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 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 A monoclonal antibody to a mitotic microtubule-associated protein blocks mitotic progression.

1990 ◽  
Vol 111 (2) ◽  
pp. 511-522 ◽  
Author(s):  
C Nislow ◽  
C Sellitto ◽  
R Kuriyama ◽  
J R McIntosh
Keyword(s):  
Monoclonal Antibody ◽  
Protein S ◽  
Dependent Manner ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Microtubule Organization ◽  
Cell Extracts ◽  
Mitotic Inhibition ◽  
Specific Localization ◽  
Dose Dependent

A monoclonal antibody raised against mitotic spindles isolated from CHO cells ([CHO1], Sellitto, C., and R. Kuriyama. 1988. J. Cell Biol. 106:431-439) identifies an epitope that resides on polypeptides of 95 and 105 kD and is localized in the spindles of diverse organisms. The antigen is distributed throughout the spindle at metaphase but becomes concentrated in a progressively narrower zone on either side of the spindle midplane as anaphase progresses. Microinjection of CHO1, either as an ascites fluid or as purified IgM, results in mitotic inhibition in a stage-specific and dose-dependent manner. Parallel control injections with nonimmune IgMs do not yield significant mitotic inhibition. Immunofluorescence analysis of injected cells reveals that those which complete mitosis display normal localization of CHO1, whereas arrested cells show no specific localization of the CHO1 antigen within the spindle. Immunoelectron microscopic images of such arrested cells indicate aberrant microtubule organization. The CHO1 antigen in HeLa cell extracts copurifies with taxol-stabilized microtubules. Neither of the polypeptides bearing the antigen is extracted from microtubules by ATP or GTP, but both are approximately 60% extracted with 0.5 M NaCl. Sucrose gradient analysis reveals that the antigens sediment at approximately 11S. The CHO 1 antigen appears to be a novel mitotic MAP whose proper distribution within the spindle is required for mitosis. The properties of the antigen(s) suggest that the corresponding protein(s) are part of the mechanism that holds the antiparallel microtubules of the two interdigitating half spindles together during anaphase.

scholarly journals Inhibition of murine erythroid colony formation in vitro by interferon gamma and correction by interferon receptor immunoadhesin

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 911-915 ◽  
Author(s):  
RT Jr Means ◽  
SB Krantz ◽  
J Luna ◽  
SA Marsters ◽  
A Ashkenazi
Keyword(s):  
Animal Model ◽  
Interferon Gamma ◽  
Colony Formation ◽  
Interleukin 1 ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Ifn Gamma ◽  
Dose Dependent

It has been previously reported that inhibition of human erythroid colony-forming units (CFU-E) in vitro by interleukin-1 (IL-1) is an indirect effect, occurring through the production of interferon gamma (IFN gamma). IFN gamma, in turn, inhibits CFU-E colony formation directly, and its inhibitory effect can be overcome by exposure to high concentrations of erythropoietin (EPO). To develop an in vitro animal model for investigating inhibition of erythropoiesis by IFN gamma, the effects of recombinant murine (rm) IFN gamma on highly purified CFU-E from the spleens of mice infected with the anemia strain of the Friend virus (FVA) were studied. rmIFN gamma inhibited CFU-E colony formation in a dose-dependent manner. This inhibition occurred with large (> or = 8 cell) colonies only; smaller colonies were not affected. The inhibitory effect was corrected to 72% of control by high EPO concentrations of 64 U/mL. Murine CFU-E were then cultured with rmIFN gamma in the presence of a soluble murine IFN gamma receptor fused to the hinge and Fc domains of the human IgG1 heavy chain (mIFN gamma R-IgG). Inhibition of CFU-E colony formation by rmIFN gamma (100 U/mL) was corrected by mIFN gamma R-IgG in a dose-dependent manner, with an approximate IC50 of 0.05 nmol/L, and complete or near complete correction at 0.5 nmol/L. Similarly, a human IFN gamma R-IgG greatly reduced the inhibitory effect of recombinant human IFN gamma on human CFU-E. These experiments provide an in vitro animal model for studying the inhibitory effects of IFN gamma on erythropoiesis and indicate that IFN gamma R-IgG may be a useful agent for reducing the toxicity of IFN gamma in vivo.

scholarly journals Activation effects of a prion protein fragment [PrP-(106-126)] on human leucocytes*

1996 ◽  
Vol 320 (2) ◽  
pp. 563-570 ◽  
Author(s):  
Luisa DIOMEDE ◽  
Silvano SOZZANI ◽  
Walter LUINI ◽  
Marina ALGERI ◽  
Luca DE GIOIA ◽  
...  
Keyword(s):  
Prion Protein ◽  
Human Lymphocytes ◽  
Cellular Prion Protein ◽  
Dependent Manner ◽  
Membrane Microviscosity ◽  
Peripheral Tissues ◽  
Cell Extracts ◽  
Dose Dependent ◽  
O2 Production

Prion-related encephalopathies are characterized by the intracerebral accumulation of an abnormal isoform of the cellular prion protein (PrPC) named scrapie prion protein (PrPSc). The pathological forms of this protein and its cellular precursor are not only expressed in the brain but also, at lower concentrations, in peripheral tissues. We recently showed that a synthetic peptide corresponding to residues 106–126 [PrP-(106–126)] of the human PrP is toxic to neurons and trophic to astrocytes in vitro. Our experiments were aimed at verifying whether PrP-(106–126) and other peptides corresponding to fragments of the amyloid protein purified from brains of patients with Gerstmann–Sträussler–Scheinker disease – namely PrP-(89–106), PrP-(106–114), PrP-(127–147) – were capable of stimulating circulating leucocytes. Native PrP expression in human lymphocytes, monocytes and neutrophils was first confirmed using PCR amplification of total RNA, after reverse transcription, and immunoblot analysis of cell extracts with anti-PrP antibodies. PrP-(106–126), but not the other peptides, increased membrane microviscosity, intracellular Ca2+ concentration and cell migration in circulating leucocytes, and O2-•production in monocytes and neutrophils. Membrane microviscosity was determined by the fluorescence polarization technique, using diphenylhexatriene as a probe, 300 s after the addition of PrP-(106–126) to the cell suspension in the concentration range 5–50 µM. The increase in intracellular Ca2+ elicited by PrP-(106–126) was dose-dependent in the range 5–500 µM. PrP-(106–126) stimulated O2-•production in monocytes and neutrophils in a dose- (10–300 µM) and time-(5–30 min) dependent manner in the presence of 10 µM dihydrocytochalasin B. Both the increase in Ca2+ concentration and the O2-•production were partially sensitive to pertussis toxin. PrP-(106–126) stimulated leucocyte migration in a dose-dependent (30–300 µM) manner and, at the highest concentration used, this migration was comparable with that elicited by 2.5 nM interleukin 8 or 10 nM fMet-Leu-Phe peptide.

scholarly journals Inhibition of murine erythroid colony formation in vitro by interferon gamma and correction by interferon receptor immunoadhesin

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 911-915 ◽  
Author(s):  
RT Jr Means ◽  
SB Krantz ◽  
J Luna ◽  
SA Marsters ◽  
A Ashkenazi
Keyword(s):  
Animal Model ◽  
Interferon Gamma ◽  
Colony Formation ◽  
Interleukin 1 ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Ifn Gamma ◽  
Dose Dependent

Abstract It has been previously reported that inhibition of human erythroid colony-forming units (CFU-E) in vitro by interleukin-1 (IL-1) is an indirect effect, occurring through the production of interferon gamma (IFN gamma). IFN gamma, in turn, inhibits CFU-E colony formation directly, and its inhibitory effect can be overcome by exposure to high concentrations of erythropoietin (EPO). To develop an in vitro animal model for investigating inhibition of erythropoiesis by IFN gamma, the effects of recombinant murine (rm) IFN gamma on highly purified CFU-E from the spleens of mice infected with the anemia strain of the Friend virus (FVA) were studied. rmIFN gamma inhibited CFU-E colony formation in a dose-dependent manner. This inhibition occurred with large (> or = 8 cell) colonies only; smaller colonies were not affected. The inhibitory effect was corrected to 72% of control by high EPO concentrations of 64 U/mL. Murine CFU-E were then cultured with rmIFN gamma in the presence of a soluble murine IFN gamma receptor fused to the hinge and Fc domains of the human IgG1 heavy chain (mIFN gamma R-IgG). Inhibition of CFU-E colony formation by rmIFN gamma (100 U/mL) was corrected by mIFN gamma R-IgG in a dose-dependent manner, with an approximate IC50 of 0.05 nmol/L, and complete or near complete correction at 0.5 nmol/L. Similarly, a human IFN gamma R-IgG greatly reduced the inhibitory effect of recombinant human IFN gamma on human CFU-E. These experiments provide an in vitro animal model for studying the inhibitory effects of IFN gamma on erythropoiesis and indicate that IFN gamma R-IgG may be a useful agent for reducing the toxicity of IFN gamma in vivo.

scholarly journals Effect of 1-amino-oxy-3-aminopropane on polyamine metabolism and growth of L1210 cells

1989 ◽  
Vol 263 (1) ◽  
pp. 215-221 ◽  
Author(s):  
R Poulin ◽  
J A Secrist ◽  
A E Pegg
Keyword(s):  
Polyamine Metabolism ◽  
Reversible Inhibitor ◽  
Dependent Manner ◽  
Intact Cells ◽  
Adenosylmethionine Decarboxylase ◽  
L1210 Cells ◽  
Cell Extracts ◽  
Drug Concentrations ◽  
Dose Dependent

1-Amino-oxy-3-aminopropane (AOAP) was reported to inhibit several mammalian polyamine-biosynthetic enzymes in vitro, including ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) [Khomutov, Hyvönen, Karvonen, Kauppinen, Paalanen, Paulin, Eloranta, Pajula, Andersson & Pösö (1985) Biochem. Biophys. Res. Commun. 130, 596-602]. In order to clarify its mechanism of action in intact cells, the inhibitory properties of AOAP on the growth and polyamine metabolism of L1210 cells were compared with those seen in a variant subline (D-R cells) which overproduces ODC. As little as 20 microM-AOAP completely blocked proliferation of L1210 cells, and this effect was reversed by the concomitant addition of exogenous putrescine or spermidine. Growth of D-R cells was not affected by AOAP at concentrations up to 0.5 mM. There was no difference in the uptake of AOAP between the L1210 and the D-R cells. Exposure of L1210 or D-R cells to AOAP greatly decreased ODC activity in undialysed cell extracts, but did not decrease AdoMetDC. Activities of both enzymes were increased severalfold by AOAP treatment when activity was measured in dialysed extracts. Treatment with AOAP depleted intracellular putrescine and spermidine contents of L1210 cells, while inducing a massive accumulation of decarboxylated AdoMet. The 8-fold higher putrescine pool present in untreated D-R cells was depleted in a dose-dependent manner by AOAP, but a significant decrease in spermidine and accumulation of decarboxylated AdoMet required 10 times higher drug concentrations, and the changes were much less dramatic than in L1210 cells. These results indicate that in L1210 cells AOAP behaves primarily as a reversible inhibitor of ODC.

scholarly journals Nitric oxide-mediated inactivation of mammalian ferrochelatase in vivo and in vitro: possible involvement of the iron-sulphur cluster of the enzyme

1995 ◽  
Vol 310 (2) ◽  
pp. 533-538 ◽  
Author(s):  
T Furukawa ◽  
H Kohno ◽  
R Tokunaga ◽  
S Taketani
Keyword(s):  
Nitric Oxide ◽  
Interferon Gamma ◽  
Biosynthetic Pathway ◽  
Raw 264.7 Cells ◽  
Dependent Manner ◽  
Macrophage Cell ◽  
Dose Dependent

To investigate the role of the iron-sulphur cluster in mammalian ferrochelatases, the terminal enzyme of the haem biosynthetic pathway, we examined the interaction of nitric oxide (NO) and ferrochelatase. When macrophage cell line RAW 264.7 cells were treated with interferon-gamma and lipopolysaccharide NO synthesis in the cells was stimulated, and a decrease in ferrochelatase activity was observed, with no change in the amount of ferrochelatase. The addition of NG-monomethyl-L-arginine, a selective inhibitor of NO synthesis, reduced the effect of interferon-gamma and lipopolysaccharide, while the effect of NG-monomethyl-L-arginine was suppressed by the addition of L-arginine, a substrate of NO synthase. When purified recombinant human ferrochelatase was treated with 3-morpholinosydnonimine, a NO-generating compound, ferrochelatase activity decreased with disappearance of characteristic absorbance spectra of the iron-sulphur cluster. S-Nitroso-N-acetylpenicillamine also reduced the activity, in a dose-dependent manner. These results indicate that ferrochelatase activity can be modulated by NO synthesis probably through disruption of the iron-sulphur cluster. We propose that inactivation of ferrochelatase mediated by NO (or NO-derived species) may play a role in the regulation of haem metabolism.

scholarly journals Widespread intradermal accumulation of mononuclear leukocytes in lepromatous leprosy patients treated systemically with recombinant interferon gamma.

1990 ◽  
Vol 172 (5) ◽  
pp. 1509-1512 ◽  
Author(s):  
C Nathan ◽  
K Squires ◽  
W Griffo ◽  
W Levis ◽  
M Varghese ◽  
...  
Keyword(s):  
Interferon Gamma ◽  
Lepromatous Leprosy ◽  
Concurrent Chemotherapy ◽  
Mononuclear Phagocytes ◽  
Mononuclear Leukocytes ◽  
Dependent Manner ◽  
Recombinant Interferon ◽  
Intradermal Administration ◽  
Leprosy Patients ◽  
Dose Dependent

Intradermal administration of recombinant interferon gamma (rIFN-gamma) to lepromatous leprosy patients has converted the local histology toward a tuberculoid pattern. However, such changes have been confined to the site of injection. In contrast, in the present study, marked, intradermal accumulation of CD3+, CD4+, CD8+, and CD1a+ T cells and Leu-M5+ mononuclear phagocytes was induced at a distance from the sites of administration, in a dose-dependent manner, by 10 daily intramuscular injections of 10-30 micrograms rIFN-gamma/m2. Mononuclear cell infiltration began within 3 d of onset of rIFN-gamma therapy and persisted at least 8 wk. Intramuscular administration of rIFN-gamma to lepromatous patients receiving concurrent chemotherapy can safely induce widespread histologic features of an upgrading reaction.

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