scholarly journals Purification from hamster cells of the multifunctional protein that initiates de novo synthesis of pyrimidine nucleotides.

1977 ◽  
Vol 252 (18) ◽  
pp. 6379-6385 ◽  
Author(s):  
P F Coleman ◽  
D P Suttle ◽  
G R Stark
Keyword(s):  
De Novo ◽  
De Novo Synthesis ◽  
Pyrimidine Nucleotides ◽  
Multifunctional Protein

scholarly journals Inhibition of De Novo Pyrimidine Nucleotide Synthesis By the Novel DHODH Inhibitor PTC299 Induces Differentiation and/or Death of AML Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5152-5152
Author(s):  
Marla Weetall ◽  
Kensuke Kojima ◽  
Sujan Piya ◽  
Christopher Trotta ◽  
John Baird ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
De Novo ◽  
Salvage Pathway ◽  
De Novo Synthesis ◽  
Dna Breaks ◽  
Advisory Committees ◽  
Pyrimidine Nucleotides ◽  
Nucleotide Synthesis ◽  
Pyrimidine Nucleotide

Background: Pyrimidine nucleotides are generated either by de novo synthesis or the salvage pathway in which pyrimidine nucleotides are obtained from the diet. Resting cells typically acquire adequate pyrimidine nucleotides from the salvage pathway. Rapidly proliferating cells, however, are dependent on the de novo synthesis of pyrimidine nucleotides. PTC299 is an inhibitor of dihydroorotate dehydrogenase (DHODH), a rate limiting enzyme for de novo pyrimidine nucleotide synthesis that had previously been in clinical trials for treatment of solid tumors. Results: Using 15N-labelled glutamine, we show that PTC299 reduces de novo pyrimidine nucleotide synthesis in PTC299-sensitive AML cell lines resulting in a depletion of total pyrimidine nucleotides. In parallel to reduction in pyrimidine nucleotides, PTC 299 leads to accumulation of DHO, the substrate of DHODH and unexpectedly, an accumulation of N-carbamoyl aspartate the metabolite above DHO in the de novo pyrimidine nucleotide synthesis pathway. PTC299 was broadly active against leukemia and lymphoma lines, with 80% of the AML lines tested showing sensitivity. Treatment of AML cell lines with PTC299 induced differentiation as shown by increased CD14 and/or reduced proliferation. Using isogenic AML lines, we show that PTC299 reduces the proliferation of both p53 wildtype and p53 deficient leukemia calls with similar potency as measured by the concentration of PTC299 required to reduce cell number by 50% (CC50). In cells expressing wildtype p53, PTC299 increases p53 activation. However, p53- wildtype cells undergo increased apoptosis whereas p53-deficience cells undergo necrosis. PTC299 induced a G1/S cell cycle arrest, also independent of p53 status. PTC299 increased H2A.X (a marker of double stranded DNA breaks) in both p53 wildtype and p53 deficient cells. These data suggest that the depletion of nucleotides results in stalling at the replication fork, and subsequent DNA-breaks. Conclusion: De novo pyrimidine nucleotide synthesis is critical for AML survival and proliferation. Depletion of nucleotides results in reduced proliferation, triggering either differentiation and/or cell death. Disclosures Weetall: PTC Therapeutics: Employment. Trotta:PTC Therapeutics: Employment. Baird:PTC Therapeutics: Employment. O'Keefe:PTC Therapeutics: Employment. Furia:PTC Therapeutics: Employment. Borthakur:PTC Therapeutics: Consultancy; Janssen: Research Funding; AbbVie: Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; NKarta: Consultancy; AstraZeneca: Research Funding; Xbiotech USA: Research Funding; Incyte: Research Funding; GSK: Research Funding; Oncoceutics, Inc.: Research Funding; Novartis: Research Funding; Agensys: Research Funding; BMS: Research Funding; Oncoceutics: Research Funding; Cantargia AB: Research Funding; Bayer Healthcare AG: Research Funding; Eisai: Research Funding; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; BioTheryX: Membership on an entity's Board of Directors or advisory committees; Polaris: Research Funding; Merck: Research Funding; Cyclacel: Research Funding; Eli Lilly and Co.: Research Funding; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Arvinas: Research Funding; Tetralogic Pharmaceuticals: Research Funding; Strategia Therapeutics: Research Funding. Spiegel:PTC Therapeutics: Consultancy.

De novo synthesis of pyrimidine nucleotides by Helicobacter pylori

1994 ◽  
Vol 77 (1) ◽  
pp. 1-8 ◽  
Author(s):  
G.L. Mendz ◽  
B.M. Jimenez ◽  
S.L. Hazell ◽  
A.M. Gero ◽  
W.J. O'Sullivan
Keyword(s):  
Helicobacter Pylori ◽  
De Novo ◽  
De Novo Synthesis ◽  
Pyrimidine Nucleotides

Characterization of Monocyte-Associated Factor V

1993 ◽  
Vol 70 (02) ◽  
pp. 273-280 ◽  
Author(s):  
Janos Kappelmayer ◽  
Satya P Kunapuli ◽  
Edward G Wyshock ◽  
Robert W Colman
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Peripheral Blood ◽  
De Novo ◽  
Factor V ◽  
Blood Monocytes ◽  
De Novo Synthesis ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Peripheral Blood Monocytes

SummaryWe demonstrate that in addition to possessing binding sites for intact factor V (FV), unstimulated peripheral blood monocytes also express activated factor V (FVa) on their surfaces. FVa was identified on the monocyte surface by monoclonal antibody B38 recognizing FVa light chain and by human oligoclonal antibodies H1 (to FVa light chain) and H2 (to FVa heavy chain) using immunofluorescence microscopy and flow cytometry. On Western blots, partially cleaved FV could be identified as a 220 kDa band in lysates of monocytes. In addition to surface expression of FVa, monocytes also contain intracellular FV as detected only after permeabilization by Triton X-100 by monoclonal antibody B10 directed specifically to the Cl domain not present in FVa. We sought to determine whether the presence of FV in peripheral blood monocytes is a result of de novo synthesis.Using in situ hybridization, no FV mRNA could be detected in monocytes, while in parallel control studies, factor V mRNA was detectable in Hep G2 cells and CD18 mRNA in monocytes. In addition, using reverse transcriptase and the polymerase chain reaction, no FV mRNA was detected in mononuclear cells or in U937 cells, but mRNA for factor V was present in Hep G2 cells using the same techniques. These data suggest that FV is present in human monocytes, presumably acquired by binding of plasma FV, and that the presence of this critical coagulation factor is not due to de novo synthesis.

Platelets Stimulate Thromboplastin Synthesis in Human Endothelial Cells

1983 ◽  
Vol 49 (02) ◽  
pp. 069-072 ◽  
Author(s):  
U L H Johnsen ◽  
T Lyberg ◽  
K S Galdal ◽  
H Prydz
Keyword(s):  
Endothelial Cells ◽  
De Novo ◽  
Umbilical Vein ◽  
Time Dependent ◽  
De Novo Synthesis ◽  
Human Endothelial Cells ◽  
Tumor Promotor ◽  
Coagulation Assay ◽  
Platelet Aggregates ◽  
Umbilical Vein Endothelial Cells

SummaryHuman umbilical vein endothelial cells in culture synthesize thromboplastin upon stimulation with phytohaemagglutinin (PHA) or the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The thromboplastin activity is further strongly enhanced in a time dependent reaction by the presence of gel-filtered platelets or platelet aggregates. This effect was demonstrable at platelet concentrations lower than those normally found in plasma, it may thus be of pathophysiological relevance. The thromboplastin activity increased with increasing number of platelets added. Cycloheximide inhibited the increase, suggesting that de novo synthesis of the protein component of thromboplastin, apoprotein III, is necessary.When care was taken to remove monocytes no thromboplastin activity and no apoprotein HI antigen could be demonstrated in suspensions of gel-filtered platelets, platelets aggregated with thrombin or homogenized platelets when studied with a coagulation assay and an antibody neutralization technique.

DE NOVO SYNTHESIS OF STEROIDS BY THE TESTES OF THE HUMAN FOETUS AT MIDGESTATION

1971 ◽  
Vol 68 (1_Supplb) ◽  
pp. S135 ◽  
Author(s):  
R. S. Mathur ◽  
N. Wiqvist ◽  
E. Diczfalusy
Keyword(s):  
De Novo ◽  
De Novo Synthesis ◽  
Human Foetus

In vivo study of the biosynthesis of long-chain fatty acids using deuterated water

1995 ◽  
Vol 269 (2) ◽  
pp. E247-E252 ◽  
Author(s):  
H. O. Ajie ◽  
M. J. Connor ◽  
W. N. Lee ◽  
S. Bassilian ◽  
E. A. Bergner ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
De Novo ◽  
Chain Elongation ◽  
Deuterated Water ◽  
De Novo Synthesis ◽  
Isotopomer Distribution ◽  
Long Chain ◽  
Mass Isotopomer

To determine the contributions of preexisting fatty acid, de novo synthesis, and chain elongation in long-chain fatty acid (LCFA) synthesis, the synthesis of LCFAs, palmitate (16:0), stearate (18:0), arachidate (20:0), behenate (22:0), and lignocerate (24:0), in the epidermis, liver, and spinal cord was determined using deuterated water and mass isotopomer distribution analysis in hairless mice and Sprague-Dawley rats. Animals were given 4% deuterated water for 5 days or 8 wk in their drinking water. Blood was withdrawn at the end of these times for the determination of deuterium enrichment, and the animals were killed to isolate the various tissues for lipid extraction for the determination of the mass isotopomer distributions. The mass isotopomer distributions in LCFA were incompatible with synthesis from a single pool of primer. The synthesis of palmitate, stearate, arachidate, behenate, and lignocerate followed the expected biochemical pathways for the synthesis of LCFAs. On average, three deuterium atoms were incorporated for every addition of an acetyl unit. The isotopomer distribution resulting from chain elongation and de novo synthesis can be described by the linear combination of two binomial distributions. The proportions of preexisting, chain elongation, and de novo-synthesized fatty acids as a percentage of the total fatty acids were determined using multiple linear regression analysis. Fractional synthesis was found to vary, depending on the tissue type and the fatty acid, from 47 to 87%. A substantial fraction (24-40%) of the newly synthesized molecules was derived from chain elongation of unlabeled (recycled) palmitate.

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