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      UMP/CMPK Is Not the Critical Enzyme in the Metabolism of Pyrimidine Ribonucleotide and Activation of Deoxycytidine Analogs in Human RKO Cells

      1 , 1 , 1 , 2 , 1 , 2 , *

      PLoS ONE

      Public Library of Science

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          Abstract

          Background

          Human UMP/CMP kinase was identified based on its enzymatic activity in vitro. The role of this protein is considered critical for the maintenance of pyrimidine nucleotide pool profile and for the metabolism of pyrimidine analogs in cells, based on the in vitro study of partially purified enzyme and recombinant protein. However, no detailed study has yet addressed the role of this protein in nucleotide metabolism in cells.

          Methodology/Principal Findings

          Two stable cell lines in which UMP/CMP kinase (mRNA: AF087865, EC 2.7.4.14) can be either up-regulated or down-regulated were developed using Tet-On Gene Expression Systems. The amount and enzymatic activity of UMP/CMP kinase extracted from these two cell lines can be induced up by 500% or down by 95–98%. The ribonucleotides of endogenous pyrimidine as well as the metabolism of exogenous natural pyrimidine nucleosides and their analogs were not susceptible to the altered amount of UMP/CMP kinase in these two stable RKO cell lines. The level of incorporation of pyrimidine nucleoside analogs, such as gemcitabine (dFdC) and troxacitabine (L-OddC), into cellular DNA and their potency in inhibiting cell growth were not significantly altered by up-regulation or down-regulation of UMP/CMP kinase expression in cells.

          Conclusions/Significance

          The UMP/CMP kinase (EC 2.7.4.14) expressed in RKO cells is not critical for the phosphorylation of (d)CMP and the maintenance of natural nucleotide pools. It also does not play an important role in the activation of dFdC and L-OddC. The increase by 500% or decrease by 95–98% in the levels of UMP/CMP kinase do not affect steady state levels of dFdC and L-OddC in RKO cells. Overall, the activity and possible mechanisms of recombinant UMP/CMP kinase expressed in the in vitro system can not be extended to that of UMP/CMP kinase expressed in a cell system or an in vivo system.

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          Most cited references 24

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          Physiological concentrations of purines and pyrimidines.

           W Traut (1994)
          The concentrations of bases, nucleosides, and nucleosides mono-, di- and tri-phosphate are compared for about 600 published values. The data are predominantly from mammalian cells and fluids. For the most important ribonucleotides, average concentrations +/- SD (microM) are: ATP, 3,152 +/- 1,698; GTP, 468 +/- 224; UTP, 567 +/- 460 and CTP, 278 +/- 242. For deoxynucleosides-triphosphate (dNTP), the concentrations in dividing cells are: dATP, 24 +/- 22; dGTP, 5.2 +/- 4.5; dCTP, 29 +/- 19 and dTTP 37 +/- 30. By comparison, dUTP is usually about 0.2 microM. For the 4 dNTPs, tumor cells have concentrations of 6-11 fold over normal cells, and for the 4 NTPs, tumor cells also have concentrations 1.2-5 fold over the normal cells. By comparison, the concentrations of NTPs are significantly lower in various types of blood cells. The average concentration of bases and nucleosides in plasma and other extracellular fluids is generally in the range of 0.4-6 microM; these values are usually lower than corresponding intracellular concentrations. For phosphate compounds, average cellular concentrations are: Pi, 4400; ribose-1-P, 55; ribose-5-P, 70 and P-ribose-PP, 9.0. The metal ion magnesium, important for coordinating phosphates in nucleotides, has values (mM) of: free Mg2+, 1.1; complexed-Mg, 8.0. Consideration of experiments on the intracellular compartmentation of nucleotides shows support for this process between the cytoplasm and mitochondria, but not between the cytoplasm and the nucleus.
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            Reconstruction of amino acid biosynthesis pathways from the complete genome sequence.

             M Kanehisa,  H Bono,  S. Goto (1998)
            The complete genome sequence of an organism contains information that has not been fully utilized in the current prediction methods of gene functions, which are based on piece-by-piece similarity searches of individual genes. We present here a method that utilizes a higher level information of molecular pathways to reconstruct a complete functional unit from a set of genes. Specifically, a genome-by-genome comparison is first made for identifying enzyme genes and assigning EC numbers, which is followed by the reconstruction of selected portions of the metabolic pathways by use of the reference biochemical knowledge. The completeness of the reconstructed pathway is an indicator of the correctness of the initial gene function assignment. This feature has become possible because of our efforts to computerize the current knowledge of metabolic pathways under the KEGG project. We found that the biosynthesis pathways of all 20 amino acids were completely reconstructed in Escherichia coli, Haemophilus influenzae, and Bacillus subtilis, and probably in Synechocystis and Saccharomyces cerevisiae as well, although it was necessary to assume wider substrate specificity for aspartate aminotransferases.
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              Post-translational modifications of recombinant proteins: significance for biopharmaceuticals.

              The production of recombinant therapeutic proteins is one of the fastest growing sectors of the pharmaceutical industry, particularly monoclonal antibodies and Fc-fusion proteins. Currently, mammalian cells are the dominant production system for these proteins because they can perform complex post-translational modifications that are often required for efficient secretion, drug efficacy, and stability. These protein modifications include misfolding and aggregation, oxidation of methionine, deamidation of asparagine and glutamine, variable glycosylation, and proteolysis. Such modifications not only pose challenges for accurate and consistent bioprocessing, but also may have consequences for the patient in that incorrect modifications and aggregation can lead to an immune response to the therapeutic protein. This mini-review describes examples analytical and preventative advances in the fields of protein oxidation, deamidation, misfolding and aggregation (glycosylation is covered in other articles in this issue). The feasibility of partially replacing traditional analytical methods such as peptide mapping with high-throughput screens and their use in clone and media selection are evaluated. This review also discusses how further technical advances could improve the manufacturability, potency, and safety of biotherapeutics.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2011
                3 May 2011
                : 6
                : 5
                Affiliations
                [1 ]Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, United States of America
                [2 ]Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan, Republic of China
                German Cancer Research Center, Germany
                Author notes

                Conceived and designed the experiments: RH Y-CC. Performed the experiments: RH WL C-HH. Analyzed the data: RH Y-CC. Contributed reagents/materials/analysis tools: RH. Wrote the paper: RH Y-CC.

                PONE-D-10-03771
                10.1371/journal.pone.0019490
                3086915
                21559290
                Hu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                Counts
                Pages: 9
                Categories
                Research Article
                Biology
                Biochemistry
                Enzymes
                Enzyme Metabolism
                Nucleic Acids
                Nucleotides
                Metabolism
                Microbiology
                Virology
                Viruses and Cancer
                Medicine
                Drugs and Devices
                Clinical Pharmacology

                Uncategorized

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