scholarly journals Parallel induction of tetrahydrobiopterin biosynthesis and indoleamine 2,3-dioxygenase activity in human cells and cell lines by interferon-γ

1989 ◽  
Vol 262 (3) ◽  
pp. 861-866 ◽  
Author(s):  
E R Werner ◽  
G Werner-Felmayer ◽  
D Fuchs ◽  
A Hausen ◽  
G Reibnegger ◽  
...  
Keyword(s):  
Cell Lines ◽  
Interferon Gamma ◽  
Interferon Γ ◽  
Human Cells ◽  
Gtp Cyclohydrolase I ◽  
Gtp Cyclohydrolase ◽  
Indoleamine 2,3 Dioxygenase ◽  
Cell Extracts ◽  
Dioxygenase Activity

In all of eight tested human cells and cell lines with inducible indoleamine 2,3-dioxygenase (EC 1.13.11.17) tetrahydrobiopterin biosynthesis was activated by interferon-gamma. This was demonstrated by GTP cyclohydrolase I (EC 3.5.4.16) activities and intracellular neopterin and biopterin concentrations. Pteridine synthesis was influenced by extracellular tryptophan. In T 24-cell extracts, submillimolar concentrations of tetrahydrobiopterin stimulated the indoleamine 2,3-dioxygenase reaction.

scholarly journals Influence of interferon-gamma and extracellular tryptophan on indoleamine 2,3-dioxygenase activity in T24 cells as determined by a non-radiometric assay

1988 ◽  
Vol 256 (2) ◽  
pp. 537-541 ◽  
Author(s):  
E R Werner ◽  
G Werner-Felmayer ◽  
D Fuchs ◽  
A Hausen ◽  
G Reibnegger ◽  
...  
Keyword(s):  
Interferon Gamma ◽  
Dependent Manner ◽  
Indoleamine 2,3 Dioxygenase ◽  
Cell Extracts ◽  
T24 Cells ◽  
Maximum Reaction ◽  
Radiometric Assay ◽  
Radioactive Assay ◽  
Dose Dependent ◽  
Dioxygenase Activity

The indoleamine 2,3-dioxygenase (EC 1.13.11.17) activity in human T24 cells has been investigated in cell extracts by using a non-radioactive assay. It is enhanced in a dose-dependent manner up to 25-fold by interferon-gamma. The maximum reaction velocity is increased rather than the Km, which remains at 4 mumol/l. Induction of activity starts 3 h after stimulation and reaches a plateau at 21-48 h. Decreased stimulation was observed in the presence of high L-tryptophan concentrations.

scholarly journals Different kynurenine pathway enzymes limit quinolinic acid formation by various human cell types

1997 ◽  
Vol 326 (2) ◽  
pp. 351-356 ◽  
Author(s):  
Melvyn P. HEYES ◽  
Cai Y. CHEN ◽  
Eugene O. MAJOR ◽  
Kuniaki SAITO
Keyword(s):  
Quinolinic Acid ◽  
Fetal Brain ◽  
Cell Types ◽  
Interferon Γ ◽  
Kynurenine Pathway ◽  
Human Cells ◽  
Hydroxylase Activity ◽  
Human Fetal Brain ◽  
Indoleamine 2,3 Dioxygenase ◽  
A Cell

Substantial increases in the tryptophan–kynurenine pathway metabolites, L-kynurenine and the neurotoxin quinolinic acid, occur in human brain, blood and systemic tissues during immune activation. Studies in vitrohave shown that not all human cells are capable of synthesizing quinolinate. To investigate further the mechanisms that limit L-kynurenine and quinolinate production, the activities of kynurenine pathway enzymes and the ability of different human cells to convert pathway intermediates into quinolinate were compared. Stimulation with interferon γ substantially increased indoleamine 2,3-dioxygenase activity and L-kynurenine production in primary peripheral blood macrophages and fetal brains (astrocytes and neurons), as well as cell lines derived from macrophage/monocytes (THP-1), U373MG astrocytoma, SKHEP1 liver and lung (MRC-9). High activities of kynurenine 3-hydroxylase, kynureninase or 3-hydroxyanthranilate 3,4-dioxygenase were found in interferon-γ-stimulated macrophages, THP-1 cells and SKHEP1 cells, and these cells made large amounts of quinolinate when supplied with L-tryptophan, L-kynurenine, 3-hydroxykynurenine or 3-hydroxyanthranilate. Quinolinate production by human fetal brain cultures and U373MG cells was restricted by the low activities of kynurenine 3-hydroxylase, kynureninase and 3-hydroxyanthranilate 3,4-dioxygenase, and only small amounts of quinolinate were synthesized when cultures were supplied with L-tryptophan or 3-hydroxyanthranilate. In MRC-9 cells, quinolinate was produced only from 3-hydroxykynurenine and 3-hydroxyanthranilate, consistent with their low kynurenine 3-hydroxylase activity. The results are consistent with the notion that indoleamine 2,3-dioxygenase is an important regulatory enzyme in the production of L-kynurenine and quinolinate. Kynurenine 3-hydroxylase and, in some cells, kynureninase and 3-hydroxyanthranilate 3,4-dioxygenase are important determinants of whether a cell can make quinolinate.

Neopterin and Nitrite in Supernatants from Interferon-γ-treated Monocytoid Cell Lines: A Tool to Identify Bacterial Pyrogens

Pteridines ◽  
1999 ◽  
Vol 10 (3) ◽  
pp. 112-118 ◽  
Author(s):  
Anja Peterbauer ◽  
Ernst R. Werner ◽  
Gabriele Werner-Felmayer
Keyword(s):  
Cell Lines ◽  
Interferon Γ ◽  
Gtp Cyclohydrolase I ◽  
Endogenous Pyrogen ◽  
Gram Positive Bacteria ◽  
Oxide Synthase ◽  
Wall Components ◽  
Necrosis Factor ◽  
Inducible Nitric Oxide

Summary The rabbit pyrogen assay identifies pyrogenic contaminations in drugs i.l1tend~d for parenteral use. In order to replace this test because of ethical and economical reasons, various in vitro methods have been developed in recent years. Here, we summarize our results on optimizing a test based on using interferony- treated monocytoid cell lines from man (THP-l) and mouse (RAW264.7). The read-out of the test is neopterin or nitrite, respectively, which is released into the supernatant in response to bacterial compounds. These are costimuli of the enzymes involved in neopterin and nitrite formation, i.e. GTP cyclohydrolase I and inducible nitric oxide synthase. The test reproducibly detects cell wall components from Gram-negative as well as from Gram-positive bacteria and mycobacteria. Furthermore, DNA and cell-free supernatants containing a number of bioactive but not further characterized proteins, both from Staphylococcus aureus, can be detected. Thus, this test is superior over the only in vitro alternative accepted in certain cases, namely the limulus amebocyte lysate assay. Results obtained by measuring neopterin or nitrite correlate well with formation of the endogenous pyrogen tumor necrosis factor-a, a read-out used by some other cell-based in vitro alternatives to the rabbit pyrogen test. We therefore think that the assay presented here has the potential to reduce or even replace the animal test.

DNA repair replication by soluble extracts from human lymphoid cell lines

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 601-604 ◽  
Author(s):  
Richard D. Wood ◽  
Peter Robins
Keyword(s):  
Dna Repair ◽  
Ultraviolet Light ◽  
Cell Lines ◽  
Lymphoid Cell ◽  
Time Course ◽  
Human Cells ◽  
Repair Synthesis ◽  
Cell Extracts ◽  
Human Lymphoid Cell ◽  
Lymphoid Cell Lines

A system is described in which extracts from human cells can perform repair replication on DNA damaged by ultraviolet light or chemical carcinogens. Whole cell extracts from lymphoid cell lines are incubated with damaged plasmid DNA circles at 30 °C in the presence of ATP and the four deoxynucleoside triphosphates. Repair synthesis is monitored by the incorporation of α-32P-dATP into closed circular plasmid molecules. Analysis of the time course of the reaction suggests that the slowest step in repair is incision, rather than polymerization or ligation. The size of repair patches inserted into ultraviolet-irradiated DNA during a reaction was estimated by substitution of thymidine triphosphate with 5-bromodeoxyuridine triphosphate and sedimentation in alkaline cesium chloride gradients. Patches with heterogeneous sizes of less than 120 bases were observed.Key words: DNA repair, human cells, ultraviolet light, cell extracts.

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