviolet radiations: action spectra  

SciTech Connect

Action spectra were determined for cell killing and mutation by monochromatic ultraviolet and visible radiations (254-434 nm) in cultured human epithelial P3 cells. Cell killing was more efficient following radiation at the shorter wavelengths (254-434 nm) than at longer wavelengths (365-434 nm). At 254 nm, for example, a fluence of 11 Jm-2 gave 37% cell survival, while at 365 nm, 17 X 10(5) Jm-2 gave equivalent survival. At 434 nm little killing was observed with fluences up to 3 X 10(6) Jm-2. Mutant induction, determined at the hypoxanthine-guanine phosphoribosyltransferase locus, was caused by radiation at 254, 313, and 365 nm. There was no mutant induction at 334 nm although this wavelength was highly cytotoxic. Mutagenesis was not induced by 434 nm radiation, either. There was a weak response at 405 nm; the mutant frequencies were only slightly increased above background levels. For the mutagenic wavelengths, log-log plots of the mutation frequency against fluence showed linear regressions with positive slopes of 2.5, consistent with data from a previous study using Escherichia coli. The data points of the action spectra for lethality and mutagenesis were similar to the spectrum for DNA damage at wavelengths shorter than 313 nm, whereas at longer wavelengths the lethality spectrum had a shoulder, and the mutagenesis spectrum had a secondary peak at 365 nm. No correlation was observed for the P3 cells between the spectra for cell killing and mutagenesis caused by wavelengths longer than 313 nm and the induction of DNA breakage or the formation of DNA-to-protein covalent bonds in these cells.

Jones, C.A.; Huberman, E.; Cunningham, M.L.; Peak, M.J.



Apoptotic induction with bifunctional E.coli cytosine deaminase-uracil phosphoribosyltransferase mediated suicide gene therapy is synergized by curcumin treatment in vitro.  


Development of novel suicide gene therapy vector with potential application in cancer treatment has a great impact on human health. Investigation to understand molecular mechanism of cell death is necessary to evaluate the therapeutic application of suicide vectors. For example, the bifunctional E.coli cytosine deaminase & uracil phosphoribosyltransferase fusion (CD-UPRT) gene expression is known to sensitize a wide range of cells toward nontoxic prodrug 5-flurocytosine (5-FC) by converting it to toxic compounds, but the exact pathway of cell death is yet to be defined. Herein, we investigated the mechanism of cell death by 5-FC/CD-UPRT suicide system in both cancer and non-cancer cells and found that the optimum 5-FC concentration led to programmed cell death in vitro. The CD-UPRT expression of transfected cells was measured by the RT-PCR analysis. Biochemical assays, such as mitochondrial activity (MTS) and lactate dehydrogenase (LDH) measurements exhibited cell death. Microscopic experiments showed characteristic onset of apoptosis which was further supported by internucleosomal DNA cleavage of BrdU labeled cellular DNA, appearance of characteristic laddering of chromosomal DNA and involvement of caspase pathway. Furthermore, the 5-FC/CD-UPRT-mediated apoptosis was potentiated with addition of a known anticancer agent curcumin. Our in vitro studies confirmed synergistic induction of apoptotic pathway in the combination treatment. Therefore, combination of 5-FC/CD-UPRT with curcumin could be a potential chemosensitization strategy for cancer treatment. PMID:18092145

Gopinath, P; Ghosh, Siddhartha Sankar



Establishment of true niacin deficiency in quinolinic acid phosphoribosyltransferase knockout mice.  


Pyridine nucleotide coenzymes are involved in >500 enzyme reactions and are biosynthesized from the amino acid L-tryptophan (L-Trp) as well as the vitamin niacin. Hence, "true" niacin-deficient animals cannot be "created" using nutritional techniques. We wanted to establish a truly niacin-deficient model animal using a protocol that did not involve manipulating dietary L-Trp. We generated mice that are missing the quinolinic acid (QA) phosphoribosyltransferase (QPRT) gene. QPRT activity was not detected in qprt(-/-)mice. The qprt(+/+), qprt(+/-), or qprt(-/-) mice (8 wk old) were fed a complete diet containing 30 mg nicotinic acid (NiA) and 2.3 g L-Trp/kg diet or an NiA-free diet containing 2.3 g L-Trp/kg diet for 23 d. When qprt(-/-)mice were fed a complete diet, food intake and body weight gain did not differ from those of the qprt(+/+) and qprt(+/-) mice. On the contrary, in the qprt(-/-) mice fed the NiA-free diet, food intake and body weight were reduced to 60% (P < 0.01) and 70% (P < 0.05) of the corresponding values for the qprt(-/-) mice fed the complete diet at d 23, respectively. The nutritional levels of niacin, such as blood and liver NAD concentrations, were also lower in the qprt(-/-) mice than in the qprt(+/+) and the qprt(+/-) mice. Urinary excretion of QA was greater in the qprt(-/-) mice than in the qprt(+/+) and qprt(+/-) mice (P < 0.01). These data suggest that we generated truly niacin-deficient mice. PMID:23096007

Terakata, Miki; Fukuwatari, Tsutomu; Sano, Mitsue; Nakao, Natsuki; Sasaki, Ryuzo; Fukuoka, Shin-Ichi; Shibata, Katsumi



Identification of Suitable Reference Genes for Real-Time PCR Analysis of Statin-Treated Human Umbilical Vein Endothelial Cells  

PubMed Central

Proper data normalization in quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) is of critical importance for reliable mRNA expression analysis. Due to a diversity in putative reference genes expression stability in different in vitro models, a validation of an internal control gene should be made for each particular tissue or cell type and every specific experimental design. A few approaches have been proposed for reference gene selection, including pair-wise comparison approach and model-based approach. In this article we have assessed the expression stability of eight putative reference genes: ACTB, B2M, GADD45A, GAPDH, HPRT1, PES1, PSMC4, YWHAZ, in human umbilical vein endothelial cells (HUVEC) treated with different statins and with TNF-?. The analysis was performed with three reference gene validation programs: geNorm, NormFinder and BestKeeper. We have shown that hypoxanthine phosphoribosyltransferase 1 gene (HPRT1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide gene (YWHAZ) are the most stably expressed genes among the analyzed ones. Furthermore, our results show that ?-actin gene (ACTB) is downregulated by statins and thus should not be used as a normalizing gene in a discussed experimental setup. A ranking of candidate reference genes stability values is provided and might serve as a valuable guide for future gene expression studies in endothelial cells. This is the first report on reference gene selection for RT-qPCR applications in statin-treated HUVEC model.

Zyzynska-Granica, Barbara; Koziak, Katarzyna



Purine nucleotide synthesis in lymphoblasts cultured from normal subjects and a patient with Lesch-Nyhan syndrome  

Microsoft Academic Search

Human lymphoblasts derived from normal and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient individuals have been maintained in permanent tissue culture, and comparative studies of their purine metabolism have been undertaken. In agreement with previous observations in fibroblasts, the HGPRT-deficient lymphoblasts (less than 2% normal HGPRT activity) demonstrate threefold increases in the production of purines by the de novo pathway and four- to

Alexander W. Wood; Michael A. Becker; J. Edwin Seegmiller



Amperometric Biosensor for Hypoxanthine Based on Immobilized Xanthine Oxidase on Iron (III) Meso?tetraphenylporphyrin Nanoparticles Modified Glassy Carbon Electrode  

Microsoft Academic Search

The preparation and performance of hypoxanthine (Hx) electrochemical biosensor, which was based on iron (III) meso?tetraphenylporphyrin (FeTPP) nanoparticles (NPs), is reported in this work. FeTPP NPs prepared by mixing solvent techniques with diameters ca. 25~45 nm and were used as a mediator. The XOD\\/FeTPPNP\\/GC electrode exhibited good amperometric signal for Hx. Based on the consumption of dissolved oxygen during the oxidation

Hong Min



DNA fragmentation, dATP pool elevation and potentiation of antifolate cytotoxicity in L1210 cells by hypoxanthine  

Microsoft Academic Search

Exogenous purines (greater than or equal to 10(-5)M) can modulate the cytotoxicity of methotrexate (MTX) in cultured cells, protecting cells at low MTX concentrations (less than or equal to 8 x 10(-8) M) and markedly potentiating its effect at higher concentrations. The ability of hypoxanthine (HX) to modulate the effects of two antifolates-ICI 198583 (an inhibitor of thymidylate synthetase) and

JBJ Kwok; MHN Tattersall



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

SciTech Connect

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.

Cao, Hongnan; Pauff, James M.; Hille, Russ (UCR)



Urate nephropathy associated with impaired kinetic properties of hypoxanthine phosphoribosyl transferase in a 45-day-old infant.  


We report a 45-day-old male infant who presented with anuric renal failure and fluid overload due to urate nephropathy consequent upon hyperuricemia with hyperuricosuria. His maternal uncle had undergone renal transplantation for chronic renal failure secondary to uric acid nephrolithiasis. The levels of hypoxanthine phosphoribosyl transferase (HPRT) and adenine phosphoribosyl transferase activity in the baby were found to be quantitatively normal. However, when the HPRT activity was measured at low substrate concentrations [phosphoribosyl pyrophosphate (PRPP) and hypoxanthine] and compared with usual assay conditions, the HPRT activity at lower PRPP was less in the propositus, suggesting altered enzyme kinetics. Apparent K (m(PRPP)) and V (max), but not K (m(hypoxanthine)), were then found to be higher in the propositus than the control range. This is the first case of urate nephropathy secondary to altered enzyme kinetics presenting as early as 45 days. Uric acid nephropathy should be considered in the differential diagnosis of unexplained acute kidney injury in infants. In such cases, quantitative tests for HPRT enzyme activity may not be sufficient and altered enzyme kinetics should also be investigated. PMID:21904906

Singal, Rashi; Krishnamurthy, Sriram; Narayanan, Parameswaran; Rajesh, Nachiappa Ganesh; Choudhary, Bharat; Jacomelli, Gabriella; Micheli, Vanna



Formation and trapping of free radicals in irradiated purines: EPR and ENDOR of hypoxanthine derivatives studied as single crystals  

NASA Astrophysics Data System (ADS)

Four different derivatives of hypoxanthine (hypoxanthine-HCl·H 2O, Na+·Inosine-·2.5H 2O, sodium inosine monophosphate, and calcium inosine monophosphate) were irradiated in the form of single crystals with the objective of identifying the radical products. To do so, magnetic resonance methods (EPR, ENDOR experiments and EPR spectrum simulations) were used to study radical products in crystals following x-irradiation at ˜10 K without warming, and under conditions of controlled warming. Also, computational chemistry methods were used in combination with the experimental methods to assist in identifying the radical products. Immediately following irradiation at 10 K, at least three different radicals were observed for hypoxanthine·HCl·H2O. R5.1 was identified at the product of electron addition followed by protonation of the parent at N3. R5.2 was identified as the product of electron loss followed by deprotonation at N7, and R5.3 was tentatively identified as the product of electron gain followed by protonation at 06. On warming to room temperature, three new radicals were observed: R6.1 and R6.3 were the products of net H addition to C8 and C2 respectively, while R6.2 was the product of OH addition to C8. At least four different radical products of Na+·Inosine - were detected immediately after irradiation at 10 K. R7.1 was identified as the electron-loss product of the parent hypoxanthine base, and R7.2 was identified as the product of net H-abstraction from C5 ' of the sugar. R7.3 and R7.4 were tentatively identified as the products of net H-addition to 06 (probably via electron addition followed by protonation), and the (doubly-negative) product of electron-gain, respectively. R7.5, the C8-H addition radical, was the only product detected on warming sodium inosine crystals to room temperature. Because the ENDOR spectra from sodium IMP irradiated at 10K were complex, it was possible to identify only two radicals. R8.1 was identified as the purine base electron-abstraction product, and R8.2 was identified as the 06 hydrogen-addition product. ENDOR spectra could be obtained from calcium IMP only at a few orientations. Thus, all radical identifications in this system are based on EPR spectrum simulations using likely radical structures based on results from other hypoxanthine-based systems.

Tokdemir, Sibel


Analysis of abnormalities in purine metabolism leading to gout and to neurological dysfunctions in man.  

PubMed Central

A modelling approach is used to analyse diseases associated with purine metabolism in man. The specific focus is on deficiencies in two enzymes, hypoxanthine:guanine phosphoribosyltransferase and adenylosuccinate lyase. These deficiencies can lead to a number of symptoms, including neurological dysfunctions and mental retardation. Although the biochemical mechanisms of dysfunctions associated with adenylosuccinate lyase deficiency are not completely understood, there is at least general agreement in the literature about possible causes. Simulations with our model confirm that accumulation of the two substrates of the enzyme can lead to significant biochemical imbalance. In hypoxanthine:guanine phosphoribosyltransferase deficiency the biochemical mechanisms associated with neurological dysfunctions are less clear. Model analyses support some old hypotheses but also suggest new indicators for possible causes of neurological dysfunctions associated with this deficiency. Hypoxanthine:guanine phosphoribosyltransferase deficiency is known to cause hyperuricaemia and gout. We compare the relative importance of this deficiency with other known causes of gout in humans. The analysis suggests that defects in the excretion of uric acid are more consequential than defects in uric acid synthesis such as hypoxanthine:guanine phosphoribosyltransferase deficiency.

Curto, R; Voit, E O; Cascante, M



Analysis of abnormalities in purine metabolism leading to gout and to neurological dysfunctions in man.  


A modelling approach is used to analyse diseases associated with purine metabolism in man. The specific focus is on deficiencies in two enzymes, hypoxanthine:guanine phosphoribosyltransferase and adenylosuccinate lyase. These deficiencies can lead to a number of symptoms, including neurological dysfunctions and mental retardation. Although the biochemical mechanisms of dysfunctions associated with adenylosuccinate lyase deficiency are not completely understood, there is at least general agreement in the literature about possible causes. Simulations with our model confirm that accumulation of the two substrates of the enzyme can lead to significant biochemical imbalance. In hypoxanthine:guanine phosphoribosyltransferase deficiency the biochemical mechanisms associated with neurological dysfunctions are less clear. Model analyses support some old hypotheses but also suggest new indicators for possible causes of neurological dysfunctions associated with this deficiency. Hypoxanthine:guanine phosphoribosyltransferase deficiency is known to cause hyperuricaemia and gout. We compare the relative importance of this deficiency with other known causes of gout in humans. The analysis suggests that defects in the excretion of uric acid are more consequential than defects in uric acid synthesis such as hypoxanthine:guanine phosphoribosyltransferase deficiency. PMID:9445373

Curto, R; Voit, E O; Cascante, M



Inhibition of Nicotinamide Phosphoribosyltransferase (NAMPT) Activity by Small Molecule GMX1778 Regulates Reactive Oxygen Species (ROS)-mediated Cytotoxicity in a p53- and Nicotinic Acid Phosphoribosyltransferase1 (NAPRT1)-dependent Manner*  

PubMed Central

Cancer cells undergo mitosis more frequently than normal cells and thus have increased metabolic needs, which in turn lead to higher than normal reactive oxygen species (ROS) production. Higher ROS production increases cancer cell dependence on ROS scavenging systems to balance the increased ROS. Selectively modulating intracellular ROS in cancers by exploiting cancer dependence on ROS scavenging systems provides a useful therapeutic approach. Essential to developing these therapeutic strategies is to maintain physiologically low ROS levels in normal tissues while inducing ROS in cancer cells. GMX1778 is a specific inhibitor of nicotinamide phosphoribosyltransferase, a rate-limiting enzyme required for the regeneration of NAD+ from nicotinamide. We show that GMX1778 increases intracellular ROS in cancer cells by elevating the superoxide level while decreasing the intracellular NAD+ level. Notably, GMX1778 treatment does not induce ROS in normal cells. GMX1778-induced ROS can be diminished by adding nicotinic acid (NA) in a NA phosphoribosyltransferase 1 (NAPRT1)-dependent manner, but NAPRT1 is lost in a high frequency of glioblastomas, neuroblastomas, and sarcomas. In NAPRT1-deficient cancer cells, ROS induced by GMX1778 was not susceptible to treatment with NA. GMX1778-mediated ROS induction is p53-dependent, suggesting that the status of both p53 and NAPRT1 might affect tumor apoptosis, as determined by annexin-V staining. However, as determined by colony formation, GMX1778 long term cytotoxicity in cancer cells was only prevented by the addition of NA to NAPRT1-expressing cells. Exposure to GMX1778 may be a novel way of inducing ROS selectively in NAPRT1-negative tumors without inducing cytotoxic ROS in normal tissue.

Cerna, David; Li, Hongyun; Flaherty, Siobhan; Takebe, Naoko; Coleman, C. Norman; Yoo, Stephen S.



Localization of the gene that codes for adenine phosphoribosyltransferase on the genetic map of chromosome 8 of mouse  

SciTech Connect

Polymorphism of electrophoretic mobility of adenine phosphoribosyltransferase (APRT) was found in Mus musculus bactrianus, a population of domestic mouse. The presence of intraspecies polymorphism allows localization of the gene that codes for this enzyme on the genetic map of the 8th chromosome. Two markers were utilized to map the Aprt gene: the plasmin esterase-1, which is coded by the gene Es-1 located at a distance of 26 morganids from the centromere; and the Robertsonian translocation Rb(8.17)1 Iem, which marks the centromere. Results of the linkage analysis showed the gene Aprt to be located on the genetic map of the 8th chromosome at a distance of 51 morganids from the centromere and 25 morganids distil of the gene Es-1. Also discussed in this work was the influence of emotional stress on the recombination process in the 8th chromosome.

Nesterova, T.B.; Borodin, P.M.; Zakiyan, S.M.



Discovery of potent and efficacious urea-containing nicotinamide phosphoribosyltransferase (NAMPT) inhibitors with reduced CYP2C9 inhibition properties.  


Potent, reversible inhibition of the cytochrome P450 CYP2C9 isoform was observed in a series of urea-containing nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. This unwanted property was successfully removed from the described inhibitors through a combination of structure-based design and medicinal chemistry activities. An optimized compound which did not inhibit CYP2C9 exhibited potent anti-NAMPT activity (17; BC NAMPT IC50=3 nM; A2780 antiproliferative IC50=70 nM), good mouse PK properties, and was efficacious in an A2780 mouse xenograft model. The crystal structure of this compound in complex with the NAMPT protein is also described. PMID:23668988

Gunzner-Toste, Janet; Zhao, Guiling; Bauer, Paul; Baumeister, Timm; Buckmelter, Alexandre J; Caligiuri, Maureen; Clodfelter, Karl H; Fu, Bang; Han, Bingsong; Ho, Yen-Ching; Kley, Nikolai; Liang, Xiaorong; Liederer, Bianca M; Lin, Jian; Mukadam, Sophie; O'Brien, Thomas; Oh, Angela; Reynolds, Dominic J; Sharma, Geeta; Skelton, Nicholas; Smith, Chase C; Sodhi, Jasleen; Wang, Weiru; Wang, Zhongguo; Xiao, Yang; Yuen, Po-wai; Zak, Mark; Zhang, Lei; Zheng, Xiaozhang; Bair, Kenneth W; Dragovich, Peter S



A highly sensitive and automated method for the determination of hypoxanthine based on lab-on-valve approach using Fe3O4/MWCNTs/?-CD modified electrode.  


A Fe(3)O(4)/multiwall carbon nanotubes/?-cyclodextrin (Fe(3)O(4)/MWCNTs/?-CD) modified electrode, for highly sensitive and automated hypoxanthine measurements, was developed in a sequential injection lab-on-valve system. The electrochemical oxidation behavior of hypoxanthine was investigated in phosphate buffer solution by cyclic voltammetry and linear sweep voltammetry. The Fe(3)O(4)/MWCNTs/?-CD modified electrode exhibits preferably analytical characteristics in electrocatalytic activity towards the oxidation of hypoxanthine. Under optimized conditions, the log oxidation peak current intensity was proportional to log hypoxanthine concentration covering the range from 5.0×10(-8) to 1.0×10(-5) mol L(-1) with a correlation coefficient of 0.9965. A detection limit of 0.3×10(-8) mol L(-1) was achieved along with a sampling frequency of 20 h(-1). This hyphenated system offers some advantages in terms of rapidness, sensitivity and ease of manipulation. Its further applications were utilized for the determination of hypoxanthine in meat samples. PMID:22967631

Wang, Yang; Wang, Lu; Tian, Tian; Yao, Guojun; Hu, Xiaoya; Yang, Chun; Xu, Qin



Progress and recent advances in fabrication and utilization of hypoxanthine biosensors for meat and fish quality assessment: a review.  


This review provides an update on the research conducted on the fabrication and utilization of hypoxanthine (Hx) biosensors published over the past four decades. In particular, the review focuses on progress made in the development and use of Hx biosensors for the assessment of fish and meat quality which has dominated research in this area. The various fish and meat freshness indexes that have been proposed over this period are highlighted. Furthermore, recent developments and future advances in the use of screen-printed electrodes and nanomaterials for achieving improved performances for the reliable determination of Hx in fish and meat are discussed. PMID:23141330

Lawal, Abdulazeez T; Adeloju, Samuel B



Examination of performance of glassy carbon paste electrode modified with gold nanoparticle and xanthine oxidase for xanthine and hypoxanthine detection.  


A composite electrode was prepared by modifying glassy carbon microparticles with gold nanoparticles (Au-nps) and xanthine oxidase enzyme (XOD) for xanthine (X) and hypoxanthine (Hx) detection. After the optimization of the system for X, the biosensor was characterized for X and Hx. A linearity was obtained in the concentration range between 5.00 x 10(-7) and 1.00 x 10(-5) M for X with equation of y=0.24 x + 0.712 and 5.00 x 10(-6) to 1.50 x 10(-4) M for Hx, with equation of y = 0.014 x + 0.575, respectively. Obtained results were compared to X and/or Hx biosensors including/not including Au-np in the structure. The developed system was also applied for detection of Hx in canned tuna fish sample and very promising results were obtained. PMID:18371660

Cubukçu, Meliha; Timur, Suna; Anik, Ulkü



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


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

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



Cyto- and genotoxicity of ultrafine TiO2 particles in cultured human lymphoblastoid cells.  


Titanium dioxide is frequently used in the production of paints, paper, plastics, welding rod-coating material, and cosmetics, because of its low toxicity. However, recent studies have shown that nano-sized or ultrafine TiO(2) (UF-TiO(2)) (<100 nm in diameter) can generate pulmonary fibrosis and lung tumor in rats. Cytotoxicity induced by UF-TiO(2) in rat lung alveolar macrophages was also observed. This generates great concern about the possible adverse effects of UF-TiO(2) for humans. The cytotoxicity and genotoxicity of UF-TiO(2) were investigated using the methyl tetrazolium cytotoxicity (MTT) assay, the population growth assay, the apoptosis assay by flow cytometry, the cytokinesis block micronucleus (CBMN) assay, the comet assay, and the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation assay. WIL2-NS cells were incubated for 6, 24 and 48 h with 0, 26, 65 and 130 microg/ml UF-TiO(2). Significant decreases in viability were seen in the MTT assay at higher doses; for example, 61, 7 and 2% relative viability at 130 microg/ml for 6, 24 and 48-h exposure (P<0.01). A dose-dependent relationship was observed, while a time-dependent relationship was seen only at the highest dose (130 microg/ml) after exposure for 24 and 48 h. Treatment with 130 microg/ml UF-TiO(2) induced approximately 2.5-fold increases in the frequency of micronucleated binucleated cells (P<0.01). In addition, a significant reduction in the cytokinesis block proliferation index was observed by the CBMN assay (P<0.05). In the comet assay, treatment with 65 microg/ml UF-TiO(2) induced approximately 5-fold increases in olive tail moment (P<0.05). In the HPRT mutation assay, treatment with 130 microg/ml UF-TiO(2) induced approximately 2.5-fold increases in the mutation frequency (P<0.05). The results of this study indicate that UF-TiO(2) can cause genotoxicity and cytotoxicity in cultured human cells. PMID:17223607

Wang, Jing J; Sanderson, Barbara J S; Wang, He



Transport of [14C]hypoxanthine by sheep choroid plexus epithelium as a monolayer in primary culture: Na+-dependent and Na+-independent uptake by the apical membrane and rapid intracellular metabolic conversion to nucleotides.  


Hypoxanthine is the main product of purine metabolic degradation and previous studies have revealed that it is present in the sheep CSF and plasma in micromolar concentrations. The aim of this study was to elucidate the transport of this molecule across the sheep choroid plexus epithelium (CPE) as a monolayer in primary culture, to explore the mechanism of uptake by the apical side of the CPE and investigate the metabolic changes inside the cell. The estimated permeability of the CPE monolayer for [14C]hypoxanthine, [14C]adenine and [14C]guanine was low and comparable to the permeability towards the extracellular space markers. The study of [14C]hypoxanthine uptake by the CPE revealed two components: Na+-dependent and Na+-independent, the latter being partially mediated by the equilibrative nucleoside transporter 2. HPLC with simultaneous detection of radioactivity revealed that the majority of [14C]hypoxanthine inside the CPE is metabolised into [14C]nucleotides and [14C]inosine. The remaining intact [14C]hypoxanthine was transported across the opposite, basolateral side of CPE and appeared in the lower chamber buffer together with [14C]inosine. These findings indicate two possible roles of hypoxanthine uptake from the CSF by the CP epithelium in vivo: to provide material for nucleotide synthesis through the salvage pathways in the CPE, as well as to transfer excess hypoxanthine from CSF to blood. PMID:18164814

Isakovic, Aleksandra J; Dencic, Sonja Misirlic; Segal, Malcolm B; Redzic, Zoran B



Preferential repair and strand-specific repair of benzo[a]pyrene diol epoxide adducts in the HPRT gene of diploid human fibroblasts.  

PubMed Central

If excision repair-proficient human cells are allowed time for repair before onset of S phase, the premutagenic lesions formed by (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (benzo[a]pyrene diol epoxide, BPDE) are lost from the transcribed strand of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene faster than from the nontranscribed strand. No change in strand distribution is seen with repair-deficient cells. These results suggest strand-specific repair of BPDE-induced DNA damage in human cells. To test this, we measured the initial number of BPDE adducts formed in each strand of the actively transcribed HPRT gene and the rate of repair, using UvrABC excinuclease in conjunction with Southern hybridization and strand-specific probes. We also measured the rate of loss of BPDE adducts from the inactive 754 locus. The frequencies of adducts formed by exposure to BPDE (1.0 or 1.2 microM) in either strand of a 20-kilobase fragment that lies entirely within the transcription unit of the HPRT gene were similar; the frequency in the 14-kilobase 754 fragment was approximately 20% lower. The rates of repair in the two strands of the HPRT fragment differed significantly. Within 7 hr after treatment with 1.2 microM BPDE, 53% of the adducts had been removed from the transcribed strand, but only 26% from the nontranscribed strand; after 20 hr, these values were 87% and 58%, respectively. In contrast, only approximately 14% of the BPDE adducts were lost from the 754 locus in 20 hr, a value even lower than the rate of loss from the overall genome (i.e., 38%). These results demonstrate strand-specific and preferential repair of BPDE adducts in human cells. They suggest that the heterogeneous repair of BPDE adducts in the human genome cannot be accounted for merely by the greatly increased rate of the repair specific to the transcribed strand of the active genes, and they point to a role for the chromatin structure. Images

Chen, R H; Maher, V M; Brouwer, J; van de Putte, P; McCormick, J J



Matrix-isolation FT-IR study and theoretical calculations of the vibrational, tautomeric and H-bonding properties of hypoxanthine  

Microsoft Academic Search

The neutral compound hypoxanthine is investigated using the technique of matrix-isolation FT-IR spectroscopy combined with density functional theory (DFT) and ab initio methods. Two theoretical methods (RHF and DFT\\/B3-LYP) are compared for vibrational frequency prediction, and four methods (RHF\\/\\/RHF, MP2\\/\\/RHF, DFT\\/\\/DFT and MP2\\/\\/DFT) for prediction of the relative energies of the tautomers and the interaction energies of the complexes. All

R. Ramaekers; G Maes; L Adamowicz; A Dkhissi



Human hybrid hybridoma  

SciTech Connect

Hybrid hybridomas are obtained by fusion of two cells, each producing its own antibody. Several authors have reported the construction of murine hybrid hybridomas with the aim to obtain bispecific monoclonal antibodies. The authors have investigated, in a model system, the feasibility of constructing a human hybrid hybridoma. They fused two monoclonal cell lines: an ouabain-sensitive and azaserine/hypoxanthine-resistant Epstein-Barr virus-transformed human cell line that produces an IgG1kappa antibody directed against tetanus toxiod and an azaserine/hypoxanthine-sensitive and ouabain-resistant human-mouse xenohybrid cell line that produces a human IgG1lambda antibody directed against hepatitis-B surface antigen. Hybrid hybridoma cells were selected in culture medium containing azaserine/hypoxanthine and ouabain. The hybrid nature of the secreted antibodies was analyzed by means of two antigen-specific immunoassay. The results show that it is possible, with the combined use of transformation and xenohybridization techniques, to construct human hybrid hybridomas that produce bispecific antibodies. Bispecific antibodies activity was measured by means of two radioimmunoassays.

Tiebout, R.F.; van Boxtel-Oosterhof, F.; Stricker, E.A.M.; Zeijlemaker, W.P.



Design of a new hypoxanthine biosensor: xanthine oxidase modified carbon film and multi-walled carbon nanotube/carbon film electrodes.  


A new and simple-to-prepare hypoxanthine biosensor has been developed using xanthine oxidase (XOD) immobilised on carbon electrode surfaces. XOD was immobilised by glutaraldehyde cross-linking on carbon film (CF) electrodes and on carbon nanotube (CNT) modified CF (CNT/CF). A comparison of the performance of the two configurations was carried out by the current response using amperometry at fixed potential; the best characteristics being exhibited by XOD/CNT/CF modified electrodes. The effects of electrolyte pH and applied potential were evaluated, and a proposal is made for the enzyme mechanism of action involving competition between regeneration of flavin adenine dinucleotide and reduction of hydrogen peroxide. Under optimised conditions, the determination of hypoxanthine was carried out at -0.2 V vs. a saturated calomel electrode (SCE) with a detection limit of 0.75 ?M on electrodes with CNT and at -0.3 V vs. SCE with a detection limit of 0.77 ?M on electrodes without CNT. The applicability of the biosensor was verified by performing an interference study, reproducibility and stability were investigated, and hypoxanthine was successfully determined in sardine and shrimp samples. PMID:23263517

Torres, A Carolina; Ghica, M Emilia; Brett, Christopher M A



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

SciTech Connect

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.

Grdina, D.J.; Hill, C.K.; Peraino, C. (Argonne National Lab., IL (USA)); Biserka, N. (Central Inst. for Tumors and Allied Diseases, Zagreb (Yugoslavia)); Wells, R.L. (Colorado State Univ., Fort Collins (USA). Dept. of Radiology and Radiation Biology)



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

PubMed Central

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 ?mol 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 ?mol gCDW-1). Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 ?mol 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.



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

SciTech Connect

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 damage will be needed.

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



Adenine Phosphoribosyltransferase (APRT) Deficiency  


... reddish-brown diaper stains. The 2,8-dihydroxyadenine crystals should be easily detected by urine microscopy. The ... enzyme activity. Furthermore, analysis of 2,8-DHA crystals and stone material may confirm the diagnosis. What ...


DNA fragmentation, dATP pool elevation and potentiation of antifolate cytotoxicity in L1210 cells by hypoxanthine.  


Exogenous purines (greater than or equal to 10(-5)M) can modulate the cytotoxicity of methotrexate (MTX) in cultured cells, protecting cells at low MTX concentrations (less than or equal to 8 x 10(-8) M) and markedly potentiating its effect at higher concentrations. The ability of hypoxanthine (HX) to modulate the effects of two antifolates-ICI 198583 (an inhibitor of thymidylate synthetase) and piritrexim (PTX, a lipophilic inhibitor of DHFR)-was investigated using cultured mouse leukaemic cells, L1210. HX (10(-4) M) was found to potentiate only the cytotoxicity of DHFR inhibitors (MTS and PTX), increasing cell kill by 20-70 fold to the level achieved by an equivalent concentration (10(-5) M) of ICI 198583 alone. Agarose gel electrophoresis of DNA extracted from cells exposed to antifolates for 24 h demonstrated that the chromatin was cleaved into multimers of 200 base pairs. This pattern of DNA cleavage indicates cell death via apoptosis. The degree of DNA fragmentation was found to be closely linked to cytotoxicity. DNA fragmentation increased from 50% in cells treated with 10(-5) M MTX or PTX to 70% when HX was added with the drugs, a level achieved by 10(-5)M ICI 198583 alone. HX potentiation of cytotoxicity was correlated with a substantial increase in dATP in conjunction with low dTTP pools. The specific potentiation of DHFR inhibitors by HX may be due to their inhibition of purine synthesis with a concurrent rise in PRPP levels. Addition of HX with MTX substantially raised intracellular purine levels via the salvage pathway as indicated by ribonucleotide pool measurements. ICI 198583, on the other hand, stimulated de novo purine synthesis with or without added HX. Treatment with MTX plus HX or ICI 198583 (with or without HX) caused a reduction of dTTP pools to 8% of untreated control and excess dATP accumulation. The subsequent elevation (to 300% of control) of the dATP pool may provide a signal for endonucleolytic fragmentation of DNA and subsequent cell death. PMID:1562458

Kwok, J B; Tattersall, M H



A human myeloma cell line suitable for the generation of human monoclonal antibodies  

Microsoft Academic Search

Ever since monoclonal antibodies were produced in 1975 with mouse myeloma cells there has been interest in developing human myeloma cultures for the production of monoclonal antibodies. However, despite multiple attempts, no human myeloma line suitable for hybridoma production has been described. Here we report the derivation of a hypoxanthine-aminopterin-thymidine-sensitive and ouabain-resistant human myeloma cell line (Karpas 707H) that contains

Abraham Karpas; Alla Dremucheva; Barbara H. Czepulkowski



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


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

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



Overexpression of the Orotate Phosphoribosyl-Transferase Gene Enhances the Effect of 5-Fluorouracil in Head and Neck Squamous Cell Carcinoma In Vitro  

PubMed Central

5-Fluorouracil (5-FU) is a widely used drug in head and neck squamous cell carcinoma (HNSCC). In the anabolic pathway of 5-FU, the first step in activation of the drug is phosphorylation of 5-FU by orotate phosphoribosyltransferase (OPRT), which directly metabolizes 5-FU to 5-fluorouridine monophosphate (FUMP) in the presence of 5-phosphoribosyl-1-pyrophosphate. To date, OPRT expression in the tumors has been related to the clinical response or survival of cancer patients receiving 5-FU-based chemotherapy. In this study, we examined whether OPRT expression correlates with the chemosensitivity to 5-FU and cell proliferation in HNSCC. We constitutively expressed an OPRT cDNA in an HNSCC cell line. The effects of OPRT expression on in vitro cell growth and 5-FU cytotoxicity were examined. OPRT transfection increases the cytotoxicity of 5-FU without affecting cell proliferation of HNSCC cells in vitro. These results indicate that OPRT expression plays an important role in the sensitivity of HNSCC to 5-FU chemotherapy.

Yasumatsu, Ryuji; Nakashima, Torahiko; Komune, Shizuo



Nicotinamide Phosphoribosyltransferase Is Essential for Interleukin-1?-mediated Dedifferentiation of Articular Chondrocytes via SIRT1 and Extracellular Signal-regulated Kinase (ERK) Complex Signaling*  

PubMed Central

Although much is known about interleukin (IL)-1? and its role as a key mediator of cartilage destruction in osteoarthritis, only limited information is available on IL-1? signaling in chondrocyte dedifferentiation. Here, we have characterized the molecular mechanisms leading to the dedifferentiation of primary cultured articular chondrocytes by IL-1? treatment. IL-1? or lipopolysaccharide, but not phorbol 12-myristate 13-acetate, retinoic acid, or epidermal growth factor, induced nicotinamide phosphoribosyltransferase (NAMPT) expression, showing the association of inflammatory cytokines with NAMPT regulation. SIRT1, in turn, was activated NAMPT-dependently, without any alteration in the expression level. Activation or inhibition of SIRT1 oppositevely regulates IL-1?-mediated chondrocyte dedifferentiation, suggesting this protein as a key regulator of chondrocytes phenotype. SIRT1 activation promotes induction of ERK and p38 kinase activities, but not JNK, in response to IL-1?. Subsequently, ERK and p38 kinase activated by SIRT1 also induce SIRT1 activation, forming a positive feedback loop to sustain downstream signaling of these kinases. Moreover, we found that the SIRT1-ERK complex, but not SIRT1-p38, is engaged in IL-1?-induced chondrocyte dedifferentiation via a Sox-9-mediated mechanism. JNK is activated by IL-1? and modulates dedifferentiation of chondrocytes, but this pathway is independent on NAMPT-SIRT1 signaling. Based on these findings, we propose that IL-1? induces dedifferentiation of articular chondrocytes by up-regulation of SIRT1 activity enhanced by both NAMPT and ERK signaling.

Hong, Eun-Hee; Yun, Hong Shik; Kim, Jongdoo; Um, Hong-Duck; Lee, Kee-Ho; Kang, Chang-Mo; Lee, Su-Jae; Chun, Jang-Soo; Hwang, Sang-Gu



Drugs effects on superoxide generation and chemiluminescence response of human leukocytes  

Microsoft Academic Search

Luminol-enhanced chemiluminescence was used to determine the effects of diethyldithiocarbamate, dipyridamole, catechin and verapamil on the generation of reactive oxygen species in human leukocytes, and on superoxide generated by chemiluminescence of the hypoxanthine xanthine-oxidase reaction. These agents reduced the luminol enhanced chemiluminescence response of activated leukocytes, most probably by inhibiting the superoxide generation reaction. On the other hand, citrate and

C. Pascual; R. González; C. Romay



Phosphoribosylpyrophosphate Synthesis in Cultured Human Cells  

Microsoft Academic Search

Phosphoribosylpyrophosphate (PRPP) concentrations and PRPP synthetase activity were studied in cultured human fibroblasts with control and mutant hypoxanthine-guanine phosphoribosylpyrophosphate (HPRT) activity. The PRPP concentrations increased 20- to 50-fold and PRPP synthetase activity 3-fold in cells from patients with the Lesch-Nyhan syndrome when aminopterin, an inhibitor of de novo purine synthesis, was added to the medium. Concentrations of PRPP and PRPP

Paul J. Benke; David Dittmar



Regionally-targeted mutagenesis by metabolically-activated steviol: DNA sequence analysis of steviol-induced mutants of guanine phosphoribosyltransferase (gpt) gene of Salmonella typhimurium TM677.  


Steviol is the aglycone of stevioside, a non-caloric sugar substitute commonly used in Japan. Steviol strongly induces mutations at the guanine phosphoribosyltransferase gene (gpt) of Salmonella typhimurium TM677 when the metabolic activation system (S9 mix) is present. However, it is completely negative in the reverse mutation assays using Escherichia coli WP2uvrA/pKM101 or S.typhimurium TA strains. In order to characterize the mutations induced by metabolically-activated steviol, the chromosomal gpt alleles of 24 induced (ST clones) and 16 spontaneous mutants (SP clones) of S.typhimurium TM677 were sequenced and the mutation spectra were compared. About 40% of the mutations of ST clones (nine out of 24) were localized in the region between nucleotides 280 and 330 from the starting codon ATG, whereas no mutations of SP clones were found in that region. The mutations identified in the region included transitions (three clones), transversions (four clones), a duplication and a deletion. There were no other marked differences between ST and SP clones: base-change mutations were dominant over frameshifts and deletions (ST clones, 20 versus three; SP clones, 16 versus two) and base change mutations occurred more frequently at G:C pairs rather than at A:T pairs (ST clones, 15 versus five; SP clones, 12 versus four). The possibility that metabolically-activated steviol gives pause to DNA synthesis around nucleotide 280, thereby stimulating the duplication, deletion and untargeted mutagenesis in the defined region of the gpt gene is discussed. PMID:8962426

Matsui, M; Sofuni, T; Nohmi, T



High-Frequency Mutagenesis of Human Cells and Characterization of a Mutant Unresponsive to both alpha and gamma Interferons  

Microsoft Academic Search

2fTGH is a human cell line containing the selectable marker guanine phosphoribosyltransferase regulated by alpha interferon (IFN-alpha). Two IFN-alpha-unresponsive mutants were isolated previously at a low frequency (ca. 10-8) by selecting mutagenized 2fTGH cells in selective medium containing 6-thioguanine and IFN-alpha. By using five rounds of mutagenesis, mutants can be isolated at an appreciably higher frequency, >3 * 10-7. Five

Roslyn McKendry; Joseph John; David Flavell; Mathias Muller; Ian M. Kerr; George R. Stark



Adenovirus-mediated Transduction of Escherichia coli Uracil Phosphoribosyltransferase Gene Sensitizes Cancer Cells to Low Concentrations of 5Fluorouracil1  

Microsoft Academic Search

fluorouracil (5-FU), although a widely used chemotherapuetic agent, has a limited effect in the treatment of human solid tumors due to their resistance to the cytotoxic effects of 5-FU. Escherichia coli uracil phos- phoribosyltransferase (UPRT) is a pyrimidine salvage enzyme that cata lyzes the synthesis of UMP from uracil and 5-phosphoribosyl-a-l-diphos- phate. The present study demonstrates that adenovirus-mediated transduction of

Fumihiko Kanai; Takayuki Kawakami; Hirofumi Hamada; Akiko Sudata; Yoko Yoshida; Torao Tanaka; Makoto Ohashi; Keisuke Tateishi; Yasushi Shiratori; Masao Ornata



Analysis of X-ray-Induced HPRT Mutations in CHO Cells: Insertion and Deletions.  

National Technical Information Service (NTIS)

Molecular alterations were examined in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene of 41 independent X-ray-induced thioguanine-resistant (TGR) Chinese hamster ovary (CHO) cell clones. Rapid screening of the clones by multiplex polymeras...

J. C. Fuscoe L. J. Zimmerman A. Fekete R. W. Setzer B. J. F. Rossiter




EPA Science Inventory

Molecular alterations were examined in the hypoxanthine guanine phosphoribosyltransferase (hprt) gene of 41 independent X-ray-induced thioguanine-resistant (TGR) Chinese hamster ovary (CHO) cell clones. Rapid screening of the clones by multiplex polymerase chain reaction (PCR) fo...


Targeted Deletion of Alkylpurine-DNA-N-Glycosylase in Mice Eliminates Repair of 1,N6-ethenoadenine and Hypoxanthine but not of 3,N4-ethenocytosine or 8-oxoguanine  

Microsoft Academic Search

It has previously been reported that 1,N6-ethenoadenine (? A), deaminated adenine (hypoxanthine, Hx), and 7,8-dihydro-8-oxoguanine (8-oxoG), but not 3,N4-ethenocytosine (? C), are released from DNA in vitro by the DNA repair enzyme alkylpurine-DNA-N-glycosylase (APNG). To assess the potential contribution of APNG to the repair of each of these mutagenic lesions in vivo, we have used cell-free extracts of tissues from

B. Hang; B. Singer; G. P. Margison; R. H. Hlder



Adenine metabolism in Plasmodium falciparum  

Microsoft Academic Search

Plasmodium falciparum lacks the de novo purine biosynthesis pathway and relies entirely on the salvage pathway to meet its purine nucleotide requirements. The entire flux for purine nucleotide biosynthesis in the parasite is believed to be through hypoxanthine guanine phosphoribosyltransferase (HGPRT), with the enzymes, adenosine kinase and adenine phosphoribosyltransferase (APRT) being unannotated in the Plasmodium genome database. This manuscript reports

Sonali Mehrotra; Monnanda P. Bopanna; Vinay Bulusu; Hemalatha Balaram



Transfer of purine metabolites between cells through the medium and via cell contacts in cocultures of HGPRT sup + and HGPRT sup minus cells  

SciTech Connect

Cells with and without hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity were used to examine the transfer of purine metabolites through the medium and via cell contacts. HGPRT{sup {minus}} Chinese hamster and human fibroblasts were able to incorporate {sup 3}H-labeled purine metabolite(s) from medium in which mouse HGPRT{sup +} B82 cells had been grown for 24 h with ({sup 3}H) hypoxanthine, but mouse A9 fibroblasts that were deficient in HGPRT, adenine phosphoribosyltransferase (APRT), and methylthioadenosine phosphorylase (MTAP) were unable to incorporate these metabolites. This suggests that in recipient cells incorporation is due to ({sup 3}H)MTA, which has been shown previously to be the major {sup 3}H-labeled purine metabolite to accumulate in B82 medium, being cleaved by MTAP to ({sup 3}H)adenine, which is phosphoribosylated by APRT to ({sup 3}H)AMP. Incorporation by recipient cells of metabolites from the medium is referred to as contact-independent metabolite transfer (CIMT). In autoradiograms of B82/A9 cocultures that were labeled with ({sup 3}H)hypoxanthine, grains were found over A9 that were not in contact with B82, although A9 did not act as recipients of CIMT. A9 were positive recipients of CDMT with only one of five cell lines tested, which suggested that these cells were selective communicators.

Bols, N.C.; Mosser, D.D.; Boliska, S.A. (Univ. of Waterloo, Ontario (Canada))



Genetics of Type II Glycogenosis: Assignment of the Human Gene for Acid alpha -glucosidase to Chromosome 17  

Microsoft Academic Search

We have studied somatic cell hybrids between thymidine kinase (EC deficient mouse cells and human diploid fibroblasts for the expression of human acid alpha -glucosidase (EC A deficiency in this enzyme is associated with the type II glycogenosis or Pompe disease. All 30 somatic cell hybrids selected in hypoxanthine\\/aminopterin\\/thymidine medium expressed human acid alpha -glucosidase and galactokinase (EC

Gian G. D'Ancona; James Wurm; Carlo M. Croce



Genotoxicity of alpha particles in human embryonic skin fibroblasts  

Microsoft Academic Search

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 ²³⁸ Pu and 250 kVp X rays. The survival curves resulting from exposure to ..cap alpha.. particles are exponential. The mean lethal dose, Dâ, is approximately 1.3 Gy for X rays and 0.25

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



Human somatic mutation assays as biomarkers of carcinogenesis  

SciTech Connect

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.

Compton, P.J.E.; Smith, M.T. (Univ. of California, Berkeley (United States)); Hooper, K. (California Dept. of Health Services, Berkeley (United States))



Human gene mapping using an X\\/autosome translocation  

Microsoft Academic Search

Human fibroblasts containing a translocation between the X chromosome and chromosome 15 were fused with the 6-thioguanine-resistant mouse cell line, IR. Resulting hybrids, selected in HAT medium, retained the X\\/15 chromosome. Hybrids which were counterselected in 6-thioguanine lost this chromosome. The X-linked markers glucose-6-phosphate dehydrogenase (G6PD), phosphoglycerate kinase (PGK), and hypoxanthine phosphoribosyl transferase (HPRT), and the non-X-linked markers pyruvate kinase

E. Solomon; M. Bobrow; P. N. Goodfellow; W. F. Bodmer; D. M. Swallow; S. Povey; B. Noël



A simple and sensitive method for estimating the concentration and synthesis of 5-phosphoribosyl 1-pyrophosphate in red blood cells.  


A method is presented for the determination of 5-phosphoribosyl 1-pyrophosphate (PRPP), which is based on the release of 14CO2 from [carboxyl-14C]-orotic acid by the consecutive action of orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase. The assay is simpler and less time-consuming than most methods currently employed and is equally sensitive. The method proved to be suitable for measuring low concentrations of PRPP such as found in human erythrocytes and fibroblasts. An increased PRPP concentration was observed in erythrocytes from patients with partial or complete deficiency of hypoxanthine-guanine phospho-ribosyltransferase. frp, sp,e (but not all) gouty patients and from a patient with deficiency of purine nucleoside phosphorylase. PRPP synthetase activity was measured with a method similar to the assay for PRPP. In erythrocytes with an increased PRPP concentration, PRPP synthetase activity was found to be normal at both optimal and suboptimal substrate concentrations. PMID:195752

Tax, W J; Veerkamp, J H



Massachusetts Institute of Technology progress report, January 1-July 14, 1986  

SciTech Connect

Progress is recorded on a coordinated research project on the biochemical basis of single nucleotide substitutions which result in a mutant hypoxanthine-guanine phosphoribosyltransferase (HGPRT). Reports from individual investigation relate progress in resolution of exon 3 of the human HGPRT gene, isolation of genomic RNA, mutant t-cell growth, restriction analysis of insertion/deletion events in HGPRT, HGPRT messenger DNA isolation, DNA sequencing of HGPRT genes, reduction of selection conditions for HGPRT mutants, and progress in improving the HGPRT assay. 4 refs., 2 figs., 1 tab. (DT)

Not Available



Epstein-Barr Virus and Human Chromosomes: Close Association of the Resident Viral Genome and the Expression of the Virus-Determined Nuclear Antigen (EBNA) with the Presence of Chromosome 14 in Human-Mouse Hybrid Cel  

Microsoft Academic Search

Fourteen hybrid clones derived from the fused cultures of human lymphoblastoid FV5 cells and 5-bromodeoxyuridine-resistant mouse fibroblastic MCB2 cells grown in hypoxanthine\\/aminopterin\\/thymidine selective medium were examined for the presence of Epstein-Barr virus (EBV) DNA, the expression of the virus-determined nuclear antigen (EBNA), and the presence of human chromosomes, in the course of serial passage in vitro. Among the hybrid clones

K. Yamamoto; F. Mizuno; T. Matsuo; A. Tanaka; M. Nonoyama; T. Osato



Quantitative assay for mutation in diploid human lymphoblasts using microtiter plates  

SciTech Connect

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.

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



Differential effects of reactive oxygen species on native synovial fluid and purified human umbilical cord hyaluronate  

Microsoft Academic Search

The ability of reactive oxygen species produced by triggered neutrophilic leukocytes, hypoxanthine\\/xanthine oxidase (HX\\/XAO), hydrogen peroxide, and hypochlorous acid\\/mycloperoxidase (HOC1\\/MPO) systems to degrade hyaluronate (HA) in human synovial fluid (SF) and purified umbilical cord HA was compared by measuring the molecular weight distribution of HA using high-performance liquid chromatography with a size-exclusion column. The exposure of noninflammatory SF to phorbol

Herkko Saari; Yrjö T. Konttinen; Claes Friman; Timo Sorsa



A novel class of unstable 6-thioguanine-resistant cells from dog and human kidneys  

Microsoft Academic Search

Thioguanine-resistant primary clones were grown from single cell suspensions obtained from dog and human kidneys by enzymatic digestion. In medium containing a relatively high concentration (10µg\\/ ml) of thioguanine, thioguanine-resistant primary clones arose from each source at frequencies ranging from 10-4 to 10-5. A reduction in total hypoxanthine uptake was found in the thioguanine-resistant primary clones which had developed in

Mitchell S. Turker; RAYMOND J. MONNAT JR; Ken-Ichiro Fukuchi; Patricia A. Johnston; Charles E. Ogburn; Richard E. Weller; JAMES F. PARKt; George M. Martin



Serum containing tumor necrosis factor is cytotoxic for the human malaria parasite Plasmodium falciparum.  

PubMed Central

Sera (BCG-lipopolysaccharide [LPS] serum) were obtained from mice infected with Mycobacterium bovis BCG 2 h after intravenous administration of bacterial endotoxin (LPS). Varying concentrations of sera were added to cultures of Plasmodium falciparum-infected human erythrocytes; parasite viability was assessed by hypoxanthine incorporation after 4 days in culture. At concentrations of 1 to 3%, cultures treated with BCG-LPS serum showed a two- to threefold increase in hypoxanthine incorporation; at higher concentrations (4 to 8%), hypoxanthine incorporation fell to 2 to 5% of that in control cultures. Concurrent assays with control sera (from untreated mice or mice treated with BCG or LPS alone) caused some stimulation but no inhibition at up to 8% concentration. Examination of cultures treated with BCG-LPS serum showed morphological, deterioration of parasites within erythrocytes. The presence of tumor necrosis factor in the BCG-LPS serum was confirmed by using a standard L-cell cytotoxicity assay. In addition, rabbit antiserum against partially purified tumor necrosis factor protected intraerythrocytic forms of P. falciparum from the toxic effects of BCG-LPS serum. These data suggest that the factor in BCG-LPS serum that is toxic to P. falciparum in human erythrocytes is antigenically similar or identical to tumor necrosis factor. This nonantibody mediator of killing may play a role in human malaria. Images

Haidaris, C G; Haynes, J D; Meltzer, M S; Allison, A C



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  

Microsoft Academic Search

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

Karl-Otto Fresen; Harald Zur Hausen



Role of nitroreductases but not cytochromes P450 in the metabolic activation of 1-nitropyrene in the HepG2 human hepatoblastoma cell line  

Microsoft Academic Search

1-Nitropyrene is an environmental contaminant that is mutagenic in many prokaryotic and eukaryotic systems, including the hypoxanthine-guanosine phosphoribosyl transferase (HGPRT) locus in the human hepatoma cell line HepG2. Metabolism and DNA adduct formation of [3H]1-nitropyrene in the HepG2 were quantified to understand the role of nitroreduction and\\/or cytochrome P450-mediated C-oxidation of 1-nitropyrene in DNA adduct formation and mutagenicity. In uninduced

Kimberly J Silvers; Letha H Couch; Ellen A Rorke; Paul C Howard



Uptake of purines in Plasmodium falciparum-infected human erythrocytes is mostly mediated by the human Equilibrative Nucleoside Transporter and the human Facilitative Nucleobase Transporter  

PubMed Central

Background Plasmodium parasites are unable to synthesize purines de novo and have to salvage them from the host. Due to this limitation in the parasite, purine transporters have been an area of focus in the search for anti-malarial drugs. Although the uptake of purines through the human equilibrative nucleoside transporter (hENT1), the human facilitative nucleobase transporter (hFNT1) and the parasite-induced new permeation pathway (NPP) has been studied, no information appears to exist on the relative contribution of these three transporters to the uptake of adenosine and hypoxanthine. Using the appropriate transporter inhibitors, the role of each of these salvage pathways to the overall purine transport in intraerythrocytic Plasmodium falciparum was systematically investigated. Methods The transport of adenosine, hypoxanthine and adenine into uninfected and P. falciparum-infected human erythrocytes was investigated in the presence or absence of classical inhibitors of the hFNT1, hENT1 and NPP. The effective inhibition of the various transporters by the classical inhibitors was verified using appropriate known substrates. The ability of high concentration of unlabelled substrates to saturate these transporters was also studied. Results Transport of exogenous purine into infected or uninfected erythrocytes occurred primarily through saturable transporters rather than through the NPP. Hypoxanthine and adenine appeared to enter erythrocytes mainly through the hFNT1 nucleobase transporter whereas adenosine entered predominantly through the hENT1 nucleoside transporter. The rate of purine uptake was approximately doubled in infected cells compared to uninfected erythrocytes. In addition, it was found that the rate of adenosine uptake was considerably higher than the rate of hypoxanthine uptake in infected human red blood cells (RBC). It was also demonstrated that furosemide inhibited the transport of purine bases through hFNT1. Conclusion Collectively, the data obtained in this study clearly show that the endogenous host erythrocyte transporters hENT1 and hFNT1, rather than the NPP, are the major route of entry of purine into parasitized RBC. Inhibitors of hENT1 and hFNT1, as well as the NPP, should be considered in the development of anti-malarials targeted to purine transport.



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

Microsoft Academic Search

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

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



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

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.

Kurka, K.; Griffin, G.D.



Genotoxic effects of sunlight-activated waste water in cultured mammalian cells  

SciTech Connect

Cultured Chinese hamster ovary cells were incubated with dilutions of an oil shale retort process water and exposed to nautral sunlight. An enhancement of sevenfold to ninefold was seen in photoinduced cytotoxicity (by a colony-forming assay) and mutagenicity (at the hypoxanthine phosphoribosyltransferase (HPRT) locus) for cells pretreated with the process water compared to effects seen in cells exposed to sunlight only. Significant photoinduced cytotoxicity was also observed in cultured human skin fibroblasts when exposed to the process water before being exposed to near UV (NUV) radiation. The mutation frequencies (determined for the HPRT locus) induced by the process water and NUV radiation were as great as those frequencies seen for far UV light alone. Increases in genotoxicity were observed in excision repair-deficient xeroderma pigmentosum skin fibroblasts when compared to the responses seen in normal cells. Risks to health resulting from the phototransformation of these oil shale retort process waste waters are unassessed at this time.

Strniste, G.F.; Chen, D.J.; Okinaka, R.T.



Genotoxic effects of sunlight-activated waste water in cultured mammalian cells.  


Cultured Chinese hamster ovary cells were incubated with dilutions of an oil shale retort process water and exposed to nautral sunlight. An enhancement of sevenfold to ninefold was seen in photoinduced cytotoxicity (by a colony-forming assay) and mutagenicity [at the hypoxanthine phosphoribosyltransferase (HPRT) locus] for cells pretreated with the process water compared to effects seen in cells exposed to sunlight only. Significant photoinduced cytotoxicity was also observed in cultured human skin fibroblasts when exposed to the process water before being exposed to near UV (NUV) radiation. The mutation frequencies (determined for the HPRT locus) induced by the process water and NUV radiation were as great as those frequencies seen for far UV light alone. Increases in genotoxicity were observed in excision repair-deficient xeroderma pigmentosum skin fibroblasts when compared to the responses seen in normal cells. Risks to health resulting from the phototransformation of these oil shale retort process waste waters are unassessed at this time. PMID:6954312

Strniste, G F; Chen, D J; Okinaka, R T



[Inherited disorders of uric acid metabolism--classification, enzymatic- and DNA-diagnosis].  


Uric acid is the end product of purine metabolism in human. Then, the enzymatic abnormalities, concerning purine metabolism, cause disorders of uric acid metabolism including hyperuricemia and hypouricemia. The superactivity of 5-phosphoribosyl-pyrophosphate (PRPP) synthetase and deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) caused hyperuricemia. In glycogen storage diseases of type I, III, V, and VII, decreased energy supply induces hyperuricemia by accelerating ATP degradation. Deficiencies of xanthine oxidase (XO), purine nucleoside phosphorylase (PNP), and PRPP were reported causing hypouricemia. Many methods for DNA-diagnosis were developed including Southern blot, Northern blot, PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism), PCR-RFLP (restriction fragment length polymorphism), and allele specific oligonucleotide hybridization etc. PMID:8976110

Iwahana, H; Itakura, M



Genotoxic effects of sunlight-activated waste water in cultured mammalian cells  

Microsoft Academic Search

Cultured Chinese hamster ovary cells were incubated with dilutions of an oil shale retort process water and exposed to nautral sunlight. An enhancement of sevenfold to ninefold was seen in photoinduced cytotoxicity (by a colony-forming assay) and mutagenicity (at the hypoxanthine phosphoribosyltransferase (HPRT) locus) for cells pretreated with the process water compared to effects seen in cells exposed to sunlight

G. F. Strniste; D. J. Chen; R. T. Okinaka




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...


Development to the blastocyst stage, the oxidative state, and the quality of early developmental stage of porcine embryos cultured in alteration of glucose concentrations in vitro under different oxygen tensions  

Microsoft Academic Search

BACKGROUND: Recent work has shown that glucose may induce cell injury through the action of free radicals generated by autooxidation or through hypoxanthine phosphoribosyltransferase inhibition. The effect of glucose during early in vitro culture (IVC) period of porcine embryos on their developmental competence, contents of reactive oxygen species (ROS) and glutathione (GSH), and the quality of the blastocysts yielded was

Ni Wayan Kurniani Karja; Kazuhiro Kikuchi; Mokhamad Fahrudin; Manabu Ozawa; Tamás Somfai; Katsuhiko Ohnuma; Junko Noguchi; Hiroyuki Kaneko; Takashi Nagai



Analysis of abnormalities in purine metabolism leading to gout and to neurological dysfunctions in man  

Microsoft Academic Search

A modelling approach is used to analyse diseases associated with purine metabolism in man. The specific focus is on deficiencies in two enzymes, hypoxanthine:guanine phosphoribosyltransferase and adenylosuccinate lyase. These deficiencies can lead to a number of symptoms, including neurological dysfunctions and mental retardation. Although the biochemical mechanisms of dysfunctions associated with adenylosuccinate lyase deficiency are not completely understood, there is



Normal Activity of Metabolic Pathways Involved in the Formation and Utilization of Phosphoribosylpyrophosphate in Erythrocytes of Patients with Primary Metabolic Gout  

Microsoft Academic Search

The activity of metabolic pathways involved in the formation and utilization of phosphoribosylpyrophosphate (PRPP) was studied in the erythrocytes of 34 patients with idiopathic metabolic gout. The activities of the oxidative pentose shunt, of the hypoxanthine-guanine and adenine phosphoribosyltransferases (HGPRT, APRT) and of PRPP synthetase, as well as the rates of PRPP generation and of adenine incorporation into nucleotides were

O. Sperling; Pnina Boer; Sara Brosh; Ella Elazar; A. Szeinberg; A. de Vries



Attenuated Variants of Lesch-Nyhan Disease  

ERIC Educational Resources Information Center

|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…

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.



Purine deoxynucleoside metabolism in human melanoma cells with a high spontaneous mutation rate.  


A human melanoma cell line (MM96L) had a spontaneous mutation rate at the HGPRT locus of approx. 7 times normal. The cells had elevated dATP and dGTP pools, lacked purine nucleoside phosphorylase (PNP) and were sensitive to killing by deoxyadenosine, deoxyinosine and related purines but not to inosine or hypoxanthine. Four other melanoma cell lines exhibited a range of nucleoside sensitivities and dNTP pool sizes. Failure of intact MM96L cells to degrade exogenous deoxyadenosine and deoxyinosine to hypoxanthine was confirmed by NMR of culture medium. Normal melanocytes were PNP+ and were insensitive to deoxyinosine. Comparison of the metabolites of [14C]deoxyinosine from MM96L and a PNP+ cell line of similar doubling time (HeLa) showed that both cell types produced 14C-labelled guanine and adenine nucleotides, with [14C]dATP and [14C]dADP being found in MM96L. This indicates that human sAMP synthetase or a similar enzyme catalyses the conversion of dIMP to dAMP, the resultant elevation of dATP causing base misincorporation and a mutator phenotype. PMID:8657185

Musk, P; Clark, J M; Thompson, D; Dunn, I S; Christopherson, R I; Szabados, E; Rose, S E; Parsons, P G



A new method for the selection of protein interactions in mammalian cells.  

PubMed Central

In the present study we present a new method that allows for the selection of protein interactions in mammalian cells. We have used this system to verify two interactions previously characterized in vitro. (1) The interaction between human TATA-binding protein 1 and nuclear factor kappaB and (2) the association of Homo sapiens nuclear autoantigen SP100B with human heterochromatin protein 1alpha, a protein implicated in chromatin remodelling. We observe for the first time that these interactions also occur in vivo. One protein was fused to the N-terminal half of ubiquitin, while the interacting partner was fused to the C-terminal half of ubiquitin, that was itself linked to guanine phosphoryltransferase 2 (gpt2) modified to begin with an arginine residue. Upon interaction of both proteins, ubiquitin is reconstituted, and its association with the Rgpt2 reporter is subsequently cleaved off by ubiquitin-processing enzymes. The presence of arginine in the Rgpt2 gene product leads to the degradation of the product by the N-end rule pathway. In the human fibroblast cell line HT1080HPRT(-) (that is deficient in the enzyme for hypoxanthine-guanine phosphoribosyltransferase) cells in which interaction between both proteins of interest occurs can then be selected for by hypoxanthine/aminopterin/thymine medium and counterselected against by 6-thioguanine medium. This method provides a suitable alternative to the yeast two-hybrid system and is generally applicable.

Rojo-Niersbach, E; Morley, D; Heck, S; Lehming, N



Monochromosomal hybrids for the analysis of the human genome  

SciTech Connect

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.

Athwal, R.S.



Generation of functional human T cell hybrids  

PubMed Central

Human T cell hybrids were generated by fusing lectin-activated normal and leukemic human T cells with an aminopterin-sensitive human T cell line. This mutant cell line, designated CEM-T15, was derived from the human T cell line CEM after chemical mutagenesis with ethane methylsulfonate and subsequent culture in medium containing 6- thioguanine. After polyethylene glycol-induced fusion, the cells were cultured in hypoxanthine-aminopterin-thymidine selective medium. More than 5 wk after fusion, evidence for successful hybridization was obtained by three independent criteria: (a) The majority of the cultures contained cells expressing the OKT3 surface antigen: this antigen is expressed on normal T cells but not on CEM-T15 cells. (b) Most of the cultures contained polyploid cells. (c) Some of the cultures provided helper activity in the generation of antibody-forming cells. This functional activity is absent from the CEM-T15 parental cell line. Evidence for functional stability of the hybrids greater than 20 wk after fusion was provided by several clones that not only continue growing exponentially but also maintain expression of OKT3 surface antigen and high levels of helper function. These T cell hybrids constructed using antigen-specific human T cells should be of considerable importance in further studies of the immunobiology of human T cells.



Effects of Stuffer DNA on Transgene Expression from Helper-Dependent Adenovirus Vectors  

PubMed Central

We have analyzed transgene (lacZ) expression from a first-generation adenovirus (Ad) vector in comparison to helper-dependent (hd) Ads deleted for various portions of the viral coding sequences and generated by using the Cre/loxP helper-dependent system (R. J. Parks et al., Proc. Natl. Acad. Sci. USA 93:13565–13570, 1996). An hd vector deleted for approximately 70% of the Ad genome (AdRP1001) provided levels and durations of transgene expression similar to those of a control first generation Ad vector containing an identical expression cassette. Deletion of all Ad sequences from the hdAd and replacement with a ?22-kb fragment of lambda DNA resulted in a decrease in the level and duration of lacZ expression which could not be reversed by the inclusion of a matrix attachment region. However, substitution of the lambda stuffer in the fully deleted hdAd with sequences from the human hypoxanthine-guanine phosphoribosyltransferase gene resulted in significantly improved transgene expression. In vitro assays for cytotoxic T lymphocytes (CTL) directed against putative peptides encoded by the vector backbone showed that, although CTL were generated against the vector containing the lambda DNA, no such CTL were generated against the vector containing the hypoxanthine-guanine phosphoribosyltransferase (HPRT) sequences. Surprisingly, the rate of loss of the HPRT- and lambda-containing vectors from mouse liver was similar, despite the differences in expression kinetics, indicating that the lambda stuffer-directed CTL were inefficient at eliminating the transduced cells. Thus, the nature of the DNA backbone of hdAds can have important effects on the functioning of the vector. Since most fully deleted vectors require “stuffer” DNA as part of the vector backbone to maintain optimum vector size, these observations must be taken into account in the design of hdAd vectors.

Parks, Robin J.; Bramson, Jonathan L.; Wan, Yonghong; Addison, Christina L.; Graham, Frank L.



Transfer of human genes conferring resistance to methylating mutagens, but not to UV irradiation and cross-linking agents, into Chinese hamster ovary cells.  

PubMed Central

Chinese hamster ovary cells were transfected by human DNA ligated to the bacterial gpt (xanthine-guanine-phosphoribosyltransferase) gene which was used either in its native form or after partial inactivation with methylnitrosourea. The gpt+ transfectants were screened for resistance to high doses of N-methyl-N'-nitro-N-nitrosoguanidine. Using this approach, we showed that Chinese hamster ovary cells can acquire N-methyl-N'-nitro-N-nitrosoguanidine resistance upon transfection with DNA from diploid human fibroblasts, that this resistance is transferable by secondary transfection and is specific for methylating mutagens, and that it is not caused by increased removal of O6-methylguanine, 3-methyladenine, and 7-methylguanine from DNA. Images

Kaina, B; Van Zeeland, A A; Backendorf, C; Thielmann, H W; Van de Putte, P



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

SciTech Connect

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.

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



The genotoxicity of alpha particles in human embryonic skin fibroblasts.  


Cell inactivation and induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus have been measured in cultured human fibroblasts (GM10) exposed to alpha particles from 238Pu (LET at the cell surface was 100 keV/microns) and 250 kVp X rays. The survival curves resulting from exposure to alpha particles are exponential. The mean lethal dose, D0, is approximately 1.3 Gy for X rays and 0.25 Gy for alpha particles. As a function of radiation dose, mutation induction at the HGPRT locus was linear for 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 alpha particles than to X rays. PMID:6494443

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



Genotoxicity of alpha particles in human embryonic skin fibroblasts  

SciTech Connect

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.

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



Monoclonal lupus autoantibody secretion by human-human hybridomas. Selection of hybrids by conventional and novel techniques.  

PubMed Central

Autoantibody-secreting hybridomas were produced by somatic cell fusion of B lymphocytes from a patient with systemic lupus erythematosus with two different human myeloma lines. Selection of hybrids formed from one of these cell lines was performed by using aminopterine-containing culture medium as this cell line was deficient in hypoxanthine-guanine-phosphoribosyl transferase (HGPRT). The second myeloma line was not HGPRT-deficient but instead was treated with diethylpyrocarbonate, which assured death of unfused myeloma cells. This novel technique has wide applicability. Hybridomas were found to secrete antibodies to native DNA and to extractable nuclear antigen. The binding specificities of one IgM anti-DNA antibody was characterized and found to be specific for double-stranded DNA and had particular binding affinity for poly(dG) . poly(dC).

Littman, B H; Muchmore, A V; Steinberg, A D; Greene, W C



Purine Salvage Pathways among Borrelia Species?  

PubMed Central

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.

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



Metabolite target analysis of human urine combined with pattern recognition techniques for the study of symptomatic gout.  


Recurrent attacks and irregularity are two important characteristics of gout disease. Uric acid as a single evaluation indicator for clinical diagnosis is insufficient considering the versatile properties of gout. The aim of this work is to identify several endogenous metabolites from urine samples for the elucidation and prediction of gout disease. Metabolite target analysis was established for human urine by high performance liquid chromatography-diode array detection (HPLC-DAD). The targeted metabolites selected included hippuric acid, uracil, phenylalanine, tryptophan, uric acid and creatinine as well as nine purine compounds. Useful information was extracted from multivariate data through Fisher Linear Discriminant Analysis (FDA) and Orthogonal Signal Correction Partial Least Squares Discriminant Analysis (OSC-PLS-DA). Uric acid, hypoxanthine, xanthosine, guanosine, inosine and tryptophan were identified as important metabolites among the acute and chronic gout and controls. Based on OSC-PLS-DA models, the regression equations obtained could discriminate gout from the controls as well as the acute from chronic. The recognition and prediction ability is respectively 100% and 85.0% for the gout, 100% and 83.3% for the acute, and 90.91% and 89.9% for the chronic. Metabolic dysfunction of tryptophan and excessive metabolism of xanthosine and hypoxanthine to xanthine were confirmed for gout disease. Metabolic dysfunction of tryptophan was also proven to be induced by allopurinol in case of Kunming mice with hyperuricemia. Potential biomarkers can be used not only to distinguish gout patients from healthy people, but also to evaluate the disease state. PMID:22932763

Liu, Yun; Yu, Pinhua; Sun, Xiaoming; Di, Duolong



Is ZMP the toxic metabolite in Lesch-Nyhan disease?  


The genetic deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT), located on the X chromosome, causes a severe neurological disorder in man, known as Lesch-Nyhan disease (LND). The enzyme HPRT is part of the savage pathway of purine biosynthesis and catalyzes the conversion of hypoxanthine and guanine to their respective nucleotides, IMP and GMP. HPRT deficiency is associated with a relatively selective dysfunction of brain dopamine systems. Several metabolites that accumulate in the patients (phosphoribosylpyrophosphate (PRPP), hypoxanthine, guanine, xanthine, and Z-nucleotides) have been proposed as toxic agents in LND. Some authors have pointed that Z-riboside, derived from the accumulation of ZMP, could be the toxic metabolite in LND. However, the available experimental data support a better hypothesis. I suggest that ZMP (and not Z-riboside) is the key toxic metabolite in LND. ZMP is an inhibitor of the bifunctional enzyme adenylosuccinate lyase, and a deficiency of this enzyme causes psychomotor and mental retardation in humans. Moreover, it has been reported that ZMP inhibits mitochondrial oxidative phosphorylation and induces apoptosis in certain cell types. ZMP is also an activator of the AMP-activated protein kinase (AMPK), a homeostatic regulator of energy levels in the cell. The AMPK has been implicated in the regulation of cell viability, catecholamine biosynthesis and cell structure. I propose that accumulation of ZMP will induce a pleiotropic effect in the brain by (1) a direct inhibition of mitochondrial respiration and the bifunctional enzyme adenylosuccinate lyase, and (2) a sustained activation of the AMPK which in turns would reduce cell viability, decrease dopamine synthesis, and alters cell morphology. In addition, a mechanism to explain the accumulation of ZMP in LND is presented. The knowledge of the toxic metabolite, and the way it acts, would help to design a better therapy. PMID:18710792

López, José M



Inhibition of salvage pathway enzymes by adenine arabinoside 5?-monophosphate (ara-AMP)  

Microsoft Academic Search

Summary The effect of 9-ß-arabinofuranosyladenine 5'-monophosphate (ara-AMP) on the purine salvage pathway has been studied. On a dose-dependant basis ara-AMP inhibits the incorporation of adenine-8-14C into nucleotides in intact erythrocytes. The partially purified enzymes of the purinc salvage pathway, the adenine phosphoribosyltransferase and the 5'-phosphoribosyl-1-pyrophosphate (PP-ribose-P) synthetase, but not the hypoxanthine-guanine phosphoribosyltransferase, are inhibited by ara-AMP in a non-competitive manner.

H. J. Becher; P. Schollmeyer



Mutagenicity of the mycotoxin patulin in cultured Chinese hamster V79 cells, and its modulation by intracellular glutathione  

Microsoft Academic Search

Because the ability of the mycotoxin patulin (PAT) to cause gene mutations in mammalian cells is still ambiguous, we have studied the mutagenicity of PAT at the hypoxanthine–guanine phosphoribosyltransferase (HPRT) gene locus in cultured Chinese hamster V79 cells with normal, depleted, and elevated glutathione (GSH) levels. PAT was more toxic to GSH-depleted cells than to normal cells and caused an

David M. Schumacher; Manfred Metzler; Leane Lehmann



Identification of a novel tissue-specific processed HPRT gene and comparison with X-linked gene transcription in the Australian marsupial Macropus robustus  

Microsoft Academic Search

The genome of the Australian marsupial Macropus robustus contains a highly conserved processed hypoxanthine phosphoribosyltransferase homologue, HPRT-2. Using the techniques of reverse transcriptase-polymerase chain reaction (RT-PCR) and protein isoelectric focusing (IEF) we have shown this processed gene to be fully functional, but liver specific. In contrast, the unprocessed X-linked parent gene HPRT-1 was expressed in all somatic tissues. Expression of

Leonie Noyce; Jason Conaty; Anita A. Piper



Molecular Analysis of X-Linked Inborn Errors of Purine Metabolism: HPRT1 and PRPS1 Mutations  

Microsoft Academic Search

Mutations of two enzyme genes, HPRT1 encoding hypoxanthine guanine phosphoribosyltransferase (HPRT) and PRPS1 encoding a catalytic subunit (PRS-I) of phosphoribosylpyrophosphate synthetase, cause X-linked inborn errors of purine metabolism. Analyzing these two genes, we have identified three HPRT1 mutations in Lesch–Nyhan families following our last report. One of them, a new mutation involving the deletion of 4224 bp from intron 4

Yasukazu Yamada; Kenichiro Yamada; Noriko Nomura; Arisa Yamano; Reiko Kimura; Misako Naiki; Daisuke Fukushi; Nobuaki Wakamatsu; Atsuo Taniguchi; Noriko Yamaoka; Kiyoko Kaneko; Shin Fujimori



Molecular Analysis of Two Enzyme Genes, HPRT1 and PRPS1, Causing X-Linked Inborn Errors of Purine Metabolism  

Microsoft Academic Search

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. On the other hand, PRPS1 mutations cause PRPP synthetase superactivity associated with hyperuricemia and gout, sometimes including neurodevelopmental abnormalities. We have identified two mutations in two Lesch-Nyhan families after our last report. One of them, a new single nucleotide substitution (130G>T) resulting in a missense

Y. Yamada; K. Yamada; N. Nomura; A. Yamano; R. Kimura; S. Tomida; M. Naiki; N. Wakamatsu



Identification of Valid Housekeeping Genes and Antioxidant Enzyme Gene Expression Change in the Aging Rat Liver  

Microsoft Academic Search

Valid housekeeping genes (HKG) are a prerequisite for accurate gene quantification. We performed real-time reverse transcription-polymerase chain reaction to investigate the gene expression of five commonly used HKGs (b-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), hypoxanthine phosphoribosyl-transferase (HPRT), and cyclo- philin A (CYPa)) and antioxidant enzymes in the liver of young and old male Fischer rats. A wide variation in

Jie Chen; David A. Rider; Runsheng Ruan



Molecular Characterization of a Deletion in the HPRT1 Gene in a Patient with Lesch–Nyhan Syndrome  

Microsoft Academic Search

Lesch–Nyhan syndrome is caused by a deficiency of hypoxanthine phosphoribosyltransferase (HPRT) encoded by HPRT1. About 20% of patients have a deletion of HPRT1 and large deletions of HPRT1 are not always fully characterized at the molecular level. Here, we report on a case of Lesch–Nyhan syndrome with a 33-kb deletion involving exon 1 of HPRT1. This novel mutation is caused

A. Taniguchi; Y. Yamada; M. Hakoda; C. Sekita; M. Kawamoto; H. Kaneko; H. Yamanaka



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

SciTech Connect

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.

Maher, V.M.; Yang, Jialing; Chen, Rueyhwa; McGregor, W.G.; Lukash, L.; Scheid, J.M.; Reinhold, D.S.; McCormick, J.J. (Michigan State Univ., East Lansing (United States))



Genotoxic damage of human adipose-tissue derived mesenchymal stem cells triggers their terminal differentiation.  


Human adipose tissue-derived mesenchymal (stromal) stem cells (AT-MSCs) and genetically modified to express cytosine deaminase:uracil phosphoribosyltransferase (CDy-AT-MSCs) were treated with hydrogen peroxide in order to induce DNA damage and subsequently evaluate their genetic stability by single cell gel electrophoresis. Both cells types (parental and transgene modified) did not differ in the sensitivity to DNA breaks induction. Potential tumorigenicity of AT-MSCs and CDy-AT-MSCs was tested by subcutaneous inoculation of cell suspension into flank of immunocompromised mice. Dose of 15x10(6) cells was not found to be tumorigenic in given experimental setup. AT-MSCs, CDy-AT-MSCs and MSCs isolated from human lipoma were treated with chemical carcinogen 4-nitroquinoline-1-oxide (4NQO) in attempts to transform them. Surviving cells after genotoxic stress were not transformed but underwent replicative senescence. Irreparable DNA damage caused triggered adipogenic terminal differentiation, rather than apoptosis induction in all kinds of cells tested. PMID:19728764

Altanerova, V; Horvathova, E; Matuskova, M; Kucerova, L; Altaner, C



Comparative anatomy of the human APRT gene and enzyme: nucleotide sequence divergence and conservation of a nonrandom CpG dinucleotide arrangement  

SciTech Connect

The functional human adenine phosphoribosyltransferase (APRT) gene is <2.6 kilobases in length and contains five exons. The amino acid sequences of APRTs have been highly conserved throughout evolution. The human enzyme is 82%, 90%, and 40% identical to the mouse, hamster, and Escherichia coli enzymes, respectively. The promoter region of the human APRT gene, like that of several other housekeeping genes, lacks TATA and CCAAT boxes but contains five GC boxes that are potential binding sites for the Sp1 transcription factor. The distal three, however, are dispensable for gene expression. Comparison between human and mouse APRT gene nucleotide sequences reveals a high degree of homology within protein coding regions but an absence of significant homology in 5' flanking, 3' untranslated, and intron sequences, except for similarly positioned GC boxes in the promoter region and a 26-base-pair region in intron 3. This 26-base-pair sequence is 92% identical with a similarly positioned sequence in the mouse gene and is also found in intron 3 of the hamster gene, suggesting that its retention may be a consequence of stringent selection. The positions of all introns have been precisely retained in the human and both rodent genes. Retention of an elevated CpG dinucleotide content, despite loss of sequence homology, suggests that there may be selection for CpG dinucleotides in these regions and that their maintenance may be important for APRT gene function.

Broderick, T.P.; Schaff, D.A.; Bertino, A.M.; Dush, M.K.; Tischfield, J.A.; Stambrook, P.J.



Establishment and characterization of human T hybrid cells secreting immunoregulatory molecules.  

PubMed Central

Hybridization of human T cells with an azaguanine-resistant human T cell line gave rise to T hybrid cell lines secreting several immunoregulatory molecules. Analyses of karyotypes, HLA phenotypes, and other surface phenotypes, such as T-cell-specific antigens or receptors for sheep erythrocytes and the patterns of mitogen responsiveness confirmed that the hypoxanthine/aminopterin/thymidine-resistant cell lines were human T-T hybridomas. One of the established hybrid clones (24-A) secreted human interleukin-2 (IL-2). The culture supernatants induced the proliferation of concanavalin A-stimulated murine T cells and supported the proliferation of an IL 2-dependent human cytotoxic T-cell line. In a clone (38-B) that did not show any IL-2 activity in culture supernatants, the addition of macrophages induced IL-2 production in the presence of phytohemagglutinin, suggesting that interleukin-1 induced IL-2 production in T hybrid cells. Hybrid cells secreting killer helper factor were also established. The culture supernatants from this clone, 55-A, helped the induction of cytotoxic T cells against UV-treated human B-blastoid cells but did not show any IL-2 activity.

Okada, M; Yoshimura, N; Kaieda, T; Yamamura, Y; Kishimoto, T



Base modification and strand breakage in isolated calf thymus DNA and in DNA from human skin epidermal keratinocytes exposed to peroxynitrite or 3-morpholinosydnonimine.  


Exposure of isolated calf thymus DNA and human skin epidermal keratinocytes to peroxynitrite or the peroxynitrite generator, 3-morpholinosydnonimine (SIN-1), led to extensive DNA base modification. Large increases in xanthine and hypoxanthine, possible deamination products of guanine and adenine, respectively, and in 8-nitroguanine were observed, but only small changes in some oxidized base products were seen. This pattern of damage suggests that hydroxyl radicals were not major contributors to base modification caused by peroxynitrite, as OH is known to cause multiple oxidative modifications to all four DNA bases. Instead, it seems that reactive nitrogen species play a much greater role in the mechanism of base damage, producing both nitration and deamination of purine bases when DNA or whole cells are exposed to peroxynitrite. If this pattern of damage is unique to peroxynitrite, it might act as a marker of cellular damage by this species in vivo. PMID:8902271

Spencer, J P; Wong, J; Jenner, A; Aruoma, O I; Cross, C E; Halliwell, B


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

PubMed Central

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.

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



Differential activation of the hprt gene on the inactive X chromosome in primary and transformed Chinese hamster cells.  

PubMed Central

We have investigated the genetic activation of the hprt (hypoxanthine-guanine phosphoribosyltransferase) gene located on the inactive X chromosome in primary and transformed female diploid Chinese hamster cells after treatment with the DNA methylation inhibitor 5-azacytidine (5azaCR). Mutants deficient in HPRT were first selected by growth in 6-thioguanine from two primary fibroblast cell lines and from transformed lines derived from them. These HPRT- mutants were then treated with 5azaCR and plated in HAT (hypoxanthine-methotrexate-thymidine) medium to select for cells that had reexpressed the hprt gene on the inactive X chromosome. Contrary to previous results with primary human cells, 5azaCR was effective in activating the hprt gene in primary Chinese hamster fibroblasts at a low but reproducible frequency of 2 x 10(-6) to 7 x 10(-6). In comparison, the frequency in independently derived transformed lines varied from 1 x 10(-5) to 5 x 10(-3), consistently higher than in the nontransformed cells. This increase remained significant when the difference in growth rates between the primary and transformed lines was taken into account. Treatment with 5azaCR was also found to induce transformation in the primary cell lines but at a low frequency of 4 x 10(-7) to 8 x 10(-7), inconsistent with a two-step model of transformation followed by gene activation to explain the derepression of hprt in primary cells. Thus, these results indicate that upon transformation, the hprt gene on the inactive Chinese hamster X chromosome is rendered more susceptible to action by 5azaCR, consistent with a generalized DNA demethylation associated with the transformation event or with an increase in the instability of an underlying primary mechanism of X inactivation.

Grant, S G; Worton, R G



Radiation-induced HPRT mutations resulting from misrejoined DNA double-strand breaks.  


DNA double-strand breaks (DSBs) are the most severe lesions induced by ionizing radiation, and unrejoined or misrejoined DSBs can lead to cell lethality, mutations and the initiation of tumorigenesis. We have investigated X-ray- and alpha-particle-induced mutations that inactivate the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene in human bladder carcinoma cells and in hTERT-immortalized human fibroblasts. Fifty to 80% of the mutants analyzed exhibited partial or total deletions of the 9 exons of the HPRT locus. The remaining mutants retained unaltered PCR products of all 9 exons but often displayed a failure to amplify the HPRT cDNA. Hybridization analysis of a 2-Mbp NotI fragment spanning the HPRT gene with a probe 200 kbp distal to the HPRT locus indicated altered fragment sizes in most of the mutants with a wild-type PCR pattern. These mutants likely contain breakpoints for genomic rearrangements in the intronic sequences of the HPRT gene that allow the amplification of the exons but prevent HPRT cDNA amplification. Additionally, mutants exhibiting partial and total deletions of the HPRT exons also frequently displayed altered NotI fragments. Interestingly, all mutations were very rarely associated with interchromosomal exchanges analyzed by FISH. Collectively, our data suggest that intrachromosomal genomic rearrangements on the Mbp scale represent the prevailing type of radiation-induced HPRT mutations. PMID:18494542

Rothkamm, Kai; Gunasekara, Kut; Warda, Salam A; Krempler, Andrea; Löbrich, Markus



Efficient delivery of RNA interference oligonucleotides to polarized airway epithelia in vitro.  


Polarized and pseudostratified primary airway epithelia present barriers that significantly reduce their transfection efficiency and the efficacy of RNA interference oligonucleotides. This creates an impediment in studies of the airway epithelium, diminishing the utility of loss-of-function as a research tool. Here we outline methods to introduce RNAi oligonucleotides into primary human and porcine airway epithelia grown at an air-liquid interface and difficult-to-transfect transformed epithelial cell lines grown on plastic. At the time of plating, we reverse transfect small-interfering RNA (siRNA), Dicer-substrate siRNA, or microRNA oligonucleotides into cells by use of lipid or peptide transfection reagents. Using this approach we achieve significant knockdown in vitro of hypoxanthine-guanine phosphoribosyltransferase, IL-8, and CFTR expression at the mRNA and protein levels in 1-3 days. We also attain significant reduction of secreted IL-8 in polarized primary pig airway epithelia 3 days posttransfection and inhibition of CFTR-mediated Cl? conductance in polarized air-liquid interface cultures of human airway epithelia 2 wk posttransfection. These results highlight an efficient means to deliver RNA interference reagents to airway epithelial cells and achieve significant knockdown of target gene expression and function. The ability to reliably conduct loss-of-function assays in polarized primary airway epithelia offers benefits to research in studies of epithelial cell homeostasis, candidate gene function, gene-based therapeutics, microRNA biology, and targeting the replication of respiratory viruses. PMID:23624792

Ramachandran, Shyam; Krishnamurthy, Sateesh; Jacobi, Ashley M; Wohlford-Lenane, Christine; Behlke, Mark A; Davidson, Beverly L; McCray, Paul B



The spectrum of inherited mutations causing HPRT deficiency: 75 new cases and a review of 196 previously reported cases.  


In humans, mutations in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) are associated with a spectrum of disease that ranges from hyperuricemia alone to hyperuricemia with profound neurological and behavioral dysfunction. Previous attempts to correlate different types or locations of mutations with different elements of the disease phenotype have been limited by the relatively small numbers of available cases. The current article describes the molecular genetic basis for 75 new cases of HPRT deficiency, reviews 196 previously reported cases, and summarizes four main conclusions that may be derived from the entire database of 271 mutations. First, the mutations associated with human disease appear dispersed throughout the hprt gene, with some sites appearing to represent relative mutational hot spots. Second, genotype-phenotype correlations provide no indication that specific disease features associate with specific mutation locations. Third, cases with less severe clinical manifestations typically have mutations that are predicted to permit some degree of residual enzyme function. Fourth, the nature of the mutation provides only a rough guide for predicting phenotypic severity. Though mutation analysis does not provide precise information for predicting disease severity, it continues to provide a valuable tool for genetic counseling in terms of confirmation of diagnoses, for identifying potential carriers, and for prenatal diagnosis. PMID:11018746

Jinnah, H A; De Gregorio, L; Harris, J C; Nyhan, W L; O'Neill, J P



Killing of human malaria parasites by macrophage secretory products.  

PubMed Central

The susceptibility of the human malaria parasite, Plasmodium falciparum, to killing in vitro by macrophage secretory products was investigated. The effect of O2 radicals and tumor necrosis factor on parasite viability was assessed both morphologically and by following the uptake of [3H]hypoxanthine. H2O2 produced by the interaction of glucose and glucose oxidase was found to reduce viability; this effect was reversed by the addition of exogenous catalase. Further studies indicated that the catalase level within the erythrocyte was not altered upon parasite invasion. O2 radicals produced during the xanthine-xanthine oxidase interaction also killed P. falciparum. The addition of various O2 radical scavengers (including catalase) did not reverse this effect; therefore, it was not possible to determine which of the O2 radicals were involved in the killing process. Samples from three different sources containing tumor necrosis factor, a nonspecific soluble mediator derived from Mycobacterium bovis BCG-activated macrophages treated with endotoxin, also killed the parasite. There was no evidence that tumor necrosis factor or the products of the xanthine-xanthine oxidase interaction caused damage to the erythrocyte membrane that could be implicated as an important aspect of the killing process. These findings all strongly suggest that such macrophage products play an important role in immunity to malaria.

Wozencraft, A O; Dockrell, H M; Taverne, J; Targett, G A; Playfair, J H



Adipose tissue-derived human mesenchymal stem cells mediated prodrug cancer gene therapy.  


Human adipose tissue-derived mesenchymal stem cells (AT-MSC) are considered to be a promising source of autologous stem cells in personalized cell-based therapies. Tumor tracking properties of MSC provide an attractive opportunity for targeted transgene delivery into the sites of tumor formation. In the present study, we addressed whether the suicide gene introduction into human AT-MSC could produce a tumor-specific prodrug converting cellular vehicle for targeted chemotherapy. We prepared yeast fusion cytosine deaminase::uracil phosphoribosyltransferase gene-expressing cells [cytosine deaminase (CD)-expressing AT-MSC (CD-AT-MSC)] by retrovirus transduction. We explored their therapeutic potential on a model of human colon cancer in the presence of prodrug 5-fluorocytosine (5-FC). Gene manipulation of human AT-MSC did not sensitize CD-AT-MSC to 5-FC, thus overcoming the inherent disadvantage of suicide effect on cellular vehicle. CD-AT-MSC in combination with 5-FC augmented the bystander effect and selective cytotoxicity on target tumor cells HT-29 in direct coculture in vitro. We confirmed directed migration ability of AT-MSC and CD-AT-MSC toward tumor cells HT-29 in vitro. Moreover, we achieved significant inhibition of s.c. tumor xenograft growth by s.c. or i.v. administered CD-AT-MSC in immunocompromised mice treated with 5-FC. We confirmed the ability of CD-AT-MSC to deliver the CD transgene to the site of tumor formation and mediate strong antitumor effect in vivo. Taken together, these data characterize MSC derived from adipose tissue as suitable delivery vehicles for prodrug converting gene and show their utility for a personalized cell-based targeted cancer gene therapy. PMID:17616689

Kucerova, Lucia; Altanerova, Veronika; Matuskova, Miroslava; Tyciakova, Silvia; Altaner, Cestmir



Development of a human T-T cell hybridoma secreting B cell growth factor  

PubMed Central

The success of long-term culture of normal human and murine B cells has been hampered by the limited availability of soluble factors capable of maintaining proliferation of activated B lymphocytes. Previous experiments using various culture-derived supernatants in a human system were unable to separate the activities of B cell growth factor (BCGF) and interleukin 2 (IL-2) by immunochemical means. Thus, purified factors with BCGF activity in the absence of IL-2 activity have not been available for study. In the present study, normal human peripheral blood T cells were fused with the hypoxanthine/aminopterin/thymidine- sensitive human T-leukemic cell line, CEM-6. Supernatants from the resulting hybrid cells were tested for the ability to maintain proliferation of normal human B cells in a recently described assay system for human BCGF. Hybrids demonstrating BCGF activity were cloned by limiting dilution. One hybrid clone, 2B11, continued to support proliferation of B cells in both long-term cultures and 6-d assays at a level significantly above that seen with conventionally produced growth factors. No IL-2 activity was found in the supernatant from hybrid 2B11. The hybridoma supernatant was fractionated by gel filtration, and maximum proliferation of B cells was supported by the 18-20,000 mol wt protein fraction. Thus, a human T-T cell hybridoma that has BCGF activity in the absence of any demonstrable IL-2 activity has been developed. Human T-T cell hybridomas secreting discrete immunoregulatory factors should prove to be powerful tools in dissecting the mechanisms of immunoregulation of human lymphocyte function.



Characterization of a human antigen specific helper factor  

SciTech Connect

While antigen (Ag) specific helper factors have been characterized in mice, similar molecules have not been identified in humans. To characterize human antigen specific helper molecules, an IL-2 dependent tetanus toxoid (T.T.) reactive T cell line was fused with a 6-thioguanine resistant CEM line, and hybrids selected in medium containing hypoxanthine and azaserine. Hybrids were screened by culturing the cells with /sup 35/S-Met then reacting the supernatants with T.T. or hepatitis vaccine immobilized on nitrocellulose. One hybrid, TT6BA-O, was identified which secreted a Met-containing molecule which bound T.T. but not hepatitis vaccine. Supernatants from TT6BA-O, but not the parent CEM line, when added to autologous peripheral blood mononuclear cells (PBMC's) stimulated secretion of T.T. specific antibodies (Abs). Specificity controls demonstrated that TT6BA-O supernatant did not induce antibodies to diphtheria toxoid, hepatitis vaccine or pneumococcal polysaccharide, and total immunoglobulin (lg) synthesis was minimally increased. In contrast, pokeweed mitogen stimulated significant lg synthesis as well as Ab's to pneumococcal polysaccharide and T.T. TT6BA-O supernatant induced anti-T.T.Ab's in autologous PBMC's but not PBMC's from 3 unrelated donors, suggesting that the activity of the helper factor is restricted, possibly by the MHC. The molecular weight of the helper factor was estimated at 100,000-150,000 by Sephacryl S-300 chromatography. Finally, the helper factor could be demonstrated to bind and elute from sephorose-immobilized T.T. and anti-DR antisera, but not anti-lg antisera or the T40/25 monoclonal antibody, which binds a nonpolymorphic determinant on the human T cell receptor. These results demonstrate that human Ag specific helper factors exist, bind antigen and bear class II MHC determinants.

Richardson, B.



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

SciTech Connect

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 hygromcying 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 lymphoblastodi cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10/sup 6/ to 10/sup 7/ independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBP-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 hypoxanthing-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2. Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10/sup 6/ total clones or one clone of EBO-pcD-Leu-2 per 2 x 10/sup 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.

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



Effects of pro-inflammatory cytokines on expression of kynurenine pathway enzymes in human dermal fibroblasts  

PubMed Central

Background The kynurenine pathway (KP) is the main route of tryptophan degradation in the human body and generates several neuroactive and immunomodulatory metabolites. Altered levels of KP-metabolites have been observed in neuropsychiatric and neurodegenerative disorders as well as in patients with affective disorders. The purpose of the present study was to investigate if skin derived human fibroblasts are useful for studies of expression of enzymes in the KP. Methods Fibroblast cultures were established from cutaneous biopsies taken from the arm of consenting volunteers. Such cultures were subsequently treated with interferon (IFN)-? 200 U/ml and/or tumor necrosis factor (TNF)-?, 100 U/ml for 48 hours in serum-free medium. Levels of transcripts encoding different enzymes were determined by real-time PCR and levels of kynurenic acid (KYNA) were determined by HPLC. Results At base-line all cultures harbored detectable levels of transcripts encoding KP enzymes, albeit with considerable variation across individuals. Following cytokine treatment, considerable changes in many of the transcripts investigated were observed. For example, increases in the abundance of transcripts encoding indoleamine 2,3-dioxygenase, kynureninase or 3-hydroxyanthranilic acid oxygenase and decreases in the levels of transcripts encoding tryptophan 2,3-dioxygenase, kynurenine aminotransferases or quinolinic acid phosphoribosyltransferase were observed following IFN-? and TNF-? treatment. Finally, the fibroblast cultures released detectable levels of KYNA in the cell culture medium at base-line conditions, which were increased after IFN-?, but not TNF-?, treatments. Conclusions All of the investigated genes encoding KP enzymes were expressed in human fibroblasts. Expression of many of these appeared to be regulated in response to cytokine treatment as previously reported for other cell types. Fibroblast cultures, thus, appear to be useful for studies of disease-related abnormalities in the kynurenine pathway of tryptophan degradation.



Mutator phenotype of Werner syndrome is characterized by extensive deletions.  

PubMed Central

Werner syndrome (WS) is a rare autosomal-recessive disorder characterized by the premature appearance of features of normal aging in young adults. The extensive phenotypic overlap between WS and normal aging suggests they may also share pathogenetic mechanisms. We reported previously that somatic cells from WS patients demonstrate a propensity to develop chromosomal aberrations, including translocations, inversions, and deletions, and that WS cell lines demonstrate a high spontaneous mutation rate to 6-thioguanine resistance. We report here the biochemical and molecular characterization of spontaneous mutations at the X chromosome-linked hypoxanthine phosphoribosyltransferase (HPRT) locus in 6-thioguanine-resistant WS and control cells. Blot hybridization analysis of 89 independent spontaneous HPRT mutations in WS and control mutants lacking HPRT activity revealed an unusually high proportion of HPRT deletions in WS as compared with control cells (76% vs. 39%). Approximately half (58%) of the deletions in WS cells consisted of the loss of greater than 20 kilobases of DNA from the HPRT gene. These results suggest that an elevated somatic mutation rate, and particularly deletions, may play pathogenetically important roles in WS and in several associated age-dependent human disease processes. Images

Fukuchi, K; Martin, G M; Monnat, R J



PfHPRT: a new biomarker candidate of acute Plasmodium falciparum infection.  


Plasmodium falciparum is a protozoan parasite that causes human malaria. This parasitic infection accounts for approximately 655,000 deaths each year worldwide. Most deaths could be prevented by diagnosing and treating malaria promptly. To date, few parasite proteins have been developed into rapid diagnostic tools. We have combined a shotgun and a targeted proteomic strategy to characterize the plasma proteome of Gambian children with severe malaria (SM), mild malaria, and convalescent controls in search of new candidate biomarkers. Here we report four P. falciparum proteins with a high level of confidence in SM patients, namely, PF10_0121 (hypoxanthine phosphoribosyltransferase, pHPRT), PF11_0208 (phosphoglycerate mutase, pPGM), PF13_0141 (lactate dehydrogenase, pLDH), and PF14_0425 (fructose bisphosphate aldolase, pFBPA). We have optimized selected reaction monitoring (SRM) assays to quantify these proteins in individual patients. All P. falciparum proteins were higher in SM compared with mild cases or control subjects. SRM-based measurements correlated markedly with clinical anemia (low blood hemoglobin concentration), and pLDH and pFBPA were significantly correlated with higher P. falciparum parasitemia. These findings suggest that pHPRT is a promising biomarker to diagnose P. falciparum malaria infection. The diagnostic performance of this marker should be validated prospectively. PMID:23339668

Thézénas, Marie L; Huang, Honglei; Njie, Madi; Ramaprasad, Abhinay; Nwakanma, Davis C; Fischer, Roman; Digleria, Katalin; Walther, Michael; Conway, David J; Kessler, Benedikt M; Casals-Pascual, Climent



Sry-negative XX sex reversal in the American cocker spaniel dog.  


The Sry gene product serves an important function in male sex determination through testis induction. However, testicular development has been reported in SRY-negative XX sex reversed humans. XX sex reversal of the American cocker spaniel, inherited as an autosomal recessive trait, may be a homolog of this disorder. The purpose of this study was to determine whether the Sry high mobility group (HMG) box is present in genomic DNA of affected dogs. Conserved Sry HMG box and hypoxanthine phosphoribosyltransferase (HPRT) sequences were used as primers in polymerase chain reactions. A 167 bp Y-specific canine Sry HMG box sequence was cloned from genomic DNA of normal male dogs. Internal primers generated a 104 bp Sry HMG box product from normal males, but not from females or XX sex reversed dogs. Parallel reactions generated an HPRT product from all dogs. Results indicate that the Sry HMG box is absent in genomic DNA of XX sex reversed dogs. We speculate that activation of the testis differentiation cascade in the absence of Sry in this model is due to a mutant autosomal gene. PMID:8588928

Meyers-Wallen, V N; Palmer, V L; Acland, G M; Hershfield, B



Generation and Characterization of Mouse Hybridomas Secreting Monoclonal Antibodies Specific for Human IgG3  

PubMed Central

Mammalians express several subclasses of the IgG molecule. In human being there are four homologous IgG subclasses, each of which is structurally unique and has different functions. Quantification of IgG subclasses is fundamental to clinical assessment and diagnosis of many diseases as such assessments depends on the availability of subclassspecific antibodies (Abs), particularly monoclonal antibodies (MAbs). In the present study, we produced and characterized two murine MAbs specific for human IgG3 molecule. These MAbs were obtained by the fusion of myeloma cells with splenocytes from Balb/c mice immunized with heavy chain of a human IgG3 myeloma protein. Fused cells were selected in hypoxanthine, aminopterine and thymidine (HAT) medium and cloned by limiting dilution assay. Ab-secreting cells were screened by enzyme-linked immunosorbent assay (ELISA) and the specificity of secreted MAbs was further analyzed, using a panel of purified myeloma proteins by ELISA and immunoblotting. Two stable hybridomas designated 1F18G7 and 1F18A11 were obtained secreting MAbs specific for Fc fragment of human IgG3. None of these MAbs showed cross-reactivity with other immunoglobulin isotypes derived from human and nine other animals, except 1F18A11 which displayed a weak cross-reactivity with only dog serum. Immunoblotting results indicate that these MAbs react with linear epitope(s) located in the heavy chain of human IgG3 molecules. The affinity constant of 1F18G7 and 1F18A11 MAbs was found to be 0.81×109 Mol ?1 and 0.71×109 Mol ?1, respectively, as measured by ELISA. These two MAbs with relatively high affinity can be useful tools for quantification of IgG3 subclass levels in human serum.

Hajighasemi, Fatemeh; Shokri, Fazel



Frameshift Mutagenesis and Microsatellite Instability Induced by Human Alkyladenine DNA Glycosylase  

PubMed Central

Human alkyladenine DNA glycosylase (hAAG) excises alkylated purines, hypoxanthine and etheno bases from DNA to form abasic (AP) sites. Surprisingly, elevated expression of hAAG increases spontaneous frameshift mutagenesis. By random mutagenesis of eight active site residues, we isolated hAAG-Y127I/H136L double mutant that induces even higher rates of frameshift mutation than the wild-type hAAG; the Y127I mutation accounts for the majority of the hAAG-Y127I/H136L induced mutator phenotype. The hAAG-Y127I/H136L and hAAG-Y127I mutants increased the rate of spontaneous frameshifts by up to 120-fold in S. cerevisiae, and also induced high rates of microsatellite instability (MSI) in human cells. hAAG and its mutants bind DNA containing 1 and 2 base pair-loops with significant affinity, thus shielding them from mismatch repair; the strength of such binding correlates with their ability to induce the mutator phenotype. This study provides important insights into the mechanism of hAAG-induced genomic instability.

Klapacz, Joanna; Lingaraju, Gondichatnahalli M.; Guo, Haiwei H.; Shah, Dharini; Moar-Shoshani, Ayelet; Loeb, Lawrence A.; Samson, Leona D.



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

NASA Astrophysics Data System (ADS)

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.

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



Enzymes of the purine interconversion system in chronic lymphatic leukemia: Decreased purine nucleoside phosphorylase and adenosine deaminase activity  

Microsoft Academic Search

Zusammenfassung Bei 25 Patienten mit chronisch-lymphatischer LeukÄmie (CLL) und 23 Kontrollpersonen wurden die AktivitÄten der Purin-Interconversionsenzyme Adenosin Deaminase (ADA), Adenosin Kinase (AK), AdeninPhosphoribosyltransferase (APRT), Hypoxanthin-Guanin-Phosphoribosyl-transferase (HGPRT) und Purin-Nucleosid-Phosphorylase (PNP) bestimmt. Dabei konnte bei den Patienten mit CLL eine statistisch signifikant verminderte PNP-AktivitÄt sowie eine grenzwertig reduzierte ADA-AktivitÄt festgestellt werden, wÄhrend die AktivitÄten der anderen untersuchten Purin-Interconversionsenzyme im Normalbereich lagen.

H. Ludwig; R. Kuzmits; H. Pietschmann; M. M. Müller



Human See, Human Do.  

ERIC Educational Resources Information Center

|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…

Tomasello, Michael



NAD+ levels control Ca2+ store replenishment and mitogen-induced increase of cytosolic Ca2+ by Cyclic ADP-ribose-dependent TRPM2 channel gating in human T lymphocytes.  


Intracellular NAD(+) levels ([NAD(+)](i)) are important in regulating human T lymphocyte survival, cytokine secretion, and the capacity to respond to antigenic stimuli. NAD(+)-derived Ca(2+)-mobilizing second messengers, produced by CD38, play a pivotal role in T cell activation. Here we demonstrate that [NAD(+)](i) modifications in T lymphocytes affect intracellular Ca(2+) homeostasis both in terms of mitogen-induced [Ca(2+)](i) increase and of endoplasmic reticulum Ca(2+) store replenishment. Lowering [NAD(+)](i) by FK866-mediated nicotinamide phosphoribosyltransferase inhibition decreased the mitogen-induced [Ca(2+)](i) rise in Jurkat cells and in activated T lymphocytes. Accordingly, the Ca(2+) content of thapsigargin-sensitive Ca(2+) stores was greatly reduced in these cells in the presence of FK866. When NAD(+) levels were increased by supplementing peripheral blood lymphocytes with the NAD(+) precursors nicotinamide, nicotinic acid, or nicotinamide mononucleotide, the Ca(2+) content of thapsigargin-sensitive Ca(2+) stores as well as cell responsiveness to mitogens in terms of [Ca(2+)](i) elevation were up-regulated. The use of specific siRNA showed that the changes of Ca(2+) homeostasis induced by NAD(+) precursors are mediated by CD38 and the consequent ADPR-mediated TRPM2 gating. Finally, the presence of NAD(+) precursors up-regulated important T cell functions, such as proliferation and IL-2 release in response to mitogens. PMID:22547068

Magnone, Mirko; Bauer, Inga; Poggi, Alessandro; Mannino, Elena; Sturla, Laura; Brini, Marisa; Zocchi, Elena; De Flora, Antonio; Nencioni, Alessio; Bruzzone, Santina



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

PubMed Central

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.

Balzarini, J; De Clercq, E



High-frequency mutagenesis of human cells and characterization of a mutant unresponsive to both alpha and gamma interferons.  


2fTGH is a human cell line containing the selectable marker guanine phosphoribosyltransferase regulated by alpha interferon (IFN-alpha). Two IFN-alpha-unresponsive mutants were isolated previously at a low frequency (ca. 10(-8)) by selecting mutagenized 2fTGH cells in selective medium containing 6-thioguanine and IFN-alpha. By using five rounds of mutagenesis, mutants can be isolated at an appreciably higher frequency, greater than 3 x 10(-7). Five new mutants have been isolated, and all are recessive, as are the two mutants we described previously. The seven mutants are in four complementation groups (U1-U4). Since several different types of mutants unresponsive to IFN-alpha have been isolated with high frequency, related approaches may succeed with other cytokines or growth factors. Mutants in the two new complementation groups U3 and U4 are unresponsive to IFN-alpha and, surprisingly, also unresponsive to IFN-gamma. They are also partially defective in response to double-stranded RNA. These results indicate that the signaling pathways for the two types of IFN and double-stranded RNA share common components or that their function depends on common enzymes or transcription factors. IFN receptors are unaffected in mutants U3A and U4A. A major defect appears to be in the synthesis or activation of E, the transcription factor mediating the primary response to type I (alpha/beta) IFNs. Band-shift complementation assays show that U3A contains the E gamma subunit but does not contain an active E alpha subunit after treatment with IFN-alpha. PMID:1837150

McKendry, R; John, J; Flavell, D; Müller, M; Kerr, I M; Stark, G R



Human Behavior.  

National Technical Information Service (NTIS)

The report is a basic presentation of human behavior theory and utilization techniques as applied to basic assumptions about human behavior and motivation, the influence of perception, the effects of stress and conflict on human reactions, the formation a...



Human Development, Human Evolution.  

ERIC Educational Resources Information Center

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…

Smillie, David


Humanity and human DNA.  


Genetics has marked the second half of the 20th century by addressing such formidable problems as the identification of our genes and their role, their interaction with the environment, and even their therapeutic uses. The identification of genes raises questions about differences between humans and non-humans, as well as about the evolution towards trans-humanism and post-humanism. In practise, however, the main question concerns the limits of prenatal genetic diagnosis, not only on account of the seriousness of the affections involved but also because of the choice to be made between following-up the medical indication and engaging in a systematic public health strategy aimed at eliminating children with certain handicaps. History reminds us that genetic science has already been misused by political forces influenced by the ideas of eugenics, particularly in the Nazi period. We may wonder whether it is reasonable to formulate a judgement on the life of a child yet to be born, merely on the basis of a DNA analysis. My experience as a practising geneticist and my involvement in French politics forces me to stress the dangers of a new eugenics hiding behind a medical mask. As demonstrated by epigenetics, human beings cannot be reduced to their DNA alone. In our society, one of the problems concerns individuals whose lives may be considered by some as simply not worth living. Another problem is the place and the social significance of the handicapped amongst us. Fortunately, recent progresses in gene therapy, biotherapy, and even pharmacology, appear to be opening up promising therapeutic perspectives. We should bear in mind that the chief vocation of medical genetics, which fully belongs to the art of medicine, is to heal and to cure. This is precisely where genetics should concentrate its efforts software. PMID:22705070

Mattei, Jean-François



Steroid sulfatase gene in XX males.  

PubMed Central

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

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



Deficiency of the purine metabolic gene HPRT dysregulates microRNA-17 family cluster and guanine-based cellular functions: a role for EPAC in Lesch-Nyhan syndrome.  


Lesch-Nyhan syndrome (LNS) is a neurodevelopmental disorder caused by mutations in the gene encoding the purine metabolic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). A series of motor, cognitive and neurobehavioral anomalies characterize this disease phenotype, which is still poorly understood. The clinical manifestations of this syndrome are believed to be the consequences of deficiencies in neurodevelopmental pathways that lead to disordered brain function. We have used microRNA array and gene ontology analysis to evaluate the gene expression of differentiating HPRT-deficient human neuron-like cell lines. We set out to identify dysregulated genes implicated in purine-based cellular functions. Our approach was based on the premise that HPRT deficiency affects preeminently the expression and the function of purine-based molecular complexes, such as guanine nucleotide exchange factors (GEFs) and small GTPases. We found that several microRNAs from the miR-17 family cluster and genes encoding GEF are dysregulated in HPRT deficiency. Most notably, our data show that the expression of the exchange protein activated by cAMP (EPAC) is blunted in HPRT-deficient human neuron-like cell lines and fibroblast cells from LNS patients, and is altered in the cortex, striatum and midbrain of HPRT knockout mouse. We also show a marked impairment in the activation of small GTPase RAP1 in the HPRT-deficient cells, as well as differences in cytoskeleton dynamics that lead to increased motility for HPRT-deficient neuron-like cell lines relative to control. We propose that the alterations in EPAC/RAP1 signaling and cell migration in HPRT deficiency are crucial for neuro-developmental events that may contribute to the neurological dysfunctions in LNS. PMID:23804752

Guibinga, Ghiabe-Henri; Murray, Fiona; Barron, Nikki; Pandori, William; Hrustanovic, Gorjan



Maternal low-protein diet alters the expression of real-time quantitative polymerase chain reaction reference genes in an age-, sex-, and organ-dependent manner in rat offspring.  


Altered perinatal environment, often manifested as low birth weight, is thought to contribute to greater susceptibility for hypertension, hyperlipidemia, and diabetes as a result of epigenetic modifications and alteration of transcriptional activity for key genes. Real-time polymerase chain reaction is a useful technique for the quantitative determination of differences in transcriptional activity. Real-time quantitative polymerase chain reaction data analyses require normalization of transcriptional activity of target genes to an endogenous control, usually a reference gene. In response to reports of altered expression of reference genes in various experimental models, we hypothesized that adverse perinatal environment alters reference gene expression. We examined the expression of the following reference genes in the offspring of a rodent maternal low-protein diet model: ?-actin, hypoxanthine phosphoribosyltransferase 1, TATA-box-binding protein, glyceraldehyde-3-phosphate dehydrogenase, and glucuronidase-? in brain, heart, kidneys, and intestines. We found altered expression in brain, heart, and kidneys for each of the reference genes measured; these effects were age, organ, and sex dependent. Glyceraldehyde-3-phosphate dehydrogenase and glucuronidase-? were found to be the least affected by these variables, whereas hypoxanthine phosphoribosyltransferase 1 was the most inconsistent. Our findings underscore the importance of empirical determination of a reliable reference gene for real-time polymerase chain reaction studies in the low-protein diet model. PMID:23507230

DuBois, Barent; Pearson, Jacob; Hastings, Bonnie; Mahmood, Tahir; Chan, Tammy; Alnakhli, Ali; Cherala, Ganesh



A phosphoenzyme mimic, overlapping catalytic sites and reaction coordinate motion for human NAMPT.  


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-BeF(3)(-) 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. PMID:19666527

Burgos, Emmanuel S; Ho, Meng-Chiao; Almo, Steven C; Schramm, Vern L



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

SciTech Connect

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.

Chuang, Yao-Yu; Liber, H.L. [Harvard School of Public Health, Boston, MA (United States)



The Human Spark: Being Human  

NSDL National Science Digital Library

This lesson plan from PBS covers a variety of biology topics related to the human as an animal. Students will view and discuss segments from the PBS program The Human Spark. In the first learning activity, the class will explore how human thought differs from that of other species. In the second learning activity, students will examine different traits and abilities, how these abilities have evolved to help humans deal with their environment, and how they distinguish humans from other animals.

WNET (Television station : New York, N.Y.)



Manipulation of Cell Physiology Enables Gene Silencing in Well-differentiated Airway Epithelia  

PubMed Central

The application of RNA interference-based gene silencing to the airway surface epithelium holds great promise to manipulate host and pathogen gene expression for therapeutic purposes. However, well-differentiated airway epithelia display significant barriers to double-stranded small-interfering RNA (siRNA) delivery despite testing varied classes of nonviral reagents. In well-differentiated primary pig airway epithelia (PAE) or human airway epithelia (HAE) grown at the air–liquid interface (ALI), the delivery of a Dicer-substrate small-interfering RNA (DsiRNA) duplex against hypoxanthine–guanine phosphoribosyltransferase (HPRT) with several nonviral reagents showed minimal uptake and no knockdown of the target. In contrast, poorly differentiated cells (2–5-day post-seeding) exhibited significant oligonucleotide internalization and target knockdown. This finding suggested that during differentiation, the barrier properties of the epithelium are modified to an extent that impedes oligonucleotide uptake. We used two methods to overcome this inefficiency. First, we tested the impact of epidermal growth factor (EGF), a known enhancer of macropinocytosis. Treatment of the cells with EGF improved oligonucleotide uptake resulting in significant but modest levels of target knockdown. Secondly, we used the connectivity map (Cmap) database to correlate gene expression changes during small molecule treatments on various cells types with genes that change upon mucociliary differentiation. Several different drug classes were identified from this correlative assessment. Well-differentiated epithelia treated with DsiRNAs and LY294002, a PI3K inhibitor, significantly improved gene silencing and concomitantly reduced target protein levels. These novel findings reveal that well-differentiated airway epithelia, normally resistant to siRNA delivery, can be pretreated with small molecules to improve uptake of synthetic oligonucleotide and RNA interference (RNAi) responses.

Krishnamurthy, Sateesh; Behlke, Mark A; Ramachandran, Shyam; Salem, Aliasger K; McCray Jr, Paul B; Davidson, Beverly L



Targeted insertion of two Mthfr promoters in mice reveals temporal- and tissue-specific regulation.  


Methylenetetrahydrofolate reductase (MTHFR), a key enzyme in folate metabolism, synthesizes 5-methyltetrahydrofolate, the main circulatory form of folate which is required for maintaining nontoxic levels of homocysteine and providing one-carbon units for methylation. A common 677C ? T variant in MTHFR confers mild MTHFR deficiency and has been associated with a number of human disorders, including neural tube defects and vascular disease. Two promoters of Mthfr, designated as upstream and downstream promoters, are located upstream of a transcription start site cluster and have previously demonstrated cell-specific activities. In this study we used a unique approach for targeted, single-copy transgene insertion to generate transgenic mice carrying a Mthfr upstream or Mthfr downstream promoter-reporter construct located 5' to the endogenous Hprt (hypoxanthine-guanine phosphoribosyltransferase) locus. The Mthfr downstream promoter demonstrated activity in the neural tube, neural crest cells, dorsal root ganglia, heart, and endothelial cells of blood vessels in 10.5-days post coitum embryos and placentas. Upstream promoter activity was absent at this developmental stage. Postnatally, both promoters demonstrated activity in the brain stem, hippocampus, and thalamus of 1-week-old brain that became stronger in the adult. The Mthfr upstream promoter also showed activity in the cerebellum and cerebral cortex. Both promoters were active in male reproductive tissues, including 1-week-old epididymides, and there was upstream promoter-specific activity in the adult testis. Our investigation of Mthfr regulation in an in vivo mouse model revealed temporal- and tissue-specific regulation that supports important roles for MTHFR in the developing embryo, and in postnatal brain and male reproductive tissues. PMID:21769670

Pickell, Laura; Wu, Qing; Wang, Xiao-Ling; Leclerc, Daniel; Friedman, Hana; Peterson, Alan C; Rozen, Rima



Expression Profiling of Crystal-Induced Injury in Human Kidney Epithelial Cells  

Microsoft Academic Search

Background: Deposition of crystals within tubular lumens is a feature of many kidney stone diseases, including crystals of calcium oxalate monohydrate (COM) in primary hyperoxaluria and of 2,8-dihydroxyadenine (DHA) in adenine phosphoribosyltransferase deficiency. Crystals are injurious to renal epithelial cells, but the molecular bases of cell injury have not been well characterized. Methods: We used a cDNA microarray to identify

Li Liang; Jianmin Chen; Ragini Vittal; Zachariah E. Selvanayagam; James A. McAteer; Li Deng; Jay Tischfield; Khew-Voon Chin; Amrik Sahota



Accumulation of 5-phosphoribosyl-1-pyrophosphate in human CCRF-CEM leukaemia cells treated with antifolates  

Microsoft Academic Search

Amido phosphoribosyltransferase (APRT) catalyzes the first step of the de novo biosynthesis of purine nucleotides, the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosylamine (PRA). APRT is a valid target for development of inhibitors as anticancer drugs. We have developed a thin layer chromatographic assay for PRPP extracted from cells. Using coupling enzymes, PRPP with excess [2-14C]orotate (OA) is quantitatively converted to

M. A. Kamal; R. I. Christopherson



Human Lactoferrin.  

National Technical Information Service (NTIS)

The invention relates to a human lactoferrin gene isolated from breast tissue and to the protein product encoded therein. The invention further relates to the promotor region of human lactoferrin gene and to methods for detecting and analyzing malignancie...

C. Teng T. J. Panella



Detection of deletion mutations in pSV2gpt-transformed cells.  

PubMed Central

We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient CHO cells that have been transformed by the plasmid vector pSV2gpt. One isolated transformant, designated AS52, carries a single copy of the Escherichia coli gpt gene stably integrated into the high-molecular-weight DNA and expresses the bacterial gene for the enzyme xanthine-guanine phosphoribosyltransferase. Mutants deficient in this enzyme can be induced in the AS52 cell line by a variety of mutagens, and spontaneous or induced mutants can be selected for resistance to 6-thioguanine (Tgr). Two Tgr clones derived from the AS52 line were analyzed by Southern blot hybridization and were found to contain deletions involving at least a portion of the gpt gene. Because of the small size and stability of the integrated pSV2gpt plasmid, and the well-defined selection protocol for mutant isolation, the AS52 line offers promise as a system suitable for the study of mutation at the molecular level in CHO cells. Images

Tindall, K R; Stankowski, L F; Machanoff, R; Hsie, A W



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

NASA Astrophysics Data System (ADS)

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/?m 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/?m. The inactivation cross-section (?i) and the action-section for mutant induction (?m) ranged from 2.2 to 92.0 ?m2 and 0.09 to 5.56 × 10-3 ?m2, respectively. The maximum values were obtained by 56Fe with an LET of 200 keV/?m. The mutagenicity (?m/?i) ranged from 2.05 to 7.99 × 10-5 with the maximum value at 150 keV/?m. 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.

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



Reaction of xanthine oxidase-derived oxidants with lipid and protein of human plasma.  


Xanthine oxidase and purines have recently been detected in the circulation during acute viral infection and following hepatotoxicity and shock. Reactions of xanthine oxidase-generated oxidants with human plasma or bovine serum albumin (BSA) and egg phosphatidylcholine (PC) liposomes have been studied by measuring protein sulfhydryl oxidation and two markers of free radical-mediated lipid peroxidation, thiobarbituric acid reactive substances (TBARS) and conjugated dienes. Plasma incubated with 5 mU/ml xanthine oxidase (XO) and 0.5 mM hypoxanthine (Hx) for 2 h at 37 degrees C had 25-53% oxidation of sulfhydryl groups, with greater than 80% of the oxidation occurring during the first 20 min of the reaction. Concentrations of BSA similar to those present in serum, when exposed to XO/Hx-mediated oxidative stress, showed an even greater decrease in sulfhydryl concentration than that of plasma. No significant increase in plasma TBARS and conjugated dienes was observed during the 2-h incubation period in the presence of XO. Egg PC liposomes, suspended to a plasma phospholipid-equivalent concentration, showed a minor increase in TBARS and conjugated dienes under similar XO/Hx incubation conditions. In the presence of 0.23 mM BSA, lipid peroxidation was completely inhibited. A similar inhibition of lipid peroxidation was induced by cysteine but not by uric acid. Electrophoretic and arsenite-mediated sulfur reduction analysis revealed that BSA was oxidized beyond the disulfide form, with sulfenic acid formed during the initial period of oxidation. Protein sulfhydryls served as sacrificial antioxidants, preventing plasma lipid peroxidation, as well as being targets for oxidative damage. Plasma protein thiol oxidation was determined to be a more sensitive and specific indication of oxidant stress to the vascular compartment than assessment of lipid oxidation byproducts. PMID:1897941

Radi, R; Bush, K M; Cosgrove, T P; Freeman, B A



Human microbiomics  

Microsoft Academic Search

The sequencing of the human genome has driven the study of human biology in a significant way and enabled the genome-wide\\u000a study to elucidate the molecular basis of complex human diseases. Recently, the role of microbiota on human physiology and\\u000a health has received much attention. The influence of gut microbiome (the collective genomes of the gut microbiota) in obesity\\u000a has

J. Rajendhran; P. Gunasekaran



Human Evolution  

NSDL National Science Digital Library

This resource from Bruce MacEvoy links to documents "summarizing the hominid fossil record and hypothesized lines of human evolution from 5 million years ago to the present." The site is divided into five parts: Chart of Human Evolution, Tour of the Human Fossil Record, The Hominid Brain, Hominid Tools, Hominid Fossil Sites and Patterns of Hominid Dispersal.

Macevoy, Bruce



Human fascioliasis  

Microsoft Academic Search

Fasciola hepatica, a zoonotic liver fluke, can cause disease in humans. Diagnosis and treatment of fascioliasis is not easy, as physicians rarely encounter this disease, and effective drugs are not available in many countries. The prevalence of human fascioliasis may be underestimated. Humans become infected after eating aquatic plants on which encysted organisms are present or by drinking contaminated water.

Metin Korkmaz



Methotrexate inhibits the first committed step of purine biosynthesis in mitogen-stimulated human T-lymphocytes: a metabolic basis for efficacy in rheumatoid arthritis?  

PubMed Central

The immunosuppressive and anti-inflammatory effects of low-dose methotrexate (MTX) have been related directly to inhibition of folate-dependent enzymes by polyglutamated derivatives, or indirectly to adenosine release and/or apoptosis and clonal deletion of activated peripheral blood lymphocytes in S-phase. In this study of phytohaemagglutinin-stimulated primary human T-lymphocytes we show that MTX (20 nM to 20 microM) was cytostatic not cytotoxic, halting proliferation at G(1). This stasis of blastogenesis was associated with an inhibition of purine ribonucleotide synthesis but a stimulation of pyrimidine biosynthesis, the normal mitogen-induced expansion of ATP and GTP pools over 72 h being restricted to concentrations of unstimulated T-cells, whereas the increment in UTP pools exceeded that of controls. Decreased incorporation of H(14)CO(3) or [(14)C]glycine into purine ribonucleotides, with no radiolabel accumulation in any de novo synthetic intermediate but enhanced H(14)CO(3) incorporation into UTP, supported these MTX-related effects. Exaggerated [(14)C]hypoxanthine salvage (which normalized the purine and UTP pools) confirmed the increased availability of 5-phosphoribosyl-1-pyrophosphate (PP-ribose-P) as the molecular mechanism underlying these disparate changes. These results provide the first substantive evidence that the immunosuppressive effects of low-dose MTX in primary blasting human T-lymphocytes relate not to the inhibition of the two folate-dependent enzymes of purine biosynthesis but to inhibition of the first enzyme, amidophosphoribosyltransferase, thereby elevating PP-ribose-P and stimulating UTP synthesis. Varying cell types or incubation conditions employed by other workers, especially malignant/activated cells with high basal metabolic rates, might mask the effects noted in primary human T-lymphocytes. The findings imply the involvement of low-dose MTX in the inhibition of T-lymphocyte proliferation and proliferation-dependent processes in rheumatoid arthritis.

Fairbanks, L D; Ruckemann, K; Qiu, Y; Hawrylowicz, C M; Richards, D F; Swaminathan, R; Kirschbaum, B; Simmonds, H A



Bis(benzyl)polyamine analogs inhibit the growth of chloroquine-resistant human malaria parasites (Plasmodium falciparum) in vitro and in combination with alpha-difluoromethylornithine cure murine malaria.  

PubMed Central

A number of bis(benzyl)polyamine analogs were found to be potent inhibitors of both chloroquine-resistant and chloroquine-sensitive strains of the human malaria parasite Plasmodium falciparum in vitro (IC50 values = 0.2-14 microM). Administration of one of the compounds, MDL 27695, which is N,N'-bis(3-[(phenylmethyl)amino]propyl)-1,7-diaminoheptane (C6H5CH2NH(CH2)3NH(CH2)7NH(CH2)3NHCH2C6H5), at 10-15 mg/kg i.p. three times per day for 3 days in combination with 2% alpha-difluoromethylornithine (DFMO; eflornithine) in drinking water effected cures of 47/54 mice infected with Plasmodium berghei. Cured mice were found to be immune upon rechallenge with the same P. berghei strain 4 months after the initial infection and drug-induced cure. MDL 27695 rapidly inhibited the incorporation of [3H]hypoxanthine into P. falciparum RNA and DNA, whereas the incorporation of [3H]isoleucine was not affected until much later. We conclude, therefore, that the major cytotoxic event may be direct binding of MDL 27695 to DNA with subsequent disruption of macromolecular biosynthesis and cell death. These compounds offer a lead in the search for new agents for chemotherapy of malaria.

Bitonti, A J; Dumont, J A; Bush, T L; Edwards, M L; Stemerick, D M; McCann, P P; Sjoerdsma, A



Structure of a natural guanine-responsive riboswitch complexed with the metabolite hypoxanthine  

Microsoft Academic Search

Riboswitches are genetic regulatory elements found in the 5' untranslated region of messenger RNA that act in the absence of protein cofactors. They are broadly distributed across bacteria and account for the regulation of more than 2% of all genes in Bacillus subtilis, underscoring their importance in the control of cellular metabolism. The 5' untranslated region of many mRNAs of

Robert T. Batey; Sunny D. Gilbert; Rebecca K. Montange



Human Relations.  

ERIC Educational Resources Information Center

Human relations units are presented in this guide designed for teachers of secondary grades. The aim is to educate students in the philosophy of humanism. Emphasis is upon social interaction in an attempt to help pupils not only realize their own potential, but moreover, to respond to the needs of others. Each of the four activity units deals with…

Dade County Public Schools, Miami, FL.


Human Abilities.  

National Technical Information Service (NTIS)

An integration and review of research on human abilities published between early 1964 and April 1968. A consideration of human abilities as traits or capacities in individuals on which performance in a variety of specific tasks depends is the general them...

E. A. Fleishman C. J. Bartlett



Human Evolution  

NSDL National Science Digital Library

The first Web site is an article from the New York Times (1) detailing some recent fossil discoveries that are shaking the paleontological world (free registration is required). Another relatively recent article from Guardian Unlimited (2) discusses a scientific debate surrounding the question of whether "a Western lifestyle now protects humanity from the forces that used to shape Homo sapiens." The third resource (3) includes a likely timeline of events in the history of hominids and a tour of the fossil record. A second timeline from the Huntarian Museum and Art Gallery at the University of Glasgow (4) is less detailed, but links to many major fossil discoveries of human and pre-human history. An "overview of the study of human evolution, and of the currently accepted fossil evidence" (5) is used to inform arguments for creationists and evolutionists. An interesting site from the University of California Santa Barbara (6) (last mentioned in the December 1, 1998 Scout Report for Social Sciences) presents 3-dimensional views of "modern primate relatives and fossil ancestors of humans." The interactive documentary from the Institute of Human Origins (7) (last mentioned in the April 20, 2001 Scout Report) is a great resource for those with the Flash plug-in and a high speed connection. Lastly, a resource from (8) focuses on human evolution in a format aimed at kids.

Lee, Amy.



Human cloning and human dignity.  


Judging from the official documents dealing with the moral and legal aspects of human reproductive cloning there seems to be a nearly worldwide consensus that reproductive cloning is incompatible with human dignity. The certainty of this judgement is, however, not matched by corresponding arguments. Is the incompatibility of reproductive with human dignity an ultimate moral intuition closed to further argument? The paper considers several ways by which the intuition might be connected with more familiar applications of the concept of human dignity, and argues that there is no such connection. It concludes that the central objections to human reproductive cloning are not objections relating to dignity but objections relating to risk, especially the risks imposed on children born in the course of testing the method's safety. PMID:15820009

Birnbacher, Dieter



Mechanism of cytotoxicity of 5,10-dideazatetrahydrofolic acid in human ovarian carcinoma cells in vitro and modulation of the drug activity by folic or folinic acid.  

PubMed Central

Inhibition of clonogenic potential by the glycinamideribonucleosyl transformylase inhibitor 5,10-dideazatetrahydrofolic acid (DDATHF, Lometrexol) was evaluated in vitro in a human ovarian carcinoma cell line, SW626. Drug-induced inhibition of clonogenic potential is a function of the dose and time of exposure and is independent of the formation of DNA single-strand breaks or de novo synthesis of protein. Simultaneous treatment with 100 microM hypoxanthine completely prevented the inhibition of clonogenic potential caused by 0.5 microM DDATHF. DDATHF blocked cells in the early-middle S-phases of the cell cycle, and there was a corresponding marked reduction in the rate of DNA synthesis after drug withdrawal. The cytotoxic potential of DDATHF was modulated by the folic acid concentration present in the medium. In a medium containing 0.22 microM folic acid, DDATHF cytotoxicity was at least 100 times that in a regular medium containing 2.22 microM folic acid, levels which, however, are about 100 times those found in human plasma. DDATHF cytotoxicity differed moderately when folic acid concentrations varied between 0.22 and 0 microM, suggesting that folic acid does not necessarily antagonise DDATHF anti-tumour activity. Folinic acid at a concentration as low as 0.1 microM can completely rescue cells when given simultaneously with 0.5 microM DDATHF. When folinic acid was given 24 h after DDATHF, a reversal of cytotoxicity was observed at 0.5 and 1 microM, but to a much lesser extent than simultaneous treatment. When folinic acid was added after 48 or 72 h of DDATHF washout, even at a high concentration and for a long time, no reduction in DDATHF cytotoxicity was found. In conclusion, the study highlights the modulation of DDATHF cytotoxicity by folic acid or by folinic acid and provides further rationale for in vivo clinical investigation with these combinations.

Erba, E.; Sen, S.; Sessa, C.; Vikhanskaya, F. L.; D'Incalci, M.



Human phosphatases  

US Patent & Trademark Office Database

The invention provides human phosphatases (HPA) and polynucleotides which identify and encode HPA. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for treating disorders associated with expression of HPA.



Human impacts  

Microsoft Academic Search

\\u000a Only in recent years have humans become aware of the enormous damage being done to natural resources. Human influence on fresh\\u000a waters is no exception. The conflict between the demand for large amounts of pure water on the one hand and the disastrous\\u000a pollution of many waters on the other is only now forcing the issue with politicians. Enormous areas

P. S. Maitland; N. C. Morgan


Human cytomegalovirus  

Microsoft Academic Search

Summary Exposure of human lung fibroblasts to human cytomegalovirus (HCMV) stimulated a rapid increase in the release of [3H] from cells prelabelled with radiolabelled arachidonic acid ([3H]AA). Maximum stimulation of [3H] release was observed at 20 min postinfection and was quantitatively similar to that induced by the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA: 10nM) or fetal calf serum (5%). The level

S. AbuBakar; I. Boldogh; T. Albrecht



Action, human.  


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

Russo, M T



Behavior, human.  


Human behavior is the collection of actions or reactions exhibited by human beings in relation to the environment, and it can be categorized as either innate or learned. In psychology, behavior became an important construct with the advent of behaviorism, a theoretical framework that required the study of only observable facts or events which can be seen or manipulated, in response to external or internal stimuli. More recently, the Relational Frame Theory (RFT) suggests that also some psychological events such as thoughts and emotions can be explained as learned responses. In TACT project, behavior is the voluntary movement of reaching and grasping in a fixed condition. PMID:20393688

Molteni, M



Human fascioliasis.  


Fasciola hepatica, a zoonotic liver fluke, can also cause disease in humans. Common symptoms are epigastric pain, upper abdominal pain and malaise. Fever and arthralgia are common in acute fascioliasis. Eosinophilia is the predominant laboratory finding, especially in patients with the acute form of the disease. Diagnosis and treatment is not easy, as physicians rarely encounter this disease, and effective drugs are not available in many countries. Human fascioliasis may be underestimated. Patients with eosinophilia and abdominal pain should be evaluated for F. hepatica infestation by parasitological, radiological and serological tests. PMID:15113313

Saba, R; Korkmaz, M; Inan, D; Mamiko?lu, L; Turhan, O; Günseren, F; Cevikol, C; Kabaalio?lu, A



Human Trafficking  

ERIC Educational Resources Information Center

|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…

Wilson, David McKay



Human Performance.  

National Technical Information Service (NTIS)

The tasking for this study was to evaluate the potential for adversaries to exploit advances in Human Performance Modification, and thus create a threat to national security. In making this assessment, we were asked to evaluate long-term scenarios. We hav...

A. Despain E. Williams H. Abarbanel M. Brenner S. Drell



Human Levitation  

Microsoft Academic Search

Human levitation occurs when the physical body rises into the air seemingly in defiance of the force of gravity. Traditionally most levitation reports have originated from seven groups: (i) mysticism, (ii) shamanism, (iii) people supposedly possessed by demonic spiritual entities, (iv) those subjected to poltergeist activity, (v) Spiritualism, (vi) people who believe they have been abducted by aliens, and (vii)




Human Effect  

NSDL National Science Digital Library

In this lesson, students will investigate changes in air quality due to human interaction particularly burning of fossil fuels, and crop burning which increase levels of carbon monoxide. Students will evaluate changes in air quality over a 6 month time frame using Air Quality-Carbon Monoxide Data and draw conclusions based on observing color plot comparison graphs.


Human torso  

NSDL National Science Digital Library

The torso is the central area of the body that all the other body parts connect to. The ribcage contains the lungs and the heart. The intestines are located below them. The pelvic region contains the human reproductive parts and parts of the digestive and waste tracts.

William Cheselden (None;)



Human Hibernation  

Microsoft Academic Search

I DID not answer your correspondent's query on human hibernation in your issue of the 5th inst. (p. 316), because I thought some one better informed than myself would answer it. However, as no one has done so, I may as well give a solution of this well-known Indian trick which I have seen, but the authority for which, I

Alfred H. Hulk



Human Hibernation  

Microsoft Academic Search

MY letter on the Hibernation of the Siberian mammoth has been followed by two others, extremely interesting, but dealing, I may say exclusively, with the question of human hibernation, and the evidence offered in support of it; this raises a very important consideration, concerning which I ask leave to offer a few remarks:-The ``fact,'' as stated by Mr. Braid, is

C. K. Bushe



Classical Humanities  

ERIC Educational Resources Information Center

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)

Goodwin, Donn; And Others



Humanizing Calculus  

ERIC Educational Resources Information Center

|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…

Cirillo, Michelle



Human Capital, (Human) Capabilities and Higher Education  

ERIC Educational Resources Information Center

|In this article I initiate a debate into the (de)merits of human capital theory and human capability theory and discuss implications of the debate for higher education. Human capital theory holds that economic growth depends on investment in education and that economic growth is the basis for improving the quality of human life. Human capable…

Le Grange, L.



Human Rights in the Humanities  

ERIC Educational Resources Information Center

|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…

Harpham, Geoffrey



Human Rights  

NSDL National Science Digital Library

The idea of "human rights" is a relatively new development in history, but as this website from Britain's National Archives notes in its discussion of the long trajectory of struggles for equality and so forth, "We could do worse than characterizing this history as the struggle for human rights." This visually compelling online exhibit uses original documents from The National Archives to take a long view of these struggles and movements. Visitors can start their journey through the site by picking a time period, and then reading an introductory essay on the period. Each time period includes a timeline and links to digitized version of relevant documents, such as The Poor Act of 1601 and a poster for a Staffordshire coal miners' union public meeting from 1831. The site is rounded out by a thorough glossary and a document index.


Human energy  

Microsoft Academic Search

In the midst of big-oil record profits and growing debate on global warming, the Chevron Corporation launched its “Human Energy”\\u000a public relations campaign. In television commercials and print advertisements, Chevron portrays itself as a compassionate\\u000a entity striving to solve the planet’s energy crisis. Yet, the first term in this corporate oxymoron misleadingly reframes\\u000a the significance of the second, suggesting that

Suzana Sawyer



Human genetics  

SciTech Connect

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.

Carlson, E.A.



Human Heredity: Genetic Mechanisms in Humans.  

ERIC Educational Resources Information Center

Discussed are some of the uncertainties in human genetic mechanisms that are often presented as dogma in Biology textbooks. Presented is a brief historical background and illustrations involving chromosome abnormality in humans and linkage studies in humans. (CW)

Blank, C. E.



Human Chromosomes  

NSDL National Science Digital Library

Representation of the 23 paired chromosomes of the human male. Chromosome: a very long DNA molecule and associated proteins, that carry portions of the hereditary information of an organism. a. Structure of a chromosome (Typical metaphase chromosome): A chromosome is formed from a single DNA molecule that contains many genes. A chromosomal DNA molecule contains three specific nucleotide sequences which are required for replication: a DNA replication origin; a centromere to attach the DNA to the mitotic spindle.; a telomere located at each end of the linear chromosome. The DNA molecule is highly condensed. The human DNA helix occupy too much space in the cell. Small proteins are responsible for packing the DNA into units called nucleosomes. b. Stained chromosomes: Chromosomes are stained with A-T (G bands) and G-C (R bands) base pair specific dyes. When they are stained, the mitotic chromosomes have a banded structure that unambiguously identifies each chromosome of a karyotype. Each band contains millions of DNA nucleotide pairs which do not correspond to any functional structure. Adapted from K.F. Jorgenson, J.H. van de Sande, and C.C. Lin, Chromosoma 68:287-302, 1978. c. Karyotype of a male: The human haploid genome contains 3,000,000,000 DNA nucleotide pairs, divided among twenty two (22) pairs of autosomes and one pair of sex chromosomes.

BEGIN:VCARD VERSION:2.1 FN:Access Excellence N:Excellence;Access REV:2005-03-12 END:VCARD



Humanities Magazine  

NSDL National Science Digital Library

Online since 1996, the Humanities Magazine is the magazine of the National Endowment for the Humanities (NEH). Designed to complement the detailed information on the operations and grant opportunities presented on the main NEH website, visitors to this website can browse and read articles from the magazine dating back to the November / December 1996 issue. Not surprising, the magazine is designed to explore the projects and various endeavors sponsored by the NEH, and visitors will find a wide range of material here, including pieces on archaeology in Guatemala and a recent documentary on the Reconstruction. Another nice feature is the profile section offered once each year on the recipients of the National Humanities Medal. The profiles from 2003 are quite compelling, as they provide information on each of the ten recipients, including Robert Ballard (who is best known for discovering the wreck of the Titanic) and Hal Holbrook, the actor who is immediately recognizable for his animated and multifaceted portrayals of Mark Twain over the past fifty years.


Human evolution.  


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

Wood, B



Remote mutations alter transition-state structure of human purine nucleoside phosphorylase.  


Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine (2'-deoxy)ribonucleosides to give the corresponding purine base and (2'-deoxy)ribose 1-phosphate as products. Human and bovine PNPs (HsPNP and BtPNP) form distinct transition states despite 87% identity in amino acid sequence. A PNP hybrid was produced by replacing K22 and H104 in HsPNP with the corresponding Glu and Arg residues found in BtPNP. We solved the transition-state structure of E:R-HsPNP (K22E:H104R mutant of HsPNP) using competitive kinetic isotope effects (KIE) and global density functional calculations. An array of PNP transition states was generated from optimized structure candidates with varied C1'-N9, C1'-Ophosphate distances, ribosyl pucker configurations and N7-protonation states. Isotopically labeled [1'-3H], [2'-3H], [1'-14C], [9-15N], [1'-14C, 9-15N] and [5'-3H2]inosines gave intrinsic KIE values of 1.210, 1.075, 1.035, 1.024, 1.065, 1.063 with E:R-HsPNP, respectively. The suite of E:R-HsPNP KIEs match a single structure from the array of PNP transition-state candidates. The transition state of E:R-HsPNP is fully dissociative, N7-protonated hypoxanthine (C1'-N9 distance >or= 3.0 A) with partial participation of phosphate (C1'-Ophosphate distance = 2.26 A), 2'-C-exo-ribosyl ring pucker and the O5'-C5'-C4'-O4' dihedral angle near 60 degrees . The transition state of E:R-HsPNP is altered from the fully dissociative DN*AN character for HsPNP to a late phosphate-associative character. E:R-HsPNP differs from native HsPNP by only two residues over 25 A away from the active site. New interactions caused by the mutations increase the catalytic efficiency of the enzyme for formation of a late transition state with increased participation of the phosphate nucleophile. Dynamic coupling motions from the remote mutations to the catalytic sites are proposed. PMID:18281957

Luo, Minkui; Li, Lei; Schramm, Vern L



Human Speedy  

PubMed Central

The decision for a cell to self-replicate requires passage from G1 to S phase of the cell cycle and initiation of another round of DNA replication. This commitment is a critical one that is tightly regulated by many parallel pathways. Significantly, these pathways converge to result in activation of the cyclin-dependent kinase, cdk2. It is, therefore, important to understand all the mechanisms regulating cdk2 to determine the molecular basis of cell progression. Here we report the identification and characterization of a novel cell cycle gene, designated Speedy (Spy1). Spy1 is 40% homologous to the Xenopus cell cycle gene, X-Spy1. Similar to its Xenopus counterpart, human Speedy is able to induce oocyte maturation, suggesting similar biological characteristics. Spy1 mRNA is expressed in several human tissues and immortalized cell lines and is only expressed during the G1/S phase of the cell cycle. Overexpression of Spy1 protein demonstrates that Spy1 is nuclear and results in enhanced cell proliferation. In addition, flow cytometry profiles of these cells demonstrate a reduction in G1 population. Changes in cell cycle regulation can be attributed to the ability of Spy1 to bind to and prematurely activate cdk2 independent of cyclin binding. We demonstrate that Spy1-enhanced cell proliferation is dependent on cdk2 activation. Furthermore, abrogation of Spy1 expression, through the use of siRNA, demonstrates that Spy1 is an essential component of cell proliferation pathways. Hence, human Speedy is a novel cell cycle protein capable of promoting cell proliferation through the premature activation of cdk2 at the G1/S phase transition.

Porter, Lisa A.; Dellinger, Ryan W.; Tynan, John A.; Barnes, Elizabeth A.; Kong, Monica; Lenormand, Jean-Luc; Donoghue, Daniel J.



The Digital Humanities as a Humanities Project  

ERIC Educational Resources Information Center

|This article argues that the digital humanities can be seen as a humanities project in a time of significant change in the academy. The background is a number of scholarly, educational and technical challenges, the multiple epistemic traditions linked to the digital humanities, the potential reach of the field across and outside the humanities

Svensson, Patrik



Human Clones and International Human Rights  

Microsoft Academic Search

The United Nations Declaration on Human Cloning calls upon member states to prohibit all forms of human cloning. However, the Declaration is nonbinding and will not put a stop to cloning around the world. Scientists will continue to clone embryos in their quest to develop stem cell therapies, ultimately, their work will facilitate the birth of human clones.;Once born, human

Kerry MacIntosh



The Effects of Azathioprine (Imuran) on Purine Synthesis in Clinical Disorders of Purine Metabolism*  

PubMed Central

Azathioprine, a purine analogue, significantly suppressed the purine synthesis de novo of two gouty patients manifesting overproduction of uric acid, as well as three of four gouty patients who showed normal uric acid production. This suppression is taken as evidence that phosphoribosyl-pyrophosphate amidotransferase, the rate-controlling step in purine synthesis de novo, has a normal sensitivity to feedback inhibitors in the patients who responded to the drug. Two children afflicted with the familial disorder of hyperuricemia, choreo-athetosis, and self-mutilation described by Lesch and Nyhan showed no reduction in the activity of the biosynthetic pathway in response to azathioprine. This inability to respond to azathioprine can be directly related to the absence in these patients of the enzyme hypoxanthine-guanine phosphoribosyltransferase which is required for conversion of the drug or its metabolites to the biochemically active ribonucleotide form.

Kelley, William N.; Rosenbloom, Frederick M.; Seegmiller, J. Edwin



Gene targeting using a mouse HPRT minigene/HPRT-deficient embryonic stem cell system: inactivation of the mouse ERCC-1 gene.  


A convenient system for gene targeting that uses hypoxanthine phosphoribosyltransferase (HPRT) minigenes as the selectable marker in HPRT-deficient mouse embryonic stem (ES) cells is described. Improvements to the expression of HPRT minigenes in ES cells were achieved by promoter substitution and the provision of a strong translational initiation signal. The use of minigenes in the positive-negative selection strategy for gene targeting was evaluated and the smaller minigenes were found to be as effective as a more conventional marker--the herpes simplex virus thymidine kinase gene. Minigenes were used to target the DNA repair gene ERCC-1 in ES cells. A new HPRT-deficient ES cell line was developed that contributes with high frequency to the germ line of chimeric animals. The ability to select for and against HPRT minigene expression in the new HPRT-deficient ES cell line will make this system useful for a range of gene-targeting applications. PMID:1440055

Selfridge, J; Pow, A M; McWhir, J; Magin, T M; Melton, D W



Autosomal dominant transmission of gouty arthritis with renal disease in a large Japanese family.  

PubMed Central

Six generations of a Japanese family had gouty arthritis and progressive nephropathy. Data on nine of 51 women (18%) and 15 of 66 men (23%) with either asymptomatic hyperuricaemia, gouty arthritis, or renal insufficiency were obtained. Renal function in four men and one woman with hyperuricaemia or gouty arthritis was also examined. Urinary excretion of uric acid was decreased in all subjects examined, including the young. Erythrocyte phosphoribosylpyrophosphate synthetase and hypoxanthine-guanine phosphoribosyltransferase activities determined in 10 patients were normal. Some patients had been treated with allopurinol to reduce serum uric acid concentrations, but the treatment did not prevent progression of renal impairment. Transmission of the disease in this large family is considered to be autosomal dominant. The data suggest that the disease in this family is the same entity as that described by other workers--that is, familial urate nephropathy. As far as is known this is the largest family with this disease so far reported.

Yokota, N; Yamanaka, H; Yamamoto, Y; Fujimoto, S; Eto, T; Tanaka, K



Mutation induction by hyperthermia  

SciTech Connect

Asynchronous Chinese hamster ovary cells were exposed to 43.5 degrees C for 60 minutes and 45.5 degrees C for 15 minutes. Mutation induction was recorded by the drug resistance system for 8-azaguanine, which selected for cells deficient in the enzyme, hypoxanthine-guanine phosphoribosyltransferase. Mutation induction was also measured for 500- and 600-rad doses of X-rays. For comparable survival levels, hyperthermia was twofold to fivefold less effective than X-rays in inducing mutations. There was no evidence of a dose response with increasing heating durations at 43.5 degrees or 45.5 degrees C. Hyperthermia mutation induction was more strongly dependent on 8-azaguanine concentration than was that of X-ray.

Hopwood, L.E.; Tanel, E.J.



Identification of mutations leading to the Lesch-Nyhan syndrome by automated direct DNA sequencing of in vitro amplified cDNA  

SciTech Connect

The Lesch-Nyhan (LN) syndrome is a severe X chromosome-linked disease that results from a deficiency of the purine salvage enzyme hypoxanthine phosphoribosyltransferase (HPRT). The mutations leading to the disease are heterogeneous and frequently arise as de novo events. The authors have identified nucleotide alterations in 15 independently arising HPRT-deficiency cases by direct DNA sequencing of in vitro amplified HPRT cDNA. They also demonstrate that the direct DNA sequence analysis can be automated, further simplifying the detection of new mutations at this locus. The mutations include DNA base substitutions, small DNA deletions, a single DNA base insertion, and errors in RNA splicing. The application of these procedures allows DNA diagnosis and carrier identification by the direct detection of the mutant alleles within individual families affected by LN.

Gibbs, R.A. (Baylor College of Medicine, Houston, TX (USA)); Nguyen, Phinga (Howard Hughes Medical Institute, Houston, TX (USA)); McBride, L.J.; Koepf, S.M. (Applied Biosystems, Foster City, CA (USA)); Caskey, C.T. (Baylor College of Medicine, Houston, TX (USA) Howard Hughes Medical Institute, Houston, TX (USA))



Building artificial humans to understand humans.  


If we could build an android as a very humanlike robot, how would we humans distinguish a real human from an android? The answer to this question is not so easy. In human-android interaction, we cannot see the internal mechanism of the android, and thus we may simply believe that it is a human. This means that a human can be defined from two perspectives: one by organic mechanism and the other by appearance. Further, the current rapid progress in artificial organs makes this distinction confusing. The approach discussed in this article is to create artificial humans with humanlike appearances. The developed artificial humans, an android and a geminoid, can be used to improve understanding of humans through psychological and cognitive tests conducted using the artificial humans. We call this new approach to understanding humans android science. PMID:17846711

Ishiguro, Hiroshi; Nishio, Shuichi



Building artificial humans to understand humans  

Microsoft Academic Search

If we could build an android as a very humanlike robot, how would we humans distinguish a real human from an android? The\\u000a answer to this question is not so easy. In human–android interaction, we cannot see the internal mechanism of the android,\\u000a and thus we may simply believe that it is a human. This means that a human can

Hiroshi Ishiguro; Shuichi Nishio



[Human metapneumovirus].  


Respiratory viral infections are the most significant cause of increased mortality and morbidity especially in immunocompromised people. These infections are increasingly recognized as being the cause of the failure of a graft or the cause of death in both solid organ and hematopoietic stem cell transplant recipients. Treatment with potent immunosuppressive medication is necessary for regulation in order to prevent rejection of solid organs and graft-versus-host disease. As a consequence of this therapy, infections are more common. Respiratory viral infections are the most frequent and serious complications after hematopoietic stem cell transplantation (HSCT). Despite increased methods of testing for viral pathogens, nearly 10% of pneumonia in HSCT recipients still remain "idiopathic pneumonia syndrome". Recently described human metapneumovirus could be one of the etiological agents of this syndrome. PMID:20809463

Stepánová, Eva; Zák, Pavel; Stepánová, Vlasta; Ryska, Pavel



Comparing Human-Human to Human-Computer Tutorial Dialogue.  

National Technical Information Service (NTIS)

Intelligent Tutoring Systems are often modeled after human tutors; however, the effectiveness of this strategy is yet to be determined. Research on media interactions suggests that behaviors with humans are similar to those with computers. Intelligent Tut...

G. E. Campbell K. M. Harrison L. S. Taylor M. O. Dzikovska N. B. Steinhauser



Temporal variations of adenosine metabolism in human blood.  


Eight diurnally active (06:00-23:00 h) subjects were adapted for 2 days to the room conditions where the experiments were performed. Blood sampling for adenosine metabolites and metabolizing enzymes was done hourly during the activity span and every 30 min during sleep. The results showed that adenosine and its catabolites (inosine, hypoxanthine, and uric acid), adenosine synthesizing (S-adenosylhomocysteine hydrolase and 5'-nucleotidase), degrading (adenosine deaminase) and nucleotide-forming (adenosine kinase) enzymes as well as adenine nucleotides (AMP, ADP, and ATP) undergo statistically significant fluctuations (ANOVA) during the 24 h. However, energy charge was invariable. Glucose and lactate chronograms were determined as metabolic indicators. The same data analyzed by the chi-square periodogram and Fourier series indicated ultradian oscillatory periods for all the metabolites and enzymatic activities determined, and 24-h oscillatory components for inosine, hypoxanthine, adenine nucleotides, glucose, and the activities of SAH-hydrolase, 5'-nucleotidase, and adenosine kinase. The single cosinor method showed significant oscillatory components exclusively for lactate. As a whole, these results suggest that adenosine metabolism may play a role as a biological oscillator coordinating and/or modulating the energy homeostasis and physiological status of erythrocytes in vivo and could be an important factor in the distribution of purine rings for the rest of the organism. PMID:8874980

Chagoya de Sánchez, V; Hernández-Muñoz, R; Suárez, J; Vidrio, S; Yáñez, L; Aguilar-Roblero, R; Oksenberg, A; Vega-González, A; Villalobos, L; Rosenthal, L; Fernández-Cancino, F; Drucker-Colín, R; Díaz-Muñoz, M



MicroRNA-mediated dysregulation of neural developmental genes in HPRT deficiency: clues for Lesch-Nyhan disease?  


Mutations in the gene encoding the purine biosynthetic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) cause the intractable neurodevelopmental Lesch-Nyhan disease (LND) associated with aberrant development of brain dopamine pathways. In the current study, we have identified an increased expression of the microRNA miR181a in HPRT-deficient human dopaminergic SH-SY5Y neuroblastoma cells. Among the genes potentially regulated by miR181a are several known to be required for neural development, including Engrailed1 (En1), Engrailed2 (En2), Lmx1a and Brn2. We demonstrate that these genes are down-regulated in HPRT-deficient SH-SY5Y cells and that over-expression of miR181a significantly reduces endogenous expression of these genes and inhibits translation of luciferase plasmids bearing the En1/2 or Lmx1a 3'UTR miRNA-binding elements. Conversely, inhibition of miR181a increases the expression of these genes and enhances translation of luciferase constructs bearing the En1/2 and Lmx1a 3'UTR miRNA-binding sequences. We also demonstrate that key neurodevelopmental genes (e.g. Nurr1, Pitx3, Wnt1 and Mash1) known to be functional partners of Lmx1a and Brn2 are also markedly down-regulated in SH-SY5Y cells over-expressing miR181a and in HPRT-deficient cells. Our findings in SH-SY5Y cells demonstrate that HPRT deficiency is accompanied by dysregulation of some of the important pathways that regulate the development of dopaminergic neurons and dopamine pathways and that this defect is associated with and possibly due at least partly to aberrant expression of miR181a. Because aberrant expression of miR181a is not as apparent in HPRT-deficient LND fibroblasts, the relevance of the SH-SY5Y neuroblastoma cells to human disease remains to be proven. Nevertheless, we propose that these pleiotropic neurodevelopment effects of miR181a may play a role in the pathogenesis of LND. PMID:22042773

Guibinga, Ghiabe-Henri; Hrustanovic, Gorjan; Bouic, Kathryn; Jinnah, Hyder A; Friedmann, Theodore



1998--Human Rights Year.  

ERIC Educational Resources Information Center

|Promotes teaching human rights at every educational level in conjunction with the United Nations-declared Human Rights Year. Provides World Wide Web addresses for resources on human rights and the Human Rights Year, including the United Nations and Amnesty International. Summarizes UN-suggested guidelines for human rights commemoration…

Mock, Karen R.



Humane Education: An Overview.  

ERIC Educational Resources Information Center

This booklet traces the historical development of human education as it has been instilled into the young people of America from colonial times to the present and provides a future prognosis of humaneness in the schools. Humane education promotes humane behavior and is an important part of the humane movement in the United States, although until…

Whitlock, Eileen S.; Westerlund, Stuart R.


Human rhinoviruses.  


Human rhinoviruses (HRVs), first discovered in the 1950s, are responsible for more than one-half of cold-like illnesses and cost billions of dollars annually in medical visits and missed days of work. Advances in molecular methods have enhanced our understanding of the genomic structure of HRV and have led to the characterization of three genetically distinct HRV groups, designated groups A, B, and C, within the genus Enterovirus and the family Picornaviridae. HRVs are traditionally associated with upper respiratory tract infection, otitis media, and sinusitis. In recent years, the increasing implementation of PCR assays for respiratory virus detection in clinical laboratories has facilitated the recognition of HRV as a lower respiratory tract pathogen, particularly in patients with asthma, infants, elderly patients, and immunocompromised hosts. Cultured isolates of HRV remain important for studies of viral characteristics and disease pathogenesis. Indeed, whether the clinical manifestations of HRV are related directly to viral pathogenicity or secondary to the host immune response is the subject of ongoing research. There are currently no approved antiviral therapies for HRVs, and treatment remains primarily supportive. This review provides a comprehensive, up-to-date assessment of the basic virology, pathogenesis, clinical epidemiology, and laboratory features of and treatment and prevention strategies for HRVs. PMID:23297263

Jacobs, Samantha E; Lamson, Daryl M; St George, Kirsten; Walsh, Thomas J



Adenine metabolism in Plasmodium falciparum.  


Plasmodium falciparum lacks the de novo purine biosynthesis pathway and relies entirely on the salvage pathway to meet its purine nucleotide requirements. The entire flux for purine nucleotide biosynthesis in the parasite is believed to be through hypoxanthine guanine phosphoribosyltransferase (HGPRT), with the enzymes, adenosine kinase and adenine phosphoribosyltransferase (APRT) being unannotated in the Plasmodium genome database. This manuscript reports on the studies carried out to explore bypass mechanisms, if any, for AMP synthesis in the intraerythrocyitc stages of the parasite life cycle. Uptake and subsequent incorporation of radiolabel adenine in the nucleotide pool of saponin released erythrocyte free parasites implicated the role of parasite encoded enzymes in adenine metabolism. To explore the route for AMP synthesis in the parasite, we have monitored adenine mediated supplementation of metabolic viability in saponin released hadacidin (N-formyl-N-hydroxyglycine) treated parasites. Our results implicate the role of an APRT like activity that enables parasite survival when the flux through the HGPRT pathway is blocked. PMID:20093117

Mehrotra, Sonali; Bopanna, Monnanda P; Bulusu, Vinay; Balaram, Hemalatha



Markerless Mutagenesis in Methanococcus maripaludis Demonstrates Roles for Alanine Dehydrogenase, Alanine Racemase, and Alanine Permease  

PubMed Central

Among the archaea, Methanococcus maripaludis has the unusual ability to use l- or d-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in these genes, we devised a method for markerless mutagenesis that builds on previously established genetic tools for M. maripaludis. The technique uses a negative selection strategy that takes advantage of the ability of the M. maripaludis hpt gene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to the base analog 8-azahypoxanthine. In addition, we developed a negative selection method to stably incorporate constructs into the genome at the site of the upt gene encoding uracil phosphoribosyltransferase. Mutants with in-frame deletion mutations in the genes for alanine dehydrogenase and alanine permease lost the ability to grow on either isomer of alanine, while a mutant with an in-frame deletion mutation in the gene for alanine racemase lost only the ability to grow on d-alanine. The wild-type gene for alanine dehydrogenase, incorporated into the upt site, complemented the alanine dehydrogenase mutation. Hence, the permease is required for the transport of either isomer, the dehydrogenase is specific for the l isomer, and the racemase converts the d isomer to the l isomer. Phylogenetic analysis indicated that all three genes had been acquired by lateral gene transfer from the low-moles-percent G+C gram-positive bacteria.

Moore, Brian C.; Leigh, John A.



The construction of cosmid libraries which can be used to transform eukaryotic cells  

Microsoft Academic Search

Cosmid vectors have been developed which carry selective markers for growth in bacteria (beta lactamase gene) and animal cells (the Herpes Simplex virus thymidine kinase gene, the transposon Tn-5 aminoglycosyl 3' phosphotransferase gene and the E. coli guanine phosphoribosyltransferase gene). The design of the cosmids allows the exchange of the eukaryotic markers in recombinant cosmids. Human and mouse cosmid libraries

F. G. Grosveld; T. Lund; E. J. Murray; A. L. Mellor; H. H. M. Dahl; R. A. Flavell



FK866, a Highly Specific Noncompetitive Inhibitor of Nicotinamide Phosphoribosyltransferase, Represents a Novel Mechanism for Induction of Tumor Cell Apoptosis  

Microsoft Academic Search

Deregulation of apoptosis, the physiological form of cell death, is closely associated with immunological diseases and cancer. Apoptosis is activated either by death receptor-driven or mitochondrial pathways, both of which may provide potential targets for novel anticancer drugs. Although sev- eral ligands stimulating death receptors have been described, the actual molecular events triggering the mitochondrial pathway are largely un- known.

Max Hasmann; Isabel Schemainda



What Are the Humanities?  

ERIC Educational Resources Information Center

|A working definition of the humanities and characteristics of a liberally educated person are specified. The humanities embrace areas of human knowledge that possess these elements: central concern for human beings rather than for the processes of nature or the structures of society; primary focus on the individual rather than on the group;…

Broderick, Francis


Human Development and Sustainability  

Microsoft Academic Search

The literatures and debates on human development on the one hand and sustainability on the other share much in common. Human development is essentially what sustainability advocates want to sustain and without sustainability, human development is not true human development. Yet the two strands of research have largely been separate and this paper shows how they can learn from each

Eric Neumayer



Adenosine kinase deficiency in tritiated deoxyadenosine-resistant mouse S49 lymphoma cell lines  

SciTech Connect

Mutant sublines were derived of S49 mouse T-lymphoma cells that were resistant to tritiated deoxyadenosine. Twenty-five isolates that were selected in 1 microCi/ml of the nucleoside were cross-resistant to 6-thioguanine, were sensitive to HAT (hypoxanthine, aminopterin, and thymidine), and contained less than 1% of hypoxanthine phosphoribosyltransferase activity in wild-type cells. One of the mutant clones, S49-dA2, was further subjected to selection in a medium containing 2 microCi/ml tritiated deoxyadenosine and 1 microgram/ml deoxycoformycin, an inhibitor of adenosine deaminase. All resistant subclones were cross-resistant to tubercidin, 6-methylmercaptopurine riboside, and arabinosyladenine. One of the subclones, S49-12, was completely devoid of adenosine kinase and was partially deficient in deoxyadenosine kinase. This subclone, however, contained wild-type levels of deoxycytidine kinase. DEAE chromatography of the wild-type cell extracts revealed two deoxyadenosine phosphorylating activities, one of which coeluted with adenosine kinase and was the enzyme missing in S49-12. The other species phosphorylated both deoxyadenosine and deoxycytidine, of which deoxycytidine was the preferred substrate.

Sastry, K.J.; Huang, C.; Chan, T.S.



Purine metabolism in Toxoplasma gondii  

SciTech Connect

We have studied the incorporation and interconversion of purines into nucleotides by freshly isolated Toxoplasma gondii. They did not synthesize nucleotides from formate, glycine, or serine. The purine bases hypoxanthine, xanthine, guanine, and adenine were incorporated at 9.2, 6.2, 5.1, and 4.3 pmol/10(7) cells/h, respectively. The purine nucleosides adenosine, inosine, guanosine, and xanthosine were incorporated at 110, 9.0, 2.7, and 0.3 pmol/10(7) cells/h, respectively. Guanine, xanthine, and their respective nucleosides labeled only guanine nucleotides. Inosine, hypoxanthine, and adenine labeled both adenine and guanine nucleotide pools at nearly equal ratios. Adenosine kinase was greater than 10-fold more active than the next most active enzyme in vitro. This is consistent with the metabolic data in vivo. No other nucleoside kinase or phosphotransferase activities were found. Phosphorylase activities were detected for guanosine and inosine; no other cleavage activities were detected. Deaminases were found for adenine and guanine. Phosphoribosyltransferase activities were detected for all four purine nucleobases. Interconversion occurs only in the direction of adenine to guanine nucleotides.

Krug, E.C.; Marr, J.J.; Berens, R.L.



Understanding the Human Estimator  

Microsoft Academic Search

Among the various forms of estimation (human-based, algorithmic, and machine learners), human-based remains the predominant methodology of choice (1). Algorithmic-based (e.g. COCOMO, FPA) approaches rely heavily upon human intervention for supplying estimates for many of the sub-components. Understanding the role of the human estimator is critical for improving the effort estimation process. Every human estimator draws upon his or her

Gary D. Boetticher; Nazim Lokhandwala; James C. Helm


Humanized Mice for Human Retrovirus Infection  

Microsoft Academic Search

Inbred mice with specific genetic defects have greatly facilitated the analysis of complex biological events. Several humanized\\u000a mouse models using the C.B.-17 scid\\/scid mouse (referred to as the SCID mouse) have been created from two transplantation protocols, and these mice have been utilized\\u000a for the investigation of human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus type I (HTLV-I)

Y. Koyanagi; Y. Tanaka; M. Ito; N. Yamamoto


Human Trafficking: A Crime Against Humanity  

Microsoft Academic Search

This chapter addresses human trafficking in light of international criminal law, with a view to considering whether and to\\u000a what extent it may be characterized, when certain conditions are met, as a crime against humanity. This conclusion may contribute\\u000a to enhancing international cooperation in this area.

Fausto Pocar


Assessment of Human Factors.  

National Technical Information Service (NTIS)

Human Factors Engineering, often referred to as Ergonomics, is a science that applies a detailed understanding of human characteristics, capabilities, and limitations to the design, evaluation, and operation of environments, tools, and systems for work an...

F. Mount T. Foley



[The human microbiome].  


The complex microbial populations that colonize the human body form the human microbiome. The human microbiota may contain 1014) cells, an order of magnitude greater than the number of the human cells, and can express 100 times more genes than the human genome. The metagenome is the collective genomes of the human and its microbial flora. Major international colLaborative efforts currently explore the diverse human microbiomes from five different body areas: the nasopharyngeal, gastrointestinal and female urogenital tracts, the oral cavity and the skin. Defining the complexity of the human microbiome in health and disease will enhance the understanding of multiple pathological mechanisms and facilitate the development of novel diagnostic tools and therapeutic interventions. PMID:21678649

Solt, Ido; Kim, Matthew J; Offer, Cohavy



The Growing Human Population.  

ERIC Educational Resources Information Center

Discusses the issue of human population. Illustrates the projections of the growing human population in terms of developed and less developed countries. Describes the family planning programs in several countries. Lists three references for further reading. (YP)

Keyfitz, Nathan



Indexing the Human Genome.  

National Technical Information Service (NTIS)

In this taped lecture methodologies for first-level analysis of human gene products are described and evaluated as tools required for indexing the human genome. In particular, he explores the utility of the technqiue of two dimensional gel electrophoresis...



Human Thelaziasis, Europe  

PubMed Central

Thelazia callipaeda eyeworm is a nematode transmitted by drosophilid flies to carnivores in Europe. It has also been reported in the Far East in humans. We report T. callipaeda infection in 4 human patients in Italy and France.

Dutto, Moreno



Human Reliability Research.  

National Technical Information Service (NTIS)

The research effort focused on two major areas, a survey and analysis of existing failure reporting systems, and the investigation of alternative indirect approaches to determining human performance and quantifying the human reliability contribution to we...

C. Beek K. Haynam G. Markisohn



Telling the Human Story.  

ERIC Educational Resources Information Center

|Proposes that one of the fundamental human attributes is telling stories. Explores the debate on whether Neanderthals possessed language ability. Discusses the role of the "human story" in teaching anthropology. (DH)|

Richardson, Miles



Human Telomerase Catalytic Subunit.  

National Technical Information Service (NTIS)

The invention provides compositions and methods related to human telomerase reverse transcriptase (hTRT), the catalytic protein subunit of human telomerase. The polynucleotides and polypeptides of the invention are useful for diagnosis, prognosis and trea...

G. B. Morin J. Lingner K. B. Chapman T. Nakamura T. R. Cech



Climate and Human Evolution  

NSDL National Science Digital Library

In this video segment adapted from NOVA: Becoming Human, learn how the analysis of rock layers and ocean sediments supports the theory that rapid climate change may have jump-started human evolution two million years ago.

Foundation, Wgbh E.



Human Research Tissues

This form must be submitted in addition to the standard PHL request form when submitting HUMAN TISSUES other than Xenografts and established, pathogen-free, human cell lines. Work will not begin until this form has been received and reviewed.


Immunology Taught by Humans  

PubMed Central

After a half-century of mouse-dominated research, human immunology is making a comeback. Informed by mouse studies and powered by new techniques, human immune research is both advancing disease treatment and providing new insights into basic biology.

Davis, Mark M.



Human melioidosis, Malawi, 2011.  


A case of human melioidosis caused by a novel sequence type of Burkholderia pseudomallei occurred in a child in Malawi, southern Africa. A literature review showed that human cases reported from the continent have been increasing. PMID:23735189

Katangwe, Thembi; Purcell, Janet; Bar-Zeev, Naor; Denis, Brigitte; Montgomery, Jacqui; Alaerts, Maaike; Heyderman, Robert Simon; Dance, David A B; Kennedy, Neil; Feasey, Nicholas; Moxon, Christopher Alan



Mining human antibody repertoires  

PubMed Central

Human monoclonal antibodies (mAbs) have become drugs of choice for the management of an increasing number of human diseases. Human antibody repertoires provide a rich source for human mAbs. Here we review the characteristics of natural and non-natural human antibody repertoires and their mining with non-combinatorial and combinatorial strategies. In particular, we discuss the selection of human mAbs from naïve, immune, transgenic and synthetic human antibody repertoires using methods based on hybridoma technology, clonal expansion of peripheral B cells, single-cell PCR, phage display, yeast display and mammalian cell display. Our reliance on different strategies is shifting as we gain experience and refine methods to the efficient generation of human mAbs with superior pharmacokinetic and pharmacodynamic properties.



The Human Body  

NSDL National Science Digital Library

We will be learning about The Human Body! Think about these questions as you go through: How does the muscular system work? Why is the skeletal system important? Hello 4A! Our next unit is learning about The Human Body! I want you to explore and come up with a burning question you have about The Human Body! Here is a fun fact about The Human Body: A baby has 300 bones at birth. An adult has ...

Bratkiewicz, Miss



Human Genome Project.  

National Technical Information Service (NTIS)

The study reviews Department of Energy supported aspects of the United States Human Genome Project, the joint National Institutes of Health/Department of Energy program to characterize all human genetic material, to discover the set of human genes, and to...

S. Koonin S. Block J. Cornwall W. Dally F. Dyson



Human genome project.  

National Technical Information Service (NTIS)

The Human Genome Project will obtain high-resolution genetic and physical maps of each human chromosome and, somewhat later, of the complete nucleotide sequence of the deoxyribonucleic acid (DNA) in a human cell. The talk will begin with an extended intro...

G. I. Bell



The Humanities' Value  

ERIC Educational Resources Information Center

Why should society support the humanities when so many people are suffering from the effects of the economic crisis? What claim do the humanities, or scholarship generally, have on increasingly limited resources? Shouldn't such pursuits be considered luxuries at a time when people should be focusing on essentials? The alleviation of human…

Harpham, Geoffrey Galt



Nonphotic Entrainment in Humans?  

Microsoft Academic Search

Although light is accepted as the dominant zeitgeber for entrainment of the human circadian system, there is evidence that nonphotic stimuli may play a role. This review critically assesses the current evidence in support of nonphotic entrainment in humans. Studies involving manipulations of sleep-wake schedules, exercise, mealtimes, and social stimuli are re-examined, bearing in mind the fact that the human

Ralph E. Mistlberger; Debra J. Skene



Humanities Curriculum Guide.  

ERIC Educational Resources Information Center

Designed as a model for a high school humanities program, this publication outlines a two-course, two-year elective in humanities for high school juniors and seniors. Introductory material includes an overview of the program and its history, credits, goals of the program, and an introduction to humanities. The major portion of the guide contains…

Stafford, Mary Ann


Human factors in mining  

SciTech Connect

This Bureau of Mines report is directed toward summarizing the application of human factors to improving safety, productivity, and the general physical and psychological working conditions of miners and toward familiarizing readers with the role of human factors in the mining industry and the benefits that car accrue by systematically applying human factors principles and data.

Sanders, M.S.; Peay, J.M.



Has Human Evolution Stopped?  

PubMed Central

It has been argued that human evolution has stopped because humans now adapt to their environment via cultural evolution and not biological evolution. However, all organisms adapt to their environment, and humans are no exception. Culture defines much of the human environment, so cultural evolution has actually led to adaptive evolution in humans. Examples are given to illustrate the rapid pace of adaptive evolution in response to cultural innovations. These adaptive responses have important implications for infectious diseases, Mendelian genetic diseases, and systemic diseases in current human populations. Moreover, evolution proceeds by mechanisms other than natural selection. The recent growth in human population size has greatly increased the reservoir of mutational variants in the human gene pool, thereby enhancing the potential for human evolution. The increase in human population size coupled with our increased capacity to move across the globe has induced a rapid and ongoing evolutionary shift in how genetic variation is distributed within and among local human populations. In particular, genetic differences between human populations are rapidly diminishing and individual heterozygosity is increasing, with beneficial health effects. Finally, even when cultural evolution eliminates selection on a trait, the trait can still evolve due to natural selection on other traits. Our traits are not isolated, independent units, but rather are integrated into a functional whole, so selection on one trait can cause evolution to occur on another trait, sometimes with mildly maladaptive consequences.

Templeton, Alan R.



Human Embryonic Stem Cells  

Microsoft Academic Search

Human embryonic stem cells hold great promise in furthering our treatment of disease and increasing our understanding of early development. This chapter describes protocols for the derivation and maintenance of human embryonic stem cells. In addition, it summarizes briefly several alternative methods for the culture of human embryonic stem cells. Thus, this chapter provides a good starting point for researchers

Hidenori Akutsu; Chad A. Cowan; Douglas Melton



Robotics of human movements.  


The construction of robotic systems that can move the way humans do, with respect to agility, stability and precision, is a necessary prerequisite for the successful integration of robotic systems in human environments. We explain human-centered views on robotics, based on the three basic ingredients (1) actuation; (2) sensing; and (3) control, and formulate detailed examples thereof. PMID:19686847

van der Smagt, Patrick; Grebenstein, Markus; Urbanek, Holger; Fligge, Nadine; Strohmayr, Michael; Stillfried, Georg; Parrish, Jonathon; Gustus, Agneta



Dogs catch human yawns  

Microsoft Academic Search

Summary This study is the first to demonstrate that human yawns are possibly contagious to domestic dogs (Canis familiaris). Twenty-nine dogs observed a human yawning or making control mouth movements. Twenty-one dogs yawned when they observed a human yawning, but control mouth movements did not elicit yawning from any of them. The presence of contagious yawning in dogs suggests that

Ramiro M. Joly-Mascheroni; Atsushi Senju; Alex J. Shepherd




EPA Science Inventory

Human activity/uptake rate data are necessary to estimate potential human exposure and intake dose to environmental pollutants and to refine human exposure models. Personal exposure monitoring studies have demonstrated the critical role that activities play in explaining and pre...



EPA Science Inventory

Human land uses may have major impacts on ecosystems, affecting biodiversity, habitat, air and water quality. The human use index (also known as U-index) is the percentage of human land use in an area, including agriculture, urban and suburban development, and mining. Low values ...



EPA Science Inventory

Human land uses may have major impacts on ecosystems, affecting biodiversity, habitat, air and water quality. The human use index (also known as U-index) is the percentage of human land use in an area, including agriculture, urban and suburban development, and mining. Low values ...


Secure distributed human computation  

Microsoft Academic Search

This paper is a preliminary exploration of secure distributed human computation. We consider the general paradigm of using large-scale distributed computation to solve difficult problems, but where humans can act as agents and provide candidate solutions. We are especially motivated by problem classes that appear to be difficult for computers to solve effectively, but are easier for humans; e.g., image

Craig Gentry; Zulfikar Ramzan; Stuart Stubblebine



Portraits of Human Greatness.  

ERIC Educational Resources Information Center

Examined is the Humanities Program at St. Anselm College, a two-year program of readings and lectures ordered chronologically from ancient to contemporary times--from the age of Classical Greek thought and the Old Testament to the twentieth century. The first year of the Humanities Program is organized in eight units on general modes of "human…

Saint Anselm's Coll., Manchester, NH.


Intervention and Human Rights.  

ERIC Educational Resources Information Center

|Defends the right of nations to criticize human rights violations within other nations. Pointing out that the human rights protections cannot be left to domestic jurisdiction, Goldberg cites numerous treaties and declarations which make human rights protection a matter of international law. (GEA)|

Goldberg, Arthur J.



Human Tail and Myelomeningocele  

Microsoft Academic Search

The human tail is rarely reported and is usually associated with underlying spina bifida occulta. A male newborn presenting a caudal appendage (human tail) with skin-covered myelomeningocele and tethered cord is described. Surgical excision of the human tail and repair of the myelomeningocele were performed 3 days after birth. After the operation, the patient had an uneventful convalescence and received

Pei-Jung Lin; Yu-Tang Chang; Hsing-I Tseng; Jan-You Lin; Yu-Sheng Huang



Human-technology Integration  

NASA Astrophysics Data System (ADS)

Human-technology integration is the replacement of human parts and extension of human capabilities with engineered devices and substrates. Its result is hybrid biological-artificial systems. We discuss here four categories of products furthering human-technology integration: wearable computers, pervasive computing environments, engineered tissues and organs, and prosthetics, and introduce examples of currently realized systems in each category. We then note that realization of a completely artificial sytem via the path of human-technology integration presents the prospect of empirical confirmation of an aware artificially embodied system.

Mullen, Katharine M.


Human Milk Banking: Considerations Related to Human ...  

Center for Biologics Evaluation and Research (CBER)

Text Version... Human Milk Banking Procedures to Reduce ... of milk containers-Batch number, name of milk bank ... (donor procedure, HIV testing, PEP electronic links ... More results from


Chimeras and human dignity.  


Discussions about whether new biomedical technologies threaten or violate human dignity are now common. Indeed, appeals to human dignity have played a central role in national and international debates about whether to allow particular kinds of biomedical investigations. The focus of this paper is on chimera research. I argue here that both those who claim that particular types of human-nonhuman chimera research threaten human dignity and those who argue that such threat does not exist fail to make their case. I first introduce some of the arguments that have been offered supporting the claim that the creation of certain sorts of chimeras threatens or violates human dignity. I next present opponents' assessments of such arguments. Finally I critically analyze both the critics' and the supporters' claims about whether chimera research threatens human dignity. PMID:19143408

de Melo-Martín, Inmaculada



Human hemoglobin genetics  

SciTech Connect

This book contains the following 10 chapters: Introduction; The Human Hemoglobins; The Human Globin Genes; Hemoglobin Synthesis and Globin Gene Expression; The Globin Gene Mutations - A. Mechanisms and Classification; The Globin Gene Mutations - B. Their Phenotypes and Clinical Expression; The Genetics of the Human Globin Gene Loci: Formal Genetics and Gene Linkage; The Geographic Distribution of Globin Gene Variation; Labortory Identification, Screening, Education, and Counseling for Abnormal Hemoglobins and Thalassemias; and Approaches to the Treatment of the Hemoglobin Disorders.

Honig, G.R.; Adams, J.G.



Human Health Risk Assessment  

Microsoft Academic Search

\\u000a Exposure of humans to contaminated sites may result in many types of health damage ranging from relatively innocent symptoms\\u000a such as skin eruption or nausea, on up to cancer or even death. Human health protection is considered as a major protection\\u000a target, both by decision-makers as well as by the general public. The first step in Human Health Risk Assessment

Frank A. Swartjes; Christa Cornelis


[Human physiology: kidney].  


The content of human physiology as an independent part of current physiology is discussed. Substantiated is the point that subjects of human physiology are not only special sections of physiology where functions are inherent only in human (physiology of intellectual activity, speech, labor, sport), but also in peculiarities of functions, specificity of regulation of each of physiological systems. By the example of physiology of kidney and water-salt balance there are shown borders of norm, peculiarities of regulation in human, new chapters of renal physiology which have appeared in connection with achievements of molecular physiology. PMID:21061667

Natochin, Iu V


Human Development Report 1997  

NSDL National Science Digital Library

The United Nations Development Programme's Human Development Report Office provides two useful selections from its Human Development Report 1997. Overview of HDR 1997 is a detailed summary of the contents of the report, discussing world poverty and a six point strategy for poverty reduction. HDR 1997 Rankings contain Human Development Index, Gender-Related Development Index and Gender Empowerment Measure rankings tables for 175 countries. More detailed information on the meaning of the HDI is contained in Analytical Tools for Human Development. Full text of the report is forthcoming, and ordering information is available at the site.



The unpatentable human being.  


On June 13, 2013, the Supreme Court placed its imprimatur on a principle that has been gathering force within patent law for several decades: human beings constitute unpatentable subject matter. In Association for Molecular Pathology v. Myriad Genetics, Inc., the court answered the question it had posed itself-"Are human genes patentable?"-decisively in the negative. This legal result was predictable, given a careful reading of the entrails of judicial decisions, congressional bills, executive branch pronouncements, and decisions in other countries about patents claiming human-related inventions, all of which have echoed the spirit of the Thirteenth Amendment by proscribing property rights-including intellectual property rights-in human beings. To understand how patent law has evolved toward this result, one may trace the legal treatment of patents claiming human embryonic stem cells, chemical products of human physiology, human thought, and now, human genes. Woven together, these strands of evidence led inexorably toward the ultimate rejection of human gene patents by the Supreme Court. PMID:24092583

Torrance, Andrew W


Antihemophilic Factor (Human), Alphanate  

Center for Biologics Evaluation and Research (CBER)

... Research (CBER) has identified promotional material for your product, Alphanate (Antihemophilic Factor (Human)), that violates ... signature -----. ... More results from


Human enhancement and perfection.  


Both, bioconservatives and bioliberals, should seek a discussion about ideas of human perfection, making explicit their underlying assumptions about what makes for a good human life. This is relevant, because these basic, and often implicit ideas, inform and influence judgements and choices about human enhancement interventions. Both neglect, and polemical but inconsistent use of the complex ideas of perfection are leading to confusion within the ethical debate about human enhancement interventions, that can be avoided by tackling the notion of perfection directly. In the recent debates, bioconservatives have prominently argued against the 'pursuit of perfection' by biotechnological means. In the first part of this paper, we show that-paradoxically-bioconservatives themselves explicitly embrace specific conceptions of human perfection and perfectionist assumptions about the good human life in order to argue against the use of enhancement technologies. Yet, we argue that the bioconservative position contains an untenable ambiguity between criticising and endorsing ideas of human perfection. Hence, they stand in need of clarifying their stance on human perfection. In the second part of the paper, we ask whether bioliberals in fact (implicitly) advocate a particular conception of perfection, or whether they are right in holding that they do not, and that discussing perfection is obsolete anyway. We show that bioliberals also rely on a specific idea of human perfection, based on the idea of autonomy. Hence, their denial of the relevance of perfection in the debate is unconvincing and has to be revised. PMID:23436909

Roduit, Johann A R; Baumann, Holger; Heilinger, Jan-Christoph



Climate and Human Migrations  

NSDL National Science Digital Library

Access to the article is free, however registration and sign-in are required. About 14,600 years ago, humans first appeared in south central Chile. But the arid regions of the Atacama desert in northern Chile were not populated for another 2000 years, and human occupation of this region subsequently remained intermittent. In his Perspective, Dillehay highlights the report of Núñez et al., whose integrative archaeological and paleoecological study shows that climate was the key factor in these human migrations. The study illustrates the power of an integrative approach to understanding the relation between human societies and climate change.

Tom D. Dillehay (University of Kentucky;Department of Anthropology)



Against human nature  

Microsoft Academic Search

Against human nature\\u000a \\u000a Tim Ingold12 \\u000a \\u000a \\u000a \\u000a (12) \\u000a Department of Anthropology, University of Aberdeen, Aberdeen, Scotland, UK\\u000a \\u000a \\u000a Abstract\\u000a Are cultural differences superimposed upon a universal human nature? The appeal to an essentialist concept of human nature\\u000a is a defensive reaction to the legacy of racist science left by Darwin’s argument in The Descent of Man. Humans are made to appear different in degree

Tim Ingold


Human target acquisition performance  

NASA Astrophysics Data System (ADS)

The battlefield has shifted from armored vehicles to armed insurgents. Target acquisition (identification, recognition, and detection) range performance involving humans as targets is vital for modern warfare. The acquisition and neutralization of armed insurgents while at the same time minimizing fratricide and civilian casualties is a mounting concern. U.S. Army RDECOM CERDEC NVESD has conducted many experiments involving human targets for infrared and reflective band sensors. The target sets include human activities, hand-held objects, uniforms & armament, and other tactically relevant targets. This paper will define a set of standard task difficulty values for identification and recognition associated with human target acquisition performance.

Teaney, Brian P.; Du Bosq, Todd W.; Reynolds, Joseph P.; Thompson, Roger; Aghera, Sameer; Moyer, Steven K.; Flug, Eric; Espinola, Richard; Hixson, Jonathan



Human Security Centre: Human Security Brief 2006  

NSDL National Science Digital Library

While the concept of human security is a relatively new one, there is a growing consensus that the subject is one that will continue to be of the utmost importance in the coming years. Generally, the term is used to describe "the complex of interrelated threats associated with civil war, genocide and the displacement of populations." Recently, the Human Security Centre (located at the University of British Columbia) published its annual Human Security Brief, and placed it online at this site. The report analyzes the findings of several datasets that track trends in such areas as organized violence against civilians and the conclusion of armed conflicts worldwide. While this ambitious work does have some positive findings to announce, there are a number of other troubling trends, such as the fact that four of the world's six regions have experienced increased numbers of conflicts since 2002.


Continuous human activity recognition  

Microsoft Academic Search

Effectively recognizing human activities requires at least 32 joint related degrees of freedom to be estimated so as to reliably track the human body in 3D. The particle filter is robust to distracting clutter by maintaining multiple hypotheses for each of these joint angles. Real-time tracking is difficult however with the computational overhead of such a large search space. This

R. D. Green; Ling Guan



Recognition of Human Gaits  

Microsoft Academic Search

We pose the problem of recognizing different types of human gait in the space of dynamical systems where each gait is represented. Established techniques are employed to track a kinematic model of a human body in motion, and the trajectories of the parameters are used to learn a represen- tation of a dynamical system, which defines a gait. Various types

Alessandro Bissacco; Alessandro Chiuso; Yi Ma; Stefano Soatto



Human Rickettsialpox, Southeastern Mexico  

PubMed Central

The detection of Rickettsia akari in 2 human patients increased the diversity of rickettsioses affecting the public health in the southeast of Mexico. Rickettsialpox should be considered in the differential diagnosis with other febrile illnesses for the correct diagnosis and accurate treatment of this potential threat to human health.

Zavala-Velazquez, Jorge E.; Peniche-Lara, Gaspar F.; Uicab, Justo E. Sulu



Environment and the Humanities.  

ERIC Educational Resources Information Center

As a conference report, the booklet is primarily devoted to abstracts of papers presented at a Conference on Environment and Humanities held in Tallahassee, Florida, April 25-27, 1976. Dr. Huston Smith of Syracuse University, the main speaker, addressed the issue of "Humanities and Environmental Awareness." Other topics discussed included: (1)…

Allen, Rodney F., Ed.; And Others


Humanizing the Secondary School.  

ERIC Educational Resources Information Center

|These papers, presented during ASCD-sponsored conference, confront educators with issues in and alternatives for making secondary schools a more humanizing experience for students. The contributors and their articles are: Norman K. Hamilton, "Alternatives in Secondary Education"; Thornton B. Monez and Norman L. Bussiere, "The High School in Human…

Hamilton, Norman K., Ed.; Saylor, J. Galen, Ed.



EPA Science Inventory

Arsenic is one of the few human carcinogens for which there is not yet a reliable animal cancer model. s such, the classification of arsenic as a carcinogen is based upon data derived from human epidemiologic studies. Although the mechanisms of action of arsenic as a toxic agent ...


Human Capital Strategy  

Microsoft Academic Search

There are two key principles in human capital strategy: (1) people are assets whose value can be enhanced through investment and (2) an organization's human capital policies must be aligned to support the organization's shared vision. Excerpts from two GAO reports provide an excellent summary of this topic.

Tom Cross


Human Adaptive Mechatronics  

Microsoft Academic Search

This article explains the background of human adaptive mechatronics (HAM) and the skill level of individuals to operate machines. It is natural to consider both human and environmental factors in the field of robotics and mechatronics. The progress of machines cannot be achieved without paying attention to these factors; a culture that accepts that this evolved through the contributions of

Satoshi Suzuki



Pushing Human Frontiers.  

National Technical Information Service (NTIS)

With human colonization of Mars, I think you will see a higher standard of civilization, just as America set a higher standard of civilization which then promulgated back into Europe. I think that if you want to maximize human potential, you need a higher...

R. Zubrin



Mapping the human genome.  

National Technical Information Service (NTIS)

The following pages aim to lay a foundation for understanding the excitement surrounding the ''human genome project,'' as well as to convey a flavor of the ongoing efforts and plans at the Human Genome Center at the Lawrence Berkeley Laboratory. Our own w...



Ecology and Human Values.  

ERIC Educational Resources Information Center

|"Ecology and Human Values" is an interdisciplinary course designed for senior year high school students in social studies and/or science. Its main thrust is the investigation of human values as they relate to the environment, although rooted in the natural sciences as a means of understanding the complexities inherent in the environment. Use is…



Humane Education Projects Handbook.  

ERIC Educational Resources Information Center

|This handbook was developed to promote interest in humane education and to encourage the adoption of humane education projects. Although specifically designed to assist Junior Leagues in developing such projects, the content should prove valuable to animal welfare organizations, zoos, aquariums, nature centers, and other project-oriented groups…

Junior League of Ogden, UT.


Human Granulocytic Anaplasmosis, Japan  

PubMed Central

We retrospectively confirmed 2 cases of human Anaplasma phagocytophilum infection. Patient blood samples contained unique p44/msp2 for the pathogen, and antibodies bound to A. phagocytophilum antigens propagated in THP-1 rather than HL60 cells. Unless both cell lines are used for serodiagnosis of rickettsiosis-like infections, cases of human granulocytic anaplasmosis could go undetected.

Gaowa; Wuritu; Kawamori, Fumihiko; Wu, Dongxing; Yoshikawa, Yuko; Chiya, Seizou; Fukunaga, Kazutoshi; Funato, Toyohiko; Shiojiri, Masaaki; Nakajima, Hideki; Hamauzu, Yoshiji; Takano, Ai; Kawabata, Hiroki; Ando, Shuji; Kishimoto, Toshio



Animation of Human Diving  

Microsoft Academic Search

The motion of a human platform diver was simulated using a dynamic model and a control system. Thedynamic model has 32 actuated degrees of freedom and dynamic parameters within the range of thosereported in the literature for humans. The control system uses algorithms for balance, jumping, andtwisting to initiate the dive, sequences of desired values for proportional--derivative servos to performthe

Wayne L. Wooten; Jessica K. Hodgins



Human Gene Therapy  

Microsoft Academic Search

Human gene therapy is a procedure that is being used in an attempt to treat genetic and other diseases. Eleven clinical protocols are under way at the present time, each with scientific and clinical objectives. Human genetic engineering raises unique safety, social, and ethical concerns.

W. French Anderson



Healthline Human Body Maps  

NSDL National Science Digital Library

This website provides a 3-D animation of the human body. The animation allows visitors to view a variety of layers of the human body organized by both system and organs. In addition, health information, articles, and structures can be found with each layer. The website is also searchable by specific structure.

Healthline (Healthline Networks Inc)



Annotated Humanities Programs.  

ERIC Educational Resources Information Center

The humanities programs offered in 1968 by 227 United States secondary schools are listed alphabetically by state, including almost 100 new programs not annotated in the 1967 listing (see TE 000 224). Each annotation presents a brief description of the approach to study used in the particular humanities course (e.g., American Studies, Culture…

Adler, Richard R.; Applebee, Arthur


Enhancing Portugal's Human Capital  

Microsoft Academic Search

The lack of human capital in Portugal has become a key obstacle to higher growth. This paper discusses the performance of education and training services in Portugal and shows that improvements are needed to narrow the significant human capital gap with other OECD countries. Despite progress in the past decades, Portuguese children spend comparatively few years in formal education, and

Stéphanie Guichard; Bénédicte Larre



in human geography  

Microsoft Academic Search

This paper presents a critical and reflexive account of using Q methodology in human geography. Q methodology has a long pedigree in psychological, political and sociological research, but is only recently beginning to be used by human geographers. We discuss, in particular, the parts of the process(es) of Q methodology that are often glossed over in the literature, through reflecting

Sally Eden; Andrew Donaldson; Gordon Walker


Aging and Human Performance  

Microsoft Academic Search

Objectives: I identify major theoretical and practical contributions to aging and human performance as reflected primarily in the pages of Human Factors.Background: Populations worldwide are aging. True experimental work on aging is not possible because age levels cannot be manipulated. Sophisticated theoretical frameworks and modeling techniques are required to reach valid inferences about age effects and age changes. Method: Citation

Neil Charness



Human Water Cycle  

NSDL National Science Digital Library

Students learn about the human water cycle, or how humans impact the water cycle by settling down in civilizations. Specifically, they learn how people obtain, use and dispose of water. Students also learn about shortages of treated, clean and safe water and learn about ways that engineers address this issue through water conservation and graywater recycling.

Integrated Teaching And Learning Program


Secure Distributed Human Computation  

Microsoft Academic Search

This paper is a preliminary exploration of secure distributed computation. We consider the general paradigm of using large-scale distributed computation to solve difficult problems, but where humans can act as agents and provide candidate solutions. We are especially motivated by problem classes that appear to be difficult for computers to solve effectively, but are easier for humans; e.g., image analysis,

Craig Gentry; Zulfikar Ramzan; Stuart G. Stubblebine



Human nonspecific suppressive lymphokines  

Microsoft Academic Search

Since the term “lymphokine” first appeared in print over 20 years ago, a tremendous number of these soluble mediators of the immune system have been described. Within the past few years, many human nonspecific suppressive lymphokines have been identified. This review discusses the historical basis of immunologic suppression and suppressor factors. Later reports describing suppressive human lymphokines are then grouped

Michael T. Halpern



Human Exploration Mission Studies.  

National Technical Information Service (NTIS)

The nation's efforts to expand human presence and activity beyond Earth orbit into the solar system was given renewed emphasis in January of 1988 when the Presidential Directive on National Space Policy was signed into effect. The expansion of human prese...

R. L. Cataldo



[Human science and medicine].  


Objective of Human Science teaching is to develop Knowledge and ability for rational analysis of bio-medical problems. The relationship between doctor and patient must be founded on dialogue, cooperation, understanding, on respect of human rights: life, health, physical integrity, privacy, autonomy, freedom and liability to guide ethical choices in clinical experience and rediscover anthropological significance of Medicine. PMID:16285093

Caporale, Maria




EPA Science Inventory

The mission of the U.S. Environmental Protection Agency (EPA) is to protect public health and safeguard the environment. Risk assessment is an integral part of this mission in that it identifies and characterizes environmentally related human health problems. The Human Health Re...



Microsoft Academic Search

he complexities and arguments both for and against human reproductive cloning (HRC) seem to have been discussed in almost every sphere imaginable. The worlds of law, medicine, science, bioethics, philosophy, and religion have all laid claim to the forum or framework in which the issues should be discussed. While public debate and the responses of national gov- ernments have been

Steven Malby



Implications for human health.  

PubMed Central

To analyze the implications for human health, the toxicologist requires four sets of data: the results of toxicity and other studies in animals; quantitative data on actual or potential human exposure; whatever information is available on effects of exposure in man; and the statistical extrapolations from the dose-response relationships in animals to the (usually) much lower levels of human exposure. Professional expertise in toxicology is essential to assess the nature and severity of the toxic effects observed in animals, including such characteristics as potential for progression, irreversibility and production of incapacity. Given sufficient data, an estimate can be arrived at of the likelihood that such effects will be elicited in human populations of differing susceptibilities. The criteria by which the overall implications for human health can be judged comprise both the direct effects on man, as well as the indirect consequences stemming from environmental impacts.

Golberg, L



[AIDS and human rights].  


AIDS and human rights are closely related issues. This paper describes the relationship between AIDS and human rights, the impact and consequences of discrimination and the importance of the defense of human rights as a cornerstone strategy in AIDS prevention. Some general ethical aspects are addressed and two dilemmas which have been raised by the epidemic are discussed: the apparent conflict between individual and community rights and the reactions of intolerance and repression from those who claim that only through coercive strategies will the epidemic be brought under control. Specific problems in Mexico are described based on data collected at CONASIDA's Human Rights Department between 1992 and 1994. Finally some conclusions are stated emphasizing that, in the AIDS epidemic, the defense of human rights is the cornerstone of any public health strategy. PMID:8599140

Rico, B; Uribe-Zúñiga, P; Panebianco-Labbé, S; del Río-Chiriboga, C


Human hepatocyte carcinogenesis  

PubMed Central

Hepatocellular carcinoma is the third most frequent cause of cancer-related death worldwide; and its incidence rate is increasing. Clinical and molecular medical analyses have revealed substantial information on hepatocarcinogenesis. Hepatocarcinogenesis is a stepwise process during which multiple genes are altered. Genetic changes and their biological consequences in human HCC can be divided into at least 4 groups: i) tumor suppressor genes (p53, retinoblastoma, phosphatase tensin homolog and runt-related transcription factor 3), ii) oncogenes (myc, K-ras, BRAF), iii) reactivation of developmental pathways (Wnt, hedgehog), and iv) growth factors and their receptors (transforming growth factor-?, insulin-like growth factor-2 receptor). An experimental model of human hepatocarcinogenesis such as in vitro neoplastic transformation of human hepatocytes has not been successfully achieved yet, but several immortalized human hepatocyte cell lines have been established. These immortalized human hepatocytes will become useful tools for the elucidation of hepatocarcinogenesis, especially for the initial step of multistep hepatocarcinogenesis.




Beliefs about Human Extinction  

SciTech Connect

This paper presents the results of a web-based survey about futures issues. Among many questions, respondents were asked whether they believe humans will become extinct. Forty-five percent of the almost 600 respondents believe that humans will become extinct. Many of those holding this believe felt that humans could become extinct within 500-1000 years. Others estimated extinction 5000 or more years into the future. A logistic regression model was estimated to explore the bases for this belief. It was found that people who describe themselves a secular are more likely to hold this belief than people who describe themselves as being Protestant. Older respondents and those who believe that humans have little control over their future also hold this belief. In addition, people who are more apt to think about the future and are better able to imagine potential futures tend to also believe that humans will become extinct.

Tonn, Bruce Edward [ORNL



Dogs catch human yawns.  


This study is the first to demonstrate that human yawns are possibly contagious to domestic dogs (Canis familiaris). Twenty-nine dogs observed a human yawning or making control mouth movements. Twenty-one dogs yawned when they observed a human yawning, but control mouth movements did not elicit yawning from any of them. The presence of contagious yawning in dogs suggests that this phenomenon is not specific to primate species and may indicate that dogs possess the capacity for a rudimentary form of empathy. Since yawning is known to modulate the levels of arousal, yawn contagion may help coordinate dog-human interaction and communication. Understanding the mechanism as well as the function of contagious yawning between humans and dogs requires more detailed investigation. PMID:18682357

Joly-Mascheroni, Ramiro M; Senju, Atsushi; Shepherd, Alex J



Archaea on Human Skin  

PubMed Central

The recent era of exploring the human microbiome has provided valuable information on microbial inhabitants, beneficials and pathogens. Screening efforts based on DNA sequencing identified thousands of bacterial lineages associated with human skin but provided only incomplete and crude information on Archaea. Here, we report for the first time the quantification and visualization of Archaea from human skin. Based on 16 S rRNA gene copies Archaea comprised up to 4.2% of the prokaryotic skin microbiome. Most of the gene signatures analyzed belonged to the Thaumarchaeota, a group of Archaea we also found in hospitals and clean room facilities. The metabolic potential for ammonia oxidation of the skin-associated Archaea was supported by the successful detection of thaumarchaeal amoA genes in human skin samples. However, the activity and possible interaction with human epithelial cells of these associated Archaea remains an open question. Nevertheless, in this study we provide evidence that Archaea are part of the human skin microbiome and discuss their potential for ammonia turnover on human skin.

Probst, Alexander J.; Auerbach, Anna K.; Moissl-Eichinger, Christine



Human Plasma Protein C  

PubMed Central

Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (Mr = 62,000) contains 23% carbohydrate and is composed of a light chain (Mr = 21,000) and a heavy chain (Mr = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human ?-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity. Images

Kisiel, Walter



Framework for Human Microbiome Research.  

National Technical Information Service (NTIS)

A variety of microbial communities and their genes (the microbiome) exist throughout the human body, with fundamental roles in human health and disease. The National Institutes of Health (NIH)-funded Human Microbiome Project Consortium has established a p...

B. A. Methe H. H. Creasy K. E. Nelson M. Pop M. G. Giglio



Redrawing Humanity's Family Tree  

NSDL National Science Digital Library

This New York Times article details two skulls, one from central Africa and the other from the Black Sea republic of Georgia, that "have shaken the human family tree to its roots, sending scientists scrambling to see if their favorite theories are among the fallen fruit." The article discusses how the two skulls have caused scientists to rethink not only how we conceive of human evolution and its chain of events, but even the geography of evolution and migration patterns of very early humans.

Wilford, John N.



Human Gyrovirus DNA in Human Blood, Italy  

PubMed Central

Human gyrovirus (HGyV) is a recent addition to the list of agents found in humans. Prevalence, biologic properties, and clinical associations of this novel virus are still incompletely understood. We used qualitative PCRs to detect HGyV in blood samples of 301 persons from Italy. HGyV genome was detected in 3 of 100 solid organ transplant recipients and in 1 HIV-infected person. The virus was not detected in plasma samples from healthy persons. Furthermore, during observation, persons for whom longitudinal plasma samples were obtained had transient and scattered presence of circulating HGyV. Sequencing of a 138-bp fragment showed nucleotide identity among all the HGyV isolates. These results show that HGyV can be present in the blood of infected persons. Additional studies are needed to investigate possible clinical implications.

Macera, Lisa; Focosi, Daniele; Vatteroni, Maria Linda; Boggi, Ugo; Antonelli, Guido; Eloit, Marc; Pistello, Mauro



Human dignity, humiliation, and torture.  


Modern human rights instruments ground human rights in the concept of human dignity, without providing an underlying theory of human dignity. This paper examines the central importance of human dignity, understood as not humiliating people, in traditional Jewish ethics. It employs this conception of human dignity to examine and criticize U.S. use of humiliation tactics and torture in the interrogation of terrorism suspects. PMID:19886522

Luban, David



PSYCHOANALYTIC ACTIVISM: Finding the Human, Staying Human  

Microsoft Academic Search

Drawing on my work in human rights abuse, I reflect on the process of dehumanization in torture, and make the links between the life-negating denial of death in trauma victims and the ethical responsibility of society vis-à-vis these victims. In examining how my own past helps me enter into my patients' posttraumatic narrative, I seek to show that the psychoanalytic

Leanh Nguyen



Human Services Budget, 1976.  

National Technical Information Service (NTIS)

Three budgets for human services in Dakota County, Minnesota, are presented: they include the program budget, the operations budget, and the appropriations budget. The program budget, which is the most comprehensive of the three, provides a basis for anal...



Teaching about Human Geography.  

ERIC Educational Resources Information Center

|Presents a sampling of items from the ERIC database concerning the teaching of human geography. Includes documents dealing with Africa, Asia, the United States, Canada, Antarctica, and geographic concepts. Explains how to obtain ERIC documents. (SG)|

Schlene, Vickie J.



Human herpesvirus 6.  


HHV-6, the first T-lymphotropic human herpesvirus, is an important novel human pathogen. It is the cause of exanthem subitum in infants and may act as an opportunistic agent in immunocompromised patients. Moreover, several lines of clinical and experimental evidence suggest that HHV-6 may accelerate the progression of HIV infection. Progress in the study of HHV-6 has been rapid, in part as a consequence of the strong current interest in human lymphotropic viruses and their relationship with the immune system. Nonetheless, the full spectrum of diseases linked to this agent is still unknown (Table 2) and animal models of infection have not yet been exploited. The next few years will be crucial for a complete understanding of the potential role of HHV-6 in human disease. PMID:7663047

Lusso, P; Gallo, R C



Human B Lymphotropic Virus.  

National Technical Information Service (NTIS)

The invention is related generally to the isolation and characterization of a new virus. More particularly, it is related to providing a biologically pure, isolated human B lymphotropic virus, molecular clones, nucleic acid, distinctive antigenic proteins...

S. Salahuddin



Antihemophilic Factor (Human)  


Your doctor has ordered antihemophilic factor (human), an antihemophilic factor, to help your blood to clot. The drug will be either injected directly into your vein or added to an intravenous fluid ...


Approaches to Human Communication.  

ERIC Educational Resources Information Center

This anthology of essays approaches human communication from the points of view of: anthropology, art biology, economics, encounter groups, semantics, general system theory, history, information theory, international behavior, journalism, linguistics, mass media, neurophysiology, nonverbal behavior, organizational behavior, philosophy, political…

Budd, Richard W., Ed.; Ruben, Brent D., Ed.


Human Genome Center  

NSDL National Science Digital Library

Human Genome Center At Lawrence Berkeley Lab (LBL), Berkeley, California: offering information about projects in Biology, Informatics and Instrumentation, photos of LBL robotic instruments, software, and online access to one LBL genomic database.


Retroviruses and human pathology  

SciTech Connect

This book contains four sections, each consisting of several papers. The section headings are: Retroviruses and the Murine Model System;Retroviruses and the Vertebrate Model System;Retroviruses and Human Pathology;and Retroviruses and Oncogenes.

Gallo, R.C.; Stehelin, D.; Varnier, O.E.



Human Planning Processes.  

National Technical Information Service (NTIS)

This report summarizes a three-year research project investigating the cognitive processes underlying human planning behavior. The project focused on problems analogous to the naval tactical planning problem: How should the decisionmaker move force units ...

B. Hayes-Roth S. Cammarata S. E. Goldin F. Hayes-Roth S. Rosenschein



Viruses and human cancer  

SciTech Connect

This book contains papers on the following topics: Immunology and Epidemiology, Biology and Pathogenesis, Models of Pathogenesis and Treatment, Simian and Bovine Retroviruses, Human Papilloma Viruses, EBV and Herpesvirus, and Hepatitis B Virus.

Gallo, R.C.; Haseltine, W.; Klein, G.; Zur Hausen, H.



Stimulating Human Services Reform.  

National Technical Information Service (NTIS)

Suggestions for stimulating reform in human services agencies are focused on persuading policymakers and managers about the need for change; on grouping community, local, and organizational agencies for change; and on coordinating the activities of the pr...

H. W. Demone



Approaches to Human Communication.  

ERIC Educational Resources Information Center

|This anthology of essays approaches human communication from the points of view of: anthropology, art biology, economics, encounter groups, semantics, general system theory, history, information theory, international behavior, journalism, linguistics, mass media, neurophysiology, nonverbal behavior, organizational behavior, philosophy, political…

Budd, Richard W., Ed.; Ruben, Brent D., Ed.


What makes us human?  

Microsoft Academic Search

The sequence of chimpanzee chromosome 22 is starting to help us to define the set of genetic attributes that are unique to humans, but interpreting the biological consequences of these remains a major challenge.

Tarjei S Mikkelsen



Evithrom, Thrombin, Topical (Human)  

Center for Biologics Evaluation and Research (CBER)

Text Version... The final product is formulated with approximately ----- calcium chloride, ---- human albumin, ---- w/v) mannitol, ---- acetate and contains no ... More results from


Human teeth model  

NSDL National Science Digital Library

Humans are omnivores, meaning they eat both meat and plants. Omnivores generally have a mix of canines to shred meat and flat teeth to grind vegetation. The canines are less sharp than those seen in strict meat-eaters.

HÃ¥kan Svensson (None;)



Detection of Human Fatigue.  

National Technical Information Service (NTIS)

Military personnel are frequently required to perform tasks for which fatigue is a common problem. Lapses in alertness associated with fatigue are major contributors to human error. We have proposed to create a system that will use unobtrusively obtained ...

J. A. Stern T. B. Brown



Human Assisted Assembly Processes  

SciTech Connect

Automatic assembly sequencing and visualization tools are valuable in determining the best assembly sequences, but without Human Factors and Figure Models (HFFMs) it is difficult to evaluate or visualize human interaction. In industry, accelerating technological advances and shorter market windows have forced companies to turn to an agile manufacturing paradigm. This trend has promoted computerized automation of product design and manufacturing processes, such as automated assembly planning. However, all automated assembly planning software tools assume that the individual components fly into their assembled configuration and generate what appear to be a perfectly valid operations, but in reality the operations cannot physically be carried out by a human. Similarly, human figure modeling algorithms may indicate that assembly operations are not feasible and consequently force design modifications; however, if they had the capability to quickly generate alternative assembly sequences, they might have identified a feasible solution. To solve this problem HFFMs must be integrated with automated assembly planning to allow engineers to verify that assembly operations are possible and to see ways to make the designs even better. Factories will very likely put humans and robots together in cooperative environments to meet the demands for customized products, for purposes including robotic and automated assembly. For robots to work harmoniously within an integrated environment with humans the robots must have cooperative operational skills. For example, in a human only environment, humans may tolerate collisions with one another if they did not cause much pain. This level of tolerance may or may not apply to robot-human environments. Humans expect that robots will be able to operate and navigate in their environments without collisions or interference. The ability to accomplish this is linked to the sensing capabilities available. Current work in the field of cooperative automation has shown the effectiveness of humans and machines directly interacting to perform tasks. To continue to advance this area of robotics, effective means need to be developed to allow natural ways for people to communicate and cooperate with robots just as they do with one another.




Human facial beauty  

Microsoft Academic Search

It is hypothesized that human faces judged to be attractive by people possess two features—averageness and symmetry—that promoted\\u000a adaptive mate selection in human evolutionary history by way of production of offspring with parasite resistance. Facial composites\\u000a made by combining individual faces are judged to be attractive, and more attractive than the majority of individual faces.\\u000a The composites possess both symmetry

Randy Thorrthill; Steven W. Gangestad



Geology and Human Health  

NSDL National Science Digital Library

This site contains a variety of educational and supporting materials for faculty teaching in the emerging field of geology and human health. You will find links to internet resources, books, teaching activities, and a group email list, as well as posters, presentations and discussions from the spring 2004 workshop on Geology and Human Health. These resources reflect the contributions of faculty members from across the country and the collections will continue to grow as materials are developed.


The Human Virome  

Microsoft Academic Search

\\u000a In this chapter we discuss changing approaches to viral discovery and human health, summarize the current understanding of\\u000a the human-associated viral community, and review contemporary methods in viral metagenomics. The virome is the community of viruses that populate an organism or ecosystem at any given time. This includes the “core” set of commensal viruses that do not give rise to

Matthew Haynes; Forest Rohwer


Paperworks: The Human Touch  

Microsoft Academic Search

Paperworks: The Human Touch\\u000aan exhibition of works by the Western Reserve Calligraphers\\u000aSOUTH EUCLID, Ohio—8 August 2012—In conjunction with Octavofest, a celebration of the book and paper arts, and Watermarks 2012, Notre Dame College’s (NDC’s) Clara Fritzsche Library will host “Paperworks: The Human Touch,” a showing of works by the Western Reserve Calligraphers. An opening reception will be held



Computational Human Body Models  

Microsoft Academic Search

Computational human body models are widely used for automotive crashsafety research and design and as such have significantly\\u000a contributed to a reduction of traffic injuries and fatalities. Currently crash simulations are mainly performed using models\\u000a based on crash-dummies. However crash dummies differ significantly from the real human body and moreover crash dummies are\\u000a only available for a limited set of

Jac Wismans; Riender Happee; J. A. W. Dommelen


Globalization and human cooperation  

Microsoft Academic Search

Globalization magnifies the problems that affect all people and that require large-scale human cooperation, for example, the overharvesting of natural resources and human-induced global warming. However, what does globalization imply for the cooperation needed to address such global social dilemmas? Two competing hypotheses are offered. One hypothesis is that globalization prompts reactionary movements that reinforce parochial distinctions among people. Large-scale

Nancy R. Buchan; Gianluca Grimalda; Rick Wilson; Marilynn Brewer; Enrique Fatas; Margaret Foddy



Engineering Human Cooperation  

Microsoft Academic Search

In a laboratory experiment, we use a public goods game to examine the hypothesis that human subjects use an involuntary eye-detector\\u000a mechanism for evaluating the level of privacy. Half of our subjects are “watched” by images of a robot presented on their\\u000a computer screen. The robot—named Kismet and invented at MIT—is constructed from objects that are obviously not human with

Terence C. Burnham; Brian Hare



Genetics of human hydrocephalus  

Microsoft Academic Search

Human hydrocephalus is a common medical condition that is characterized by abnormalities in the flow or resorption of cerebrospinal\\u000a fluid (CSF), resulting in ventricular dilatation. Human hydrocephalus can be classified into two clinical forms, congenital\\u000a and acquired. Hydrocephalus is one of the complex and multifactorial neurological disorders.\\u000a \\u000a A growing body of evidence indicates that genetic factors play a major role

Jun Zhang; Michael A. Williams; Daniele Rigamonti



Mapping the human genome  

SciTech Connect

This article is a review of the book Mapping the Human Genome: Using Law and Ethics as Guides, edited by George C. Annas and Sherman Elias. The book is a collection of essays on the subject of using ethics and laws as guides to justify human gene mapping. It addresses specific issues such problems related to eugenics, patents, insurance as well as broad issues such as the societal definitions of normality.

Annas, G.C.; Elias, S. (eds.)



Human cardiac stem cells  

PubMed Central

The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. The characterization of human cardiac stem cells (hCSCs) would have important clinical implications for the management of the failing heart. We have established the conditions for the isolation and expansion of c-kit-positive hCSCs from small samples of myocardium. Additionally, we have tested whether these cells have the ability to form functionally competent human myocardium after infarction in immunocompromised animals. Here, we report the identification in vitro of a class of human c-kit-positive cardiac cells that possess the fundamental properties of stem cells: they are self-renewing, clonogenic, and multipotent. hCSCs differentiate predominantly into cardiomyocytes and, to a lesser extent, into smooth muscle cells and endothelial cells. When locally injected in the infarcted myocardium of immunodeficient mice and immunosuppressed rats, hCSCs generate a chimeric heart, which contains human myocardium composed of myocytes, coronary resistance arterioles, and capillaries. The human myocardium is structurally and functionally integrated with the rodent myocardium and contributes to the performance of the infarcted heart. Differentiated human cardiac cells possess only one set of human sex chromosomes excluding cell fusion. The lack of cell fusion was confirmed by the Cre-lox strategy. Thus, hCSCs can be isolated and expanded in vitro for subsequent autologous regeneration of dead myocardium in patients affected by heart failure of ischemic and nonischemic origin.

Bearzi, Claudia; Rota, Marcello; Hosoda, Toru; Tillmanns, Jochen; Nascimbene, Angelo; De Angelis, Antonella; Yasuzawa-Amano, Saori; Trofimova, Irina; Siggins, Robert W.; LeCapitaine, Nicole; Cascapera, Stefano; Beltrami, Antonio P.; D'Alessandro, David A.; Zias, Elias; Quaini, Federico; Urbanek, Konrad; Michler, Robert E.; Bolli, Roberto; Kajstura, Jan; Leri, Annarosa; Anversa, Piero



Immunogenetics of Human Placentation  

PubMed Central

Natural killer (NK) cells are a population of lymphocytes that function in both immune defense and reproduction. Diversifying NK cell phenotype and function are interactions between NK cell receptors and major histocompatibility complex (MHC) class I ligands. As a consequence of strong and variable selection these ligand-receptor systems are polymorphic, rapidly evolving, and considerably species-specific. Counterparts to the human system of HLA class I ligands and killer cell immunoglobulin-like receptors (KIR) are present only in apes and Old World monkeys. HLA-C, the dominant ligand for human KIR and the only polymorphic HLA class I expressed by trophoblast, is further restricted to humans and great apes. Even then, the human system appears qualitatively different from that of chimpanzees, in that it has evolved a genetic balance between particular groups of receptors and ligands that favor reproductive success and other groups of receptors and ligands that have been correlated with disordered placentation. Human populations that have survived successive episodes of epidemic disease and population bottlenecks maintain a breadth of diversity for KIR and HLA class I, implying that loss of such diversity disfavors long-term survival of a human population.

Parham, Peter; Norman, Paul J.; Abi-Rached, Laurent; Hilton, Hugo G.; Guethlein, Lisbeth A.



Human Development Report 2005  

NSDL National Science Digital Library

The notion of human development is one that is quite old, even if it has not always gone by that name. The United Nations understands human development to be about Âcreating an environment in which people can develop their full potential and lead productive, creative lives in accord with their needs and interestsÂ. In this way, of course, many ancient city-states and other such communities of shared interests have always been about creating such an environment. This latest report on the state of human development affords curious individuals an insight into the United Nations work in this area. It offers some insights into the nature of inequalities which affect the potentiality of human development schemes and also looks at other related processes such as the nature of international trade and violent conflict. The report is divided into five primary chapters, and is supplemented by a list of human development indicators and a bibliography. The site also contains an animated file that offers a visualization of the trend of human development.



Glycobiology of human milk.  


Glycans are characteristic components of milk, and each species has unique patterns of specific carbohydrates. Human milk is unusually rich in glycans, with the major components being lactose and oligosaccharides, representing approximately 6.8 and 1% of the milk, respectively. Other sources of glycans in human milk include monosaccharides, mucins, glycosaminoglycans, glycoproteins, glycopeptides, and glycolipids. In human milk, the presence and patterns of these glycans vary depending upon the stage of lactation and the maternal genes and their genetic polymorphisms that control glycosyl transferases. The synthesis of milk glycans utilizes a significant portion of the metabolic energy that the mother expends when producing her milk, but other than lactose, these glycans contribute little to the nutritional needs of the infant. The data herein support several functions. 1) Many human milk glycans inhibit pathogens from binding to the intestinal mucosa. 2) Human milk glycans attenuate inflammation. 3) Glycans also directly stimulate the growth of beneficial (mutualist) bacteria of the microbiota (formerly considered commensal microflora of the intestine); these mutualists and their fermentation products can, in turn, (a) inhibit pathogens, (b) modulate signaling and inflammation, and (c) the fermentation products can be absorbed and utilized as a source of dietary calories. These functions can help direct and support intestinal postnatal growth, development, and ontogeny of colonization. The many functions of the milk glycans may synergistically protect infants from disease. Hence, human milk glycans and their homologs may serve as novel prophylactic or therapeutic agents for a diverse range of deleterious conditions. PMID:24010840

Newburg, D S



Human Factors Review Plan  

SciTech Connect

''Human Factors'' is concerned with the incorporation of human user considerations into a system in order to maximize human reliability and reduce errors. This Review Plan is intended to assist in the assessment of human factors conditions in existing DOE facilities. In addition to specifying assessment methodologies, the plan describes techniques for improving conditions which are found to not adequately support reliable human performance. The following topics are addressed: (1) selection of areas for review describes techniques for needs assessment to assist in selecting and prioritizing areas for review; (2) human factors engineering review is concerned with optimizing the interfaces between people and equipment and people and their work environment; (3) procedures review evaluates completeness and accuracy of procedures, as well as their usability and management; (4) organizational interface review is concerned with communication and coordination between all levels of an organization; and (5) training review evaluates training program criteria such as those involving: trainee selection, qualification of training staff, content and conduct of training, requalification training, and program management.

Paramore, B.; Peterson, L.R. (eds.)



Genomics of human longevity  

PubMed Central

In animal models, single-gene mutations in genes involved in insulin/IGF and target of rapamycin signalling pathways extend lifespan to a considerable extent. The genetic, genomic and epigenetic influences on human longevity are expected to be much more complex. Strikingly however, beneficial metabolic and cellular features of long-lived families resemble those in animals for whom the lifespan is extended by applying genetic manipulation and, especially, dietary restriction. Candidate gene studies in humans support the notion that human orthologues from longevity genes identified in lower species do contribute to longevity but that the influence of the genetic variants involved is small. Here we discuss how an integration of novel study designs, labour-intensive biobanking, deep phenotyping and genomic research may provide insights into the mechanisms that drive human longevity and healthy ageing, beyond the associations usually provided by molecular and genetic epidemiology. Although prospective studies of humans from the cradle to the grave have never been performed, it is feasible to extract life histories from different cohorts jointly covering the molecular changes that occur with age from early development all the way up to the age at death. By the integration of research in different study cohorts, and with research in animal models, biological research into human longevity is thus making considerable progress.

Slagboom, P. E.; Beekman, M.; Passtoors, W. M.; Deelen, J.; Vaarhorst, A. A. M.; Boer, J. M.; van den Akker, E. B.; van Heemst, D.; de Craen, A. J. M.; Maier, A. B.; Rozing, M.; Mooijaart, S. P.; Heijmans, B. T.; Westendorp, R. G. J.



Potentiality and human embryos.  


Consideration of the potentiality of human embryos to develop characteristics of personhood, such as intellect and will, has figured prominently in arguments against abortion and the use of human embryos for research. In particular, such consideration was the basis for the call of the US President's Council on Bioethics for a moratorium on stem cell research on human embryos. In this paper, I critique the concept of potentiality invoked by the Council and offer an alternative account. In contrast to the Council's view that an embryo's potentiality is determined by definition and is not affected by external conditions that may prevent certain possibilities from ever being realized, I propose an empirically grounded account of potentiality that involves an assessment of the physical and decisional conditions that may restrict an embryo's possibilities. In my view, some human embryos lack the potentiality to become a person that other human embryos have. Assuming for the sake of argument that the potential to become a person gives a being special moral status, it follows that some human embryos lack this status. This argument is then used to support Gene Outka's suggestion that it is morally permissible to experiment on 'spare' frozen embryos that are destined to be destroyed. PMID:17845464

Lizza, John P



The human telomere  

SciTech Connect

An ultimate goal of human genetics is the generation of a complete physical and ''functional'' map of the human genome. Twenty-five percent of human DNA, however, consists of repetitive DNA sequences. These repetitive DNA sequences are thought to arise by many mechanisms, from direct sequence amplification by the unequal recombination of homologous DNA regions to the reverse flow of genetic information. A general outline of the chromosomal organization of these repetitive sequences will be discussed. Our working hypothesis is that certain classes of human repetitive DNA sequences ''encode'' the information necessary for defining long-range genomic structure. Evidence will be presented that the first goal of this research, the identification and cloning of the human telomere, has been achieved. A human repetitive DNA library was constructed from randomly sheared, reassociated, and oligo(G/center dot/C)-tailed DNA, a method that minimizes the potential loss of sequences devoid of a given restriction enzyme site. Sequences too large to clone efficiently in cosmid or /lambda/ vectors, such as centromeric repeats, or telomeric sequences with an end incompatible for cloning, should be present in this library. In order to isolate highly conserved repetitive DNA sequences, this library was screened with radiolabeled hamster Cot50 repetitive DNA. Two clones, containing tandem arrays of the sequence (TTAGGG), were isolated by this method. 30 refs., 1 fig., 2 tabs.

Moyzis, R.K.



The state human trafficking and human rights issues in Africa  

Microsoft Academic Search

Internal factors in Africa which include limited autonomy of African states, the states’ various degrees of lack of capacity, as well as inept and parasitic leadership make human trafficking and human rights abuses in Africa inevitable. Regardless of the connections suggested to exist between globalization and human trafficking, internal factors in Africa are more fundamental than globalization in explaining human

Browne Onuoha



Infants' Responses to Real Humans and Representations of Humans  

ERIC Educational Resources Information Center

|Infants' responses to typical and scrambled human body shapes were assessed in relation to the realism of the human body stimuli presented. In four separate experiments, infants were familiarized to typical human bodies and then shown a series of scrambled human bodies on the test. Looking behaviour was assessed in response to a range of…

Heron, Michelle; Slaughter, Virginia



Effect of Human Biases on Human-Agent Teams  

Microsoft Academic Search

As human-agent teams get increasingly deployed in the real-world, agent designers need to take into account that humans and agents have different abilities to specify preferences. In this paper, we focus on how human biases in specifying preferences for resources impacts the performance of large, heterogeneous teams. In particular, we model the inclination of humans to simplify their preference functions

Praveen Paruchuri; Pradeep Reddy VARAKANTHAM; Katia P. Sycara; Paul Scerri



Humans in the Loop: Human-Computer Interaction and Security  

Microsoft Academic Search

The security field suffers from an endemic problem: despite our best efforts, the current infrastructure is continually full of security vulnerabilities. The systems that comprise this infrastructure also are full of boundaries and interfaces where humans and systems must interact: most secure systems exist to serve human users and carry out human-oriented processes, and are designed and built by humans.

Sean W. Smith



Developing Human Performance Measures  

SciTech Connect

Through the reactor oversight process (ROP), the U.S. Nuclear Regulatory Commission (NRC) monitors the performance of utilities licensed to operate nuclear power plants. The process is designed to assure public health and safety by providing reasonable assurance that licensees are meeting the cornerstones of safety and designated crosscutting elements. The reactor inspection program, together with performance indicators (PIs), and enforcement activities form the basis for the NRC’s risk-informed, performance based regulatory framework. While human performance is a key component in the safe operation of nuclear power plants and is a designated cross-cutting element of the ROP, there is currently no direct inspection or performance indicator for assessing human performance. Rather, when human performance is identified as a substantive cross cutting element in any 1 of 3 categories (resources, organizational or personnel), it is then evaluated for common themes to determine if follow-up actions are warranted. However, variability in human performance occurs from day to day, across activities that vary in complexity, and workgroups, contributing to the uncertainty in the outcomes of performance. While some variability in human performance may be random, much of the variability may be attributed to factors that are not currently assessed. There is a need to identify and assess aspects of human performance that relate to plant safety and to develop measures that can be used to successfully assure licensee performance and indicate when additional investigation may be required. This paper presents research that establishes a technical basis for developing human performance measures. In particular, we discuss: 1) how historical data already gives some indication of connection between human performance and overall plant performance, 2) how industry led efforts to measure and model human performance and organizational factors could serve as a data source and basis for a framework, 3) how our use of modeling and simulation techniques could be used to develop and validate measures of human performance, and 4) what the possible outcomes are from this research as the modeling and simulation efforts generate results.

Jeffrey Joe; Bruce Hallbert; Larry Blackwood; Donald Dudehoeffer; Kent Hansen



[Antiviral humanized and human monoclonal antibodies].  


The paper considers the characteristics of monoclonal antibodies, methods for their experimental preparation, problems of their production, and possibilities of their use for the emergency prevention of viral infections and for the treatment of chronic diseases caused by human immunodeficiency virus, hepatitis B and C viruses, and herpes viruses. The future of experimentally produced or clinically trialed monoclonal antibodies is mainly determined by commercial considerations. It is possible that simplification of industrial production technologies and a reduction in the cost of evidence-based methods for evaluation of clinical effectiveness will allow monoclonal antibodies to be extensively used for the prevention and treatment of viral infections. PMID:22171470

Lashkevich, V A


Living Human Rights students raise awareness of ‘Human Trafficking’  

Microsoft Academic Search

Students from the unit Living Human Rights, from The University of Notre Dame Australia’s Fremantle Campus, recently held an expo to raise awareness of human trafficking.\\u000aThe unit, Living Human Rights, introduces students to human rights from a number of interrelated perspectives: global and local; professional and personal; present and historical. It explores how human rights need to form an

Cassidy Rebecca



Human-mouse cell hybrid with human multiple Y chromosomes  

Microsoft Academic Search

HUMAN-mouse cell hybrids usually exhibit preferential loss of human chromosomes1,2. After a time interval, which depends in part on the cell types used, most or sometimes all the human chromosomes segregate out from the hybrid cells3. In the course of investigation of the phenomenon of preferential loss of human chromosomes from hybrids between the mouse cell line RAG and human




Human evolution: taxonomy and paleobiology  

Microsoft Academic Search

This review begins by setting out the context and the scope of human evolution. Several classes of evidence, morphological, molecular, and genetic, support a particularly close relationship between modern humans and the species within the genus Pan, the chimpanzee. Thus human evolution is the study of the lineage, or clade, comprising species more closely related to modern humans than to




Making IBM's Computer, Watson, Human  

ERIC Educational Resources Information Center

|This essay uses the recent victory of an IBM computer (Watson) in the TV game, "Jeopardy," to speculate on the abilities Watson would need, in addition to those it has, to be human. The essay's basic premise is that to be human is to behave as humans behave and to function in society as humans function. Alternatives to this premise are considered…

Rachlin, Howard



Translucency of Human Dental Enamel  

Microsoft Academic Search

Translucency of human dental enamel was determined by total transmittance of wavelengths from 400 to 700 nm. The transmission coefficient at 525 nm was 0.481 mm-1. Total transmission of light through human dental enamel increased with increasing wavelength. Human tooth enamel is more translucent at higher wavelengths. The translucency of wet human enamel and enamel after dehydration was also measured

R. H. W. Brodbelt; W. J. OBrien; P. L. Fan; J. G. Frazer-Dib; R. Yu



Neuron mechanism of human languages  

Microsoft Academic Search

Human brain is exceptionally complex and simple at the same time. Its extremely composite biological structure results in human everyday behavior. Many people might consider this behavior rather simple than complex. In our research we will concentrate on the ways with which the human brain can process English and other human languages because taken in general sense, the ability to

Alexander Borzenko



Human values and natural systems  

Microsoft Academic Search

. Ecological valu~s contribute positively to human experiences but also seem to be there apart from humans. In an evolutionary ecosystem, the centtal goods of the biosys­ temic Earth were in place before humans arrived. Humans cash in on and spend what is naturally given. In many respects, though by no means all, the earthen setup is \\

Holmes Rolston III



Human Security and Global Health  

Microsoft Academic Search

The authors consider the ways in which the concept of human security expands understanding of the links between health and human development. After discussing the emergence of the concept of human security, they examine the ways in which human security is linked to global health, particularly as regards violence and conflict, global infectious diseases, and poverty and inequality. The authors

Lincoln Chen; Vasant Narasimhan



Archaic human genomics.  


For much of the 20th century, the predominant view of human evolutionary history was derived from the fossil record. Homo erectus was seen arising in Africa from an earlier member of the genus and then spreading throughout the Old World and into the Oceania. A regional continuity model of anagenetic change from H. erectus via various intermediate archaic species into the modern humans in each of the regions inhabited by H. erectus was labeled the multiregional model of human evolution (MRE). A contrasting model positing a single origin, in Africa, of anatomically modern H. sapiens with some populations later migrating out of Africa and replacing the local archaic populations throughout the world with complete replacement became known as the recent African origin (RAO) model. Proponents of both models used different interpretations of the fossil record to bolster their views for decades. In the 1980s, molecular genetic techniques began providing evidence from modern human variation that allowed not only the different models of modern human origins to be tested but also the exploration demographic history and the types of selection that different regions of the genome and even specific traits had undergone. The majority of researchers interpreted these data as strongly supporting the RAO model, especially analyses of mitochondrial DNA (mtDNA). Extrapolating backward from modern patterns of variation and using various calibration points and substitution rates, a consensus arose that saw modern humans evolving from an African population around 200,000 years ago. Much later, around 50,000 years ago, a subset of this population migrated out of Africa replacing Neanderthals in Europe and western Asia as well as archaics in eastern Asia and Oceania. mtDNA sequences from more than two-dozen Neanderthals and early modern humans re-enforced this consensus. In 2010, however, the complete draft genomes of Neanderthals and of heretofore unknown hominins from Siberia, called Denisovans, demonstrated gene flow between these archaic human species and modern Eurasians but not sub-Saharan Africans. Although the levels of gene flow may be very limited, this unexpected finding does not fit well with either the RAO model or MRE model. More thorough sampling of modern human diversity, additional fossil discoveries, and the sequencing of additional hominin fossils are necessary to throw light onto our origins and our history. PMID:23124308

Disotell, Todd R



The Human Serum Metabolome  

PubMed Central

Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at

Psychogios, Nikolaos; Hau, David D.; Peng, Jun; Guo, An Chi; Mandal, Rupasri; Bouatra, Souhaila; Sinelnikov, Igor; Krishnamurthy, Ramanarayan; Eisner, Roman; Gautam, Bijaya; Young, Nelson; Xia, Jianguo; Knox, Craig; Dong, Edison; Huang, Paul; Hollander, Zsuzsanna; Pedersen, Theresa L.; Smith, Steven R.; Bamforth, Fiona; Greiner, Russ; McManus, Bruce; Newman, John W.; Goodfriend, Theodore; Wishart, David S.



The human serum metabolome.  


Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at PMID:21359215

Psychogios, Nikolaos; Hau, David D; Peng, Jun; Guo, An Chi; Mandal, Rupasri; Bouatra, Souhaila; Sinelnikov, Igor; Krishnamurthy, Ramanarayan; Eisner, Roman; Gautam, Bijaya; Young, Nelson; Xia, Jianguo; Knox, Craig; Dong, Edison; Huang, Paul; Hollander, Zsuzsanna; Pedersen, Theresa L; Smith, Steven R; Bamforth, Fiona; Greiner, Russ; McManus, Bruce; Newman, John W; Goodfriend, Theodore; Wishart, David S



Human herpesvirus 6.  

PubMed Central

Human herpesvirus 6 variant A (HHV-6A) and human herpesvirus 6 variant B (HHV-6B) are two closely related yet distinct viruses. These visuses belong to the Roseolovirus genus of the betaherpesvirus subfamily; they are most closely related to human herpesvirus 7 and then to human cytomegalovirus. Over 95% of people older than 2 years of age are seropositive for either or both HHV-6 variants, and current serologic methods are incapable of discriminating infection with one variant from infection with the other. HHV-6A has not been etiologically linked to any human disease, but such an association will probably be found soon. HHV-6B is the etiologic agent of the common childhood illness exanthem subitum (roseola infantum or sixth disease) and related febrile illnesses. These viruses are frequently active and associated with illness in immunocompromised patients and may play a role in the etiology of Hodgkin's disease and other malignancies. HHV-6 is a commensal inhabitant of brains; various neurologic manifestations, including convulsions and encephalitis, can occur during primary HHV-6 infection or in immunocompromised patients. HHV-6 and distribution in the central nervous system are altered in patients with multiple sclerosis; the significance of this is under investigation.

Braun, D K; Dominguez, G; Pellett, P E



The Human Aerodynamic Wake  

NASA Astrophysics Data System (ADS)

The wake that trails behind a walking person in still air is, in effect, that of an irregular 3-D cylinder. At a brisk walking speed of 1.3 m/s (3 mph), the human wake is characterized by a Reynolds number of about 50,000. It is thus turbulent with underlying large-scale vortex motion. We show that buoyancy plays no role at this Reynolds number, even though it is dominant in the plume of a standing person. Computational Navier-Stokes solutions and laser-light-sheet experiments with a human subject reveal a large recirculation zone behind the torso and flow between the legs. The decay of a passive scalar introduced on the human body is found to be exponential with downstream distance. The volume flux in the human wake is roughly constant with downstream distance until the recirculation closes, whence it grows due to turbulent entrainment. Further experiments reveal the development of the wake from the human thermal plume as the Reynolds number (proportional to walking speed) is increased from zero to 50,000. These results pertain to the sensing of chemical traces in the wakes of walking persons for aviation security. Supported by FAA Grant 99-G-040.

Settles, Gary; Moyer, Zachary; Paterson, Eric; Edge, Brian



The Human Genome Program  

SciTech Connect

Early in 1986, Charles DeLisi, then head of the Office of Health and Environmental Research at the Department of Energy (DOE) requested the Los Alamos National Laboratory (LANL) to organize a workshop charged with inquiring whether the state of technology and potential payoffs in biological knowledge and medical practice were such as to justify an organized program to map and sequence the human genome. The DOE's interest arose from its mission to assess the effects of radiation and other products of energy generation on human health in general and genetic material in particular. The workshop concluded that the technology was ripe, the benefits would be great, and a national program should be promptly initiated. Later committees, reporting to DOE, to the NIH, to the Office of Technology Assessment of the US Congress, and to the National Academy of Science have reviewed these issues more deliberately and come to the same conclusion. As a consequence, there has been established in the United States, a Human Genome Program, with funding largely from the NIH and the DOE, as indicated in Table 1. Moreover, the Program has attracted international interest, and Great Britain, France, Italy, and the Soviet Union, among other countries, have been reported to be starting human genome initiatives. Coordination of these programs, clearly in the interests of each, remains to be worked out, although an international Human Genome Organization (HUGO) is considering such coordination. 5 refs., 1 fig., 2 tabs.

Bell, G.I.